CN112167241A - Stem cell freezing medium and stem cell freezing and recovering method - Google Patents
Stem cell freezing medium and stem cell freezing and recovering method Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a stem cell cryopreservation liquid for clinic and a stem cell cryopreservation and recovery method.
Description
Technical Field
The invention belongs to the technical field of biological material cryopreservation, and particularly relates to a clinically usable stem cell cryopreservation solution and a stem cell cryopreservation and recovery method.
Background
Mesenchymal stem cells are widely present in the connective tissue and the organ interstitium of the whole body, and are contained in the bone marrow tissue
It is most abundant, and can be separated from fetal umbilical cord blood, and is also present in placenta, amniotic fluid, umbilical vein subendothelial layer, peripheral blood, liver, fat, muscle, skin, etc. The mesenchymal stem cells have the potential of high proliferation, self-renewal and multidirectional differentiation, can differentiate into various tissues and cells, are easy to separate, culture and amplify, are easy to introduce and express exogenous genes, always maintain the potential of multidirectional differentiation in the in vitro long-term culture process, and have quite stable genetic background.
In 1995, mesenchymal stem cells are first applied to clinical application, in 2012, the company Osiris declares that mesenchymal stem cells are approved by FDA in canada as a medicine to be marketed, and the clinical application of mesenchymal stem cells reaches a historical height. Mesenchymal stem cells have been the first choice in biomedical research by replacing placental stem cells, and are widely used in basic and clinical research of various diseases. After 10 to 20 years, stem cells will become a basic medical technology, and help people to solve diseases which cannot be overcome at present.
At present, the way of treating diseases by stem cells adopts intravenous infusion, and the product belongs to injection in medicines. The development of stem cell preparations as pharmaceuticals is the best way in which stem cell products can really be used in humans. Therefore, many large-scale biological companies have started to develop stem cells into stem cell preparations, and for pharmaceutical products, the stability and effectiveness of products need to be considered, because stem cells belong to bioactive substances, and how to stably store stem cells is a key process for transformation for developing drugs.
Most of cryopreservation means for stem cells in a laboratory adopt dimethyl sulfoxide, bovine serum, culture solution and the like which are mixed according to a certain proportion for cryopreservation of cells, but the bovine serum is an animal-derived substance and cannot be applied to clinical products. Other products are developed by companies, for example, in a patent CN103070161B entitled fat mesenchymal stem cell cryopreservation liquid and cryopreservation method, the fat mesenchymal stem cell cryopreservation liquid and cryopreservation method adopts components such as medicine, human autologous serum, a DMEM-LG culture medium and the like, the human autologous serum has great difference and can be used in clinical products, but the human autologous serum has quality inconsistency as a product, which causes great risk to human bodies in later application.
At present, some frozen stock solutions are developed and dedicated to clinical application, for example, patent publication No. CN101990888B entitled "frozen stock solution of stem cells and method for freezing stem cells" provides a frozen stock solution of stem cells and method for freezing stem cells, and patent publication No. CN201010246409.3 entitled "frozen stock solution of mesenchymal stem cells for direct intravenous application", but the main component in the frozen stock solution is human AB serum or plasma, the use of non-product components of human origin has limited access, and the non-product components are not clinically available products, such as stem cell preparations and clinical use, and have the risks of uncontrollable source, undefined quality standard and uncontrollable product stability.
The stem cell cryopreservation liquid prepared in the invention patent with the authorization number of CN201110419684.5, namely mesenchymal stem cell cryopreservation liquid and a preparation method thereof, achieves a good cell cryopreservation effect, the cell activity can reach more than 90%, but the cell cryopreservation liquid does not refer to the dispersion degree of cells, and the dispersion degree of the cells can influence the safety of product clinical application.
From the above analysis, although there are related cell frozen stocks, there are some problems in applying these frozen stocks to stem cell clinical applications.
Disclosure of Invention
Aiming at the defects and shortcomings of the stem cell cryopreservation liquid in the prior art in clinical application, the invention provides the stem cell cryopreservation liquid for clinical application and a stem cell cryopreservation and recovery method.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
in a first aspect of the present invention, a stem cell cryopreservation solution is provided, which comprises the following components: human serum albumin injection, low molecular weight heparin calcium, compound amino acid injection, compound electrolyte injection, dextran-glucose injection and antioxidant.
Preferably, the volume fraction of the human serum albumin injection is 10-30. More preferably, the human serum albumin injection has a volume fraction of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
Preferably, the compound amino acid injection comprises 10-30 parts by volume. More preferably, the compound amino acid injection has 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 parts by volume.
Preferably, the compound electrolyte injection has the volume part of 20-50. More preferably, the compound electrolyte injection has 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 42, 45, 48 or 50 parts by volume.
Preferably, the dextran-glucose injection liquid has 20-50 parts by volume. More preferably, the dextran-glucose injection liquid volume fraction is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 42, 45, 48 or 50.
Preferably, the concentration of the low molecular heparin calcium is 2000-10000IU/ml, and the volume part of the low molecular heparin calcium is 2-5 parts. More preferably, the volume fraction of the low molecular heparin calcium is 2, 3, 4 or 5 parts.
Preferably, the stem cell freezing medium contains an antioxidant, and the volume part of the antioxidant is 1-5. More preferably, the antioxidant is present in 1, 2, 3, 4 or 5 parts by volume.
Preferably, the antioxidant is vitamin C injection.
Preferably, the stem cell freezing medium further comprises DMSO, and the volume part of the DMSO is 2-10. More preferably, the volume fraction of DMSO is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In a second aspect of the present invention, a stem cell cryopreservation and recovery method is provided, which comprises the following steps:
1) preparing a stem cell freezing medium, and preparing the freezing medium according to the following proportion: according to the volume parts, uniformly mixing 10-20 parts of human serum albumin injection, 2 parts of low molecular weight heparin calcium with the concentration of 2000-10000IU/ml, 15 parts of compound amino acid injection, 35 parts of compound electrolyte injection, 25 parts of dextran-glucose injection and 3 parts of vitamin C injection to obtain a stem cell frozen stock solution;
2) freezing and storing: taking 3 groups of stem cells, placing the 3 groups of stem cells in cryopreservation tubes, wherein the first group is 1E 6/tube, the second group is 5E 6/tube, the third group is 1E 7/tube, adding 1ml of the stem cell cryopreservation liquid in the step 1) into each tube of each group, slowly blowing, uniformly mixing, cooling to-80 ℃ by adopting a program cooling box, standing for 24h at-80 ℃, and transferring into a liquid nitrogen tank for cryopreservation;
3) and (3) resuscitation: taking out the stem cells cryopreserved in the step 2) from the liquid nitrogen tank, quickly placing the stem cells in a 37-DEG water bath, quickly vibrating, and completing resuscitation within 1 minute to obtain resuscitated stem cells.
Preferably, in step 1), the stem cell cryopreservation solution further comprises DMSO, and the volume fraction of the DMSO is 2 to 10.
Preferably, the stem cell is a mesenchymal stem cell.
Compared with the prior art, the invention has the advantages and positive effects that:
the components adopted by the invention are all clinically applied medicines and pharmaceutic adjuvants approved by United states pharmacopoeia, and are products capable of intravenous infusion, and the products have definite product quality standards, so the injection is safe and effective for human body.
The frozen stock solution of the invention is added with the component for preventing the mesenchymal stem cells from aggregating and agglomerating, thereby improving the safety of clinical application of the mesenchymal stem cells.
The invention adds antioxidant, nutrient components and products for adjusting osmotic pressure, can reduce the damage of stem cells in the freezing process and obviously improve the survival rate and the function of the stem cells.
Drawings
FIG. 1A is a photomicrograph of the stem cells of example 1 with a density of 1E6 after cryopreservation and recovery for 0h of culture;
FIG. 1B is a photomicrograph of the stem cells of example 1 with a density of 5E6 after cryopreservation and recovery for 0h of culture;
FIG. 1C is a photomicrograph of the cells dispersed in example 1 after thawing frozen stem cells at a density of 1E7 and culturing for 0 h;
FIG. 2A is a photomicrograph of the cells dispersed in example 1 after the freeze-storage recovery of stem cells with a density of 1E6 and after 1h of culture;
FIG. 2B is a micrograph of the stem cells of 5E6 density cultured for 1h after they were frozen and thawed in example 1;
FIG. 2C is a photomicrograph of the cells dispersed during 1h of the freeze-storage resuscitation of the stem cells with density of 1E7 in example 1;
FIG. 3A is a photomicrograph of the stem cells cultured for 24 hours after the cryopreservation recovery of the stem cells with the density of 1E6 in example 1;
FIG. 3B is a photomicrograph of the stem cells cultured for 24 hours after the cryopreservation recovery of the stem cells with the density of 5E6 in example 1;
FIG. 3C is a photomicrograph of the stem cells cultured for 24 hours after the cryopreservation recovery of the stem cells with the density of 1E7 in example 1;
FIG. 4A is a photomicrograph of the dispersion of cells cultured for 48 hours after the freeze-storage recovery of the stem cells with the density of 1E6 in example 1;
FIG. 4B is a photomicrograph of the dispersion of cells cultured for 48h after the freeze-storage recovery of the stem cells with the density of 5E6 in example 1;
FIG. 4C is a photomicrograph of the dispersion of cells cultured for 48h after the freeze-storage recovery of the stem cells with the density of 1E7 in example 1;
FIG. 5 shows the results of the 9-month resuscitation with frozen stem cells in example 2 to detect CD 90;
FIG. 6 shows the results of the 9-month resuscitation with frozen stem cells for detecting CD105 in example 2;
FIG. 7 shows the results of the 9-month resuscitation test for CD73 on stem cells in example 2;
FIG. 8 shows the results of the 9-month resuscitation test for CD34 on stem cells in example 2;
FIG. 9 shows the results of the 9-month resuscitation test for CD45 on the stem cells in example 2;
FIG. 10 shows the results of the 9-month resuscitation with frozen stem cells in example 2 for detecting CD 14;
FIG. 11 is the results of HLA-DR testing of the 9-month resuscitation of stem cells in example 2;
FIG. 12 shows the results of the 9-month resuscitation with frozen stem cells of example 2 for detecting CD 19;
FIG. 13 shows the results of adipogenic differentiation after 9-month resuscitation of the stem cells in example 2;
FIG. 14 shows the results of osteogenic differentiation after 9-month resuscitation of the stem cells in example 2.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1
The embodiment provides a stem cell cryopreservation solution which comprises, by volume, 10 parts of human serum albumin injection, 2 parts of low molecular weight heparin calcium with the concentration of 2000-10000IU/ml, 15 parts of compound amino acid injection, 35 parts of compound electrolyte injection, 25 parts of dextran-glucose injection and 3 parts of vitamin C injection.
The embodiment further provides a stem cell cryopreservation solution and a cryopreservation method, and the stem cell cryopreservation solution comprises the following steps: 1) preparing a stem cell freezing medium, and preparing the freezing medium according to the following proportion: according to the volume parts, 10 parts of human albumin injection, 2 parts of low molecular heparin calcium with the concentration of 2000-10000IU/ml, 15 parts of compound amino acid injection, 35 parts of compound electrolyte injection, 25 parts of dextran-glucose injection and 3 parts of vitamin C injection are taken; uniformly mixing to obtain a stem cell frozen stock solution;
2) freezing and storing: taking 3 groups of stem cells, placing the 3 groups of stem cells in cryopreservation tubes, wherein the first group is 1E 6/tube, the second group is 5E 6/tube, the third group is 1E 7/tube, adding 1ml of the stem cell cryopreservation liquid in the step 1) into each tube of each group, slowly blowing, uniformly mixing, cooling to-80 ℃ by adopting a program cooling box, standing for 24h at-80 ℃, and transferring into a liquid nitrogen tank for cryopreservation;
3) and (3) resuscitation: taking out the stem cells cryopreserved in the step 2) from the liquid nitrogen tank, quickly placing the stem cells in a 37-DEG water bath, quickly vibrating, and completing resuscitation within 1 minute to obtain resuscitated stem cells. Counting was performed using trypan blue staining, the viability was calculated and the average value of each set of results is shown in table 1. The degree of cell dispersion is shown in FIGS. 1A-2C. The recovered cells are 8000 cells/cm2After inoculation, the cells were cultured in a culture medium, and the morphology of the cells is shown in FIGS. 3A to 4C. The prepared stem cell frozen stock solution is light yellow, and the storage temperature is 2-8 ℃. The stem cells described in this example are mesenchymal stem cells.
TABLE 1 survival rate after cryopreservation and recovery of cells of different densities
| Group of | 1E6/ml | 5E6/ml | 1E7/ml |
| Rate of cell viability | 98% | 97.3% | 96.9% |
Example 2
The embodiment provides a stem cell cryopreservation solution which comprises, by volume, 20 parts of human serum albumin injection, 2 parts of low molecular weight heparin calcium with the concentration of 2000-10000IU/ml, 15 parts of compound amino acid injection, 35 parts of compound electrolyte injection, 25 parts of dextran-glucose injection and 3 parts of vitamin C injection.
The embodiment further provides a stem cell cryopreservation solution and a cryopreservation method, and the stem cell cryopreservation solution comprises the following steps:
1) preparing a stem cell freezing medium, and preparing the freezing medium according to the following proportion: according to the volume parts, taking 10 parts of human albumin injection, 2 parts of low molecular weight heparin calcium with the concentration of 2000-10000IU/ml, 15 parts of compound amino acid injection, 35 parts of compound electrolyte injection, 25 parts of dextran-glucose injection, 3 parts of vitamin C injection and 10 parts of DMSO, and uniformly mixing to obtain a stem cell frozen stock solution;
2) freezing and storing: taking 10 tubes of umbilical cord mesenchymal stem cells, adding 1ml of the stem cell cryopreservation solution into each tube with 5E6/ml, slowly blowing, uniformly mixing, cooling to-80 ℃ by adopting a programmed cooling box, and transferring into a liquid nitrogen tank;
3) and (3) resuscitation: freezing the frozen stem cells obtained in the step 2) for 0 day, recovering after 1 month, 3 months, 6 months and 9 months, taking out the frozen stem cells from a liquid nitrogen tank, quickly placing the frozen stem cells into a 37-DEG water bath kettle, quickly vibrating, completing the recovery within 1 minute, counting by trypan blue staining, calculating the survival rate, and taking the average value of each group of results as shown in the table 2. After 9 months of recovery, the cells were subjected to mesenchymal stem cell surface marker detection, and the results are shown in FIGS. 5-12. The number of cells was 8000/cm2After inoculation, the culture medium is added for culture, and osteogenesis and adipogenesis induction are carried out. The results are shown in FIGS. 13-14.
TABLE 2 survival rate of cells after different time of cryopreservation
| Group of | |
1 month | 3 months old | 6 months old | 9 months old |
| Rate of cell viability | 98.2% | 97.3% | 96.9% | 96.5% | 96% |
Claims (10)
1. The stem cell frozen stock solution is characterized by comprising human serum albumin injection, low molecular heparin calcium, compound amino acid injection, compound electrolyte injection, dextran-glucose injection and antioxidant.
2. The stem cell cryopreservation liquid of claim 1, wherein the volume fraction of the human serum albumin injection is 10-30.
3. The stem cell cryopreservation solution of claim 1, wherein the compound amino acid injection comprises 10 to 30 parts by volume.
4. The stem cell cryopreservation liquid as claimed in claim 1, wherein the compound electrolyte injection is 20-50 parts by volume.
5. The stem cell cryopreservation solution of claim 1, wherein the dextran-glucose injection is 20-50 parts by volume.
6. The stem cell cryopreservation solution of claim 1, wherein the concentration of the low molecular weight heparin calcium is 2000-10000IU/ml, and the volume fraction of the low molecular weight heparin calcium is 2-5.
7. The stem cell cryopreservation solution of claim 1, wherein the stem cell cryopreservation solution comprises 1-5 volume parts of an antioxidant.
8. The stem cell cryopreservation solution of claim 1, wherein the antioxidant is vitamin C injection.
9. The stem cell cryopreservation solution of claim 1, further comprising DMSO, wherein the volume fraction of DMSO is 2-10.
10. A stem cell cryopreservation and recovery method is characterized by comprising the following steps:
1) preparing a stem cell freezing medium, and preparing the freezing medium according to the following proportion: according to the volume parts, uniformly mixing 10-20 parts of human serum albumin injection, 2 parts of low molecular weight heparin calcium with the concentration of 2000-10000IU/ml, 15 parts of compound amino acid injection, 35 parts of compound electrolyte injection, 25 parts of dextran-glucose injection and 3 parts of vitamin C injection to obtain a stem cell frozen stock solution;
2) freezing and storing: taking 3 groups of stem cells, placing the 3 groups of stem cells in cryopreservation tubes, wherein the first group is 1E 6/tube, the second group is 5E 6/tube, the third group is 1E 7/tube, adding 1ml of the stem cell cryopreservation liquid in the step 1) into each tube of each group, slowly blowing, uniformly mixing, cooling to-80 ℃ by adopting a program cooling box, standing for 24h at-80 ℃, and transferring into a liquid nitrogen tank for cryopreservation;
3) and (3) resuscitation: taking out the stem cells cryopreserved in the step 2) from the liquid nitrogen tank, quickly placing the stem cells in a 37-DEG water bath, quickly vibrating, and completing resuscitation within 1 minute to obtain resuscitated stem cells.
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