RU2836320C1 - Composition for storage or transportation of mesenchymal stem cells and methods for preparation and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for preparing a composition for storage and transportation of mesenchymal stem cells, where said mesenchymal stem cells are mesenchymal stem cells of the amniotic membrane of the umbilical cord. Also disclosed is a composition for storage and transportation of mesenchymal stem cells. Invention also relates to a method for transporting mesenchymal stem cells and to the use of a composition for storing and transporting mesenchymal stem cells for preparing a pharmaceutical composition for treating a subject suffering from a disease, where the disease is a wound.

EFFECT: invention provides efficient storage and transportation of mesenchymal stem cells.

27 cl, 33 dwg, 5 tbl, 9 ex

Description

LINK TO RELATED APPLICATIONS

This invention claims the benefit of U.S. Provisional Patent Application No. 62/912,368, filed October 8, 2019, the contents of which are incorporated herein by reference for all purposes.

SEQUENCE LISTINGS

This patent application contains a sequence listing in machine-readable form, which is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a composition for storing or transporting mesenchymal stem cells, a method for preparing a composition for storing or transporting mesenchymal stem cells, and methods for using a composition for storing or transporting mesenchymal stem cells. Such methods include a method for transporting mesenchymal stem cells in this composition for storing or transporting, and a method for treating a subject suffering from a disease, where the method includes local administration of mesenchymal stem cells that are stored or transported in this composition for storing or transporting. A single dose of mesenchymal stem cells is also contemplated.

PRIOR ART

Mesenchymal stem cells isolated from the umbilical cord amniotic membrane and their wound healing properties were first reported in U.S. Patent Application No. 2006/0078993 (from which U.S. Patent Nos. 9,085,755, 9,737,568, and 9,844,571 were issued) and the corresponding International Patent Application WO 2006/019357. Since then, umbilical cord tissue has received attention as a source of multipotent cells; due to its wide availability, umbilical cord tissue and, in particular, stem cells isolated from the umbilical cord amniotic membrane (also referred to as "cord-lining stem cells") are considered as an excellent alternative cell source for regenerative medicine. See, Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27. At the same time, a population of such mesenchymal stem cells from the amniotic cord membrane is described in US patent application 20181/27721 or in the corresponding international patent application WO 2018/067071.

The mesenchymal stem cell population described in US Patent Application No. 20181/27721 or the corresponding International Patent Application WO 2018/067071 has the advantage that 99% or more of the stem cells in this population express the three MSC markers CD73, CD90, and CD 105, but do not express CD34, CD45, and HLA-DR. This extremely homogeneous and well-defined cell population thus represents an ideal candidate for clinical trials and cell therapy methods, since it, for example, fully satisfies the criteria generally accepted for human MSCs used for cell therapy, as defined, for example, in Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review", Stem Cell Research & Therapy 2013, 4:66), Vonk et al., Stem Cell Research & Therapy (2015) 6:94, or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75-79. As described in the international patent application WO 2018/067071, this population of mesenchymal stem cells can, for example, be used in its undifferentiated state for wound healing applications such as burn treatment.

However, stem cells, such as the mesenchymal stem cells described above, are generally not used/administered to patients at the site where they are obtained. Often, sufficient cell passages are performed between the collection of the cells and their subsequent use. Thus, there is a need to provide storage or transport compositions that keep the cells viable and healthy for the period of time typically used to transport or store the cells.

Accordingly, the object of the present invention is to provide a composition suitable for storing and/or transporting mesenchymal stem cells that satisfies this need.

SUMMARY OF THE INVENTION

This task is accomplished by means of methods, a composition for storing or transporting mesenchymal stem cells and a single dose having the features set out in the independent claims of the invention.

In a first aspect, the invention provides a method for preparing a composition for storing or transporting mesenchymal stem cells that contains from about 0.5 to about 10 million mesenchymal stem cells, wherein the method comprises

a) suspending mesenchymal stem cells in a predetermined volume of a crystalloid solution, wherein the crystalloid solution comprises from about 0.5% or about 1% to about 5% (w/v) serum albumin, thereby obtaining a first cell suspension,

b) determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a composition containing from about 0.5 to about 10 million mesenchymal stem cells,

c) mixing a determined volume of the first cell suspension with a volume of a liquid carrier, wherein said liquid carrier comprises from about 0.5% or about 1% to about 5% (w/v) serum albumin, and

1) Trolox;

2) Na ⁺ ;

3) K+;

4) Ca ²⁺ ,

5) Mg2 ⁺ ;

6) Cl ^- ;

7) H ₂ PO ₄ ^- ;

8) HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);

9) lactobionate;

10) sucrose;

11) mannitol;

12) glucose;

13) dextran-40;

14) adenosine and

15) glutathione,

thereby obtaining a composition for storing or transporting mesenchymal stem cells comprising from about 0.5 to about 10 million mesenchymal stem cells.

In a second aspect, the invention provides a composition for storing or transporting mesenchymal stem cells obtained by the method defined herein.

In a third aspect, the invention provides a composition for storing or transporting mesenchymal stem cells, which can be obtained by the method defined herein.

In a fourth aspect, the invention provides a method for transporting mesenchymal stem cells, comprising transporting said mesenchymal stem cells in a composition for storing or transporting mesenchymal stem cells as defined herein.

In a fifth aspect, the invention provides a method of treating a subject suffering from a disease, comprising local administration of mesenchymal stem cells that are stored or transported in a composition for storing or transporting mesenchymal stem cells as defined herein.

In a sixth aspect, the invention provides a single dose of mesenchymal stem cells that is prepared using the method defined herein.

Brief description of graphic materials

The invention will be better understood with reference to the detailed description taken in conjunction with the non-limiting examples and drawings, in which:

Fig. 1 shows a technical information sheet from Lonza for Dulbecco's modified Eagle's medium, including the catalog number of DM EM used to prepare an illustrative example of the medium according to the invention (RTT-6) in the experimental section;

Fig. 2 shows the technical information sheet from Lonza for Ham's F12 medium;

Fig. 3 shows a technical information sheet from Lonza for DMEM:F12 (1:1) medium, including the catalog number of DMEM:F12 (1:1) medium used to prepare an illustrative example of a medium according to the invention (PTT-6) in the experimental section;

Fig. 4 shows a technical information sheet from Life Technologies Corporation for the M171 medium, including the catalog number of the M171 medium used to prepare an illustrative example of the medium according to the invention (RTT-6) in the experimental section;

Fig. 5 shows the list of ingredients, including their commercial supplier and catalog number, used in the experimental section to prepare the RTT-6 medium. When RTT-6 medium is used in a GMP (Good Manufacturing Practice) manufacturing process, it does not contain antibiotics in order to meet the US FDA (Food and Drug Administration) manufacturing guidelines for biological substances.

Figure 6 shows the results of flow cytometry experiments in which mesenchymal stem cells isolated from umbilical cord were analyzed for the expression of mesenchymal stem cell markers CD73, CD90, and CD 105. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media, followed by passaging the mesenchymal stem cells in the corresponding media. The following three culture media were used in these experiments: a) 90% (v/v) DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% (v/v) FBS (fetal bovine serum), b) the PTT-4 culture medium described in US Patent Application 2006/0078993 and the corresponding International Patent Application WO 2006/019357, which consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO 2006/019357), and c) the PTT-6 culture medium of the present invention, the composition of which is described herein. In this flow cytometric assay, two distinct samples of the umbilical cord lining mesenchymal stem cell (CLMC) population were analyzed for each of the three culture media used. The results are shown in Fig. 6a through Fig. 6c. In detail, Fig. 6a shows the percentage of isolated umbilical cord lining mesenchymal stem cells that express the stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in DMEM/10% FBS, Fig. 6b shows the percentage of isolated umbilical cord lining mesenchymal stem cells that express the stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in PTT-4, and Fig. 6c shows the percentage of isolated umbilical cord-lining mesenchymal stem cells that express the stem cell markers CD73, CD90, and CD 105 after isolation from umbilical cord tissue and culture in RTT-6.

Fig. 7 shows the results of flow cytometry experiments in which umbilical cord-derived mesenchymal stem cells were analyzed for their expression of stem cell markers (CD73, CD90, CD 105, CD34, CD45, and HLA-DR (human leukocyte antigen-related antigen D) used to determine the suitability of multipotent human mesenchymal stem cells for cell therapy) and compared with the expression of these markers by bone marrow-derived mesenchymal stem cells. For this experiment, amniotic membrane umbilical cord mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in the inventive PTT-6 culture medium, while bone marrow-derived mesenchymal stem cells were isolated from human bone marrow using a standard protocol.

Figure 7a shows the percentage of isolated umbilical cord mesenchymal stem cells that express the stem cell markers CD73, CD90, and CD 105 and do not express CD34, CD45, and HLA-DR after isolation from umbilical cord tissue and culture in PTT-6 medium, whereas Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and do not express CD34, CD45, and HLA-DR.

Fig. 8 shows a schematic of an experiment for comparing different carriers. A first population of the mesenchymal stem cells described herein was grown in cell culture flasks. The number of living mesenchymal stem cells was counted, and then 2 million cells/flask were stored for various periods of time in Plasmalyte-A or HypoThermosol™-FRS. After storage, cells were counted in a sample of less than or equal to 50 μl daily for days 1-5 (total volume of fluid extracted was 250 μl) and checked for viability by staining the cells with trypan blue. In addition, on days 1, 3, and 5, a sample of less than or equal to 80 μl was removed and analyzed. After storage for 1, 3 and 5 days, 100,000 MSCs at each time point were then grown in PTT-6 medium for 48 h, and supernatants were collected for cytokine analysis: PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF and TGFpl by measurement using the FLEXMAP 3D system.

Fig. 9 summarizes the viability data. As can be seen in the left graph, 73% of the total cells (approximately 95%) from the start of storage were still viable 7 days after storage in HypoThermosol™. In contrast, after 7 days of storage in Plasmalyte-A, only 42% of the total cells (approximately 94%) from the start of storage were still viable. All counts are based on recording in duplicate within 10% of each other (according to SOP (standard operating procedures) CR D2.600.1). At the time of counting, the cells stored in HypoThermosol™ were distinctly smaller with smooth and well-defined edges. In contrast, the cells in Plasmalyte-A were in a range of sizes. HypoThermosol™ significantly maintained membrane integrity and presumably survival for 6 days. Similar results are also shown in the graph on the right side.

Fig. 10 shows the results obtained by measuring the cell diameter. The population of mesenchymal stem cells described in this paper had a smaller range of diameters when stored in HypoThermosol™ compared to cells stored in Plasmalyte-A. The comparison was made after 3 days of storage.

Figure 11 shows the concentration of TGF61 in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A after 48 hours of storage. As can be seen in the graph on the right side, the cells secreted approximately the same amount of TGFB1 when stored in HypoThermosol™ as when stored in Plasmalyte-A. Overall, the amount of TGF61 secreted decreased over time (graph on the right side).

Fig. 12 and 13 show control experiments. Here, PDGF-BB and IL-10 concentrations were measured in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 h. Since PDGF-BB or IL-10 are not normally secreted by the mesenchymal stem cell population described herein, neither PDGF-BB nor IL-10 were detected in any of the samples.

Figure 14 shows the VEGF concentration in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 h. As can be seen in the graph on the right side, the cells secreted approximately the same amount of VEGF when stored in HypoThermosol™ as when stored in Plasmalyte-A on day 0. On days 1 and 5, the cells secreted more VEGF when stored in Plasmalyte-A. Notably, when the cells were stored for 3 days, they secreted more VEGF when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, HypoThermosol™ outperformed Plasmalyte-A on day 3 of storage. The more VEGF that is detected, the healthier the culture. Thus, due to the higher secretion of VEGF after 3 days of storage in HypoThermosol™ than during storage in Plasmalyt-A, the cells were healthier in HypoThermosol™ than in Plasmalyt-A. From day 5 onwards, storage in Plasmalyt seems to become more favourable, since at this point in the storage of cells in Plasmalyt-A, they secreted more VEGF. Overall, the amount of secreted VEGF decreased over time (graph on the right side).

Figure 15 shows the PDGF-AA concentration in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 h. As can be seen in the graph on the right side, the cells secreted approximately the same amount of PDGF-AA when stored in HypoThermosol™ as when stored in Plasmalyte-A at day 0. On days 1 and 5, the cells secreted more PDGF-AA when stored in Plasmalyte-A. Notably, when the cells were stored for 3 days, they secreted more PDGF-AA when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, the cells stored in HypoThermosol™ are healthier than the cells stored in Plasmalyte-A after 3 days of storage. From day 5 of storage onwards, Plasmalyte appears to be a more favourable carrier, as from this point onwards, cells stored in Plasmalyte-A secreted more PDGF-AA. Overall, the amount of PDGF-AA secreted decreased over time (graph on the right side).

Figure 16 shows the concentration of Ang-1 in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 hours. As can be seen in the graph on the right side, the cells secreted approximately the same amount of Ang-1 when stored in HypoThermosol™ as when stored in Plasmalyte-A on days 0 and 3. On day 5, the cells secreted more Ang-1 when stored in Plasmalyte-A. Notably, when the cells were stored for 1 day, they secreted significantly more Ang-1 when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, the cells that were stored in HypoThermosol™ appeared healthier than those stored in Plasmalyte-A for at least 48 hours up to 3 days of storage. From day 5 onwards, Plasmalyte appears to be a more favourable carrier, as from this point onwards, cells stored in Plasmalyte-A secreted more Ang-1. Overall, the amount of Ang-1 secreted decreased over time (graph on the right side).

Figure 17 shows the concentration of HGF in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A after 48 hours of storage. As can be seen in the graph on the right side, the cells secreted approximately the same amount of HGF when stored in HypoThermosol™ as when stored in Plasmalyte-A at day 0. On days 3 and 5, the cells secreted more HGF when stored in Plasmalyte-A. Notably, when the cells were stored for 1 day, they secreted significantly more HGF when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, the cells that were stored in HypoThermosol™ appeared to be healthier than the cells that were stored in Plasmalyte-A from at least day 1 (48 hours) to day 3 of storage. From day 3 onwards, Plasmalyte-A appears to be a more favourable carrier, as at time points 3 and 5 days, cells stored in Plasmalyte-A secreted more HGF. Overall, the amount of HGF secreted decreased over time (graph on the right side).

Fig. 18 shows photographs obtained in a preclinical study of the mesenchymal stem cell population of the present invention in pigs. The pigs were induced to become diabetic with streptozotocin at a dose of 120 mg/kg and allowed to recover for 45 days, after which six full-thickness wounds of 5 cm × 5 cm were made on their backs. The pigs (n=2) were treated twice weekly with 10 ⁵ cells/cm ² of the human mesenchymal stem cell population described herein for 4 weeks. Two control pigs were treated with PBS. The wounds were photographed at postoperative day 0 (PODay 0) and every seven days up to postoperative day 35. Wounds were analyzed for surface area size using Image J. By 35 days, addition of the mesenchymal stem cell population described herein resulted in healing of 10 of 12 diabetic wounds (83%) compared to only 3 of 12 (25%) PBS-treated control wounds. The wound healing rate was 0.8 ^cm2 /day for the mesenchymal stem cell population described herein compared to 0.6 ^cm2 /day in control animals, a 33% improvement.

Fig. 19 Technical description of Trolox, available from Tocris.

Fig. 20 shows a data sheet for NaCl available from Sigma Aldrich.

Fig. 21 shows the data sheet for KH2PO4 available from Sigma Aldrich.

Fig. 22 shows the technical description of HEPES from Sigma Aldrich.

In fig. 23 shows the product description of sodium lactobionate from COMBI-BLOCKS.

Fig. 24 shows a description of the sucrose product from Sigma Aldrich.

In fig. Figure 25 shows a product description of mannitol from avantor.

Fig. 26 shows a description of the glucose product from Sigma Aldrich.

In fig. 27 shows a product description of dextran-40 from Sigma Aldrich.

In fig. 28 shows a product description of adenosine from Sigma Aldrich.

In fig. 29 shows a glutathione product description from Sigma Aldrich.

In fig. 30 shows the product description of HypoThermosol™-FRS (HTS-FRS) from STEMCELL Technologies.

In fig. 31 shows a description of the product CaC1 from Sigma Aldrich.

In fig. 32 shows a description of the MgCl product from Sigma Aldrich.

Fig. 33 shows the results of stability testing performed on the umbilical cord lining mesenchymal stem cell population as described herein seeded in the composition of the present invention (Plasmalyte/HSA/HypoThermosol) for up to 3 days. Fig. 33a shows the results of viability testing of MSCs after storage in the composition of the present invention. MSCs were stored at 2-8°C for 1-3 days to simulate the transport and storage of the product prior to application to wounds. The results demonstrate that the cells did not show significant loss of viability for up to 3 days under these conditions. Fig. 33b shows the morphology of MSCs after storage in the composition of the present invention at 2-8°C. MSCs were photographed after removal from Aseptic Technologies (AT)-sealed vials and cultured for 24 hours at 37°C. As can be seen, the cells obtained up to 2 days of refrigerated storage were able to adhere to the tissue culture plates and form typical spindle-shaped structures. After storage for 2.5 days at 2-8°C, the cells showed an increase in spheroid shapes, indicative of cell death. Fig. 33c shows the proliferation and metabolism of MSCs after storage in the composition of the present invention. MSCs from the same cultures analyzed in Fig. 33a were examined for lactate production as an indicator of metabolism and growth over a 48-hour period in culture at 37°C. Lactate is a product of glucose metabolism that has been validated as directly proportional to the cell growth rate of MSCs. Cells stored for 24 hours at 2-8°C were equivalent in metabolism and growth to cells stored for 0 hours, and cells stored for 36 hours exhibited 86% of the control in lactate production. After 72 hours at 2-8°C, the cells showed only 46% of their metabolism during subsequent culture. Figure 33d shows the lactate production of MSCs that were stored for 0, 1, 1.5, 2, 2.5 or 3 days in the composition of the present invention and then measured after 24 hours and 48 hours in culture. It can be seen that the lactate production after 24 hours and 48 hours in MSCs that were stored in the composition of the present invention for 24 hours (day 1) was identical to MSCs that were not stored (day 0). By day 3, lactate production had dropped by 40-45%. Figure 33e shows the cytokine production measured in the same cultures analyzed in Figure 33c after 24 hours at 37°C. According to the metabolism data, the ability of MSCs to produce angiopoietin 1 (Ang-1), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) was within 10-20% relative to controls (day 0) when the cells were stored in the composition of the present invention at 2-8°C for 24 hours. Fig. 33f shows the cytokine production measured in another culture after 24 hours. The results demonstrate that the ability of MSCs to produce VEGF, angiopoietin-1, TGF-β and HGF was maintained when the cells were stored in the composition of the present invention at 2-8°C for 24 hours, but decreased by approximately 50% when stored for more than 2 days.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, in a first aspect the invention relates to a method for preparing a composition for storing or transporting mesenchymal stem cells comprising from about 0.5 to about 10 million mesenchymal stem cells, wherein the method comprises

a) suspending mesenchymal stem cells in a predetermined volume of a crystalloid solution, wherein the crystalloid solution comprises from about 0.5 to about 5% (w/v) serum albumin, thereby obtaining a first cell suspension,

b) determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a composition containing from about 0.5 to about 10 million mesenchymal stem cells,

c) mixing a determined volume of the first cell suspension with a volume of a liquid carrier, wherein said liquid carrier comprises from about 0.5 to about 5% (w/v) serum albumin, and

1) Trolox;

2) Na ⁺ ;

3) K+;

4) Ca ²⁺ ,

5) Mg2 ⁺ ;

6) Cl ^- ;

7) H ₂ PO ₄ ^- ;

8) HEPES;

9) lactobionate;

10) sucrose;

11) mannitol;

12) glucose;

13) dextran-40;

14) adenosine and

15) glutathione,

thereby obtaining a composition for storing or transporting mesenchymal stem cells comprising from about 0.5 to about 10 million mesenchymal stem cells.

Surprisingly, it has been found in the present invention that the use of the composition for storing or transporting mesenchymal stem cells described herein stabilizes the proliferation and metabolism of MSCs during storage/transportation, resulting in increased viability of MSCs for up to 72 hours. For example, after 3 days of storing mesenchymal stem cells in the composition for storing or transporting mesenchymal stem cells according to the present invention, approximately 90% of the cells were still viable (see Fig. 33a). In contrast, after 3 days of storage in ^{Plasmalyte®,} only approximately 66% of the cells were still viable (see examples of hemocytometer measurements and Fig. 9). Thus, the use of the composition for storing or transporting mesenchymal stem cells described herein enables the transportation/storage of stem cells for a certain period of time without significant loss of cell viability. In particular, storage in the composition for storing or transporting mesenchymal stem cells of the present invention for a shorter period of time of 3 days or less appears to be particularly advantageous, since the stem cells generally secrete more factors than after storage in Plasmalyte-A, as described in detail in the experimental section. Furthermore, it was unexpectedly found that using the composition for storing or transporting mesenchymal stem cells described herein provides for recovery of more than 95% of the MSCs from the storage/transport vessel, thereby ensuring that the desired dose of cells can be administered to the patient.

As used herein, the term "transportation" or "transportation" refers to any transportation. Such transportation may be carried out in any vehicle, such as a car, train, and airplane, or by a person carrying/transporting a container containing stem cells in contact with a liquid carrier from one place to another. In one embodiment, the transportation is from the place of interest for producing mesenchymal stem cells (or a population of mesenchymal stem cells, as both terms are used interchangeably herein) to the place of administration of the stem cells (e.g., from a GMP facility where the stem cells, respectively the population of stem cells of interest, are obtained, to the place of administration of the stem cells or the population of stem cells, such as a clinic or a physician's office). However, it is also intended that the term "transportation" refers to storing the cells in the same place for a certain period of time. For example, stem cells can be stored after collection until they are used on a subject in one place. The container in which the stem cells can be stored or transported can be any container suitable for the method of the present invention.

The preparation of a composition for storing or transporting mesenchymal stem cells includes resuspending MSCs in a predetermined volume of a crystalloid solution. In the present invention, any volume of a crystalloid solution suitable for sufficiently resuspending the MSCs can be used as the predetermined volume. For example, the predetermined volume can be in the range of about 0.5 ml to about 15 ml. In one example, the predetermined volume can be in the range of about 1 ml to about 10 ml. In an illustrative example, the predetermined volume of a crystalloid solution can be about 1 ml, about 2 ml, about 3 ml, about 4 ml, or about 5 ml. By resuspending the MSCs in the predetermined volume of a crystalloid solution, a first cell suspension is obtained. Resuspension is typically performed after collection of the mesenchymal stem cells/mesenchymal stem cell population after culturing for pharmaceutical administration.

After determining the concentration of MSCs in the first cell suspension and determining the volume of the first cell suspension required to prepare a composition containing from about 0.5 to about 10 million mesenchymal stem cells, the first cell suspension is mixed with a volume of a liquid carrier. The volume of the first cell suspension mixed with the liquid carrier can be from about 0.5 ml to about 10 ml. In an illustrative example of a certain volume of the first cell suspension with a volume of a liquid carrier, the total volume of the composition for storing or transporting mesenchymal stem cells is about 1 ml. An amount of from 0.5 to about 10 million mesenchymal stem cells is selected for preparing a unit dose that contains from 0.5 to about 10 million mesenchymal stem cells, preferably in a predetermined volume, such as 1 ml, 2 ml, etc. In the present invention, a predetermined volume of a crystalloid solution contains from about 0.1 to about 15 million viable MSCs. In one example, the predetermined volume of crystalloid solution contains from about 0.5 to about 10 million MSCs. In an illustrative example, the composition for storing or transporting mesenchymal stem cells contains about 1 million MSCs, about 2 million MSCs, about 3 million MSCs, about 4 million MSCs, about 5 million MSCs, or about 6 million MSCs. As used herein, the term "about" in relation to the amount of mesenchymal stem cells may mean that the numerical value may vary by a certain percentage. For example, "about" may mean a numerical variation/deviation of ±1% to about ±15%. Thus, "about" may also mean ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. It is obvious to those skilled in the art that such variations occur in the case where, in particular, the composition for storing or transporting mesenchymal stem cells is prepared manually (which is a common approach for preparing such compositions based on living cells) for subsequent storage and/or transport of the composition to an administration site, such as a wound care clinic or a doctor's office.

In the present invention, MSCs can be collected directly from a tissue culture containing MSCs or from a culture of isolated MSCs or a population of MSCs before resuspension in a crystalloid solution. In any case, the culturing of MSCs can be carried out in a cell culture vessel. Therefore, the MSCs used in the present invention can be collected from a cell culture vessel before resuspension of the MSCs in a predetermined volume of a crystalloid solution.

Both the crystalloid solution and the liquid carrier of the present invention are supplemented with serum albumin. Without wishing to be bound by theory, it is believed that serum albumin improves the viability of the mesenchymal stem cells/mesenchymal stem cell population and may also improve the recovery of stem cells from the vessel in which they are stored for transporting the stem cells to the site of administration. The concentration of serum albumin may be the same or different in the crystalloid solution and in the liquid carrier. Preferably, the concentrations of serum albumin are the same in both the crystalloid solution and the liquid carrier. In this context, any concentration of serum albumin that is suitable for improving the viability of MSCs, for example, may be used. For example, both the crystalloid solution and the liquid carrier can comprise from about 0.5% (w/v), from about 0.6% (w/v), from about 0.7% (w/v), from about 0.8 (w/v), from about 0.9% (w/v), or from about 1.0% (w/v) to about 5% (w/v) serum albumin. In one such example, the crystalloid solution and the liquid carrier can comprise from about 1% (w/v) to about 3% (w/v) serum albumin. In an illustrative example, both the crystalloid solution and the liquid carrier comprise about 1% (w/v) serum albumin. Any pharmaceutically acceptable serum albumin can be used herein, for example, bovine or human serum albumin. In an illustrative example, both the crystalloid solution and the liquid carrier may comprise human serum albumin (HSA). The serum albumin used herein is ideally of pharmaceutically acceptable grade. An example of such pharmaceutical grade serum albumin is a 25% (w/v) solution of human serum albumin commercially available under the trade name ^Plasbumin® from Grifols Therapeutics LLC, Clayton, North Carolina, USA.

The crystalloid solution may also contain one or more components suitable for supporting the growth and/or proliferation of MSCs. Such a component may be an inorganic substance such as sodium, potassium, iron, magnesium, zinc, selenium, chloride, or a combination thereof. In one example, the crystalloid solution comprises sodium, potassium, magnesium, and chloride. The crystalloid solution may be a commercially available solution comprising an additional component suitable for supporting the growth and/or proliferation of MSCs. In one example, the crystalloid solution may be Plasmalyte or Ringer's lactate. In the composition of the present invention, the total amount of crystalloid solution may be limited to a specific percentage. For example, a composition for storing or transporting mesenchymal stem cells may contain no more than about 50%, no more than about 40%, no more than about 30%, no more than about 20%, no more than about 10%, or no more than about 5% crystalloid solution. In an illustrative example, a composition for storing or transporting mesenchymal stem cells may contain no more than about 30%, or about 20%, or about 10% Plasmalyte.

The transportation/storage may be carried out for any period of time. For example, the transportation/storage may be carried out for about 7 days or less. It is also envisaged that the transportation/storage may be carried out for about 6, 5, 4, 3, 2, 1 days or less. Thus, the transportation/storage is carried out for about 48 hours or about 24 hours or less.

It is also contemplated that the transport/storage is carried out at any temperature suitable for the method of the present invention. For example, the transport/storage can be carried out at a temperature of from about -5°C to about 15°C. Thus, it is also envisaged that the transport/storage can be carried out at a temperature of from about 2°C to about 8°C. The transport can also be carried out at a temperature of greater than about -5°C, greater than about -10°C, greater than about -15°C, or greater than about -20°C. Additionally, it is contemplated that the transport/storage can be carried out at a temperature of less than 20°C, less than 18°C, less than 15°C, less than 12°C, or less than 10°C.

The method of the present invention also contemplates that the population of stem cells (or mesenchymal stem cells) is stored or transported in any suitable concentration. As noted above, the terms "mesenchymal stem cells" and "mesenchymal stem cell population" may be used interchangeably herein. It is also possible that when reference is made herein to "mesenchymal stem cells", these stem cells refer to the same population of mesenchymal stem cells. For example, all of the mesenchymal stem cells may belong to a population of mesenchymal stem cells in which about 97% or more, about 98% or more, or about 99% or more of the cells thereof express CD73, CD90, and CD105, but do not express CD34, CD45, and HLA-DR. It is noted herein that if the term "carrier" or "liquid carrier" can be used in the context of a solution containing MSCs, Plasmalyte, HSA and Hyothermosol, then it can also mean a composition for storing or transporting mesenchymal stem cells according to the present invention. Thus, the terms "carrier" or "liquid carrier" and "composition for storing or transporting stem cells" can also be used interchangeably if the solution contains MSCs, Plasmalyte, HSA and Hyothermosol. The stem cell populations used herein can, for example, be transported/stored at a concentration of about 70 million cells per 1 ml of carrier, about 60 million cells per 1 ml of carrier, about 50 million cells per 1 ml of carrier, about 40 million cells per 1 ml of carrier, about 30 million cells per 1 ml of carrier, about 20 million cells per 1 ml of carrier, about 10 million cells per 1 ml of carrier, about 5 million cells per 1 ml of carrier, about 4 million cells per 1 ml of carrier, about 3 million cells per 1 ml of carrier, about 2 million cells per 1 ml of carrier, about 1 million cells per 1 ml of carrier, about 0.5 million cells per 1 ml of carrier, about 0.1 million cells per 1 ml of carrier, or less than 0.1 million cells per 1 ml of carrier. Thus, the stem cell population can be transported/stored at a concentration of from about 10 million cells per ml of carrier to about 1 million cells per 1 ml of carrier.

The method of the present invention concerns the transportation/storage of stem cells. In principle, any stem cell can be used in the method of the present invention. One of the characteristic features of stem cells is their ability to self-renew. "Self-renewal" is the ability to undergo multiple cell division cycles while maintaining an undifferentiated state. Methods for testing the ability of cells to self-renew are known to those skilled in the art. For example, self-renewal can be tested by passaging the cells for more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more passages. Passaging involves separating the cells before re-applying them as a single cell suspension. An additional feature of stem cells is their multipotency or pluripotency, as also described herein. In principle, multipotency or pluripotency can be tested by differentiating said stem cells into different lineages.

In particular, the stem cell population used in the method of the present invention may be a population of embryonic stem cells, a population of adult stem cells, a population of mesenchymal stem cells, or a population of induced pluripotent stem cells.

As used herein, an "embryo stem cell population" is a "pluripotent stem cell population". As used herein, a pluripotent cell refers to a cell type that has the ability to self-renew and the ability to differentiate into different cell types. Pluripotent stem cells can differentiate into almost all cells, i.e., cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm. The term "pluripotent stem cell" also encompasses stem cells derived from the inner cell mass of the early embryonic stage known as the blastocyst. It is noted that recent advances in embryonic stem cell research have made it possible to generate new embryonic stem cell lines without destroying embryos, for example by using a blastomere biopsy-based method that does not disrupt the embryo's ability to develop (Klimanskaya (2006) "Embryonic stem cells from blastomeres maintaining embryo viability." Semin Reprod Med. 2013 Jan;31(l):49-55). In addition, there are a large number of established embryonic stem cell lines in the art. Thus, it is possible to work with embryonic stem cells without the need to destroy the embryo. Pluripotent stem cells may be embryonic stem cells that are not obtained by destroying a human embryo. Thus, pluripotent stem cells are embryonic stem cells obtained from an embryo without destroying the embryo.

As used herein, the term "adult stem cell population" is a population of multipotent stem cells. A population of multipotent stem cells may be a source of a limited number of cell types, so that their somatic fate is limited. For example, a neural stem cell may give rise to neural and glial cells. Adult stem cells have the ability to self-renew and may be obtained from any suitable source. For example, an adult stem cell may be obtained from bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, skin and digestive epithelium, cornea, retina, liver, or pancreas.

The population of stem cells used in the method of the present invention may also be a population of mesenchymal stem cells. In this context, it is noted that the culture medium described herein (e.g., PTT-6) allows for the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from the amniotic membrane under conditions that allow for the cellular proliferation of mesenchymal stem/progenitor cells without differentiation of the mesenchymal stem/progenitor cells. Thus, after isolation of mesenchymal stem cells from the amniotic membrane described herein, the isolated population of mesenchymal stem/progenitor cells has the ability to differentiate into a variety of cell types, as described, for example, in U.S. Patent Application 2006/0078993, U.S. Patent 9,085,755, International Patent Application WO 2006/019357, U.S. Patent 8,287,854, or WO 2007/046775. As described, for example, in U.S. Patent Application 2006/0078993, the umbilical cord amniotic membrane mesenchymal stem cells have a spindle shape, express the following genes: POU5fl, Bmi-1, leukemia inhibitory factor (LIF), and secrete activin A and follistatin. The mesenchymal stem cells isolated in the present invention can, for example, differentiate into any type of mesenchymal cells, such as, but not limited to, adipocytes, skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, mucin-producing cells, endocrine gland-derived cells such as insulin-producing cells (e.g., islet beta cells), or neuroectodermal cells. The stem cells isolated according to the method described herein can be differentiated in vitro for subsequent use in differentiating cells for medical purposes. An illustrative example of such an approach is the differentiation of mesenchymal stem cells into insulin-producing islet beta cells, which can then be administered, for example by implantation, to a patient suffering from insulin deficiency, such as diabetes mellitus (in this connection, see also WO 2007/046775). Alternatively, the mesenchymal stem cells described herein can be used in their undifferentiated state for cell therapy, for example for the purpose of wound healing, such as the treatment of wounds or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells according to the invention can either promote wound healing by interacting with the surrounding diseased tissue, or can also differentiate into a corresponding skin cell (see also, for example, WO 2007/046775).

In this context, it is noted that the MSCs may be derived from any mammalian tissue or body compartment/part known to contain MSCs. In illustrative examples, the MSCs may be umbilical cord MSCs, placental MSCs, cord-placental junction MSCs, cord blood MSCs, bone marrow MSCs, or adipose-derived MSCs. The umbilical cord MSCs may be (derived) from any compartment of the umbilical cord tissue that contains MSCs, such as the amnion, perivascular MSCs, Wharton's jelly MSCs, amniotic membrane MSCs, and mixed umbilical cord MSCs, which means MSCs that include stem cells from two or more than two of these compartments. The mesenchymal stem cell population described herein can be isolated and cultured (i.e., obtained) from any umbilical cord tissue, as well as umbilical cord tissue that contains the amniotic membrane (also referred to herein as "umbilical cord lining"). Accordingly, the mesenchymal stem cell population can be isolated from (pieces of) the entire umbilical cord, as described in the experimental section of the present invention application. Thus, this umbilical cord tissue can contain, in addition to the amniotic membrane, any other umbilical cord tissue/compartment. As shown, for example, in Fig. 16 of US Patent Application 2006/0078993 or International Patent Application WO 2006/019357, the amniotic membrane of the umbilical cord is the outermost portion covering the umbilical cord. Additionally, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-poor blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's jelly, a gelatinous substance composed primarily of mucopolysaccharides. Accordingly, the umbilical cord tissue used herein may also contain this one vein, two arteries, and Wharton's jelly. The use of such an intact section of umbilical cord has the advantage that the amniotic membrane does not need to be separated from the other components of the umbilical cord. This allows for fewer isolation steps, and thus the method described herein is simpler, faster, less error-prone, and more cost-effective - all of which are important aspects of GMP manufacturing required for the therapeutic use of mesenchymal stem cells. Thus, the isolation of mesenchymal stem cells may begin with a tissue explant, which may be followed by subculturing (culturing) the isolated mesenchymal stem cells if larger quantities of mesenchymal stem cells are desired, e.g., for use in clinical trials. Alternatively, it is also possible to first separate the amniotic membrane from the other components of the umbilical cord and to isolate the umbilical cord-lining mesenchymal stem cells from the amniotic membrane by culturing the amniotic membrane in a culture medium, e.g., PTT-6. This culturing may also be accomplished by obtaining a tissue explant, possibly followed by passaging the isolated mesenchymal stem cells. In this context, the term "tissue explant" or "process for obtaining a tissue explant" is used in its usual meaning in the art, which means a process in which the tissue after collection or a piece of tissue is placed in a cell culture dish containing a culture (growth) medium, and from which, over time, stem cells migrate from the tissue to the surface of the dish. These primary stem cells can then be further grown and transferred to fresh dishes by micropropagation (subculturing), as also described herein. In this context, it is noted that, from the point of view of obtaining cells for therapeutic purposes, in the first step of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a master bank of isolated mesenchymal stem cells is formed, while in subsequent subculturing a working bank of cells can be obtained. Thus, in specific embodiments, the population of stem cells is a population of mesenchymal stem cells. A population of mesenchymal stem cells can be isolated from the amniotic membrane of the umbilical cord by a method involving culturing the umbilical cord tissue in a culture medium containing DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (Medium 171) and FBS (fetal bovine serum). The use of such a medium makes it possible to isolate a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord in which more than 90%, or even 99% or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90 and CD105, while at the same time, these stem cells do not express CD34, CD45 and HLA-DR (see experimental section), which means that 99% or even more than 99% of the cells of this population express the stem cell markers CD73, CD90 and CD 105, while at the same time, not expressing the markers CD34, CD45 and HLA-DR. Such an extremely homogeneous and well-defined population of cells was first reported in copending U.S. Patent Application No. 15/725,913, filed October 5, 2018 (published as US 2018/127721), which claims priority to U.S. Provisional Patent Application No. 62/404,582, filed October 5, 2017, the contents of which are incorporated herein by reference), and in copending PCT Application PCT/SG2017/050500 (published as WO 2018/067071), also filed October 5, 2018, which claims priority to U.S. Provisional Patent Application No. 62/404,582, filed October 5, 2017, and represents an ideal candidate for clinical trials and cell therapy, since this population of stem cells, for example, fully satisfies the criteria generally accepted for human mesenchymal stem cells used for cell therapy, as defined, for example, in Dominici et al, "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review", Stem Cell Research & Therapy 2013, 4:66), Vonket al., Stem Cell Research & Therapy (2015) 6:94, or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75-79. Also, using a bioreactor such as the Quantum Cell Expansion System, it is possible to obtain large quantities of mesenchymal stem cells, such as 300-700 million mesenchymal stem cells in a single cycle (see also the experimental section). Thus, the present invention provides for the transportation/storage of stem cells in an amount that is necessary for therapeutic use, such as their use in wound healing, in a cost-effective manner. Additionally, all components used to prepare the culture medium of the present invention are commercially available in GMP quality. Accordingly, the present invention opens the way to the transportation/storage of a GMP-derived and highly homogeneous population of mesenchymal stem cells from the amniotic membrane of the umbilical cord.

Thus, in some embodiments, the population of mesenchymal stem cells is a population of isolated umbilical cord amniotic membrane mesenchymal stem cells. It is further contemplated that at least about 90% or more of the cells of the isolated population of mesenchymal stem cells express each of the following markers: CD73, CD90, and CD 105. For example, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated population of mesenchymal stem cells express each of CD73, CD90, and CD105. Additionally or alternatively, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells do not express the following markers: CD34, CD45, and HLA-DR (human leukocyte antigen-related antigen D). In further examples, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the MSCs express each of CD73, CD90 and CD105, while at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the MSCs may not express CD34, CD45 and HLA-DR. In specific examples, about 97% or more, about 98% or more, or about 99% or more of the MSCs express CD73, CD90, and CD 105, but do not express CD34, CD45, and HLA-DR.

The CD73 marker is known to those skilled in the art. In this regard, CD73 belongs to the cluster of differentiation 73, also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase. The sequence of the human CD73 protein may be SEQ ID NO. 1. The CD90 marker is known to those skilled in the art. In this regard, CD90 belongs to the cluster of differentiation 90, also known as thymocyte differentiation antigen 1 (Thy-1). The sequence of the human CD90 protein may be SEQ ID NO: 2. The CD 105 marker is known to those skilled in the art. CD 105 is also known as endoglin (ENG). The sequence of the human CD105 protein may be SEQ ID NO: 3.

If the population of mesenchymal stem cells according to the invention (in particular a population of mesenchymal stem cells in which at least about 98% or 99% express each of the markers CD73, CD90 and CD10 and do not express each of the markers: CD34, CD45 and HLA-DR) is used for clinical trials or as an improved therapeutic application, then a cell population of a working cell bank is typically used for this purpose. As explained, the population of mesenchymal stem cells may not express the following markers: CD34, CD45 and HLA-DR. In this context, it is noted that the marker CD34, CD45 and HLA-DR are known to those skilled in the art. The human CD34 protein may have the sequence of SEQ ID NO. 4. The human CD45 protein may have the sequence of SEQ ID NO: 5. The human HLA-DR protein may have the sequence of SEQ ID NO: 6.

Both the stem cell population from the isolation stage (which may form the master cell bank) and the stem cell population from the subculture stage (which may form the working cell bank) may, for example, be stored in cryopreserved form.

As mentioned above, the method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord according to the present invention has the advantage that all components used in the culture medium according to the invention are available as GMP, and thus provide the possibility of isolating mesenchymal stem cells under GMP conditions for subsequent therapeutic administration.

Thus, the stem cell population may also be a population of induced pluripotent stem cells. As used herein, the term "induced pluripotent stem cells" refers to adult somatic cells that are genetically reprogrammed to a state similar to embryonic stem cells by enhancing the expression of genes and factors important for maintaining the defining properties of embryonic stem cells. Thus, induced pluripotent stem cells can be obtained/generated from a non-pluripotent cell.

Induced pluripotent stem cells represent an important advancement in stem cell research because they provide the ability to generate pluripotent stem cells without the use of embryos. Mouse iPSCs (induced pluripotent stem cells) were first reported in 2006 (Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors". Cell 126 (4): 663-76), and human iPSCs (hiPSCs) were first reported in 2007 (Takahashi et al. (2007) "Induction of pluripotent stem cells from adult human fibroblasts by defined factors." Cell; 131(5):861-72). Mouse iPSCs display important characteristics of pluripotent stem cells, including expression of tumor-forming stem cell markers that contain cells from all three germ layers and are capable of contributing to a variety of tissues when injected into mouse embryos at a very early stage of development. Human iPSCs also express stem cell markers and are capable of giving rise to cells characteristic of all three germ layers. Such stem cell markers may include Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP), and stem cell-specific angigene 3 and 4 (SSEA3/4). Also, the chromatin methylation patterns of iPSCs resemble those of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014) "Induction of pluripotency by defined factors." Proc. Jpn. Acad., 2014, Ser. B 90).

Additionally, iPSCs are capable of self-renewal in vitro and differentiate into all three germ layers. The pluripotency, or ability to differentiate into different cell types, of iPSCs can be tested, for example, by in vitro differentiation into neural or glial cells, or by producing germline chimeric animals via blastocyst injection.

Methods for generating human induced pluripotent stem cells are well known to those skilled in the art and are, for example, described in WO 2009115295, WO 2009144008 or EP2218778. Thus, a person skilled in the art can obtain iPSCs using any method. In principle, induced pluripotent stem cells can be obtained from any adult somatic cell (in a subject). Examples of somatic cells include peripheral blood mononuclear cells (PBMCs) or fibroblasts obtained from skin tissue biopsies.

The present invention relates, inter alia, to a composition for storing or transporting MSCs that is obtainable using the method described herein, as well as to a composition for storing or transporting MSCs that can be obtained using the method described herein. Furthermore, the present invention relates to transporting MSCs, comprising transporting said MSCs in the composition defined herein for storing or transporting mesenchymal stem cells. In this context, the present invention comprises contacting the population of stem cells described herein with a liquid carrier. It is contemplated that in the method of the present invention, the population of stem cells described herein is contacted with the carrier prior to transport/storage. Additionally or alternatively, the population of stem cells is contacted with the carrier after its isolation. How the isolation can be carried out is described in detail herein, as well as in the experimental section. For example, the stem cell population may be contacted with the carrier at about 0 minutes, at about 1 minute, at about 5 minutes, at about 10 minutes, at about 30 minutes, at about 45 minutes, at about 60 minutes, or a longer time after its isolation.

The isolation may include separating the stem cell population from the culture medium, such as from the RTT-6. Suitable methods for such separation are known to those skilled in the art. For example, the separation may be accomplished by centrifuging the stem cells in the culture medium and decanting the culture medium.

The stem cell population is brought into contact with a liquid carrier, wherein the liquid carrier contains

1) Trolox;

2) Na ⁺ ;

3) K+;

4) Ca ²⁺ ,

5) Mg2 ⁺ ;

6) Cl ^- ;

⁷ ) _{H2PO4- ;}

8) HEPES;

9) lactobionate;

10) sucrose;

11) mannitol;

12) glucose;

13) dextran-40;

14) adenosine and

15) glutathione,

Trolox refers to 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, which has the CAS number 53188-07-1. It is a water-soluble analogue of vitamin E and is thought to reduce oxidative stress or damage. Figure 19 shows the technical data sheet for Trolox, available from Tocris. It is also commercially available from Sigma Aldrich (product number: 238813).

Both Na ⁺ and Cl ^- are well-known ions. One skilled in the art knows how to prepare them. For example, these ions can be added to the carrier as a salt, NaCl. GMP grade NaCl can be obtained from Sigma Aldrich. Fig. 20 shows the technical description of NaCl available from Sigma Aldrich.

Ca2 ⁺ and Mg2 ⁺ are also well known ions. One skilled in the art knows how to prepare them. For example, these ions can be added to the carrier in the form of a salt _{of CaCl2} or _MgCl2 . Fig. 31 shows a data sheet for _CaCl2 available from Sigma Aldrich and Fig. 32 shows a data sheet for _MgCl2 available from Sigma Aldrich.

K ⁺ and _H2PO4- ⁽ dihydrogen _phosphate ) are also well known to those skilled in the art. They can be used, for example, in the form _{of KH2PO4} obtained from SigmaAldrich. Fig. 21 shows a technical description _{of KH2PO4} available from Sigma Aldrich.

HEPES, also called 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9), is commonly used as a zwitterionic organic chemical buffering agent. A person skilled in the art also knows how to prepare HEPES, which is commercially available.

For example, one skilled in the art can obtain it from Sigma Aldrich; a corresponding technical description is presented in Fig. 22.

Lactobionate is the carboxylate anion of lactobionic acid. Lactobionate (4-(3-β-galactopyranosyl-B-gluconic acid) is a sugar acid. Lactobionate can be used in various ways. When used as potassium lactobionate, it can, for example, provide osmotic support and prevent cellular swelling, and when combined with sodium, it can perform a preservative function. Alternatively, inorganic salts of lactobionic acid can be used as an inorganic additive. For pharmaceutical use, the antibiotic erythromycin can often be used, among other things, as the erythromycin lactobionate salt. A person skilled in the art also knows how to prepare lactobionate, for example sodium lactobionate (Cas number: 27297-39-8), namely, for example, from COMBI-BLOCKS, see product description in Fig. 23.

Sucrose, also known as D-Glc-(1→2)-β-D-Fru, α-D-glucopyranosyl β-D-fructofuranoside, β-D-fructofuranosyl-α-D-glucopyranoside, D(+)-sucrose or sugar (CAS number 57-50-1), may be other commercially available substances, and a person skilled in the art also knows where to buy it. A corresponding description of the sucrose product from Sigma Aldrich is shown in Fig. 24.

Mannitol is a type of sugar alcohol (CAS registry number: 69-65-8). A person skilled in the art knows how to obtain mannitol. For example, it can be obtained from Avantor. The corresponding product description is presented in Fig. 25.

Glucose (CAS number: 50-99-7) is also well known to those skilled in the art and is commercially available. The corresponding product description from Sigma Aldrich is shown in Fig. 26.

Dextran is a branched glucan consisting of linear α(1→6) linked glucose monomers and α(1→3) linked initiated branches. Dextran ranges from 10,000 to 150,000 kDa. Dextrans are used in a variety of applications as bulking agents, stabilizers, matrix components, binders, lubricants, and physical structure components. Dextran 40 (CAS No. 9004-54-0), used herein as a carrier, is typically used in the development of new and improved preservative solutions for organ transplantation. Dextran 40 can be used to determine cell density and permeability parameters between cell layers. Dextran 40 can also be used as a colloidal agent that expands blood plasma volume. Dextran-40 is commercially available and can be obtained from, among others, Sigma Aldrich (product description is shown in Fig. 27).

Adenosine (CAS number 58-61-7) is a purine nucleoside consisting of an adenine molecule attached to a ribose sugar moiety (ribofuranose) by a β-N ₉ -glycosidic linkage. Adenosine is commercially available from Sigma-Aldrich, among others (see Fig. 28 for the product description).

Glutathione is also known as (2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid. This component is commercially available and can be obtained from Sigma Aldrich, among others (the corresponding product description is presented in Fig. 29).

In principle, any liquid carrier containing the substances listed in 1)-15) above can be used in the method of the present invention. The carrier is a liquid carrier. Thus, it is possible for the substances listed in 1)-15) to be dissolved in a liquid to form a solution/suspension. The liquid can be any suitable liquid. For example, the liquid can be a culture medium, water, a buffer, etc.

The carrier may further comprise pH adjusting buffers, energetic substrates, free radical scavengers and osmotic/oncotic stabilizers, all of which are known to those skilled in the art. Furthermore, the liquid carrier may be serum-free and/or protein-free. The liquid carrier may be free of a dipolar aprotic solvent, such as, for example, DMSO (dimethyl sulfoxide). In particular, the liquid carrier may be the carrier described in WO 2010/064054. This carrier may be HypoThermosol™ or HypoThermosol™-FRS (HTS-FRS). HypoThermosol™-FRS (HTS-FRS) may be purchased from STEMCELL Technologies (according to the product description provided in Fig. 30).

It is further contemplated that the carrier is a transport/storage medium or an excipient. The transport/storage medium may be a natural medium that consists exclusively of naturally occurring biological fluids that additionally contain the substances listed in 1)-15) described herein. The medium may also be a medium containing the substances listed in 1)-15) described herein and with the addition of (additional) nutrients (organic and inorganic), vitamins, salts, gas phases _O2 and _CO2 , blood serum proteins, carbohydrates and/or cofactors. In specific embodiments, the medium does not contain blood serum and/or protein.

The carrier may also be an excipient. An "excipient" is a substance formulated with the active ingredient of a drug. In the method of the present invention, the active ingredient is a population of stem cells.

The carrier may further comprise biocompatible matrices or microcarriers. The matrices or microcarriers may, for example, be biodegradable polymeric substances, most preferably poly(D,L copolymer of lactic acid and glycolic acid) (PLGA)). Alternatively, the matrices or microcarriers may be smooth macroporous or microporous structures containing substances including poly-L-lactide (PLLA), collagen, fibronectin, glycosaminoglycans (GAG), fibrin, starch, cellulose arabinogalactan (larch gum), alginic acid, agar, carrageenan, chitin, hyaluronic acid, dextran, gellan gum, pullulan, hydroxyapatite, polyhydroxyalkanoates (PHA), hydrogels or other self-assembling materials such as peptide-based nanostructured fibrous matrices.

In principle, any number of stem cells can be contacted with any amount of liquid carrier. In this regard, contacting can be accomplished by suspending the stem cell population at a density of about 70 million/mL, about 60 million/mL, about 50 million/mL, about 40 million/mL, about 30 million/mL, about 20 million/mL, about 10 million/mL, about 5 million/mL, about 4 million/mL, about 3 million/mL, about 2 million/mL, about 1 million/mL, about 0.5 million/mL, about 0.1 million/mL, or less than 0.1 million cells in 1 ml of carrier. In some embodiments, contacting is accomplished by suspending the stem cell population at a density of about 10 million/1 ml of carrier.

After contacting the stem cell population with the composition for storing or transporting mesenchymal stem cells, the stem cells contacting the composition for storing or transporting mesenchymal stem cells can be aliquoted into vials in a volume of about 50 ml, about 20 ml, about 10 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, about 1 ml, about 0.5 ml, about 0.25 ml, or less than 0.25 ml of the composition for storing or transporting mesenchymal stem cells. For example, the stem cells contacting the composition for storing or transporting mesenchymal stem cells can be aliquoted into vials in a volume of about 1 ml.

It is further contemplated that the method of the present invention does not include a thawing or freezing step. The latter may be due to the fact that after the selection of the stem cell population, they are transported/stored without the need for freezing and thawing the stem cell population.

The carrier described herein for use in the method for transporting/storing a population of stem cells is particularly suitable for this purpose. One advantage of this carrier is that essentially all of the stem cells transported/stored in it remain viable. A "viable cell" is a cell that is capable of living. A person skilled in the art knows how to detect viable cells. One such method is to stain the cells with trypan blue. Viable cells do not stain with trypan blue.

In this regard, in the method of the present invention, no more than about 50%, about 40%, about 30%, about 20%, about 10%, or less than about 10% of the stem cell population may die during transportation/storage compared to the number/amount of viable stem cells before transportation/storage.

The method of the present invention also assumes that the stem cell populations have any cell diameter after transportation/storage. A person skilled in the art knows how to measure the cell diameter. For example, the cell size/diameter can be determined by recording an image from a microscope and using secondary software to measure the cell diameter. Thus, most of the stem cells in the stem cell population can have a cell diameter of about 9 μm to about 20 μm after transportation/storage. It is also assumed that most of the stem cells in the stem cell population have a cell diameter of about 12 μm to about 16 μm after transportation.

Stem cells that have been transported/stored in the carrier described herein secrete the same proteins/factors as viable stem cells. For example, the method of the present invention contemplates that after transport/storage, the population of (mesenchymal) stem cells can secrete approximately the same amount of TGF beta 1 as before transport/storage. TGF beta 1 (transforming growth factor beta, TGF-β1) is known to those skilled in the art and can comprise the sequence presented in SEQ ID NO. 7. Additionally or alternatively, after transport/storage, the population of (mesenchymal) stem cells can secrete approximately the same amount of VEGF (vascular endothelial growth factor), PDGF-AA (platelet-derived growth factor subunit AA), Ang-1 (angiogenin-1) and/or HGF (hepatocyte growth factor) as before transport/storage. All of VEGF, PDGF-AA, Ang-1 and/or HGF are known to those skilled in the art for their involvement in wound healing. In particular, VEGF may comprise the sequence presented in SEQ ID NO. 8, PDGF-AA may have the sequence presented in SEQ ID NO. 9, Ang-1 may have the sequence presented in SEQ ID NO. 10, while HGF may have the sequence presented in SEQ ID NO. 11. Additionally or alternatively, substantially none of PDGF-BB and/or IL-10 is detectable before and/or after transport. PDGF-BB (platelet-derived growth factor subunit BB) and/or IL-10 (interleukin-10) are also known to those skilled in the art. PDGF-BB may comprise the sequence presented in SEQ ID NO.

12, whereas IL-10 may comprise the sequence presented in SEQ ID NO:

13. The secretion of these factors can be determined by any suitable method, for example by measuring the amount of protein (i.e., for example, PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF or TGFβ1) that the stem cells secrete into the carrier. The amount of protein can be measured using commercially available antibodies/immunoassays automatically using, for example, a system such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA). In this context, it is noted that the involvement of the proteins angiopoietin 1 (Ang-1), TGF-β1, VEGF and HGF in the wound healing process is known to those skilled in the art. With respect to the involvement of angiopoietin 1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4:113 "Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing." For the involvement of hepatocyte growth factor (HGF) in wound healing, particularly chronic/non-healing wound healing, see, for example, Yoshida et al., "Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation. J. Invest. Dermatol. 120:335–343, 2003, Li, Jin-Feng et al. "HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through p 1-Integrin/ILK Pathway." BioMed Research International 2013 (2013): 470–418 or Conway et al, "Hepatocyte growth factor regulation: An integral part of why wounds become chronic". Wound Rep Reg (2007) 15 683–692. For the involvement of HGF in wound healing, in particular chronic/non-healing wound healing, see, for example, Yoshida et al., "Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation. J. Invest. Dermatol. 120:335–343, 2003, Li, Jin-Feng et al. "HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through p 1-Integrin/ILK Pathway." BioMed Research International 2013 (2013): 470–418 or Conway et al, "Hepatocyte growth factor regulation: An integral part of why wounds become chronic". Wound Rep Reg (2007) 15 683–692. vascular endothelial growth factor (VEGF) in wound healing, particularly chronic/non-healing wound healing, see, for example, Froget et al., Eur. Cytokine Netw., Vol. 14, March 2003, 60 64 or Bao et al., "The Role of Vascular Endothelial Growth Factor in Wound Healing" J Surg Res. 2009 May 15; 153(2): 347-358.

For the involvement of transforming growth factor beta (including TGF-β1, TGF-β2 and TGF-β3) in wound healing, particularly chronic/non-healing wound healing, see, for example, Ramirez et al. "The Role of TGFb Signaling in Wound Epithelialization" Advances hi Wound Care, Volume 3, Number 7, 2013, 482-491 or Pakyari et al., Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing, Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

Returning to the culture medium used in the present invention, the culture medium may comprise, for the isolation or culture of umbilical cord lining mesenchymal stem cells, DMEM at a final concentration of about 55-65% (v/v), F12 at a final concentration of about 5-15% (v/v), M171 at a final concentration of about 15-30% (v/v), and FBS at a final concentration of about 1-8% (v/v). As used herein, the value "% (v/v)" refers to the volume of an individual component relative to the final volume of the culture medium. The latter means that if, for example, DMEM is present in the culture medium at a final concentration of about 55-65% (v/v), then 1 liter of the culture medium contains about 550-650 ml of DMEM.

In other embodiments, the culture medium may comprise DMEM at a final concentration of about 57.5-62.5% (v/v), F12 at a final concentration of about 7.5-12.5% (v/v), M171 at a final concentration of about 17.5-25.0% (v/v), and FBS at a final concentration of about 1.75-3.5% (v/v). In further embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v/v), F12 at a final concentration of about 11.8% (v/v), M171 at a final concentration of about 23.6% (v/v), and FBS at a final concentration of about 2.5% (v/v).

In addition to the above components, the culture medium may contain additives that are beneficial for culturing umbilical cord-lining mesenchymal stem cells. The culture medium of the present invention may, for example, contain epidermal growth factor (EGF). If EGF is present, it may be present in the culture medium at a final concentration of about 1 ng/ml to about 20 ng/ml. In some of these embodiments, the culture medium may contain EGF at a final concentration of about 10 ng/ml.

The culture medium may also contain insulin. If insulin is present, it may be present at a final concentration of about 1 μg/mL to 10 μg/mL. In some of these embodiments, the culture medium may contain insulin at a final concentration of about 5 μg/mL.

The culture medium may further comprise at least one of the following additives: adenine, hydrocortisone, and sodium salt of 3,3',5-triiodo-L-thyronine (T3). In such embodiments, the culture medium may comprise all three of adenine, hydrocortisone, and sodium salt of 3,3',5-triiodo-L-thyronine (T3). In these embodiments, the culture medium may comprise adenine at a final concentration of from about 0.05 to about 0.1 μg/mL, hydrocortisone at a final concentration of from about 1 to about 10 μg/mL, and/or sodium salt of 3,3',5-triiodo-L-thyronine (T3) at a final concentration of from about 0.5 to about 5 ng/mL.

In one embodiment, mesenchymal stem cells are grown in a PTT6 medium to obtain the highly purified mesenchymal stem cell population described and used herein. In this context, it is noted that the PTT6 medium described herein is a medium that is prepared by mixing to obtain a final volume of 500 ml of culture medium:

1. 250 ml DMEM

2. 118 mlM171

3. 118 ml DMEM/F12

4. 12.5 ml fetal bovine serum (FBS) to achieve a final concentration of 2.5% (v/v)

5. EGF at a final concentration of 10 ng/ml

6. Insulin at a final concentration of 5 mcg/ml.

7. Insulin 0.175 ml (final concentration 5 mcg/ml).

By "DMEM" is meant Dulbecco's modified Eagle's medium, which was developed in 1969, and is a modification of the basic Eagle's medium (BME) (see Fig. 1 showing the technical description of DMEM, available from Lonza). The original formula of DMEM contains 1000 mg/L glucose and was first used for culturing mouse embryonic cells. DMEM then became a standard cell culture medium that is commercially available from various sources, such as ThermoFisher Scientific (catalog number 11965-084), Sigma Aldrich (catalog number D5546), or Lonza, to name a few suppliers. Thus, any commercially available DMEM can be used in the present invention. In preferred embodiments, the DMEM used herein is the DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g/L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is DMEM medium from Sigma Aldrich, catalog number D5546, which contains 1000 mg/L glucose and sodium bicarbonate, but without L-glutamine.

By "F12" medium is meant Ham's F12 medium. This medium is also a standard cell culture medium and is a nutrient mixture originally developed for culturing a wide variety of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin (see Fig. 2, showing the technical description of Ham's F12 medium from Lonza). Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (catalog number 11765-054), Sigma Aldrich (catalog number N4888), or Lonza, to name a few suppliers) can be used in the present invention. In preferred embodiments, Ham's F12 medium from Lonza is used.

By "DMEM/F12" or "DMEM:F12" is meant a 1:1 mixture of DMEM with Ham's F12 culture medium (see Fig. 3 showing the technical specification for DMEM: F12 (1:1) medium from Lonza). DMEM/F12 (1:1) medium is a widely used basal medium for supporting the growth of a variety of different mammalian cells and is commercially available from various suppliers such as ThermoFisher Scientific (catalog number 11330057), Sigma Aldrich (catalog number D6421) or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In preferred embodiments, the DMEM:F12 medium used herein is DMEM/F12 (1:1) medium available from Lonza under catalog number 12-719F (which is DMEM: F12 with L-glutamine, 15 mM HEPES, and 3.151 g/L glucose).

By "M171" is meant culture medium 171, which was developed as a basic medium for culturing and growing normal human epithelial cells (see Fig. 4 showing the technical specification for medium M171 from Life Technologies Corporation). This basic medium is widely used and is commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In preferred embodiments, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalog number M171500.

"FBS" refers to fetal bovine serum (also referred to herein as "fetal calf serum"), which is the fraction of blood that remains after the blood has undergone natural clotting followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for in vitro eukaryotic cell culture because it has very low levels of antibodies and contains a higher amount of growth factors, providing versatility in a variety of cell culture applications. FBS is preferably obtained from a member of the International Serum Industry Association (ISIA), whose primary focus is the safe use of serum and animal products through proper traceability, proper labeling, and appropriate standardization and oversight. Suppliers of FBS that are members of ISIA include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTD A, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In preferred embodiments, FBS is obtained from GE Healthcare under catalog number A15-151.

As mentioned above, the method for preparing a culture medium for isolating a population of mesenchymal stem cells used in the invention comprises mixing to obtain a final volume of 500 ml of culture medium:

1. 250 ml DMEM

2. 118 mlM171

3. 118 ml DMEM/F12

4. 12.5 ml fetal bovine serum (FBS) to achieve a final concentration of 2.5% (v/v).

As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Thus, 118 ml of DMEM/F12 medium contains 59 ml of DMEM and 59 ml of F12. Accordingly, when using this method of preparing the culture medium, the final concentrations (v/v) per 500 ml of total volume are as follows:

- DMEM: 250 ml + 59 ml = 309 ml, corresponds to 309/500 = 61.8% (vol/vol)

- M171: 118 ml, corresponds to 118/500=23.6% (vol/vol)

- F12: 59 ml, corresponds to 59/500=11.8% (vol/vol).

Embodiments of this method for preparing the culture medium further include the addition of

5. 1 ml of concentrated EGF solution (5 μg/ml) to achieve a final EGF concentration of 10 ng/ml, and

6. 0.175 ml of concentrated insulin solution (14.28 mg/ml) to achieve a final insulin concentration of 5 μg/ml.

It is noted herein that in these embodiments, the above-mentioned volumes of these components 1-6 result in a final volume of 499.675 ml of culture medium. If no additional components are added to the culture medium, the remaining 0.325 ml (to bring the volume to 500 ml) can be, for example, any of the components 1-4, meaning DMEM, M171, DMEM/F12 or FBS. Alternatively, the concentration of the concentrated EGF or insulin solution can of course be adjusted such that the total volume of the culture medium is 500 ml. Additionally, it is also noted that components 1-4 do not necessarily have to be added in the order in which they are listed, but it is of course also possible to use any order of mixing these components to prepare the culture medium of the present invention. The latter means that, for example, M171 and DMEM/F12 can be mixed together and then combined with DMEM and FBS to achieve the final concentration described herein, i.e., a final DMEM concentration of approximately 55 - 65% (v/v), a final F12 concentration of approximately 5 - 15% (v/v), a final M171 concentration of approximately 15 - 30% (v/v), and a final FBS concentration of approximately 1 - 8% (v/v).

In other embodiments, the method further comprises adding to the DMEM a volume of 0.325 ml of one or more of the following additives: adenine, hydrocortisone, sodium salt of 3,3',5-triiodo-L-thyronine (T3), thereby achieving a total volume of 500 ml of culture medium. In this embodiment, the final concentrations of these additives in the DMEM may be as follows:

approximately 0.05 - 0.1 μg/ml adenine, for example, approximately 0.025 μg/ml adenine,

approximately 1-10 mcg/ml hydrocortisone,

approximately 0.5 - 5 ng/ml sodium salt of 3,3',5-triiodo-L-thyronine (T3), for example, 1.36 ng/ml sodium salt of 3,3',5-triiodo-L-thyronine (T3).

In light of the above description, the cell culture medium used herein may be obtained or prepared using the method for preparing the medium described herein.

Additionally, a method for isolating mesenchymal stem cells from umbilical cord amniotic membrane comprises culturing amniotic membrane tissue in a culture medium prepared using the method described herein.

Thus, the present invention also relates to (the use of) a cell culture medium(s) comprising:

- DMEM at a final concentration of approximately 55 - 65% (v/v),

- F12 at a final concentration of approximately 5 - 15% (v/v),

- M171 at a final concentration of approximately 15 - 30% (v/v) and

- FBS at a final concentration of approximately 1 - 8% (v/v).

In some embodiments of the culture medium described herein, the culture medium comprises DMEM at a final concentration of about 57.5-62.5% (v/v), F12 at a final concentration of about 7.5-12.5% (v/v), M171 at a final concentration of about 17.5-25.0% (v/v), and FBS at a final concentration of about 1.75-3.5% (v/v). In other embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v/v), F12 at a final concentration of about 11.8% (v/v), M171 at a final concentration of about 23.6% (v/v), and FBS at a final concentration of about 2.5% (v/v).

Additionally, the culture medium may further comprise epidermal growth factor (EGF) at a final concentration of about 1 ng/mL to about 20 ng/mL. In some embodiments, the culture medium comprises EGF at a final concentration of about 10 ng/mL. The culture medium described herein may further comprise insulin at a final concentration of about 1 μg/mL to 10 μg/mL. In such embodiments, the culture medium may comprise insulin at a final concentration of about 5 μg/mL.

The cell culture medium may further comprise at least one of the following additives: adenine, hydrocortisone, and sodium salt of 3,3',5-triiodo-L-thyronine (T3). In some embodiments, the culture medium comprises all three of adenine, hydrocortisone, and sodium salt of 3,3',5-triiodo-L-thyronine (T3). If these components are present, the culture medium may contain adenine at a final concentration of from about 0.01 to about 0.1 μg/mL adenine or from about 0.05 to about 0.1 μg/mL adenine, hydrocortisone at a final concentration of from about 0.1 to about 10 μg/mL hydrocortisone or from about 1 to about 10 μg/mL hydrocortisone, and/or sodium salt of 3,3',5-triiodo-L-thyronine (T3) at a final concentration of from about 0.5 to about 5 ng/mL.

In embodiments of the cell culture medium, 500 ml of the cell culture medium of the present invention contains:

1. 250 ml DMEM

2. 118 mlM171

3. 118 ml DMEM/F12

4. 12.5 ml fetal bovine serum (FBS) (final concentration 2.5%)

In additional embodiments, the cell culture medium may further comprise

5. EGF at a final concentration of 10 ng/ml, and

6. Insulin at a final concentration of 5 mcg/ml.

Both insulin and EGF can be added to the culture medium using a selected concentrated solution such that the total volume of the culture medium does not exceed 500 ml.

In a specific example, components 1-6 of the culture medium used in the present invention are those indicated in Fig. 5, which means that they are obtained from the respective manufacturers using the catalog number indicated in Fig. 5. The medium that is obtained by mixing components 16 as indicated in Fig. 5 is also referred to herein as "PTT-6". In this context, it is also noted that components 1-6, as well as any other ingredient, such as an antibiotic from any other commercial supplier, can be used in the preparation of the medium of the present invention.

Additionally, the cell culture medium according to the invention may comprise adenine at a final concentration of from about 0.01 to about 0.1 μg/ml adenine or from about 0.05 to about 0.1 μg/ml adenine, hydrocortisone at a final concentration of about 0.1 - 10 μg/ml, from about 0.5 to about 10 μg/ml, or from about 1 to about 10 μg/ml hydrocortisone and/or sodium salt of 3,3',5-triiodo-L-thyronine (T3) at a final concentration of from about 0.1 to about 5 ng/ml or from about 0.5 to about 5 ng/ml.

To obtain the mesenchymal stem cell population described herein, umbilical cord tissue may be cultured until a suitable number of (primary) umbilical cord lining mesenchymal stem cells have expanded from the tissue. In exemplary embodiments, the umbilical cord tissue is cultured until the amniotic membrane mesenchymal stem cell expansion reaches about 70 to about 80% confluency. It is noted herein that the term "confluency" or "confluency" is used in its conventional art meaning with respect to cell culture and is understood to be an estimate/indicator of the amount of adherent cells in a culture dish or flask, indicating the proportion of the surface area covered by cells. For example, 50 percent confluency means that approximately half of the surface area is covered and there is still room for cells to grow. 100 percent confluency means that the surface is completely covered with cells and there is no more space left for the cells to grow as a monolayer.

After obtaining a suitable number of primary cells (umbilical cord-lining mesenchymal stem cells) from the umbilical cord-lining tissue using a tissue explant, these mesenchymal stem cells are removed from the culture container. In this way, a master cell bank containing (primary) isolated amniotic membrane mesenchymal stem cells can be formed. Typically, since the mesenchymal stem cells are adherent cells, removal is performed using standard enzymatic treatment. For example, the enzymatic treatment may include trypsinization as described in US International Patent Application No. 2006/0078993, WO International Patent Application No. 2006/019357, or WO International Patent Application No. 2007/046775, which means that the expanded cells can be collected by trypsinization (0.125% trypsin/0.05% EDTA) for further expansion. After collecting the mesenchymal stem cells, for example, for use in forming a master cell bank, these cells can also be cryopreserved and stored for further use, as explained herein below.

After collection, the mesenchymal stem cells can be transferred to a subculture container. Subculture can also be initiated from frozen primary cells, i.e., from a master cell bank. For subculture, any suitable number of cells can be seeded into a culture container, such as a cell culture plate. For this purpose, the mesenchymal stem cells can be suspended in a suitable medium (typically PTT-6 culture medium) for subculture at a concentration of, for example, from about 0.5×10 ⁶ cells/mL to about 5.0×10 ⁶ cells/mL. In one embodiment, the cells are suspended for subculture at a concentration of about 1.0×10 ⁶ cells/mL. Subculturing can be carried out by culturing in simple culture flasks, but also, for example, in a multi-layer system such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA), which can be installed in incubators. Alternatively, subculturing can also be carried out in a closed system such as a bioreactor. Various bioreactor designs are known to those skilled in the art, such as plate-parallel, hollow fiber or microfluidic bioreactors, see, for example, Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review" above. An illustrative example of a commercially available hollow fiber bioreactor is the ^Quantum® Cell Expansion System (Terumo BCT, Inc), which is used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell population of the present invention is the Xuri Cell Expansion System, available from GE Heathcare. Cultivation of a mesenchymal stem cell population in an automated system such as the ^Quantum® Cell Expansion System is particularly advantageous when a working cell bank for therapeutic use must be generated under GMP (Good Manufacturing Practice) conditions and large quantities of cells are required.

Subculturing of the umbilical cord mesenchymal stem cells described herein is performed in a culture medium described herein, such as PTT-6 medium. Accordingly, a culture medium such as PTT-6 can be used to isolate mesenchymal stem cells from the amniotic membrane and then culture the isolated primary cells by subculturing. Also, for subculturing, the mesenchymal stem cells can be cultured to a suitable cell number. In exemplary embodiments, the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach about 70 to about 80% confluence.

The isolation/cultivation of the population of umbilical cord mesenchymal stem cells can be carried out under standard conditions for culturing mammalian cells. Typically, the method according to the invention for isolating a population of umbilical cord mesenchymal stem cells is typically carried out under conditions (temperature, atmosphere) that are typically used for culturing cells of the species from which the cells are obtained.

For example, human umbilical cord tissue and umbilical cord-lining mesenchymal stem cells, respectively, are typically grown at 37°C in an air atmosphere containing 5% _CO2 . In this context, it is noted that the mesenchymal cells can be obtained from any mammalian species, such as a mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, with mesenchymal stem cells of human origin being preferred in one embodiment.

Once the desired/appropriate number of umbilical cord lining mesenchymal stem cells have been obtained from the subculture, the mesenchymal stem cells can be collected by removing them from the container used for subculture. Collection of the mesenchymal stem cells is typically again accomplished by enzymatic treatment involving trypsinization of the cells. The isolated mesenchymal stem cells are then collected and used directly or stored for further use. Preservation is typically accomplished by cryopreservation. The term "cryopreservation" is used herein in its usual meaning to describe the process by which the mesenchymal stem cells are preserved by cooling to sub-zero temperatures, such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation may be accomplished as known to one skilled in the art and may include the use of cryopreservatives such as dimethyl sulfoxide (DMSO) or glycerol, which retard the formation of ice crystals in umbilical cord cells.

The isolated population of umbilical cord lining mesenchymal stem cells, which is a population prepared using the isolation method described herein, is well defined and homogeneous. In typical embodiments of the method, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105. Additionally, in these embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may not express the following markers: CD34, CD45, and HLA-DR. In particular embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cell population express CD73, CD90, and CD105, but do not express CD34, CD45, and HLA-DR.

Thus, in light of the above description, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD 105. In preferred embodiments, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population are CD73+, CD90+ and CD105+, which means that this percentage of the isolated cell population expressing each of CD73, CD90 and CD105 can be used herein (see the experimental section of the present invention application). Additionally, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may not express the following markers. In particular embodiments, about 97% or more, about 98% or more, or about 99% or more of the cells of the isolated mesenchymal stem cell population express CD73, CD90, and CD 105, but do not express CD34, CD45, and HLA-DR. Such a highly homogeneous population of mesenchymal stem cells derived from the amniotic cord membrane was first reported in U.S. Provisional Patent Application No. 62/404,582, filed October 5, 2016, and in co-pending U.S. Patent Application No. 15/725,913, filed October 5, 2017, and in co-pending PCT application PCT/SG2017/050500, also filed October 5, 2017, and meets the criteria for mesenchymal stem cells intended for use in cell therapy (see also the experimental section and, e.g., Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review," above). In this context, it is noted that this population of mesenchymal stem cells may be a population obtained using the isolation method of the present invention, and also, if necessary, using a different method, such as cell sorting.

A method for preparing a culture medium for isolating the mesenchymal stem cells described herein may include mixing to obtain a final volume of 500 ml of culture medium:

1. 250 ml DMEM

2. 118 mlM171

3. 118 ml DMEM/F12

4. 12.5 ml fetal bovine serum (FBS) to achieve a final concentration of 2.5% (v/v).

As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium.

Thus, 118 ml of DMEM/F12 medium contains 59 ml of DMEM and 59 ml of F12. Accordingly, when using this method of preparing the culture medium, the final concentrations (v/v) per 500 ml of total volume are as follows:

DMEM: 250 ml + 59 ml = 309 ml, equals 309/500 = 61.8% (vol/vol)

Ml71: 118 ml, corresponds to 118/500=23.6% (vol/vol)

F12: 59 ml, corresponds to 59/500=11.8% (vol/vol).

The present invention also relates to a method of treating a subject suffering from a disease, wherein the method comprises locally administering to the subject mesenchymal stem cells that are stored or transported in a solution for storing or transporting mesenchymal stem cells, or a population described herein, wherein the mesenchymal stem cells or population of stem cells are administered within about 96 hours of collecting the population of mesenchymal stem cells. This method of treating a subject can be carried out as described in international patent application WO 2019/199229 "A Method Of Transporting Mesenchymal Stem Cells By Means Of A Transporting Solution And A Method Of Administering Stem Cells To Wounds", published after the priority date of this PCT invention application and incorporated herein in its entirety for all purposes.

Similarly, the present invention also relates to the mesenchymal stem cell population described herein for use in a method of treating a disease in a subject, wherein the mesenchymal stem cell population is administered locally within about 96 hours of harvesting the mesenchymal stem cell population.

The subject to be treated may be any suitable subject. The subject may be a vertebrate animal, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, dogs, horses, mice, and rats. The mammal may also be a human, a dog, a cat, a cow, a pig, a mouse, a rat, etc. Thus, in one embodiment, the subject is a vertebrate animal. The subject may also be a human subject. Thus, the subject may be a subject in need of treatment. Such a subject may suffer from a disease described herein. In some embodiments, the subject suffers from type I or type II diabetes with chronic foot ulcers. Preferably, the subject is negative for HLA antibodies against a population of mesenchymal stem cells.

The mesenchymal stem cell population may be administered in any dose. The dose may be therapeutically effective. A "therapeutically effective amount/dose" may vary depending on factors including, but not limited to, the activity of the cells used, the stability of the cells in the patient's body, the severity of the conditions to be alleviated, the age and sensitivity of the patient being treated, adverse events, etc., as will be understood by one skilled in the art. The amount of administration may be adjusted according to changes in various factors over time.

The dose in which the mesenchymal stem cells are used can also be a single dose. For example, the population of mesenchymal stem cells can be used in a single dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells, or less than 0.25 million cells. In one example, the mesenchymal stem cells can be used in a dose of about 3, about 5, or about 10 million cells. In a particular embodiment, the population of mesenchymal stem cells is used in a dose of about 10 million cells.

The mesenchymal stem cells may be administered multiple times to the same subject. For example, the stem cells may be administered once, twice, three times, or more than three times per week. In principle, any single dose of mesenchymal stem cells may be administered as many times as are appropriate to treat or alleviate the disease. For example, the mesenchymal stem cell population may be administered once, twice, three times, or more than three times per week. The mesenchymal stem cell population may also be administered for one, two, three, four, five, six, seven, eight, nine, ten, eleven weeks, or more.

Thus, a single dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells or less than 0.25 million cells is administered once or twice a week. A single dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells or less than 0.25 million cells can also be administered once or twice a week for a period of three weeks, four weeks or five weeks, or six weeks, or seven weeks, or eight weeks or ten weeks or more weeks.

In the method of treatment according to the present invention, it is also contemplated that the mesenchymal stem cells or the population of mesenchymal stem cells are applied at a dose of from about 1000 cells/cm ² to about 5 million cells/cm ² . As used herein, the term cm ² means the area of the wound/skin to which the stem cells are applied. It is also contemplated that the population of mesenchymal stem cells is applied at a dose of about 100,000 cells/cm ² , 300,000 cells/cm ² or 500,000 cells/cm ² . The population of mesenchymal stem cells can also be applied twice a week for about 8 weeks at a dose of about 100,000 cells/cm ² , about 300,000 cells/cm ² or about 500,000 cells/cm ² .

The mesenchymal stem cell population is administered within about 96 hours of collection of the mesenchymal stem cell population. The method of collection is described herein. It is also possible that the mesenchymal stem cells or the mesenchymal stem cell population are used within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours or less of collection of the mesenchymal stem cell population. Between collection and use, the mesenchymal stem cell population can be transported or stored in a composition for storing or transporting mesenchymal stem cells as described herein. Thus, the aspects described for transporting/storing in a composition for storing or transporting mesenchymal stem cells according to the present invention, with appropriate amendments, equally apply to a method of treating a subject comprising administering MCS that have been stored in a composition for storing or transporting mesenchymal stem cells according to the present invention.

The method of treating a subject according to the present invention serves to alleviate a disease from which the subject suffers. In principle, any disease that can be treated using the mesenchymal stem cell population described herein is meant herein. In particular, the disease can be a skin disease or a wound. The wound can be caused by any cause, such as a burn, a bite, an injury, a surgical intervention or a disease. The wound can also be caused by a diabetic disease. Thus, the wound can also be a diabetic wound. The wound can also be an ulcer caused by diabetic foot syndrome. It is noted that the mesenchymal stem cell population can, for example, be placed directly on a wound, such as a burn or a diabetic wound (see international patent application WO 2007/046775).

According to the present document, from the collection of the mesenchymal stem cell population described herein to their application to the subject, the cells may be transported/stored in the carrier defined herein. Thus, the method of treating a subject according to the present invention may also include the step of separating the mesenchymal stem cell population from the carrier before administering the mesenchymal stem cell population to the subject. One skilled in the art knows how to perform the separation of cells from the carrier. For example, separating the mesenchymal stem cell population from the carrier may include centrifugation. Additionally or alternatively, separating the mesenchymal stem cell population from the carrier may include extracting the cell population from the vial using a syringe.

After separating the stem cells from the composition for storing or transporting mesenchymal stem cells, or after collecting the mesenchymal stem cells, or after obtaining the mesenchymal stem cell population described herein by any other method, these cells are applied to the subject locally. In principle, any route of local administration is contemplated herein. The mesenchymal stem cell population can be administered by means of a syringe. However, it is also possible to contact the mesenchymal stem cells with a cream, ointment, gel, suspension or any other suitable substance before applying the mesenchymal stem cells to the subject. The mesenchymal stem cell population can be held in place by a film or bandage after application to the subject. An example of such a film or bandage can be a dressing, such as a ^Tegaderm® dressing, and an elastic bandage for covering the ^Tegaderm® dressing. For additional distribution of the cells, the area of application can be gently massaged.

The present invention also relates to a single dose of mesenchymal stem cells that are obtained or obtainable using the method described herein. For example, a single dose can contain about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells, or less than 0.25 million cells of the mesenchymal stem cell population described herein in a volume of 1 ml.

It is also contemplated that a single dose contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.25, or about 0.1 million cells. In one example, a single dose may contain about 1 million, about 3 million, or about 5 million cells. Preferably, a single dose contains about 10 million cells. It is further contemplated that a single dose contains from about 1000 cells to about 5 million cells. A single dose may be administered at a dose of about 100,000 cells, 300,000 cells, or 500,000 cells. The single dose described herein may be administered topically. For example, a single dose may be administered topically to 1 cm ² .

A single dose may be applied once, twice, three times or more than three times per week. For example, a single dose may be applied for one, two, three, four, five, six, seven, eight, nine, ten, eleven weeks or more. A single dose containing about 100,000 cells, about 300,000 cells or about 500,000 cells may be applied twice per week for 8 weeks, preferably per 1 cm ² .

The single dose may be contained in any suitable container. For example, the single dose may be contained in a 1 ml vial. In such cases, for example, 0.1 ml from the vial may be applied to the subject, preferably per 1 cm ² . The single dose may alternatively be contained in a syringe.

A single dose of cells of the present invention may be in contact with a liquid carrier as defined herein. In this case, the mesenchymal stem cells are then separated from the carrier prior to administration. For example, the cells may be centrifuged and isolated prior to administration to a subject. The carrier may comprise or be any carrier described herein, such as HypoThermosol™ or Hypothermosol™-FRS.

The single dose of the present invention may comprise umbilical cord MSCs. As described above, umbilical cord MSCs may be (obtained) from any compartment of the umbilical cord tissue that contains MSCs. Thus, the single dose may comprise amnion MSCs, perivascular MSCs, Wharton's jelly MSCs, umbilical cord amniotic membrane MSCs. The umbilical cord amniotic membrane MSCs may be well-defined and homogeneous. Thus, in one embodiment, the present invention uses a single dose that may comprise MSCs as described in international patent application WO 2018/067071. Thus, in typical examples of the method, a single dose may comprise MSCs exhibiting at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more MSCs expressing each of the following markers: CD73, CD90, and CD105. In addition, a single dose can comprise MSCs exhibiting at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the MSCs that do not express the following markers: CD34, CD45, and HLA-DR. In specific examples, a single dose comprises about 97% or more, about 98% or more, or about 99% or more of the MSCs expressing CD73, CD90, and CD105, but not expressing CD34, CD45, and HLA-DR. In further examples, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the MSCs express each of CD73, CD90 and CD105, while at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the MSCs may not express CD34, CD45 and HLA-DR. In specific examples, about 97% or more, about 98% or more, or about 99% or more of the MSCs express CD73, CD90, and CD 105, but do not express CD34, CD45, and HLA-DR.

The method of treatment and the single dose of the present invention may include the use of viable cells. The present document describes how viability can be tested.

The invention will be further illustrated by means of the following non-limiting experimental examples.

The sequences used in this document are presented in Table 1 below.

Experimental examples

1. Cryopreservation of umbilical cord tissue before isolation of mesenchymal stem cells

Umbilical cord tissue (umbilical cords were obtained with informed consent from the mother) was processed for subsequent isolation of mesenchymal stem cells from the amniotic membrane of the umbilical cord as follows.

1.1 Washing the umbilical cord tissue sample:

a. The protective film is removed with a scalpel.

b. The cord is held securely with tweezers and the cord is cut into 10 cm long pieces using a scalpel. Unused cord is placed back into the original tissue cups.

c. Transfer 10 cm pieces of umbilical cord to a new 150 mm culture dish. The 150 mm culture dish can be used instead of the cups.

g.Use the lid of a 150mm culture dish as a storage place for tweezers and scalpel.

d. Using a 30 ml syringe, remove 25 ml of Plasmalyte A (Baxter, catalog number 2B2543Q). Hold the syringe at a 45° angle using one hand and apply the Plasmalyte A directly onto the umbilical cord tissue.

e. Hold the culture dish at a slight angle to remove Plasmalyte A using a 30 ml syringe and blunt needle.

g. Used Plasmalyte A is collected in a 300 ml carrying container, which serves as a waste container and is disposed of in a biohazard waste tank.

h. If necessary, repeat the washing procedure using a new culture dish for each wash. Ensure that all blood clots are removed from the surface. Additional Plasmalyte A may be used to cleanse the tissue if necessary.

i. Place the tissue in a new labeled tissue culture dish to continue cutting the tissue. Place 20 ml Plasmalyte A in the dish to prevent the tissue from drying out while cutting.

k. The umbilical cord is cut into equal slices approximately 1 cm thick, resulting in a total of 10 slices.

l. Additionally, cut each of the 1 cm thick slices into smaller pieces measuring approximately 0.3 cm x 0.3 cm to 0.5 cm x 0.5 cm per slice.

m. All remaining Plasmalyte A is removed from the cup.

n. Using a 30 ml syringe, add 25 ml of Plasmalyte A from the original Plasmalyte A container and distribute directly onto the pieces of umbilical cord tissue.

o. Hold the culture dish at an angle to collect all of the Plasmalyte A used to wash the tissue on one side and remove it using a syringe and blunt needle.

p. Rinsing is repeated once more. No clots should remain.

NOTE: If the umbilical cord is not to be frozen immediately, the umbilical cord tissue should be kept in Plasmalyte A until ready to freeze.

1.2 Cryopreservation of umbilical cord tissue:

a. Prepare a solution for cryopreservation:

1. Prepare 50 ml of freezing solution consisting of 60% Plasmalyte A, 30% 5% human serum albumin, and 10% dimethyl sulfoxide (DMSO).

2. Label the 150 ml transfer container with "Tissue Freezing Solution" and attach the plasma transfer accessories to the port using aseptic technique.

3. Remove 30 ml Plasmalyte A using a 30 ml syringe from the original Plasmalyte A container and transfer it to a transfer container labeled “tissue freezing solution” with the time and date the solution was prepared.

4. Remove 15 ml of 5% human serum albumin using a 20 ml syringe and transfer it to a labeled transfer container.

5. Add 5 ml DMSO to the transfer container.

b. Mix well and record the mixing of the freezing solution.

6. Plasmalyte A is removed from the tissue before adding the freezing solution.

c. Using a 60 ml syringe, withdraw all 50 ml of freezing solution into the syringe, and add approximately 30 ml of freezing solution to the 150 mm cell culture dish containing the umbilical cord tissue. A blunt needle is placed on the syringe to maintain sterility.

g. Stir the contents of the culture dish containing the tissue and freezing solution every minute for 10 minutes.

d. Using tweezers, randomly select 8 sections and place them in each of four 4 ml cryovials. Randomly select 4 sections and place them in one 1.8 ml cryovial. These sections should not contain blood clots.

e. Fill each of the cryovials containing the umbilical cord tissue with the remaining freezing solution to the 3.6 mL fill line for 4 mL tubes and the 1.8 mL line for the 1.8 mL Nunc vial.

g. Label one bottle of Bactec Lytic/10 - Anaerobic/F and one bottle of Bactec Plus Aerobic/F with a tissue identification number (ID).

h. Using a syringe and blunt needle, remove 20 ml of freezing solution from the culture dish, then wipe the Bactec vials with an alcohol wipe, replace the blunt needle with an 18g needle, and inoculate the aerobic and anaerobic Bactec vials with 10 ml of each solution.

i. Start the freezer with a controlled freezing rate.

k. After controlled rate freezing is completed, the pieces are placed in a continuously temperature controlled liquid nitrogen freezer for further use.

2. Isolation of umbilical cord-lining mesenchymal stem cells from umbilical cord tissue

2.1. Preparation of media for the isolation of MSCs from umbilical cord tissue:

a. To prepare 500 ml of RTT6 (culture/growth medium), add the following components in the indicated sequence:

1. DMEM, 250 ml

2. M171 118 ml

3. DMEMF12 118 ml

4. FBS 12.5 ml (final concentration 2.5%)

5. EGF 1 ml (final concentration 10 ng/ml)

b. Insulin 0.175 ml (final concentration 5 mcg/ml)

The above volumes of components 1-6 result in a final volume of 499.675 ml of culture medium. If no other components are added to the culture medium, then the remaining 0.325 ml (added to a volume of 500 ml) can, for example, be any of components 1-4, i.e., DMEM, M171, DMEM/F12, or FBS. Alternatively, the concentration of the concentrated EGF or insulin solution can of course be adjusted so that the total volume of the culture medium is 500 ml. Alternatively, a concentrated solution of an antibiotic, such as penicillin-streptomycin-amphotericin, can be added, resulting in a final volume of 500 ml. It is also possible to add to the culture medium a volume of 0.325 ml of one or more of the following additives: adenine, hydrocortisone, sodium salt of 3,3',5-triiodo-L-thyronine (T3), thus achieving a total volume of 500 ml of culture medium.

7. The vial is labeled "RTT6" with the date of preparation of the media, the operator's initials, and the phrase "best before" followed by the expiration date. The expiration date is the shortest expiration date of any of the components or 1 month from the date of preparation, whichever comes first.

b. To prepare wash media (Hanks' buffered saline solution (HBSS) without calcium or magnesium and with 5% FBS), add 2.5 ml FBS to 47.5 ml HBSS in a 50 ml centrifuge tube. Label the tube "Wash Media" with the operator's initials and the date the media was prepared.

c. All media are tested for sterility using Bactec Lytic/10 - Anaerobic/F (Becton Dickinson & Company) and Bactec Pluc+Aerobic/F (Becton Dickinson & Company). 20 ml of the prepared media are injected into each vial.

2.2 Thawing of umbilical cord tissue for MSC collection:

a. Once the operator is ready to process the specimen in the clean room, initiate thawing. Do not thaw more than 1 vial at a time unless the vials contain material from the same donor.

b. Wipe the water bath with disinfectant, then with 70% isopropanol and fill with 1 liter of sterile water. Heat the water bath to 36-38°C.

c. In a clean room, prepare 10 ml of washing medium consisting of 70% - 90% Plasmalyte A inside a biological safety cabinet. The solution is sterile filtered through a 0.2 µm syringe filter attached to a 10 ml syringe, and the solution is kept frozen until use.

g.A 50 ml conical tube is labeled according to the processing process.

d. Make sure that the temperature of the water bath is 36-38°C.

e. Remove tissue vial(s) from liquid nitrogen storage and rapidly thaw in a 37°C water bath filled with 1 L of sterile water. The Mr. Frosty Nalgene Cryo 1°C Freezing Container Vial Holder floats with vials in one place and can be used as a floating rack when thawing samples.

f. Remove the vial from the water bath and spray it with 70% isopropanol solution. The correct time to remove the vial from the water bath is when small ice crystals can be seen floating in the vial, indicating that the internal temperature of the vial is less than 37°C.

z. The vial is passed through the transfer window and a specialist is called for processing in a clean room.

2.3 Preparation of fabric for processing:

a. Umbilical cord tissue processing should be performed in a clean room with controlled environment (CE): complete cleaning of the room and exhaust at the end of each step.

b. Prepare/clean the biological safety cabinet.

c. Viable particle counting is performed while working in a biological safety cabinet.

g. All necessary consumables are collected in a biological safety cabinet, each of which is checked for integrity of packaging and expiration dates. When working with dispensers, serological pipettes, sterile tweezers, scalpels, tissue plates and needles, it is necessary to avoid touching any surface that comes into contact with a sterile product. Only the outside of the syringe barrel, test tubes, plunger tip and/or needle cap or sheath can be touched safely. The consumable must be disposed of if the surface is touched or if a non-sterile surface is touched.

d. Register batch numbers and expiration dates (if taken into account) of all reagents and consumables used.

e. The defrosted vial is cleaned with a lint-free cloth moistened with 70% alcohol before being transferred to a biological safety cabinet.

g. Using an aspiration needle and syringe, remove all the liquid from the vial. Avoid sucking in tissue.

z. Using sterile tweezers, transfer the tissue into a 100 mm sterile dish.

Petri.

i. 5 ml of washing medium is added to the tissue fragments.

k. The contents are stirred for 15-30 seconds, then the washing medium is collected with a pipette or a syringe with an aspiration needle. This washing process is repeated twice.

l. 2 ml of washing medium is added to the tissue to avoid drying out the tissue.

2.4. Initiation of MSC proliferation from tissue:

a. Label the bottom of a 6-well "Overgence 1" plate with the MSC batch number or cord tissue identification number and the date of outgrowth initiation. If using a 60 mm tissue culture dish, then divide the dish into 4 quadrants by applying a grid to the bottom of the dish.

b. Using sterile disposable tweezers, place one piece of tissue measuring 3 x 3 mm to 5 x 5 mm into each well. If using a 60 mm tissue culture dish, place the tissue in the center of each quadrant to ensure spatial separation of the tissues (more than 1 cm apart).

c. Each well is filled with 3 ml of RTT6.

g. Using an aspirating needle attached to a 30 ml syringe, withdraw enough medium to just cover the tissue. Do not tilt the plate. Do not touch the bottom of the well with the aspirating needle.

d. Using an inverted light microscope, cell proliferation is observed daily (24±6 hours). The real-time cell culture imaging system can be used instead of a light microscope.

e. Change the media daily. Allow the media to equilibrate to room temperature before use.

1. The environment is taken away.

2. Add 3 ml of RTT6.

3. Continue until the tissue is barely immersed in the medium.

g. When cell proliferation from the tissue is detected, the tissue is transferred to a new 6-well plate using the same procedure as described in 4.a - 4.d above, except that the dish is labeled "Outgrowth 2". Cell proliferation in the Outgrowth 1 plate is maintained by adding 2 ml of PTT6 to each well. Confluency is monitored daily. Media is replaced every 2-3 days (ensure that media has equilibrated to room temperature before use).

h. When cell proliferation is detected in the "Growth 2" plate, then steps 4.a - 4.d are repeated except that the plate is labeled "Growth 3." Cell proliferation in the "Growth 2" plate is maintained by adding 2 ml of PTT6 to each well. Confluence is monitored daily. Media are replaced every 2-3 days (ensure that media have equilibrated to room temperature before use).

i. When overgrowth is detected in the "Overgrowth 3" plate, then the tissue is removed. If the tissue is very small and does not appear to affect cell growth, then the tissue is removed by subculture.

k. When the cells reach 40-50% confluency, the cells are then monitored every day to prevent excessive proliferation.

l. When the cells reach 70-80% confluence, the cells are then subcultured. The cells are not allowed to grow to a confluence greater than 80%.

When the size of the tissue explants is approximately 1-3 mm and the tissue explant/cell culture is performed in 175 mm square culture dishes, then the average number of mesenchymal stem cells collected from the explant is typically approximately 4000-6000 cells/explant. Accordingly, when the mesenchymal stem cells are cultured in 48 explants at a time, then approximately 300,000 cells can be obtained during the collection. These 300,000 mesenchymal stem cells collected from the explants can then be used for subculture by seeding into a 175 ^cm2 cell culture flask these 300,000 cells as described in the following Example 2.5 (this can be called passage 1). The mesenchymal stem cells obtained after this passage 1 can then be used to reseed 175 ^cm2 flasks (passage 2) and expand the cells as described in the following Example 2.5. The cells obtained after passage 1 and passage 2 can be "banked" by cryopreservation, with the mesenchymal stem cells obtained after passage 2 being considered as constituting a master cell bank, which is intended for further expansion of mesenchymal stem cells, for example in a bioreactor, as explained below in Example 2.7.

2.5. Subculturing of MSCs in cell culture dishes

a. Viable particles are handled in a biological safety cabinet. All media are equilibrated to room temperature before use.

b. When cell proliferation reaches approximately 70-80% confluency, the cells are then subcultured.

1. Remove RTT6 from the Petri dish.

2. Wash with HBSS without calcium or magnesium.

3. Add 0.2 ml IX TrypLE-EDTA and mix for 1-2 minutes.

4. Tilt the dish at 30-45° to allow the cells to slide down by gravity. Gently tapping the side of the plate will speed up cell separation.

5. Add 1 ml of PTT6. The cells are gently aspirated/pushed out of the pipette tip and then transferred to a 15 ml centrifuge tube.

A clean pipette tip is used for each well. Cells from all 6 wells can be combined into a single 15 ml tube.

6. Centrifuge for 10 minutes at 1200 rpm.

7. The supernatant is removed and the cells are resuspended with 5 ml of RTT6. c. Subculturing of MSCs

1. Aliquot 50 µl of the cell suspension and measure TNC and viability using trypan blue exclusion assay.

2. Cells are counted using a hemocytometer. Expect a count of 20-100 cells/quadrant. If the count exceeds 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer.

3. Calculate cell viability/ml and total cell viability:

1. Viable cells/ml=number of viable cells×dilution factor×10 ⁴

2. Total viable cell count = viable cell count x dilution factor x total volume x 10 ⁴

4. Calculate the % viability:

1.% viability = number of viable cells x 100 / (number of viable cells + number of dead cells)

5. The cell suspension is diluted to 1.0×10 ⁶ cells/ml:

1. "X" volume = total viable cells/10 ⁶ cells/ml

2. For example, if the total number of viable cells is 1.0×10 ⁷ ;

3. "X"=10 ⁷ /10 ⁶ cells/ml or 10 ml, thus, it is possible to bring the total volume of cells to 10 ml by adding 5 ml to the cell suspension (same for 5 ml).

6. If the cell suspension is less than 10 ⁶ /ml, then determine the volume required to seed 2×10 ⁶ cells for each 150 mm Petri dish or 175 cm ² flask.

1. Volume for 2× ¹⁰⁶ cells = 2× ¹⁰⁶ cells number of viable cells/ml

2. For example, if the viable cell count/ml is 8×10 ⁵ cells/ml, then 2×10 ⁶ cells+8×10 ⁵ cells/ml or 2.5 ml is required.

7. Leave 0.5 ml for analysis of MSC markers.

8. Sow 2×10 ⁶ cells in each 150 mm Petri dish or 175 cm ² flask with 30 ml of PTT6.

9. Monitor cell attachment, colony formation, and confluency every three days. When cells reach 40-50% confluency, monitor cells every one to two days to prevent overgrowth. DO NOT allow cells to grow to greater than 80% confluency. A real-time cell culture monitoring system may be used instead of a light microscope.

10. The media are changed every 2-3 days.

2.6 Cryopreservation of MSC cells

a. Viable particles are handled in a biological safety cabinet. All media are equilibrated to room temperature before use.

b. When the cells reach 70-80% confluency, the cells are then detached using 2 ml IX TrypLE-EDTA for each 150 mm Petri dish or 175 ^cm2 flask.

1. RTT6 is removed from the Petri dish.

2. Wash with 5 ml HBSS or PBS without calcium or magnesium.

3. Add 2 ml IX TrypLE-EDTA and mix for 1-2 minutes.

4. Tilt the dish at 30-45° to allow the cells to slide down by gravity. Gently tapping the side of the plate will speed up cell separation.

5. Add 10 ml PTT6 to inactivate TrypLE. Mix well to dissociate cell clumps.

6. The cells are transferred into a 15 ml centrifuge tube using a Pasteur pipette.

7. Centrifuge for 10 minutes at 1200 rpm.

8. The medium is removed and the cells are resuspended in 10 ml of PTT6.

9. Remove a 50 µl aliquot and determine the total viable cell count and % viability as above. Cell counts must be completed within 15 minutes as cells may begin to form agglomerates.

c. Preparation of cells for cryopreservation

1. Prepare media for cell suspension and media for cryopreservation and freezing of cells.

2.7. Subcultivation (reproduction) of MSCs in the Quantum bioreactor (Terumo BTC, Inc.)

It is also possible to use the Quantum Bioreactor for MSC expansion. The initial cell count for cultivation in the Quantum Bioreactor should be in the range of 20 to 30 million cells per run. Typical yield per run is 300 to 700 million MSCs after collection. The bioreactor is operated according to the manufacturer's protocol. The mesenchymal stem cells thus obtained are usually cryopreserved (see below) and serve as a working cell bank.

Materials/reagents:

1. Quantum Expansion Kit

2. Quantum waste container

3. Quantum Environment Container

4. Quantum Entry Container

5. RTT6

6. PBS

7. Fibronectin

8. TrypLE

9. 3 ml syringe

10. Glucose test strips

11. Lactate Test Strips

12. 60 ml cell culture plate or equivalent

13. Gas mixture 5% _CO2 for medical use

14. 50ml Combi-tips Equipment:

1. Biological safety cabinet

2. Glucose meter (Bayer Healthcare/Ascensia Contour Blood Glucose Meter)

3. Lactate Plus (Nova Biomedical)

4. Peristaltic pump with tubing block

5. Centrifuge Eppendorf 5810

6. Sterile tubular connector

7. Step pipette dispenser M4

8. RF sealer

Procedure:

1. Preparing the Quantum Bioreactor

a) Initiate the Quantum bioreactor

b) Close the bioreactor:

1) A fibronectin solution is prepared in a biological safety cabinet.

1) Allow lyophilized fibronectin to come to room temperature (≥15 min at room temperature)

2) Add 5 ml of sterile distilled water; do not stir or shake.

3) Allow fibronectin to enter the solution phase for 30 minutes.

4) Using a 10 ml syringe fitted with an 18g needle, transfer the fibronectin solution into a cell inlet container containing 95 ml PBS.

2) The container is connected to the "reagent" line.

3) Check for bubbles (bubbles can be removed using "Remove 1C Air" or "Remove EC Air" and using "Flush" as the input source.

4) Open or set the program to close the bioreactor (Fig. 1, stages 3-5).

5) Launch the program

6) While the program is running, to close the bioreactor, prepare a container for the medium with 4 liters of RTT6 medium.

7) Connect the medium container to the 1C Media line using a sterile tubular connector.

8) When the bioreactor sealing steps are completed, then the cell inlet container used for the fibronectin solution is disconnected using the RF sealer.

a) Excess fibronectin is washed away

b) Condition the bioreactor with media

2. Cell cultivation in the Quantum bioreactor

a) Loading and attachment of cells with a homogeneous suspension:

b) Filling and culturing cells

1) Select the flow rate of the medium for loading cells.

2) Every day, samples are taken for lactate and glucose analysis.

3) Adjust the flow rate of the medium as lactate levels increase. The actual maximum allowable lactate concentration will be determined by the culture flask from which the cells are derived. Determine if the medium container contains the appropriate PTT6 medium. If necessary, replace the PTT6 medium container with fresh PTT6 medium.

4) When the flow rate reaches the desired value, the lactate level is measured every 8-12 hours. If the lactate level does not decrease or if the lactate level continues to increase, the cells are harvested.

3. Collection of cells from the Quantum bioreactor

a) When the lactate concentration does not decrease, then the cells are collected after the last sample collection for lactate and glucose analysis.

b) Collection of cells:

1) Connect the cell inlet container filled with 100 ml TrypLE to the "reagent" lines using a sterile tubing connector.

2) Confirm that there is sufficient PBS in the PBS container. If there is not enough, connect a new container with at least 1.7 liters of PBS to the "Flush" line using a sterile tubing connector.

3) Launch the collection program.

4. Cryopreservation of cells

1) After collecting the cells, they are transferred to a 50 ml centrifuge tube to pellet the cells.

2) Resuspend with 25 ml of chilled cell suspending solution. Cells are counted using a Sysmex or Biorad cell counter. The cell count report is attached to the appropriate Quantum Processing Batch report.

3) Bring the cell concentration to 2×10 ⁷ /ml

4) Add an equal volume of cryopreservation solution and mix well (do not shake or vortex)

5) Using a step pipette dispenser, add 1 ml of the cell suspension in cryopreservative to each 1.8 ml vial. Cryopreserve using the CRF program as described in SOP D6.100 CB Cryopreservation using a controlled rate freezer.

6) The vials are stored in a designated storage space in liquid nitrogen.

7) The CRF program report is attached to the corresponding P3-Quantum Processing Batch Record MSC batch report form.

3. Analysis of stem cell marker expression in populations of umbilical cord lining mesenchymal stem cells isolated from umbilical cord tissue using different culture media

Flow cytometry experiments are performed to analyze umbilical cord-derived mesenchymal stem cells for expression of mesenchymal stem cell markers CD73, CD90, and CD 105.

For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media, followed by passaging the mesenchymal stem cells in the corresponding media, as presented in Example 2.

The following three culture media were used in these experiments: a) 90% (v/v) DMEM supplemented with 10% (v/v) FBS, b) the PTT-4 culture medium described in US Patent Application No. 2008/0248005 and the corresponding International Patent Application No. WO 2007/046775, which consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO 2007/046775), and c) the inventive PTT-6 culture medium, the composition of which is described herein. In this flow cytometric assay, two distinct samples of cord-lined mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used.

The following protocol was used for flow cytometric analysis.

Materials and methods

Way

a) Cells isolated and grown from the umbilical cord lining membrane

1. Tissue explant samples were incubated in a cell culture plate and immersed in appropriate media, then left in a CO2 incubator at 37°C as explained in Example 2.

2. The medium was changed every 3 days.

3. Cell proliferation from tissue culture explants was monitored by light microscopy.

4. At approximately 70% confluency, cells were detached from the dish by trypsinization (0.0125% trypsin/0.05% EDTA) and used in flow cytometry experiments.

b) Trypsinization of cells for experiments

1. Remove the medium from the cell culture plate.

2. Wash gently with sterile IX PBS to remove traces of FBS, since FBS interferes with the enzymatic action of trypsin.

3. IX Trypsin is added to the cell culture plate and incubated for 3-5 min at 37°C.

4. The cells are observed under a microscope to ensure that they are detached. Trypsin is neutralized by adding complete media containing FBS (DMEM with 10% FBS).

5. Use a pipette to break up cell clumps by pipetting cells in the media onto the wall of the plate. Collect the cell suspension and transfer to 50 ml centrifuge tubes.

6. Sterile IX PBS is added to the plate and washed, the cell suspension is collected in the same centrifuge tube.

7. The test tube is centrifuged at 1800 rpm for 10 minutes.

8. The supernatant is removed and the cell pellet is resuspended in PBA medium.

c) Cell counting

1. Ensure that the hemocytometer and its coverslip are clean and dry, preferably by rinsing them with 70% ethanol, and allow them to dry before wiping them with Kim wipes (lint-free paper).

2. Aliquot a small number of cells in suspension into a microcentrifuge tube and remove from the biological safety cabinet.

3. The cells are stained in suspension with an equal volume of trypan blue, for example, for 500 µl of suspension add 500 µl of trypan blue (dilution factor = 2X, resulting in a 0.2% trypan blue solution).

4. Avoid exposing cells to trypan blue for more than 30 min, as trypan blue is toxic and will increase the number of non-viable cells and result in cell count errors.

5. Add 20 µl of the cell suspension mixture to each chamber of the hemocytometer and examine under a light microscope.

a. The number of viable cells is counted (luminous cells; non-viable cells readily absorb trypan blue and are therefore dark) in each quadrant of the hemocytometer for a total of 8 quadrants in the upper and lower chamber.

Total cell counts are given as (mean cell count/quadrant)× ¹⁰⁴ cells/mL.

d) Cell staining

1. Preparation before cell staining

- The cell suspension is aliquoted into 3 tubes (CD73, CD90, CD105) in two parallels and into 2 tubes (negative control), each containing 50,000 cells.

2._Primary antibody staining (Ab)

- Add 1 µl [0.5 mg/ml Ab] of primary antibody to 100 µl of cell suspension. Incubate at 4°C for 45 min.

- Bring up to 1 ml using RVA.

- Centrifuge at 8000 rpm at 4°C for 5 minutes.

- Super natant is removed.

- Add 1 ml of RVA and resuspend the sediment.

- Centrifuge at 8000 rpm at 4°C for 5 minutes.

- Super natant is removed.

- Resuspend in 100 µl of RVA.

3._Secondary Ab staining - in the dark

- Add 1 µl [0.5 mg/ml ab] secondary antibody to 100 µl cell suspension. Incubate at 4°C for 30 min.

- Bring up to 1 ml using RVA.

- Centrifuge at 8000 rpm at 4°C for 5 minutes.

- Super natant is removed.

- Add 1 ml of RVA and resuspend the sediment.

- Centrifuge at 8000 rpm at 4°C for 5 minutes.

- Super natant is removed.

- Resuspend in 200-300 µl of RVA for flow cytometric analysis.

- Cells are transferred to a FACS tube for flow cytometry analysis on a BD FACS CANDO.

The results of the flow cytometry analyses are shown in Fig. 6a through Fig. 6c. Fig. 6a shows the percentage of isolated umbilical cord mesenchymal stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in DMEM/10% FBS. Fig. 6b shows the percentage of isolated umbilical cord mesenchymal stem cells expressing the stem cell markers CD73, CD90, and CD 105 after isolation from umbilical cord tissue and cultured in PTT-4, and Fig. 6c shows the percentage of isolated umbilical cord mesenchymal stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in PTT-6. As can be seen in Fig. 6a, the cell population isolated using DMEM/10% FBS as the culture medium has approximately 75% CD73+ cells, 78% CD90+ cells and 80% CD 105+ cells (average of two experiments), whereas after isolating/cultured umbilical cord tissue using PTT-4 culture medium (see Fig. 6b), the number of mesenchymal stem cells that are CD73-positive, CD90-positive and CD105-positive is approximately 87% (CD73+ cells), 93% (CD90+ cells) and 86% (CD105+ cells) on average of two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0% for all three markers (CD73, CD90, CD 105), meaning that the purity of this cell population is significantly higher than when culturing using the PTT-4 medium or DMEM/10% FBS. Additionally, and even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially a 100% pure and defined stem cell population. This results in the stem cell population of the present invention being an ideal candidate for stem cell-based therapies. Thus, this population of umbilical cord-lining mesenchymal stem cells may represent a gold standard for such stem cell-based therapeutic approaches.

The experimental data shown in Fig. 6 were further supported by the flow cytometric analysis results shown in Fig. 7a and Fig. 7b. Fig. 7a shows the percentage of isolated umbilical cord lining mesenchymal stem cells (umbilical cord amniotic membrane mesenchymal stem cells) that express the stem cell markers CD73, CD90, and CD 105 and do not express CD34, CD45, and HLA-DR after isolation from umbilical cord tissue and culture in PTT-6 medium. As shown in Fig. 7a, the mesenchymal stem cell population contains 97.5% viable cells that express 100% each of CD73, CD90, and CD 105 (see arrows "CD73+CD90+" and "CD73+CD105+"), whereas 99.2% of the stem cell population does not express CD45, and 100% of the stem cell population does not express CD34 and HLA-DR (see arrows "CD34-CD45- and "CD34-HLA-DR-"). Thus, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and defined stem cell population that meets the criteria that mesenchymal stem cells used for cell therapy must meet (95% or more of the stem cell population expresses CD73, CD90, and CD 105, whereas 98% or more of the stem cell population does not express CD34, CD45, and HLA-DR, see Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review," above). It is noted in this document that the amniotic membrane mesenchymal stem cells disclosed herein adhere to plastic under standard culture conditions and differentiate in vitro into osteoblasts, adipocytes and chondroblasts, see US Patent 9,085,755, US Patent 8,287,854 or WO 2H007/046775, and thus satisfy the generally accepted criteria for the use of mesenchymal stem cells in cell therapy.

Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and do not express CD34, CD45, and HLA-DR. As shown in Figure 7b, the bone marrow mesenchymal stem cell population contains 94.3% of viable cells that express 100% each of CD73, CD90, and CD 105 (see arrows "CD73+CD90+" and "CD73+CD105+"), whereas only 62.8% of the bone marrow stem cell population does not express CD45 and 99.9% of the stem cell population does not express CD34 and HLA-DR (see arrows "CD34-CD45- and "CD34-HLA-DR-"). Thus, bone marrow mesenchymal stem cells, which are considered as the gold standard of mesenchymal stem cells, are significantly less homogeneous/pure in terms of stem cell markers than the mesenchymal stem cell population (amniotic cord membrane) according to the present invention. These experimental data also demonstrate that the stem cell population of the present invention may be an ideal candidate for stem cell-based therapies and may represent the gold standard for stem cell-based therapeutic approaches.

4. Experiments demonstrating that the mesenchymal stem cell population according to the invention can be transported/stored in HypoThermosol™:

To analyze the health and viability of the mesenchymal stem cells described herein in different storage or transport media, two different media were compared with each other. Specifically, the HypoThermosol™-FRS media was compared with the Plasmalyte-A media. Both are commercially available. The product description for HypoThermosol™ -FRS is shown in Fig. 30 and its composition is described herein. Each 100 mL of Plasmalyte contains 526 mg sodium chloride, USP (United States Pharmacopoeia) (NaCl); 502 mg sodium gluconate (C ₆ H ₁₁ NaO ₇ ); 368 mg sodium acetate trihydrate, USP (C ₂ H ₃ O ₂ ⋅3H ₂ O); 37 mg potassium chloride, USP (KCl); and 30 mg magnesium chloride, USP (MgCl ₂ ⋅6H ₂ O). Plasmalyte does not contain antimicrobial agents. The pH of plasmalyte is adjusted with sodium hydroxide to 7.4 (from 6.5 to 8.0).

The schematic of the comparison experiment is shown in Fig. 8. First, the mesenchymal stem cell population described herein was grown in cell culture flasks. The number of living mesenchymal stem cells was counted, and then 2 million cells/flask were stored for various periods of time in Plasmalyte-A or Hypothermosol™-FRS. After storage, cells were counted in a ≤50 μL sample daily for days 1-5 (total fluid extraction volume 250 μL) and checked for viability by cell staining with trypan blue. In addition, on days 1, 3, and 5, a ≤80 μL sample was collected and analyzed. Additionally, the supernatant was obtained and frozen. Then, PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, and TGFβ1 were measured using the FLEXMAP 3D system.

Fig. 9 summarizes the viability data. As can be seen in the left graph, 73% of the total cells (approximately 95%) from the start of storage were still viable 7 days after storage in HypoThermosol™. In contrast, after 7 days of storage in Plasmalyte-A, only 42% of the total cells (approximately 94%) from the start of storage were still viable. All counts are based on recording in duplicates that are within 10% of each other (according to SOP (standard operating procedures) CR D2.600.1). At the time of counting, the cells stored in HypoThermosol™ were clearly smaller with smooth and well-defined edges. In contrast, the cells in Plasmalyte-A appeared to be of variable size. HypoThermosol™ significantly maintained membrane integrity and presumably survival for a week (6 days). Similar results are also shown in the graph on the right side.

Fig. 10 shows the results obtained by measuring the cell diameter. The population of mesenchymal stem cells described in this paper had a smaller range of diameters when stored in HypoThermosol™ compared to cells stored in Plasmalyte-A. The comparison was made after 3 days of storage.

Figure 11 shows the concentration of TGF61 in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A after 48 hours of storage. As can be seen in the graph on the right side, the cells secreted approximately the same amount of TGFB1 when stored in HypoThermosol™ as when stored in Plasmalyte-A. Overall, the amount of TGFB1 secreted decreased over time (graph on the right side).

Fig. 12 and 13 show control experiments. Here, PDGF-BB and IL-10 concentrations were measured in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 h. Since PDGF-BB or IL-10 are not normally secreted by the mesenchymal stem cell population described herein, neither PDGF-BB nor IL-10 were detected in any of the samples.

Figure 14 shows the VEGF concentration in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 h. As can be seen in the graph on the right side, the cells secreted approximately the same amount of VEGF when stored in HypoThermosol™ as when stored in Plasmalyte-A on day 0. On days 1 and 5, the cells secreted more VEGF when stored in Plasmalyte-A. Notably, when the cells were stored for 3 days, they secreted more VEGF when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, HypoThermosol™ outperformed Plasmalyte-A on day 3 of storage. The more VEGF that is detected, the healthier the culture. Thus, by secreting more VEGF after 3 days of storage in HypoThermosol™ than when stored in Plasmalyte-A, the cells were healthier in HypoThermosol™ than in Plasmalyte-A. From day 5 of storage, Plasmalyte seemed to become a more favorable carrier, since when the cells were stored in Plasmalyte-A, they secreted more VEGF. Overall, the amount of VEGF secreted decreased over time (graph on the right side).

Figure 15 shows the PDGF-AA concentration in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 h. As can be seen in the graph on the right side, the cells secreted approximately the same amount of PDGF-AA when stored in HypoThermosol™ as when stored in Plasmalyte-A at day 0. On days 1 and 5, the cells secreted more PDGF-AA when stored in Plasmalyte-A. Notably, when the cells were stored for 3 days, they secreted more PDGF-AA when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, the cells stored in HypoThermosol™ are healthier than the cells stored in Plasmalyte-A after 3 days of storage. From day 5 of storage onwards, plasmalyte appears to be a more favourable carrier, as from this point onwards, cells stored in Plasmalyte-A secreted more PDGF-AA. Overall, the amount of PDGF-AA secreted decreased over time (graph on the right side).

Figure 16 shows the concentration of Ang-1 in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A for 48 hours. As can be seen in the graph on the right side, the cells secreted approximately the same amount of Ang-1 when stored in HypoThermosol™ as when stored in Plasmalyte-A on days 0 and 3. On day 5, the cells secreted more Ang-1 when stored in Plasmalyte-A. Notably, when the cells were stored for 1 day, they secreted significantly more Ang-1 when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, the cells that were stored in HypoThermosol™ appeared healthier than those stored in Plasmalyte-A for at least 48 hours up to 3 days of storage. From day 5 onwards, plasmalyte appears to be a more favourable carrier, as from this point onwards, cells stored in Plasmalyte-A secreted more Ang-1. Overall, the amount of secreted Ang-1 decreased over time (graph on the right).

Figure 17 shows the concentration of HGF in the supernatant of the mesenchymal stem cell population described herein that was stored in HypoThermosol™ or Plasmalyte-A after 48 hours of storage. As can be seen in the graph on the right side, the cells secreted approximately the same amount of HGF when stored in HypoThermosol™ as when stored in Plasmalyte-A at day 0. On days 3 and 5, the cells secreted more HGF when stored in Plasmalyte-A. Notably, when the cells were stored for 1 day, they secreted significantly more HGF when stored in HypoThermosol™ than when stored in Plasmalyte-A. Thus, the cells that were stored in HypoThermosol™ appeared to be healthier than the cells that were stored in Plasmalyte-A from at least day 1 (48 hours) to day 3 of storage. From day 3 onwards, Plasmalyte-A appears to be a more favourable carrier, as at time points 3 and 5 days, cells stored in Plasmalyte-A secreted more HGF. Overall, the amount of HGF secreted decreased over time (graph on the right side).

In summary of the above data, it can be concluded that storage of the mesenchymal stem cell population according to the present invention in HypoThermosol™ is superior to storage in Plasmalyte-A, especially during the first 3 days of storage.

5. Experiments demonstrating that the mesenchymal stem cell population according to the invention has wound healing properties when locally treated in pigs:

Preclinical studies were also performed using 10-week-old female Yorkshire Landrace pigs (50 kg). Treatments were performed at the SingHealth Experimental Medicine Centre, Singapore. Pigs were induced to become diabetic with 120 mg/kg streptozotocin and allowed to recover for 45 days before receiving six 5 cm × 5 cm full-width wounds on their backs (see Fig. 18). Pigs (n=2) were treated twice weekly with the herein described population of ¹⁰⁵ human mesenchymal stem cells/ ^cm2 for 4 weeks. Two control pigs were treated with PBS. Wounds were photographed at postoperative day 0 (PODay 0) and every seven days up to postoperative day 35. Wounds were analysed for surface area using ImageJ. At 35 days, addition of the mesenchymal stem cell population described herein resulted in closure of 10 of 12 diabetic wounds (83%) compared to only 3 of 12 (25%) in PBS-treated control wounds. The wound healing rate was 0.8 ^cm2 /day using the mesenchymal stem cell population described herein compared to 0.6 ^cm2 /day in control animals, representing a 33% improvement. The results of this study are summarized in Fig. 18.

The porcine model is not spontaneous, since the skin architecture most closely resembles that of humans. These data indicate that the population of umbilical cord mesenchymal stem cells of the present invention improves wound healing without the risk of serious side effects. Thus, these data strongly support the hypothesis that the population of human umbilical cord mesenchymal stem cells described herein can promote chronic wound healing by suppressing inflammation and stimulating angiogenesis. In addition, there is apparently no evidence of inflammation when xenogeneic mesenchymal stem cells are used in mice or pigs, and thus the likelihood that allogeneic mesenchymal stem cells will cause any adverse side effects in humans is very low.

6. Experiments demonstrating that the mesenchymal stem cells described herein are effective for local treatment in humans:

Experiments demonstrating that the mesenchymal stem cells described herein are effective in topical treatment of humans are described in WO 2007/046775. In particular, as explained in Examples 23-26 of WO 2007/046775, umbilical cord mesenchymal stem cells (UCMC) can alleviate deep burns (Example 23), superficial wounds (Example 24), non-healing radiation wounds (Example 25), as well as non-healing diabetic wounds and non-healing diabetic foot wounds (Example 26). It is noted that according to Example 2 in WO 2007/046775, the mesenchymal stem cells were resuspended in PTT-4 medium.

It is noted that, as shown in Fig. 6b and 6c, the population of stem cells obtained by culturing using the PTT6 growth medium (as used herein) is significantly more homogeneous than the population of cells obtained using the PTT4 medium (used in WO 2007/046775). Since PTT-4 was used as a medium for mesenchymal stem cells in Examples 23-26 in WO 2007/046775, it is evident that even a more homogeneous population of mesenchymal stem cells isolated after culturing in PTT-6 (as used herein) will have the same beneficial effects when applied to wound healing, such as deep burns, superficial wounds, non-healing radiation wounds, and non-healing diabetic wounds and non-healing diabetic foot wounds.

7. Experiments demonstrating that the mesenchymal stem cells described herein are effective for local treatment in humans:

This study is a planned dose escalation study of the mesenchymal stem cell population generated as described herein, conducted at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The purpose of this study was to determine the safe dose of the mesenchymal stem cell population (human umbilical cord lining mesenchymal stem cells) described herein. This study was a single-center, dose escalation study with five subjects enrolled at each of three dose levels for a total of fifteen subjects. The first group of 5 patients received 100,000 MSCs/ ^cm2 (skin/wound area) twice weekly for 8 weeks. The second group of 5 patients received 300,000 MSCs/cm2 ^twice weekly for 8 weeks. The third group of 5 patients received 500,000 MSCs/cm2 ^twice weekly for 8 weeks. This regimen was continued until the highest dose was reached or until at least 2 subjects at a dose level experienced a grade ≥2 allergic reaction considered to be related to the mesenchymal stem cell population derived herein, or 2 or more subjects at a dose level experienced unexpected treatment-related serious adverse events or dose-limiting toxicities within 14 days following administration of the initial dose of the mesenchymal stem cell population derived herein. All of these patients were assessed at 30 days post-treatment for anti-HLA antibody production and wound healing. At this time, we do not consider anti-HLA antibody production to be an absolute contraindication to a specific dose, but rather as a factor in the overall safety assessment. This is an open-label study in which all subjects received study drug and all study personnel were aware of the dose each subject was receiving. The secondary endpoint of this study is significant wound healing. This result is based on the rate of wound closure, the percentage of successful wound area closure, and the percentage of completely healed wounds as measured using the Silhouette Wound Measurement and Documentation System. This device is FDA cleared for this purpose.

Subject Population: Patients with type I or II diabetes mellitus with chronic foot ulcers that had not healed after at least 30 days of conventional therapy and were negative for HLA antibodies to the mesenchymal stem cell population described herein. Patients continued to receive conventional wound care for the first 2 weeks of treatment entry, after which they were assessed for the presence of a diabetic foot ulcer that had not healed within 30 days. At this time, photographic documentation and wound measurement were initiated. Conventional dressing changes were performed twice weekly for the first 2 weeks, after which the mesenchymal stem cell population described herein was applied to the wound at a defined concentration twice weekly. Wounds treated with the mesenchymal stem cell population described herein were also closed with ^Tegaderm® and a fixation bandage.

Dose Levels. The objective of this study was to determine a safe dose of the human umbilical cord mesenchymal stem cells described herein for further investigation. Patients were treated with one of three doses: 100,000 MSCs/ ^cm2 skin/wound area, 300,000 MSCs/ ^cm2 , or 500,000 MSCs/cm2 ^twice weekly for 8 weeks. Each 100,000 cell dose represents 0.1 mL of the mesenchymal stem cell population described herein from a vial containing 1 million cells/mL in HypoThermosol.

Dosing regimen. This study was a safety and tolerability study of escalating doses of the mesenchymal stem cells described herein. The objective of this study was to determine a safe dose of the human umbilical cord mesenchymal stem cells described herein for further investigation. Five subjects were enrolled for each of three dose levels. The first group of 5 patients received 100,000 MSCs/ ^cm2 skin/wound area twice weekly for 8 weeks. The second group of 5 patients received 300,000 MSCs/cm2 ^twice weekly for 8 weeks. The third group of 5 patients received 500,000 MSCs/cm2 ^twice weekly for 8 weeks. This regimen was continued until the highest dose was reached, or until at least 2 subjects at a dose level experienced a grade ≥2 allergic reaction thought to be related to the mesenchymal stem cell population described herein, or 2 or more subjects at a dose level experienced unexpected treatment-related serious adverse events or dose-limiting toxicities within 30 days following administration of the initial dose of the mesenchymal stem cell population described herein. All of these patients were assessed at 30 days post-treatment for anti-HLA antibody production and wound healing. At this time, we do not consider anti-HLA antibody production to be an absolute contraindication to a specific dose, but rather as a factor in the overall safety assessment. This is an open-label, placebo-controlled study in which all subjects received study drug and all study personnel were aware of the dose each subject received.

Route of administration: The mesenchymal stem cell population described herein is applied topically to cleansed diabetic foot ulcers and held in place with Tegaderm ^® dressing.

Dosing Procedure: After appropriate cleansing, if necessary, the patient is placed in a prone position and the affected leg is flexed at a 90° angle. The vial containing the mesenchymal stem cell population described herein is gently mixed to ensure uniform distribution of the cells. The elevated foot is then treated by withdrawing 100,000 (0.1 ml) to 500,000 (0.5 ml) cells per ^cm2 from the vial using a sterile syringe and applying them to the center of the wound. The wound is then covered with a Tegaderm ^® membrane and gently massaged to ensure uniform distribution of the cells. The foot is maintained in the elevated position for five minutes to allow the cells to settle and adhere. The foot is then wrapped in a fixation bandage covering the Tegaderm ^® bandage.

8. Preparation of a composition for storing or transporting mesenchymal stem cells (containing a population of stem cells in which more than 99% of the cells express each of CD73, CD90 and CD105 and do not express CD34 and HLA-DR)

Preparation for processing after the fourth cell passaging (stage 4 of processing):

Stage 4 processing is typically performed in a clean room with controlled environment (CE). The required solutions and laboratory glassware should be prepared in advance for use.

Cells are transferred from the cryovial to a labeled 50 ml centrifuge tube.

Complete RTT6 medium is used within 5 minutes of removal from the refrigerator (record the time of removal of RTT6 media from the refrigerator). Slowly add 9 ml of complete RTT6 medium to the cells with gentle agitation to facilitate mixing.

Centrifuge at 1200 rpm at room temperature (15-25°C) for 5 minutes to pellet the cells. Record the centrifuge usage on the Centrifuge Periodic Preventive Maintenance Form - CR and confirm the operating parameters according to the Centrifuge Preventive Maintenance SOP.

The supernatant is removed and the cells are resuspended in sufficiently complete PTT6 medium for counting.

Count:

To determine the cell concentration (with or without trypan blue), cells are counted using TC20. In most cases, no sample dilution is required since TC20 works with a wide range of cell concentrations (from 5×10 ⁴ to 1×10 ⁷ cells/mL).

To determine viability, count using a hemocytometer according to the same SOP. Ensure that at least 200 cells are counted in total.

If it is desired to determine both cell viability and cell concentration using a hemocytometer, then the suspension can be diluted to fit the hemocytometer range (20-100 cells per outer quadrant). If approximately 10 million thawed cells are to be resuspended, then a volume of 6 ml should provide this range. Therapeutic culture (P4):

300,000 live cells are seeded per 175 ^cm2 30 ml flask of complete RTT6 medium and incubated at 35-39°C with 4-6% _CO2 . It is necessary to ensure that preventive maintenance of the incubator has been performed in a timely manner in accordance with the SOP Incubator Preventative Maintenance and General Use. Flasks are labeled with the MSC PI-P4 process label.

Once the majority of cells have adhered (preferably overnight), perform a quick inspection of the sentinel vial under a Nikon inverted microscope located in a clean room to determine if there is an area of the vial that contains a noticeably higher cell density. If so, use this area for continuous monitoring with CytoSmart. If multiple vials are seeded, one vial can be used as the representative "sentinel" vial. As a CytoSmart feature, email alerts are set at 60%, 70%, and 80% confluence for the "sentinel" vial.

The media are changed to 30 ml of pre-heated fresh complete RTT6 per vial every 2-3 days and incubation is continued.

When cell proliferation reaches 80%±10% confluency, MSCs are collected as follows:

Each vial is rinsed with 10 ml HBSS without Ca ²⁺ or Mg ²⁺ .

Add 5 ml IX TrypLE per vial. Tilt the vial to cover the entire surface and immediately aspirate TrypLE by tilting the vial and removing most of the TrypLE with a sterile serological pipette, leaving just enough TrypLE to cover the surface. Discard the aspirated TrypLE.

Allow the cells to detach (10-20 minutes at 15-25°C). Tilt the vial 30-45° to allow the cells to slide down by gravity. Gently tapping the side of the plate accelerates cell detachment. Inspect the vial under an inverted microscope to ensure that all cells have detached.

Add 5 ml of HBSS without Ca ²⁺ or Mg ²⁺ to the first vial. The cells are gently aspirated/pushed out of the pipette tip, then the cell suspension is transferred to the next vial. Repeat until all cells have been collected from all vials and transferred to a 50 ml centrifuge tube labeled with a process label.

Replace with 5 ml fresh HBSS without Ca ²⁺ or Mg ²⁺ and combine with the suspension.

Under a microscope, they confirm that all cells have been removed and, if necessary, repeat the cell collection from all vials a third time.

Centrifuge the combined cell suspension for 5 minutes at 1200 rpm at 15-25°C. Record centrifuge usage on the Centrifuge Periodic Preventive Maintenance Form - CR and confirm operating parameters.

Preparation of suspension of collected cells:

The supernatant is removed without disturbing the pellet and the cells are resuspended in 1.0 ml of complete RTT6 medium per collection flask using a serological pipette of the appropriate size. There is no need to preheat the medium.

Cells resuspended in complete PPT6 medium were pelleted at 1200 rpm for 5 min at room temperature.

All the supernatant of RTT6 is removed without disturbing the pellet and the pellet is carefully resuspended in 1.0 ml of "1% HSA in Plasmalyte" per collected vial using a serological pipette of the appropriate size. A suspension of the collected cells is obtained. From this point on, the suspension of the collected cells is stored in a cooling block.

Counting of collected cell suspension:

Before each sample collection for cell counting, the collected cell suspension is thoroughly mixed.

To determine the cell concentration (with or without trypan blue), counts are performed using the TC20 according to the SOP Cell Counting and Viability Assay. In most cases, no sample dilution is required since the TC20 works with a wide range of cell concentrations (from 5×10 ⁴ to 1×10 ⁷ cells/mL).

To determine viability, count using a hemocytometer according to the same SOP. Ensure that at least 200 cells are counted in total.

Prepare suspension for loading vials (keep cooled in a 50-volume conical tube in a cooling block):

Based on the previous count of the collected cell suspension, the volume of collected cell suspension and "1% HSA in HypoThermosol" required to prepare the required patient dose is determined. The conical tube is labeled with the appropriate label. The conical tubes containing the suspension for vial loading are stored in a pre-cooled cooling block.

HypoThermosol is used and the prepared "1% HSA in HypoThermosol" is stored and used in a refrigerated temperature range (2-8°C) such that the vial loading suspension is maintained in the refrigeration unit.

Record the volume of each component (HSA, Plasmalyte-A, and HypoThermosol-FRS) used to prepare this final suspension. Based on this volume, also record the volumes of HSA, Plasmalyte, and HypoThermosol contained in each AT-sealed vial.

Counting cells in suspension for loading vials:

Before each sample is taken from the vial loading suspension for counting, ensure that the cells are well mixed.

To determine the cell concentration (with or without trypan blue), count using TC20 according to SOP Cell Counting and Viability Assay.

To determine cell viability, cells are counted using a hemocytometer according to the same SOP. It is necessary to ensure that at least 200 cells are counted in total. Viability analysis can only be performed once by VLS.

Load AT-closed vials as follows:

The syringes and needles previously placed in the refrigerator are removed and placed in a biological safety cabinet (BSC).

The AT-sealed vials previously placed in the CoolRackSV10/XT Cooling Core are removed from the refrigerator and transferred to the BSC box. The timer is started to ensure that the loading is completed within 30 minutes.

The injection hole is wiped with an alcohol wipe.

Before loading the vial, a sterile 22G needle is inserted into the stopper near the center to vent the vial (to avoid excess pressure in the vial during filling).

The suspension for loading the vial is mixed, then slowly sucked into a syringe without bubbles. The center of the stopper is pierced with a syringe and 1.0 ml is injected into each AT-closed vial (registered according to the graduation on the syringe), being careful not to cause bubbles to form.

Remove the loading syringe, then remove the venting needle.

Close the opening of the bottle with the appropriate cap and press it tightly. Return to CoolRackSVIO and store at 2-8°C for transportation to the destination.

Sample collection for cytokine analysis after P4 (cytokine analysis is performed at least once for each lot number, i.e. identical CBU # and lot # of donor tissue):

Based on the above concentration of the vial loading suspension, add a sufficient volume of the vial loading suspension to at least one well of a 6-well plate such that a total of 100,000 (live + dead) cells are added per well. Add the vial loading suspension directly to a sufficient amount of complete PTT6 medium previously added to each well such that the total volume in each well is 2 ml. Mark the start time of incubation.

Incubate for 48 hours ±1 hour. After incubation is complete:

A single typical CytoSmart image is acquired randomly at approximately the center of each well.

Measure the lactate in each well and record it on the Lactate Test Results Form.

Aspirate medium from each well and centrifuge at 1200 rpm at room temperature for 5 minutes. Record centrifuge usage on the CR Centrifuge Periodic Preventive Maintenance Form and confirm operating parameters.

The supernatants of the media are added to cryovials and frozen within 1 hour after collection. The storage location is marked in the batch recorder.

9. Study of the stability of proliferation and metabolism of MSCs during storage/transportation

Umbilical cord tissues and cells from early passages are stored at -195°C and tested for stability.

Initial stability testing of the umbilical cord lining mesenchymal stem cells (MSCs) described herein, which are greater than 99% pure, for positive and negative markers (see Fig. 7 in this regard) was performed with a final product consisting of viable MSCs in HypoThermosol ^® alone. During actual manufacturing operations, it was found that the adhesion of the mesenchymal stem cells was due to the intrinsic properties of the mesenchymal stem cells and the viscosity of HypoThermosol ^® .

In order to reduce cell loss due to MSC transfer into HypoThermosol ^® during dispensing, MSC adhesion to plastic containers at various stages of step 4 processing and to maximize drug product release from the vial used during dispensing, it was found that plasmalyte and human serum albumin (HSA), which are two pharmaceutically inactive ingredients, when added optimize the amount of final drug product.

A new stability study was performed to confirm that the addition of these two pharmaceutically inactive ingredients did not adversely affect the stability of the final drug product. The results are shown in Fig. 33.

Viability Analysis

Mesenchymal stem cells were seeded into AT-Closed Vials ^® at 10 ⁶ cells per vial in 1 ml Plasmalyte/HSA/HypoThermosol ^® . Samples from individual vials were collected at different time points, viability was measured manually using trypan blue (hemocytometer) and total cell numbers were counted using an automated system (TC20).

MSCs were stored at 2-8°C for 1-3 days to simulate the transportation and storage of the product prior to application to wounds. As shown in Fig. 33a, the cells did not show significant loss of viability up to 3 days under these conditions.

Analysis of appearance

MSCs were photographed after removal from AT-sealed flasks and grown for 24 hours at 37°C. As can be seen below, cells obtained up to 2 days of refrigerated storage were able to adhere to tissue culture plates and form typical spindle-shaped structures. After storage for 2.5 days at 2-8°C, cells showed an increase in spheroid shapes, indicating cell death. The results are shown in Fig. 33b. Proliferation and Metabolic Assay

MSCs from the same cultures shown in Fig. 33a were examined for lactate production as an index of metabolism and growth over a 48-hour period in culture at 37°C. Cells stored for 24 hours at 2-8°C were equivalent in metabolism and growth to cells stored for 0 hours, and cells stored for 36 hours exhibited 86% of the control in lactate production. After 72 hours at 2-8°C, cells exhibited only 46% of the metabolism in subsequent cultures. The results are shown in Fig. 33c.

Individual vials were further tested at days 0, 1, 1.5, 2, 2.5, and 3 based on a recognized day 3 viability threshold assay. Trypan blue viability assays were performed immediately after removal of cells from sealed vials, and there was no significant loss of viability over 2.5 days (range 92-98%). Cells were also plated at 10 ⁵ cells/cm ² in standard PTT6 medium, and lactate production was measured at 24 and 48 h. Lactate is a product of glucose metabolism that we have confirmed is directly proportional to the growth rate of MSCs. In Fig. Figure 33d shows the lactate production of MSCs stored for 0, 1, 1.5, 2, 2.5 or 3 days in Plasmalyte/HSA/HypoThermosol ^® and then measured after 24 hours and 48 hours in culture. Lactate production of MSCs after 24 hours and 48 hours of storage in Plasmalyte/HSA/HypoThermosol ^® for 24 hours (day 1) was identical to that of MSCs that were not stored (day 0). After 3 days, lactate production dropped by 40-45%.

Analysis of cytokine production

Cytokine production was measured from the same cultures after 24 h at 37°C. Consistent with the metabolism data, the capacity of MSCs to produce Ang-1, TGF β, VEGF, and HGF was 10-20% relative to controls (day 0) when cells were stored at 2-8°C for 24 h. The results shown in Fig. 33e indicate that the capacity of MSCs to produce VEGF, angiopoietin-1, TGF-β, and HGF was maintained when cells were stored in Plasmalyte/HSA/HypoThermosol ^® at 2-8°C for 24 h. However, the capacity of MSCs to produce VEGF and angiopoietin-1 was reduced by approximately 50% after storage for >2 days. Results for HGF were similarly maintained for 24 hours but dropped by >70% when stored for >2 days. Results for TGF-β demonstrate that the capacity of MSCs to produce TGF-β was maintained at approximately 75% when stored for >2 days in Plasmalyte/HSA/HypoThermosol ^® at 2-8°C.

Another analysis of cytokine production in MSCs stored for 0, 1, 1.5, 2, 2.5, or 3 days in Plasmalyte/HSA/HypoThermosol ^® confirmed the results obtained in the first cytokine analysis (Fig. 33e). The results demonstrate that the ability of MSCs to produce VEGF, angiopoietin-1, and TGF-β was maintained when the cells were stored in Plasmalyte/HSA/HypoThermosol ^® at 2-8°C for 24 hours. Furthermore, the level of VEGF and angiopoietin-1 secretion was reduced by approximately 50% when stored for >2 days, while the level of TGF-β secretion was reduced by approximately 25%.

Overall, based on the analysis of the viability, appearance, metabolism and cytokine production exhibited by the cells in these studies, a shelf life of 72 hours from the time the product vial is closed can be established. Thus, since in principle any location in the (developed) world can be reached by air travel within 72 hours, the storage and transport composition according to the invention essentially enables the transport of live MSCs from the MSC production facility to essentially any location in the world where the MSCs are administered to a subject. Thus, the storage and/or transport composition of the present invention essentially reduces the complexity of GMP manufacturing and the supply chain of pharmaceutically acceptable mesenchymal stem cells/stem cell populations, thereby resulting in mesenchymal stem cell based therapies becoming more readily available to a larger population.

The invention is further characterized by the following points:

1. A method for preparing a composition for storing or transporting mesenchymal stem cells, wherein the composition comprises from about 0.5 to about 10 million mesenchymal stem cells, wherein the method comprises:

a) suspending mesenchymal stem cells in a predetermined volume of a crystalloid solution, wherein the crystalloid solution comprises from about 0.5% to about 5% (w/v) serum albumin, thereby obtaining a first cell suspension,

b) determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a composition containing from about 0.5 to about 10 million mesenchymal stem cells,

c) mixing a determined volume of the first cell suspension with a volume of a liquid carrier, wherein said liquid carrier comprises from about 0.5% to about 5% (w/v) serum albumin, and

1) Trolox;

2) Na ⁺ ;

3) K+;

4) Ca ²⁺ ,

5) Mg2 ⁺ ;

6) Cl ^- ;

7) H ₂ PO ₄ ^- ;

8) HEPES;

9) Lactobionate;

10) sucrose;

11) mannitol;

12) glucose;

13) dextran-40;

14) adenosine and

15) glutathione,

thereby obtaining a composition for storing or transporting mesenchymal stem cells comprising from about 0.5 to about 10 million mesenchymal stem cells.

2. The method of claim 1, wherein the predetermined volume of crystalloid solution used to suspend the mesenchymal stem cells is from about 1 ml to about 10 ml.

3. The method according to claim 1 or 2, wherein, after mixing a certain volume of the first cell suspension with a volume of liquid carrier, the total volume of the composition for storing or transporting mesenchymal stem cells is approximately 1 ml.

4. The method of claim 2, wherein the composition comprises from about 0.5 to about 10 million viable mesenchymal stem cells.

5. The method according to any one of claims 1-4, wherein the composition comprises about 1, about 3, or about 5 million mesenchymal stem cells.

6. The method according to any one of claims 1 to 5, wherein "about" in relation to the amount of mesenchymal stem cells means ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9% or ±10%.

7. The method according to any one of claims 1-6, wherein the mesenchymal stem cells are collected from a cell culture vessel before resuspending said mesenchymal stem cells in a predetermined volume of crystalloid solution.

8. The method according to any one of claims 1 to 7, wherein both the crystalloid solution and the liquid carrier contain the same concentration of serum albumin.

9. The method of claim 8, wherein both the crystalloid solution and the liquid carrier comprise from about 0.5% to about 5% (w/v) serum albumin.

10. The method of claim 8 or 9, wherein both the crystalloid solution and the liquid carrier comprise from about 1% to about 5% (w/v) serum albumin.

11. The method of any one of claims 8-10, wherein both the crystalloid solution and the liquid carrier contain from about 1% to about 3% (w/v) serum albumin.

12. The method of any one of claims 8-11, wherein both the crystalloid solution and the liquid carrier contain approximately 1% (w/v) serum albumin.

13. The method according to any one of claims 1-12, wherein the serum albumin is human serum albumin.

14. The method according to any one of claims 1-13, wherein the crystalloid solution contains sodium, potassium, magnesium and chloride.

15. The method according to any one of claims 1-14, wherein the crystalloid solution is PlasmaLyte or Ringer's lactate.

16. The method according to claim 15, wherein the composition for storing or transporting mesenchymal stem cells contains no more than 20% plasmalyte.

17. The method according to any one of claims 1-16, wherein the mesenchymal stem cells are mesenchymal stem cells selected from the group consisting of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord-placental junction mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose tissue-derived mesenchymal stem cells.

18. The method according to claim 17, wherein the umbilical cord mesenchymal stem cells are selected from the group consisting of amnion mesenchymal stem cells, perivascular mesenchymal stem cells, Wharton's jelly mesenchymal stem cells, and umbilical cord amniotic membrane mesenchymal stem cells.

19. The method of claim 17 or 18, wherein the umbilical cord amniotic membrane mesenchymal stem cells are a population of mesenchymal stem cells, wherein at least approximately 90% or more of the cells of the population of mesenchymal stem cells express each of the following markers: CD73, CD90, and CD 105.

20. The method of claim 19, wherein at least about 90% or more of the cells of the mesenchymal stem cell population do not express the following markers: CD34, CD45, and HLA DR (human leukocyte antigen-related antigen D).

21. The method of claim 19 or 20, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the mesenchymal stem cell population express each of CD73, CD90, and CD 105 and do not express each of CD34, CD45, and HLA-DR.

22. A composition for storing or transporting mesenchymal stem cells obtained by the method defined in any of paragraphs 1-21.

23. A composition for storing or transporting mesenchymal stem cells, which can be obtained by the method defined in any of paragraphs 1-21.

24. A method for transporting mesenchymal stem cells, comprising transporting said mesenchymal stem cells in a composition for storing or transporting mesenchymal stem cells, as defined in paragraph 22 or paragraph 23.

25. The method according to claim 24, wherein the transportation is carried out over a period of approximately 7 days or less.

26. The method according to claim 24 or 25, wherein the transportation is carried out for about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or less than about 1 day.

27. The method according to any one of paragraphs 24-26, wherein the transportation is carried out for about 48 hours or about 24 hours or less.

28. The method according to any one of paragraphs 24-27, wherein the transportation is carried out at a temperature from about -5°C to about 15°C.

29. The method according to any one of paragraphs 24-28, wherein the transportation is carried out at a temperature from about 2°C to about 8°C.

30. The method according to any one of paragraphs. 24-29, wherein the transportation is carried out at a temperature of more than about -5°C, more than about -10°C, more than about -15°C, or more than about -20°C.

31. A method for treating a subject suffering from a disease, comprising local administration of mesenchymal stem cells that are stored or transported in a composition for storing or transporting mesenchymal stem cells as defined in paragraph 22 or paragraph 23.

32. The method according to claim 31, wherein the mesenchymal stem cells are administered to the subject after separating the mesenchymal stem cells from the composition for storing or transporting the mesenchymal stem cells.

33. The method according to claim 32, wherein separating the mesenchymal stem cells from the composition for storing or transporting the mesenchymal stem cells comprises centrifugation.

34. The method according to claims 32 and 33, wherein separating the mesenchymal stem cells from the composition for storing or transporting the mesenchymal stem cells comprises extracting the cell population from the vial using a syringe.

35. The method according to any one of claims 31-34, comprising introducing mesenchymal stem cells via a syringe.

36. The method according to any one of claims 31-35, wherein the mesenchymal stem cells are used in a dose of about 3, about 5, or about 10 million cells.

37. The method according to any one of claims 31-36, wherein the population of mesenchymal stem cells is used within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours or less from the time of collection of the population of mesenchymal stem cells.

38. The method of claim 37, wherein the mesenchymal stem cells are used within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours or less from the time of collection of the mesenchymal stem cells.

39. The method according to any one of claims 31-38, wherein the disease is a skin disease or a wound.

40. The method according to claim 39, wherein the wound is caused by a burn, bite, injury, surgery or disease.

41. The method according to claim 40, wherein the wound is caused by a diabetic disease, wherein the wound is preferably a diabetic wound.

42. The method according to claim 41, wherein the wound is an ulcer caused by diabetic foot syndrome.

43. The method according to any one of claims 31-42, wherein a dose of about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells, or less than 0.25 million cells is administered once or twice a week.

44. The method of claim 43, wherein a dose of about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells, or less than 0.25 million cells is administered once or twice a week for a period of three weeks, four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or ten weeks, or more than ten weeks.

45. The method according to any one of paragraphs 31-44, wherein the mesenchymal stem cells are used locally and covered with a film or bandage.

46. The method according to any one of claims 31-45, wherein the mesenchymal stem cells are used at a dose of from about 1000 cells/ ^cm2 to about 5 million cells/ ^cm2 .

47. The method according to any one of claims 31-46, wherein the mesenchymal stem cells are used at a dose of about 100,000 cells/ ^cm2 , about 300,000 cells/ ^cm2 , or about 500,000 cells/ ^cm2 .

48. The method according to any one of claims 31-47, wherein the mesenchymal stem cells are used once, twice or more times per week.

49. The method according to any one of claims 31-48, wherein the mesenchymal stem cells are used for one, two, three, four, five, six, seven, eight, nine, ten, eleven weeks or more than eleven weeks.

50. The method of any one of claims 31-49, wherein the mesenchymal stem cells are used twice a week for about 8 weeks at a dose of about 100,000 cells/ ^cm2 , about 300,000 cells/ ^cm2 , or about 500,000 cells/ ^cm2 .

51. A single dose of mesenchymal stem cells obtained by the method defined in any of paragraphs 1-21.

52. A single dose of mesenchymal stem cells that can be obtained by the method defined in any of paragraphs 1-21.

53. A single dose according to claim 51 or 52, wherein the single dose contains from about 0.5 to about 10 million mesenchymal stem cells in a volume of 1 ml.

54. A single dose according to claim 53, wherein the single dose contains about 1 million, about 3 million, or about 5 million cells.

55. A single dose according to any one of claims 52-54, wherein the umbilical cord mesenchymal stem cells are selected from the group consisting of amnion mesenchymal stem cells, perivascular mesenchymal stem cells, Wharton's jelly mesenchymal stem cells, and umbilical cord amniotic membrane mesenchymal stem cells.

56. A single dose according to claim 55, wherein the amniotic cord membrane mesenchymal stem cells are a population of mesenchymal stem cells, wherein at least about 90% or more of the cells of the population of mesenchymal stem cells express each of the following markers: CD73, CD90, and CD 105.

57. A single dose according to claim 56, wherein at least about 90% or more of the cells of the mesenchymal stem cell population do not express the following markers: CD34, CD45 and HLA DR.

58. A single dose according to claim 56 or 57, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the mesenchymal stem cell population express each of CD73, CD90, and CD105 and do not express each of CD34, CD45, and HLA-DR (human leukocyte antigen-related antigen D).

It will be apparent to one skilled in the art that various changes and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of levels of skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "comprising," etc. are to be construed broadly and without limitation. Furthermore, the terms and expressions used herein have been used as terms of description and not limitation, and by the use of such terms and expressions there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is understood that various modifications are possible within the scope of the claimed invention. It is therefore to be understood that, although the present invention has been particularly disclosed by means of preferred embodiments and optional features, modifications and variations of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention. The invention is described herein broadly and generally. Each of the more specific species and subgeneric groups falling within the general description also forms part of the invention. The latter includes a general description of the invention with a qualification or negative limitation eliminating any item of the kind, whether or not the material to be eliminated is specifically stated. Furthermore, when features or aspects of the invention are described in terms of Markush groups, then those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group. Additional embodiments of the invention will become apparent from the following claims.

It is understood that the term "about" as used herein means that there may be a variation in the relevant value or range (such as pH, concentration, percentage, molarity, amount of amino acids, time, etc.), which may be up to 5%, up to 10%, up to 15%, or up to 20% and including 20% of the specified value. For example, if a composition contains from about 5 mg/mL of a compound, it is understood that this means that the composition may contain from 4 to 6 mg/mL, preferably from 4.25 to 5.75 mg/mL, more preferably from 4.5 to 5.5 mg/mL, and even more preferably from 4.75 to 5.25 mg/mL, with 5 mg/mL being most preferred. As used herein, a range that is designated as "(from) X to Y" is the same as the range designated as "X-Y". Both intervals specifically include an upper limit as well as a lower limit. The latter means that, for example, the interval "from 5 mg/ml to 10 mg/ml" or "5 mg/ml -10 mg/ml" includes concentrations of 5, 6, 7, 8, 9 and 10 mg/ml, as well as any intermediate value.

--->

SEQUENCE LIST

<110> CellResearch Corporation Pte Ltd

The Regents of the University of Colorado

<120> COMPOSITION FOR STORAGE OR TRANSPORTATION OF MESENCHYMAL STEM

CELLS AND METHODS OF ITS PREPARATION AND APPLICATION

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35 40 45

Phe Ser Asn Val Ser Thr Asn Val Ser Tyr Gln Glu Thr Thr Thr Pro

50 55 60

Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly Asn

65 70 75 80

Glu Ala Thr Thr Asn Ile Thr Glu Thr Thr Val Lys Phe Thr Ser Thr

85 90 95

Ser Val Ile Thr Ser Val Tyr Gly Asn Thr Asn Ser Ser Val Gln Ser

100 105 110

Gln Thr Ser Val Ile Ser Thr Val Phe Thr Thr Pro Ala Asn Val Ser

115 120 125

Thr Pro Glu Thr Thr Leu Lys Pro Ser Leu Ser Pro Gly Asn Val Ser

130 135 140

Asp Leu Ser Thr Thr Ser Thr Ser Leu Ala Thr Ser Pro Thr Lys Pro

145 150 155 160

Tyr Thr Ser Ser Ser Pro Ile Leu Ser Asp Ile Lys Ala Glu Ile Lys

165 170 175

Cys Ser Gly Ile Arg Glu Val Lys Leu Thr Gln Gly Ile Cys Leu Glu

180 185 190

Gln Asn Lys Thr Ser Ser Cys Ala Glu Phe Lys Lys Asp Arg Gly Glu

195 200 205

Gly Leu Ala Arg Val Leu Cys Gly Glu Glu Gln Ala Asp Ala Asp Ala

210 215 220

Gly Ala Gln Val Cys Ser Leu Leu Leu Ala Gln Ser Glu Val Arg Pro

225 230 235 240

Gln Cys Leu Leu Leu Val Leu Ala Asn Arg Thr Glu Ile Ser Ser Lys

245 250 255

Leu Gln Leu Met Lys Lys His Gln Ser Asp Leu Lys Lys Leu Gly Ile

260 265 270

Leu Asp Phe Thr Glu Gln Asp Val Ala Ser His Gln Ser Tyr Ser Gln

275 280 285

Lys Thr Leu Ile Ala Leu Val Thr Ser Gly Ala Leu Leu Ala Val Leu

290 295 300

Gly Ile Thr Gly Tyr Phe Leu Met Asn Arg Arg Ser Trp Ser Pro Thr

305 310 315 320

Gly Glu Arg Leu Gly Glu Asp Pro Tyr Tyr Thr Glu Asn Gly Gly Gly

325 330 335

Gln Gly Tyr Ser Ser Gly Pro Gly Thr Ser Pro Glu Ala Gln Gly Lys

340 345 350

Ala Ser Val Asn Arg Gly Ala Gln Glu Asn Gly Thr Gly Gln Ala Thr

355 360 365

Ser Arg Asn Gly His Ser Ala Arg Gln His Val Val Ala Asp Thr Glu

370 375 380

Leu

385

<210> 5

<211> 1304

<212> PRT

<213> human

<400> 5

Met Tyr Leu Trp Leu Lys Leu Leu Ala Phe Gly Phe Ala Phe Leu Asp

1 5 10 15

Thr Glu Val Phe Val Thr Gly Gln Ser Pro Thr Pro Ser Pro Thr Gly

20 25 30

Leu Thr Thr Ala Lys Met Pro Ser Val Pro Leu Ser Ser Asp Pro Leu

35 40 45

Pro Thr His Thr Thr Ala Phe Ser Pro Ala Ser Thr Phe Glu Arg Glu

50 55 60

Asn Asp Phe Ser Glu Thr Thr Thr Ser Leu Ser Pro Asp Asn Thr Ser

65 70 75 80

Thr Gln Val Ser Pro Asp Ser Leu Asp Asn Ala Ser Ala Phe Asn Thr

85 90 95

Thr Gly Val Ser Ser Val Gln Thr Pro His Leu Pro Thr His Ala Asp

100 105 110

Ser Gln Thr Pro Ser Ala Gly Thr Asp Thr Gln Thr Phe Ser Gly Ser

115 120 125

Ala Ala Asn Ala Lys Leu Asn Pro Thr Pro Gly Ser Asn Ala Ile Ser

130 135 140

Asp Val Pro Gly Glu Arg Ser Thr Ala Ser Thr Phe Pro Thr Asp Pro

145 150 155 160

Val Ser Pro Leu Thr Thr Thr Leu Ser Leu Ala His His Ser Ser Ala

165 170 175

Ala Leu Pro Ala Arg Thr Ser Asn Thr Thr Ile Thr Ala Asn Thr Ser

180 185 190

Asp Ala Tyr Leu Asn Ala Ser Glu Thr Thr Thr Leu Ser Pro Ser Gly

195 200 205

Ser Ala Val Ile Ser Thr Thr Thr Ile Ala Thr Thr Pro Ser Lys Pro

210 215 220

Thr Cys Asp Glu Lys Tyr Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn

225 230 235 240

Lys Glu Thr Lys Leu Phe Thr Ala Lys Leu Asn Val Asn Glu Asn Val

245 250 255

Glu Cys Gly Asn Asn Thr Cys Thr Asn Asn Glu Val His Asn Leu Thr

260 265 270

Glu Cys Lys Asn Ala Ser Val Ser Ile Ser His Asn Ser Cys Thr Ala

275 280 285

Pro Asp Lys Thr Leu Ile Leu Asp Val Pro Pro Gly Val Glu Lys Phe

290 295 300

Gln Leu His Asp Cys Thr Gln Val Glu Lys Ala Asp Thr Thr Ile Cys

305 310 315 320

Leu Lys Trp Lys Asn Ile Glu Thr Phe Thr Cys Asp Thr Gln Asn Ile

325 330 335

Thr Tyr Arg Phe Gln Cys Gly Asn Met Ile Phe Asp Asn Lys Glu Ile

340 345 350

Lys Leu Glu Asn Leu Glu Pro Glu His Glu Tyr Lys Cys Asp Ser Glu

355 360 365

Ile Leu Tyr Asn Asn His Lys Phe Thr Asn Ala Ser Lys Ile Ile Lys

370 375 380

Thr Asp Phe Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe Cys Arg Ser

385 390 395 400

Glu Ala Ala His Gln Gly Val Ile Thr Trp Asn Pro Pro Gln Arg Ser

405 410 415

Phe His Asn Phe Thr Leu Cys Tyr Ile Lys Glu Thr Glu Lys Asp Cys

420 425 430

Leu Asn Leu Asp Lys Asn Leu Ile Lys Tyr Asp Leu Gln Asn Leu Lys

435 440 445

Pro Tyr Thr Lys Tyr Val Leu Ser Leu His Ala Tyr Ile Ile Ala Lys

450 455 460

Val Gln Arg Asn Gly Ser Ala Ala Met Cys His Phe Thr Thr Lys Ser

465 470 475 480

Ala Pro Pro Ser Gln Val Trp Asn Met Thr Val Ser Met Thr Ser Asp

485 490 495

Asn Ser Met His Val Lys Cys Arg Pro Pro Arg Asp Arg Asn Gly Pro

500 505 510

His Glu Arg Tyr His Leu Glu Val Glu Ala Gly Asn Thr Leu Val Arg

515 520 525

Asn Glu Ser His Lys Asn Cys Asp Phe Arg Val Lys Asp Leu Gln Tyr

530 535 540

Ser Thr Asp Tyr Thr Phe Lys Ala Tyr Phe His Asn Gly Asp Tyr Pro

545 550 555 560

Gly Glu Pro Phe Ile Leu His His Ser Thr Ser Tyr Asn Ser Lys Ala

565 570 575

Leu Ile Ala Phe Leu Ala Phe Leu Ile Ile Val Thr Ser Ile Ala Leu

580 585 590

Leu Val Val Leu Tyr Lys Ile Tyr Asp Leu His Lys Lys Arg Ser Cys

595 600 605

Asn Leu Asp Glu Gln Gln Glu Leu Val Glu Arg Asp Asp Glu Lys Gln

610 615 620

Leu Met Asn Val Glu Pro Ile His Ala Asp Ile Leu Leu Glu Thr Tyr

625 630 635 640

Lys Arg Lys Ile Ala Asp Glu Gly Arg Leu Phe Leu Ala Glu Phe Gln

645 650 655

Ser Ile Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg Lys

660 665 670

Pro Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu Pro Tyr Asp

675 680 685

Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser Asn

690 695 700

Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe Lys Glu Pro Arg Lys Tyr

705 710 715 720

Ile Ala Ala Gln Gly Pro Arg Asp Glu Thr Val Asp Asp Phe Trp Arg

725 730 735

Met Ile Trp Glu Gln Lys Ala Thr Val Ile Val Met Val Thr Arg Cys

740 745 750

Glu Glu Gly Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro Ser Met Glu

755 760 765

Glu Gly Thr Arg Ala Phe Gly Asp Val Val Val Lys Ile Asn Gln His

770 775 780

Lys Arg Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn Lys

785 790 795 800

Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile Gln Phe Thr Ser

805 810 815

Trp Pro Asp His Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu

820 825 830

Arg Arg Arg Val Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val

835 840 845

Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr Ile Gly Ile

850 855 860

Asp Ala Met Leu Glu Gly Leu Glu Ala Glu Asn Lys Val Asp Val Tyr

865 870 875 880

Gly Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu Met Val Gln Val

885 890 895

Glu Ala Gln Tyr Ile Leu Ile His Gln Ala Leu Val Glu Tyr Asn Gln

900 905 910

Phe Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro Tyr Leu His

915 920 925

Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro Ser Pro Leu Glu Ala

930 935 940

Glu Phe Gln Arg Leu Pro Ser Tyr Arg Ser Trp Arg Thr Gln His Ile

945 950 955 960

Gly Asn Gln Glu Glu Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile

965 970 975

Pro Tyr Asp Tyr Asn Arg Val Pro Leu Lys His Glu Leu Glu Met Ser

980 985 990

Lys Glu Ser Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp Ser Asp

995 1000 1005

Ser Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser Phe Ile Met Ser

1010 1015 1020

Tyr Trp Lys Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys

1025 1030 1035

Glu Thr Ile Gly Asp Phe Trp Gln Met Ile Phe Gln Arg Lys Val

1040 1045 1050

Lys Val Ile Val Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu

1055 1060 1065

Ile Cys Ala Gln Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp

1070 1075 1080

Ile Glu Val Asp Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr

1085 1090 1095

Leu Arg Val Phe Glu Leu Arg His Ser Lys Arg Lys Asp Ser Arg

1100 1105 1110

Thr Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser Val Glu Gln Leu

1115 1120 1125

Pro Ala Glu Pro Lys Glu Leu Ile Ser Met Ile Gln Val Val Lys

1130 1135 1140

Gln Lys Leu Pro Gln Lys Asn Ser Ser Glu Gly Asn Lys His His

1145 1150 1155

Lys Ser Thr Pro Leu Leu Ile His Cys Arg Asp Gly Ser Gln Gln

1160 1165 1170

Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu Leu Glu Ser Ala Glu

1175 1180 1185

Thr Glu Glu Val Val Asp Ile Phe Gln Val Val Lys Ala Leu Arg

1190 1195 1200

Lys Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr Gln Phe

1205 1210 1215

Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln

1220 1225 1230

Val Lys Lys Asn Asn His Gln Glu Asp Lys Ile Glu Phe Asp Asn

1235 1240 1245

Glu Val Asp Lys Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu

1250 1255 1260

Gly Ala Pro Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly

1265 1270 1275

Ser Glu Pro Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn

1280 1285 1290

Gly Pro Ala Ser Pro Ala Leu Asn Gln Gly Ser

1295 1300

<210> 6

<211> 254

<212> PRT

<213> human

<400> 6

Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val

1 5 10 15

Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Lys Glu Glu His Val Ile

20 25 30

Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met

35 40 45

Phe Asp Phe Asp Gly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys

50 55 60

Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu

65 70 75 80

Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Glu

85 90 95

Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn Val Pro Pro

100 105 110

Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg Glu Pro Asn

115 120 125

Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val Val Asn Val

130 135 140

Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr

145 150 155 160

Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu

165 170 175

Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His

180 185 190

Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro

195 200 205

Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu Gly Leu

210 215 220

Thr Val Gly Leu Val Gly Ile Ile Ile Gly Thr Ile Phe Ile Ile Lys

225 230 235 240

Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu

245 250

<210> 7

<211> 503

<212> PRT

<213> human

<400> 7

Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val

1 5 10 15

Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr

20 25 30

Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys

35 40 45

Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys

50 55 60

Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg

65 70 75 80

Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr

85 90 95

Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro

100 105 110

Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala

115 120 125

Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu Met

130 135 140

Val Tyr Ile Cys His Asn Arg Thr Val Ile His His Arg Val Pro Asn

145 150 155 160

Glu Glu Asp Pro Ser Leu Asp Arg Pro Phe Ile Ser Glu Gly Thr Thr

165 170 175

Leu Lys Asp Leu Ile Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly

180 185 190

Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln

195 200 205

Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp

210 215 220

Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg

225 230 235 240

Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His

245 250 255

Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr

260 265 270

Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu His Gly Ser Leu

275 280 285

Phe Asp Tyr Leu Asn Arg Tyr Thr Val Thr Val Glu Gly Met Ile Lys

290 295 300

Leu Ala Leu Ser Thr Ala Ser Gly Leu Ala His Leu His Met Glu Ile

305 310 315 320

Val Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser

325 330 335

Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu

340 345 350

Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile Ala

355 360 365

Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu

370 375 380

Asp Asp Ser Ile Asn Met Lys His Phe Glu Ser Phe Lys Arg Ala Asp

385 390 395 400

Ile Tyr Ala Met Gly Leu Val Phe Trp Glu Ile Ala Arg Arg Cys Ser

405 410 415

Ile Gly Gly Ile His Glu Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val

420 425 430

Pro Ser Asp Pro Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln

435 440 445

Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu

450 455 460

Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala

465 470 475 480

Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln Leu Ser

485 490 495

Gln Gln Glu Gly Ile Lys Met

500

<210> 8

<211> 232

<212> PRT

<213> human

<400> 8

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1 5 10 15

Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30

Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln

35 40 45

Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu

50 55 60

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu

65 70 75 80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

85 90 95

Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His

100 105 110

Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys

115 120 125

Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val

130 135 140

Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr

145 150 155 160

Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp

165 170 175

Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys

180 185 190

His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn

195 200 205

Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr

210 215 220

Cys Arg Cys Asp Lys Pro Arg Arg

225 230

<210> 9

<211> 1089

<212> PRT

<213> human

<400> 9

Met Gly Thr Ser His Pro Ala Phe Leu Val Leu Gly Cys Leu Leu Thr

1 5 10 15

Gly Leu Ser Leu Ile Leu Cys Gln Leu Ser Leu Pro Ser Ile Leu Pro

20 25 30

Asn Glu Asn Glu Lys Val Val Gln Leu Asn Ser Ser Phe Ser Leu Arg

35 40 45

Cys Phe Gly Glu Ser Glu Val Ser Trp Gln Tyr Pro Met Ser Glu Glu

50 55 60

Glu Ser Ser Asp Val Glu Ile Arg Asn Glu Glu Asn Asn Ser Gly Leu

65 70 75 80

Phe Val Thr Val Leu Glu Val Ser Ser Ala Ser Ala Ala His Thr Gly

85 90 95

Leu Tyr Thr Cys Tyr Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu

100 105 110

Glu Gly Arg His Ile Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe

115 120 125

Val Pro Leu Gly Met Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp

130 135 140

Ser Ala Ile Ile Pro Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr

145 150 155 160

Leu His Asn Ser Glu Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln

165 170 175

Gly Phe Asn Gly Thr Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr

180 185 190

Val Lys Gly Lys Lys Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu

195 200 205

Lys Ala Thr Ser Glu Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val

210 215 220

Tyr Lys Ser Gly Glu Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn

225 230 235 240

Glu Val Val Asp Leu Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys

245 250 255

Gly Ile Thr Met Leu Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val

260 265 270

Tyr Thr Leu Thr Val Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr

275 280 285

Glu Cys Ala Ala Arg Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys

290 295 300

Val Thr Ile Ser Val His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr

305 310 315 320

Phe Ser Gln Leu Glu Ala Val Asn Leu His Glu Val Lys His Phe Val

325 330 335

Val Glu Val Arg Ala Tyr Pro Pro Pro Arg Ile Ser Trp Leu Lys Asn

340 345 350

Asn Leu Thr Leu Ile Glu Asn Leu Thr Glu Ile Thr Thr Asp Val Glu

355 360 365

Lys Ile Gln Glu Ile Arg Tyr Arg Ser Lys Leu Lys Leu Ile Arg Ala

370 375 380

Lys Glu Glu Asp Ser Gly His Tyr Thr Ile Val Ala Gln Asn Glu Asp

385 390 395 400

Ala Val Lys Ser Tyr Thr Phe Glu Leu Leu Thr Gln Val Pro Ser Ser

405 410 415

Ile Leu Asp Leu Val Asp Asp His His Gly Ser Thr Gly Gly Gln Thr

420 425 430

Val Arg Cys Thr Ala Glu Gly Thr Pro Leu Pro Asp Ile Glu Trp Met

435 440 445

Ile Cys Lys Asp Ile Lys Lys Cys Asn Asn Glu Thr Ser Trp Thr Ile

450 455 460

Leu Ala Asn Asn Val Ser Asn Ile Ile Thr Glu Ile His Ser Arg Asp

465 470 475 480

Arg Ser Thr Val Glu Gly Arg Val Thr Phe Ala Lys Val Glu Glu Thr

485 490 495

Ile Ala Val Arg Cys Leu Ala Lys Asn Leu Leu Gly Ala Glu Asn Arg

500 505 510

Glu Leu Lys Leu Val Ala Pro Thr Leu Arg Ser Glu Leu Thr Val Ala

515 520 525

Ala Ala Val Leu Val Leu Leu Val Ile Val Ile Ile Ser Leu Ile Val

530 535 540

Leu Val Val Ile Trp Lys Gln Lys Pro Arg Tyr Glu Ile Arg Trp Arg

545 550 555 560

Val Ile Glu Ser Ile Ser Pro Asp Gly His Glu Tyr Ile Tyr Val Asp

565 570 575

Pro Met Gln Leu Pro Tyr Asp Ser Arg Trp Glu Phe Pro Arg Asp Gly

580 585 590

Leu Val Leu Gly Arg Val Leu Gly Ser Gly Ala Phe Gly Lys Val Val

595 600 605

Glu Gly Thr Ala Tyr Gly Leu Ser Arg Ser Gln Pro Val Met Lys Val

610 615 620

Ala Val Lys Met Leu Lys Pro Thr Ala Arg Ser Ser Glu Lys Gln Ala

625 630 635 640

Leu Met Ser Glu Leu Lys Ile Met Thr His Leu Gly Pro His Leu Asn

645 650 655

Ile Val Asn Leu Leu Gly Ala Cys Thr Lys Ser Gly Pro Ile Tyr Ile

660 665 670

Ile Thr Glu Tyr Cys Phe Tyr Gly Asp Leu Val Asn Tyr Leu His Lys

675 680 685

Asn Arg Asp Ser Phe Leu Ser His His Pro Glu Lys Pro Lys Lys Glu

690 695 700

Leu Asp Ile Phe Gly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr

705 710 715 720

Val Ile Leu Ser Phe Glu Asn Asn Gly Asp Tyr Met Asp Met Lys Gln

725 730 735

Ala Asp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys Glu Val Ser

740 745 750

Lys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp Arg Pro Ala Ser Tyr

755 760 765

Lys Lys Lys Ser Met Leu Asp Ser Glu Val Lys Asn Leu Leu Ser Asp

770 775 780

Asp Asn Ser Glu Gly Leu Thr Leu Leu Asp Leu Leu Ser Phe Thr Tyr

785 790 795 800

Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Lys Asn Cys Val His

805 810 815

Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Ala Gln Gly Lys Ile Val

820 825 830

Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Met His Asp Ser Asn

835 840 845

Tyr Val Ser Lys Gly Ser Thr Phe Leu Pro Val Lys Trp Met Ala Pro

850 855 860

Glu Ser Ile Phe Asp Asn Leu Tyr Thr Thr Leu Ser Asp Val Trp Ser

865 870 875 880

Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Thr Pro Tyr

885 890 895

Pro Gly Met Met Val Asp Ser Thr Phe Tyr Asn Lys Ile Lys Ser Gly

900 905 910

Tyr Arg Met Ala Lys Pro Asp His Ala Thr Ser Glu Val Tyr Glu Ile

915 920 925

Met Val Lys Cys Trp Asn Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr

930 935 940

His Leu Ser Glu Ile Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys

945 950 955 960

Ser Tyr Glu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro Ala

965 970 975

Val Ala Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly Val Thr

980 985 990

Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu Gly Gly Leu Asp

995 1000 1005

Glu Gln Arg Leu Ser Ala Asp Ser Gly Tyr Ile Ile Pro Leu Pro

1010 1015 1020

Asp Ile Asp Pro Val Pro Glu Glu Glu Asp Leu Gly Lys Arg Asn

1025 1030 1035

Arg His Ser Ser Gln Thr Ser Glu Glu Ser Ala Ile Glu Thr Gly

1040 1045 1050

Ser Ser Ser Ser Thr Phe Ile Lys Arg Glu Asp Glu Thr Ile Glu

1055 1060 1065

Asp Ile Asp Met Met Asp Asp Ile Gly Ile Asp Ser Ser Asp Leu

1070 1075 1080

Val Glu Asp Ser Phe Leu

1085

<210> 10

<211> 498

<212> PRT

<213> human

<400> 10

Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His

1 5 10 15

Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg

20 25 30

Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro

35 40 45

Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr

50 55 60

Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser

65 70 75 80

Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp

85 90 95

Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met

100 105 110

Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu

115 120 125

Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys

130 135 140

Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu

145 150 155 160

Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln

165 170 175

Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser

180 185 190

Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu

195 200 205

Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr

210 215 220

Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala

225 230 235 240

Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp

245 250 255

Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu

260 265 270

Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp

275 280 285

Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile

290 295 300

Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn

305 310 315 320

Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp

325 330 335

Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser

340 345 350

Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln

355 360 365

Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg

370 375 380

Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn

385 390 395 400

Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser

405 410 415

Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn

420 425 430

Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp

435 440 445

Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala

450 455 460

Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys

465 470 475 480

Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu

485 490 495

Asp Phe

<210> 11

<211> 728

<212> PRT

<213> human

<400> 11

Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu

1 5 10 15

Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln

20 25 30

Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr

35 40 45

Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val

50 55 60

Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu

65 70 75 80

Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys

85 90 95

Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe

100 105 110

Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys

115 120 125

Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys

130 135 140

Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His

145 150 155 160

Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr

165 170 175

Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser

180 185 190

Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu

195 200 205

Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp

210 215 220

His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro

225 230 235 240

His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp

245 250 255

Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr

260 265 270

Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys

275 280 285

Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu

290 295 300

Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile

305 310 315 320

Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu

325 330 335

His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn

340 345 350

Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr

355 360 365

Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp

370 375 380

Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met

385 390 395 400

Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp

405 410 415

Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala

420 425 430

Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His

435 440 445

Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys

450 455 460

Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu

465 470 475 480

Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val

485 490 495

Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg

500 505 510

Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp

515 520 525

Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr

530 535 540

Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys

545 550 555 560

Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly

565 570 575

Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp

580 585 590

Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu

595 600 605

Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn

610 615 620

Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu

625 630 635 640

Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu

645 650 655

Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp

660 665 670

Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu

675 680 685

Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly

690 695 700

Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile

705 710 715 720

Leu Thr Tyr Lys Val Pro Gln Ser

725

<210> 12

<211> 241

<212> PRT

<213> human

<400> 12

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg

1 5 10 15

Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met

20 25 30

Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu

35 40 45

His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met

50 55 60

Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg

65 70 75 80

Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu

85 90 95

Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp

100 105 110

Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln

115 120 125

Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr

130 135 140

Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg

145 150 155 160

Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu

165 170 175

Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser

180 185 190

Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val

195 200 205

Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg

210 215 220

Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly

225 230 235 240

Ala

<210> 13

<211> 178

<212> PRT

<213> human

<400> 13

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn

<---

Claims (64)

1. A method for preparing a composition for storing and transporting mesenchymal stem cells, where said mesenchymal stem cells are mesenchymal stem cells of the amniotic membrane of the umbilical cord, where said composition contains from 0.5 to 10 million mesenchymal stem cells, where the method includes: a) suspending mesenchymal stem cells in a predetermined volume of a crystalloid solution suitable for sufficiently resuspending the mesenchymal stem cells, wherein said crystalloid solution is PlasmaLyte and contains from 0.5 to 3% w/v serum albumin, thereby obtaining a cell suspension, b) determining the concentration of mesenchymal stem cells in the cell suspension and determining the volume of the cell suspension required to prepare a composition containing from 0.5 to 10 million mesenchymal stem cells, c) mixing a certain volume of cell suspension with a volume of liquid carrier, where after mixing the composition for storing and transporting mesenchymal stem cells contains no more than 20% plasmalyte, where said liquid carrier contains from 0.5 to 3% w/v serum albumin, and 1) Trolox; 2) Na ⁺ ; 3) K ⁺ ; 4) Ca ²⁺ , 5) Mg2 ⁺ ; 6) Cl ^- ; ⁷ ) _{H2PO4- ;} 8) HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 9) lactobionate; 10) sucrose; 11) mannitol; 12) glucose; 13) dextran-40; 14) adenosine and 15) glutathione, thereby obtaining a composition for storing and transporting mesenchymal stem cells containing from 0.5 to 10 million mesenchymal stem cells. 2. The method according to claim 1, wherein the predetermined volume of crystalloid solution used to suspend the mesenchymal stem cells is from 1 to 10 ml. 3. The method according to claim 1 or 2, wherein after mixing a certain volume of cell suspension with a volume of liquid carrier, the total volume of the composition for storing and transporting mesenchymal stem cells is 1 ml. 4. The method according to claim 2, wherein the composition contains from 0.5 to 10 million viable mesenchymal stem cells. 5. The method according to any one of claims 1-4, wherein the composition comprises 1, 3 or 5 million mesenchymal stem cells. 6. The method according to any one of claims 1-5, wherein the mesenchymal stem cells are collected from the cell culture vessel before resuspending the mesenchymal stem cells in a predetermined volume of crystalloid solution. 7. The method according to any one of claims 1-6, wherein both the crystalloid solution and the liquid carrier contain the same concentration of serum albumin. 8. The method of claim 7, wherein both the crystalloid solution and the liquid carrier contain from 1 to 3% w/v serum albumin. 9. The method according to any one of claims 7 or 8, wherein both the crystalloid solution and the liquid carrier contain 1% w/v serum albumin. 10. The method according to any one of claims 1 to 9, wherein the serum albumin is human serum albumin. 11. The method according to any one of claims 1-10, wherein the amniotic membrane mesenchymal stem cells are a population of mesenchymal stem cells, wherein at least 90% or more of the cells of the population of mesenchymal stem cells express each of the following markers: CD73, CD90 and CD105. 12. The method according to claim 11, wherein at least 90% or more of the cells of said mesenchymal stem cell population do not express the following markers: CD34, CD45 and HLA-DR (human leukocyte antigen - related antigen D). 13. The method according to claim 11 or 12, wherein at least 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the cells of said mesenchymal stem cell population express each of CD73, CD90 and CD105 and do not express each of CD34, CD45 and HLA-DR. 14. A composition for storing and transporting mesenchymal stem cells, which is obtained or can be obtained by the method defined in any of claims 1 to 13, wherein said composition contains from 0.5 to 10 million mesenchymal stem cells of the amniotic membrane of the umbilical cord and a mixture of a crystalloid solution and a liquid carrier, wherein said crystalloid solution is PlasmaLyte and contains from 0.5 to 3% w/v serum albumin, wherein said liquid carrier comprises from 0.5 to 3% w/v serum albumin, and 1) Trolox; 2) Na ⁺ ; 3) K ⁺ ; 4) Ca ²⁺ , 5) Mg2 ⁺ ; 6) Cl ^- ; ⁷ ) _{H2PO4- ;} 8) HEPES; 9) lactobionate; 10) sucrose; 11) mannitol; 12) glucose; 13) dextran-40; 14) adenosine and 15) glutathione, and wherein said composition contains no more than 20% plasmalite. 15. A method for transporting mesenchymal stem cells, comprising transporting said mesenchymal stem cells in a composition for storing and transporting mesenchymal stem cells, as defined in paragraph 14. 16. The method according to item 15, wherein transportation is carried out within 7 days or less. 17. The method according to item 15 or 16, wherein transportation is carried out at a temperature from -5°C to 15°C. 18. Use of a composition for storing and transporting mesenchymal stem cells as defined in claim 14 for the manufacture of a pharmaceutical composition for treating a subject suffering from a disease, wherein the disease is a wound, and wherein the use comprises local administration of mesenchymal stem cells that are stored or transported in a composition for storing and transporting mesenchymal stem cells as defined in claim 14, wherein the use comprises separating said mesenchymal stem cells from said composition for storing and transporting mesenchymal stem cells and administering said mesenchymal stem cells to said subject. 19. The use according to claim 18, wherein the separation of mesenchymal stem cells from the composition for storing and transporting mesenchymal stem cells comprises centrifugation. 20. The use according to claim 18 or 19, wherein separating the mesenchymal stem cells from the composition for storing and transporting the mesenchymal stem cells comprises extracting the cell population from the vial using a syringe. 21. The use according to any one of claims 18-20, wherein the mesenchymal stem cells are used in a dose of 3, 5 or 10 million cells. 22. The use according to any one of claims 18-21, wherein the population of mesenchymal stem cells is used within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours or less from the moment of collection of the population of mesenchymal stem cells. 23. Use according to any of paragraphs 18-22, where the wound is caused by a burn, bite, trauma, surgery or disease. 24. The use according to claim 23, wherein the wound is caused by a diabetic disease, wherein the wound is preferably a diabetic wound. 25. Use according to claim 24, wherein the wound is an ulcer caused by diabetic foot syndrome. 26. The use according to any one of claims 18-25, wherein a dose of 10 million cells, 5 million cells, 4 million cells, 3 million cells, 2 million cells, 1 million cells, 0.5 million cells, 0.25 million cells or less than 0.25 million cells is administered once or twice a week. 27. The use according to claim 26, wherein a dose of 10 million cells, 5 million cells, 4 million cells, 3 million cells, 2 million cells, 1 million cells, 0.5 million cells, 0.25 million cells or less than 0.25 million cells is administered once or twice a week for a period of three weeks, four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or ten weeks, or more than ten weeks.

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