JP2006055106A - Human bone marrow-derived mesenchymal stem cell culture method using human serum medium - Google Patents

Abstract

A method for safely and efficiently culturing mesenchymal stem cells for transplantation into humans from human bone marrow fluid is provided.
A method of preparing human mesenchymal stem cells for collecting human bone marrow fluid, culturing mesenchymal stem cells derived from bone marrow, and transplanting them to humans, comprising the following steps: How to:
(i) a step of adding 80-120% v / v heparin / buffer solution at a concentration of about 5 to 15 U / mL with respect to the collected amount of bone marrow fluid;
(ii) Bone marrow fluid to which heparin / buffer solution obtained in step (i) is added, human serum from 10 to 20
Culturing in an α-MEM medium containing% v / v and removing blood cell components floating in the culture medium by changing the medium at least twice during the culture; and
(iii) A cell group mainly composed of mesenchymal stem cells is selectively detached from the culture container using a cell dissociating agent, and adherent cells other than the mesenchymal stem cells are separated from the detached cell group, and then separated into another container. Culturing the mesenchymal stem cells selectively in the step.
[Selection] Figure 1

Description

  The present invention relates to a cell culture method for efficiently and safely culturing a large amount of human tissues / cells for use in regenerative medicine, cell therapy and the like.

  Regenerative medicine that cures injuries and diseases by culturing human cells in a clean cell culture room and transplanting them into the human body is being researched and put into practical use. In this case, it is required to culture the cells efficiently and safely, and conventionally fetal bovine serum has been used as the medium. Although fetal bovine serum is said to contain extremely abundant nutrients, including various growth factors, it is difficult to ensure safety with respect to species differences in culture methods using sera from different species. . Despite these concerns, fetal bovine serum has actually been used for human cell culture because no other suitable material has been found.

However, with the occurrence of BSE (Bovine Spongiform Encephalopathy) from several years ago, ensuring the safety of fetal bovine serum has become an urgent issue, and development of a serum-free medium that does not use fetal bovine serum has been promoted. We have not achieved satisfactory results in terms of cell growth rate.

  In recent years, several patent applications have been filed for techniques for adding human serum to a culture medium.

For example, Republished Patent (A1): International Publication No. WO2002 / 024875 (Patent Document 1) is an invention relating to a culture solution obtained by adding growth factors and the like together with human serum to a culture solution of human mature hepatocytes. Kai 2002-78484 (Patent Document 2) is an invention relating to a method of adding a growth factor such as chondrogenic growth factor together with human serum in a method for producing cultured chondrocytes, and published patent publication: JP 2003-52360 (Patent Document). 3) is an invention relating to a method of adding 2-10% human serum when culturing mesenchymal stem cells in the presence of a basement membrane extracellular matrix. Published patent publication: JP 2003-235548 (Patent Document 4) ) Is a patent application characterized in that human culture medium contains human serum and growth factors.

  That is, in the prior art, when human serum is used as a medium, it is required to contain a growth factor, or to be cultured in the presence of a special substrate. Although the mechanism of action of these growth factors has been clarified to some extent, it is still in the process of elucidating the behavior of these affected cells in the medium and long term in the human body. is there. Since these cells are transplanted by humans, it is necessary to be safe and secure.

Furthermore, in published patent gazette: JP-T-11-506010 (Patent Document 5), cell culture as part of a therapeutic drug test method for osteoporosis patients, which is a continuous enzyme using biopsy sections of patient iliac bones It is characterized by using added serum such as fetal bovine serum, human serum, bovine serum albumin or Ultrocell in the range of 1-12% when culturing osteoblast precursor cells after treatment and isolation The object and contents of the present invention are clearly different.
One of the inventors, Ogushi is Hiroyuki Funaoka, Hajime Ogushi: Bone Regenerative Medicine, Rheumatology, 30: 430-435, 2003 (Non-Patent Document 1). Has been announced. Similarly, Yonsei Medical Journal, vol. 45, p61-67, 2004 (Non-patent Document 2) has published a paper on basic research results using fetal bovine serum.
WO2002 / 024875 JP2002-78484 JP2003-52360 JP2003-235548 11-506010 Hiroyuki Funaoka, Ogushi Hajime: Bone Regenerative Medicine, Rheumatology, 30: 430-435, 2003 Yonsei Medical Journal, vol. 45, p61-67, 2004

  An object of the present invention is to provide a method for culturing mesenchymal stem cells for transplantation into humans safely and efficiently from human bone marrow fluid.

When collecting and culturing the patient's own bone marrow cells and transplanting them into the original patient, it is ideal to use as much of the patient's own serum as possible, that is, a medium containing autologous serum, without using any sera from different animals. Is. When the amount of serum required for culture cannot be collected due to the patient's symptoms, it is safer to use the serum of a healthy person such as a close relative of the patient than to use fetal bovine serum. In addition, when culturing mesenchymal stem cells derived from bone marrow, in order to increase the therapeutic effect, it is also important to differentiate to a target tissue (for example, bone tissue). However, it is significant to use a medium using human serum, particularly autologous serum as much as possible.
The present inventor examined the optimal conditions for culturing bone marrow-derived mesenchymal stem cells, and increased the proliferation rate of stem cells, removal of unnecessary cells other than stem cells, and further the activity of stem cells (proliferation ability, differentiation inducing ability, An excellent culture method was established with a high survival rate.
That is, the present invention relates to the following method.
1. A method of collecting human bone marrow fluid, culturing mesenchymal stem cells derived from bone marrow, and preparing human mesenchymal stem cells for transplantation to humans, the method comprising the following steps:
(i) a step of adding 80-120% v / v heparin / buffer solution at a concentration of about 5 to 15 U / mL with respect to the collected amount of bone marrow fluid;
(ii) Bone marrow fluid to which heparin / buffer solution obtained in step (i) is added, human serum from 10 to 20
Culturing in an α-MEM medium containing% v / v and removing blood cell components floating in the culture medium by changing the medium at least twice during the culture; and
(iii) A cell group mainly composed of mesenchymal stem cells is selectively detached from the culture container using a cell dissociating agent, and adherent cells other than the mesenchymal stem cells are separated from the detached cell group, and then separated into another container. Culturing the mesenchymal stem cells selectively in the step.
2. The bone marrow fluid to which the heparin / buffer solution obtained in step (i) was added was rotated at 500 to 1500 rpm.
Centrifuge at rpm (40 to 390 g by centrifugal gravity), separate from the lower layer into three layers, the red blood cell layer, the nucleated cell layer and the plasma layer, and the mixture of the red blood cell layer and the nucleated cell layer excluding the plasma layer, Item 2. The method according to Item 1, which is used in step (ii).
3. Item 3. The method according to Item 1 or 2, wherein the heparin / buffer solution is a heparin / PBS (Phosphate buffered saline) solution.
4). Item 4. The method according to any one of Items 1 to 3, wherein α-MEM comprises the following components at the following concentrations (mg / L):
CaCl ₂ (anhyd.) : 160-240
KCL: 320-480
MgSO ₄ (anhyd.): 78.4-117.6
NaCl: 5440-8160
NaHCO ₃ : 1760-2640
NaH ₂ PO ₄・ H ₂ O: 112-168
D-Glucose: 800-1200
Lipoic Acid: 0.16-0.24
Sodium Pyruvate ： 88-132
L-Alanine: 20-30
L-Arginine · HCl: 101.6-152.4
L-Asparagine ・ H ₂ O ： 40-60
L-Aspartic Acid: 24-36
L-Cystine · 2HCl: 24.8-37.2
L-Cysteine / HCl / H ₂ O: 80-120
L-Glutamic Acid: 60-90
L-Glutamine: 233.6-350.4
Glycine: 40-60
L-Histidine HCl · H ₂ O: 33.6-50.4
L-Isoleucine: 41.6-62.4
L-Leucine: 41.6-62.4
L-Lysine · HCl: 58.4-87.6
L-Methionine: 12-18
L-Phenylalanine: 25.6-38.4
L-Proline: 32-48
L-Serine: 20-30
L-Threonine: 38.4-57.6
L-Tryprophan: 8-12
L-Tyrosine (disodium salt): 41.6-62.4
L-Valine: 36.8-55.2
L-Ascorbic Acid: 40-60
Biotin: 0.08-0.12
D-Ca Pantothenate: 0.8-1.2
Choline Chloride: 0.8-1.2
Folic Acid: 0.8-1.2
i-Inositol: 1.6-2.4
Niacinamide: 0.8-1.2
Pyridoxal HCl: 0.8-1.2
Riboflavin: 0.08-0.12
Thiamine HCl: 0.8-1.2
Vitamin B ₁₂ : 1.12-1.68
Adenosine: 8-12
Cytidine: 8-12
Guanosine: 8-12
Uridine: 8-12
2′Deoxyadenosine: 8-12
2′Deoxycytidine HCl: 8.8-13.2
2′Deoxyguanosine: 8-12
Thymidine: 8-12
5. In the step (iii), the 0.05% trypsin / 0.53 mM EDTA solution or an enzyme such as protease having the same action is used as a cell dissociating agent, and the reaction is performed in the culture vessel for 1 to 10 minutes. Item 5. The method according to any one of Items 1 to 4, wherein the leaf stem cells are selectively detached.
6). The cell concentration of the mesenchymal stem cells separated and purified after detachment in step (iii) is adjusted to 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL, seeded on a scaffold, and 8 to 12 mM β-Glycerophosphate. Culturing in human serum-containing α-MEM medium supplemented with 16 to 24 μg / mL Vitamin C and 80 to 120 nM dexamethasone for 7 to 28 days to differentiate into osteoblasts or progenitor cells thereof Item 6. The method according to any one of Items 1 to 5.
7). The subcultured cells are treated with trypsin or a cell dissociating agent such as protease having the same action, and the cell concentration is adjusted to 1 × 10 ⁶ to 1 × 10 ⁸ cells / mL. Item 7. The method according to any one of Items 1 to 6, wherein the scaffold is seeded and cultured for 1 to 24 hours.
8). Item 8. The method according to Item 6 or 7, wherein the scaffold is any one of porous calcium apatite, calcium phosphate ceramics such as porous calcium triphosphate, porous calcium carbonate, and alumina having a surface porous structure.
9. The scaffold is made of a biomaterial, or a biomaterial made of titanium and coated with a bioactive glass or an inorganic material composed mainly of calcium phosphate such as hydroxyapatite. Item 8. The method according to Item 6 or 7.
10. Item 10. The method according to any one of Items 1 to 9, wherein the human serum is the same human serum as bone marrow fluid, that is, autoserum.

  According to the present invention, when autologous cell culture is performed, a cell viability of 90% or more can be obtained even when a medium using autologous serum instead of fetal bovine serum is used, and can be performed safely and efficiently. It has become possible to completely eliminate concerns about the use of different species of sera and bovine-derived infectious diseases, and has demonstrated significant effects such as ensuring the safety of regenerative medicine and maintaining patient comfort. Yes.

  Further, as shown in FIG. 5, cells cultured by this method have a survival rate of 90% or more in an environment other than the culture environment at least 24 hours, and should be stored or transported for a long time outside the culture environment. Became possible.

  Furthermore, a culture method for safely and efficiently differentiating bone marrow-derived mesenchymal stem cells into bone tissue outside the body has been established, and a method using an artificial bone or artificial joint as a scaffolding material has been developed. This makes it possible to achieve close contact with the patient at an early stage, greatly contributing to the improvement of treatment results.

In the present invention, human serum is not particularly limited, but it is desirable to use autoserum when bone marrow fluid is collected from a patient and transplanted to the patient. Safety that can deny virus infections from healthy relatives such as relatives if autologous serum cannot be used or the necessary amount of autologous serum cannot be obtained due to the severity of the patient's disease, general condition, etc. It is desirable to use human serum with a high level.
In addition, when the mesenchymal stem cells cultured by the method of the present invention are used to repair or regenerate bone tissue, the mesenchymal stem cells obtained before transplantation can be induced into bone tissue or cells that can induce bone tissue (for example, bone Blast cells or their precursors). Alternatively, the mesenchymal stem cells obtained by the method of the present invention can be directly transplanted into the stem depending on the transplant target (for example, muscle, organ, etc.) and differentiated in the same manner as the surrounding cells at the transplant site. .
Human serum (preferably autoserum) can be prepared by, for example, centrifuging (patient) blood 250-400 ml under the condition of 3000 rpm (centrifugal gravity 1580 g). The human serum is preferably added to the α-MEM solution at 10 to 20% v / v. The α-MEM solution preferably has the following concentration range (mg / L).

Components and concentration of α-MEM solution (mg / L)
CaCl ₂ (anhyd.) : 160-240
KCL: 320-480
MgSO ₄ (anhyd.): 78.4-117.6
NaCl: 5440-8160
NaHCO ₃ : 1760-2640
NaH ₂ PO ₄・ H ₂ O ： 112-168
D-Glucose: 800-1200
Lipoic Acid: 0.16-0.24
Sodium Pyruvate ： 88-132
L-Alanine: 20-30
L-Arginine · HCl: 101.6-152.4
L-Asparagine ・ H ₂ O ： 40-60
L-Aspartic Acid: 24-36
L-Cystine · 2HCl: 24.8-37.2
L-Cysteine / HCl / H ₂ O: 80-120
L-Glutamic Acid: 60-90
L-Glutamine: 233.6-350.4
Glycine: 40-60
L-Histidine HCl · H ₂ O: 33.6-50.4
L-Isoleucine: 41.6-62.4
L-Leucine: 41.6-62.4
L-Lysine · HCl: 58.4-87.6
L-Methionine: 12-18
L-Phenylalanine: 25.6-38.4
L-Proline: 32-48
L-Serine: 20-30
L-Threonine: 38.4-57.6
L-Tryprophan: 8-12
L-Tyrosine (disodium salt): 41.6-62.4
L-Valine: 36.8-55.2
L-Ascorbic Acid: 40-60
Biotin: 0.08-0.12
D-Ca Pantothenate: 0.8-1.2
Choline Chloride: 0.8-1.2
Folic Acid: 0.8-1.2
i-Inositol: 1.6-2.4
Niacinamide: 0.8-1.2
Pyridoxal HCl: 0.8-1.2
Riboflavin: 0.08-0.12
Thiamine HCl: 0.8-1.2
Vitamin B ₁₂ : 1.12-1.68
Adenosine: 8-12
Cytidine: 8-12
Guanosine: 8-12
Uridine: 8-12
2′Deoxyadenosine: 8-12
2′Deoxycytidine HCl: 8.8-13.2
2′Deoxyguanosine: 8-12
Thymidine: 8-12
In a particularly preferred embodiment of the present invention, 88 mL of autoserum is added to 500 mL of α-MEM solution to obtain a cell culture medium having a 15% v / v autoserum concentration. In addition, this autoserum concentration is 10% or more and 20% or less, and a desirable concentration is 15% in order to perform cell culture safely, effectively and efficiently.

In addition, if the culture solution is substantially equivalent to the component system of the α-MEM solution, the culture solution can also be a component of the present invention.
After collecting the bone marrow fluid, heparin buffer solution, preferably heparin phosphate buffer solution (heparin / PBS solution) in an amount of about 80% or more and about 120% or less with respect to the bone marrow fluid is usually about 5 to 15 U / mL, preferably Add about 10 U / mL for storage or transportation. The heparinized bone marrow fluid is centrifuged at about 500 rpm (centrifugal gravity 40 g) or more and about 1500 rpm (centrifugal gravity 390 g) or less, preferably about 1000 rpm (centrifugal gravity 140 g) for 1 to 20 minutes. After centrifugation, it is confirmed that the blood is separated into three layers of red blood cells, nucleated cell layers, and plasma from the bottom, and then plasma is removed. Culture in about 30 mL of autologous serum medium for 2 to 3 mL in total of two layers of erythrocytes and nucleated cell layer, and replace serum medium (preferably α-MEM medium supplemented with autoserum) almost every 2 days. .
Even if the bone marrow itself is cultured without centrifuging, the mesenchymal stem cells of the adhesion type can be cultured although the efficiency is lowered. Furthermore, PBS is usually used to adjust the heparin / buffer solution, but not limited to PBS, physiological saline may be used, and 5-15 U / ml heparin is added to various culture solutions containing MEM. Also good.
During the serum medium exchange, the suspended blood cell components are removed. By repeating this medium exchange, blood cell components are sequentially removed, and the blood cell components are completely removed by medium exchange at least twice, preferably at least three times or at least four times. Only adherent cells (ie, mesenchymal cells) centering on mesenchymal stem cells selectively proliferate.
By the above operation, only adherent cells selectively grow on the bottom surface of the culture vessel, but cells other than mesenchymal stem cells sometimes appear in the adherent cells. Adherent cells other than mesenchymal stem cells are easily distinguishable because they have a completely different appearance from mesenchymal stem cells. Furthermore, this cell has been confirmed to be CD34, 45, positive by cell surface antigen analysis, and from this, it has been confirmed that it is a cell derived from the blood cell lineage. As a method for efficiently separating mesenchymal stem cells and other adherent cells, 0.05% trypsin / 0.53 mM EDTA is used by utilizing the difference in the detachment effect by the cell dissociating agent, that is, the difference in the cell adhesion force to the bottom of the culture vessel. By reacting a solution or a protease having the same effect in a culture vessel for 1 to 10 minutes, on average for 3 minutes, mesenchymal stem cells are detached, and other adherent cells are attached and remain on the bottom surface. (Fig. 2b). A cell group mainly composed of exfoliated mesenchymal stem cells is cultured in a separate culture vessel. By repeating this once or twice, mesenchymal stem cells can be separated and purified with high purity and proliferated (FIG. 2a).

  Using a cell dissociator such as trypsin or a protease with the same action, all the adherent cells are detached from the bottom surface, and the mesenchymal stem cells using a cell separator (cell sorter) in a floating state where the adhesion between the cells is released. It is also possible to cultivate a cell group mainly composed of mesenchymal stem cells by separating cells other than those and culturing mesenchymal stem cells again in another culture vessel.

  When bone marrow-derived mesenchymal stem cells are further differentiated into bone tissue, stem cells adhering to the bottom of the culture dish are separated from the bottom using trypsin or a protease with equivalent action, and the cells adhere to each other. Operate in a floating state that has been solved.

When differentiating into bone tissue, porous hydroxyapatite and porous tricalcium phosphate are used as the scaffold, and the bone tissue is differentiated in or near the pores of these ceramics. Used for transplantation to bone defects. Furthermore, for the purpose of accelerating the engraftment between the artificial joint and the surrounding bone that supports it, mesenchymal stem cells are seeded at the bone contact portion of the artificial joint and differentiated into bone tissue before transplantation. As an application example to an artificial joint, a porous structure is formed by baking a large number of alumina bead balls having a diameter of 0.8 mm as shown in FIG. A medicinal stem cell is seeded on the surface of the scaffold material that has been formed, differentiated into bone tissue, and then transplanted to obtain good therapeutic results.

Stem cells to be seeded on these scaffolds are uniformly seeded by adjusting the cell concentration to 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL. Then, it culture | cultivates using the following culture medium. That is, the concentration in autoserum is about 8-12 mM, preferably about 10 mM for β-Glycerophosphate, about 16-24 μg / mL for vitamin C, preferably about 20 μg / mL, and about 80-120 nM for dexamethasone, preferably about Cultivate for 7 days or more and less than 28 days, regularly changing the medium in autoserum medium added and adjusted to 100 nM, and periodically observing and examining the progress of differentiation into bone tissue When the differentiation into predetermined bone cells is confirmed, the patient is transplanted. In addition, the cell concentration is adjusted to a relatively high concentration of 1 × 10 ⁶ to 1 × 10 ⁸ cells / mL, seeded on a scaffold such as ceramics or polymer, and cultured for a short period of 1 to 24 hours. After attaching the scaffold to the scaffold at a high density, it is directly transplanted into the bone to differentiate into bone tissue at the site where the mesenchymal stem cells are transplanted. This method has an advantage in that when the mesenchymal stem cells are obtained in a relatively large amount, the culture time until transplantation can be shortened.

  In the case of infusion of mesenchymal stem cells themselves in ischemic myocardial recovery, dilated cardiomyopathy treatment, or chronic peripheral arterial occlusive disease (diabetic necrosis, arteriosclerotic obstruction, Buerger disease, etc.) Are used as mesenchymal stem cells.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Example 1: 250-400 ml of blood was collected from a patient having a cultured bone defect of mesenchymal stem cells, and centrifuged at 3000 rpm for 10 minutes to obtain serum. The autoserum contains α-MEM having the specific composition described above.
To 500 mL of the solution, 88 mL of autoserum was added to obtain a cell culture medium having a 15% v / v autoserum concentration.
Next, after collecting bone marrow fluid from the patient, 10 U / mL heparin phosphate buffer was added in an amount equivalent to bone marrow fluid (corresponding to 100%). This heparinized bone marrow was centrifuged at 1000 rpm for 10 minutes. After centrifuging, the plasma was removed after confirming that it was separated into three layers of red blood cells, nucleated cell layers, and plasma from the bottom. Culture in 30 mL of autologous serum medium per 2 to 3 mL in total of two layers of erythrocytes and nucleated cell layer, and replace α-MEM medium supplemented with autologous serum almost every 2 days. The floating blood cell component was removed. This medium exchange was performed 4 times. When the medium was exchanged four times, the blood cell component was completely removed.

The cells adhered to the bottom of the culture dish are treated with 0.05% trypsin / 0.53 mM EDTA solution for 3 minutes to peel mesenchymal stem cells, which are transferred to another container, and the autoserum-containing α-MEM medium is added to the cells. And further cultured. The cell detachment treatment using 0.05% trypsin / 0.53 mM EDTA solution was repeated twice to separate and purify mesenchymal stem cells.
Cell photographs when human bone marrow-derived cells are cultured in an autoserum-containing α-MEM medium (FIG. 1-a, b) and photograph cells when cultured in fetal bovine serum-containing α-MEM medium (FIG. 1-c, When d) was compared, it was confirmed that the culture in the autoserum-containing α-MEM medium was clearly superior in activity (growth rate, differentiation-inducing ability, viability, etc.) and the number of proliferated cells. .

  Moreover, as shown in Table 1, in 13 actual cases, the number of cultured cells of autologous serum and fetal bovine serum was divided by the number of culture days to determine the degree of cell proliferation. The results showed that the cell growth was equivalent or better.

    FIG. 2a shows the morphology of mesenchymal stem cells by selectively detaching adherent cells from the mesenchymal stem cells by trypsin treatment and culturing the detached cells in a separate container, ie, mesenchymal stem cells. The adherent cells remain in and remain attached to the bottom surface of the culture container after trypsinization and detachment of mesenchymal stem cells.

  Furthermore, the surface antigens of mesenchymal stem cells cultured using autoserum were analyzed. As shown in FIG. 4, these cells were negative for blood cell markers CD34 and CD45, and also included mesenchymal cells. CD44 and CD90 were positive, indicating that the cultured cells are non-hemoclastic and mesenchymal stem cell lines.

Furthermore, as shown in FIG. 5, cells cultured by this method have a survival rate of 90% or more in an environment other than the culture environment at least 24 hours, and should be stored or transported for a long time outside the culture environment. Became possible.
Example 2: Induction of differentiation into bone tissue A 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL culture solution was prepared from the mesenchymal stem cells obtained in Example 1, and this was 0.8 mm in diameter as shown in FIG. Self-serum supplemented with β-Glycerophosphate (10 mM), Vitamin C (20 μg / mL) and dexamethasone (100 nM) seeded on a scaffold material made of alumina ceramics having a porous structure made by coating a large number of alumina beads The cells were cultured in α-MEM medium for 7 to 28 days.

  By this culture, it was confirmed that differentiation into predetermined bone cells was achieved, and a transplant material for transplantation into the bone defect of the patient himself was obtained.

  The present invention greatly contributes to the application of cell / tissue engineering to regenerative medicine, and has an important meaning / role for practical application and industrialization of cell / tissue engineering.

FIG. 1a shows a stem cell derived from human bone marrow cultured in autologous serum medium, and shows a 100-fold magnified image of the cell morphology after 9 days, FIG. 1b shows a 40-fold magnified image, and FIG. A 100-fold magnified image of the morphology of the cells after 9 days in culture in a medium supplemented with serum is shown, and FIG. It can be seen that human bone marrow-derived mesenchymal stem cells are more active when cultured in autologous serum medium than when cultured in fetal bovine serum medium. FIG. 2a shows isolated human mesenchymal stem cells, and FIG. 2b shows adherent cells remaining after detachment of human mesenchymal stem cells by trypsin treatment. FIG. 3 shows a porous structure made by coating a large number of alumina beads having a diameter of 0.8 mm on the surface of an alumina ceramic. Bone tissue is formed by seeding and culturing mesenchymal stem cells in such a porous structure on the surface in contact with the bone tissue of the artificial joint. FIG. 4 is a cell surface antigen pattern (FACS analysis) of human mesenchymal cells cultured in autoserum. FIG. 4a is a pattern of CD34, a blood cell marker, and FIG. 4b is a pattern of CD45, a blood cell marker. , FIG. 4c is a pattern of CD44 which is a marker including mesenchymal cells, and FIG. 4d is also a pattern of CD90. This suggests that cells cultured using autoserum are non-hemocytic and mesenchymal stem cells. FIG. 5 shows cell viability of human mesenchymal cells cultured in autoserum for 24 hours outside a carbon dioxide incubator. The cultured cells were stored in physiological saline, physiological saline (PBS) containing a phosphate buffer, and the culture solution, and all showed a cell survival rate of 90% or more.

Claims (10)

A method of collecting human bone marrow fluid, culturing mesenchymal stem cells derived from bone marrow, and preparing human mesenchymal stem cells for transplantation to humans, the method comprising the following steps:
(i) a step of adding 80-120% v / v heparin / buffer solution at a concentration of about 5 to 15 U / mL with respect to the collected amount of bone marrow fluid;
(ii) Bone marrow fluid to which heparin / buffer solution obtained in step (i) is added, human serum from 10 to 20
Culturing in an α-MEM medium containing% v / v and removing blood cell components floating in the culture medium by changing the medium at least twice during the culture; and
(iii) A cell group mainly composed of mesenchymal stem cells is selectively detached from the culture container using a cell dissociating agent, and adherent cells other than the mesenchymal stem cells are separated from the detached cell group, and then separated into another container. Culturing the mesenchymal stem cells selectively in the step. The bone marrow fluid added with the heparin / buffer obtained in step (i) is rotated at 500 to 1500 rpm.
Is centrifuged (40 to 390 g by centrifugal gravity) to separate the erythrocyte layer, nucleated cell layer and plasma layer from the lower layer into three layers, and the mixture of the erythrocyte layer and nucleated cell layer excluding the plasma layer is a step. The method according to claim 1, wherein the method is provided in (ii). The method according to claim 1 or 2, wherein the heparin / buffer solution is a heparin / PBS (Phosphate buffered saline) solution. The method according to any one of claims 1 to 3, wherein α-MEM comprises the following components at the following concentrations (mg / L):
CaCl ₂ (anhyd.) : 160-240
KCL: 320-480
MgSO ₄ (anhyd.): 78.4-117.6
NaCl: 5440-8160
NaHCO ₃ : 1760-2640
NaH ₂ PO ₄・ H ₂ O: 112-168
D-Glucose: 800-1200
Lipoic Acid: 0.16-0.24
Sodium Pyruvate ： 88-132
L-Alanine: 20-30
L-Arginine · HCl: 101.6-152.4
L-Asparagine ・ H ₂ O ： 40-60
L-Aspartic Acid: 24-36
L-Cystine · 2HCl: 24.8-37.2
L-Cysteine / HCl / H ₂ O: 80-120
L-Glutamic Acid: 60-90
L-Glutamine: 233.6-350.4
Glycine: 40-60
L-Histidine HCl · H ₂ O: 33.6-50.4
L-Isoleucine: 41.6-62.4
L-Leucine: 41.6-62.4
L-Lysine · HCl: 58.4-87.6
L-Methionine: 12-18
L-Phenylalanine: 25.6-38.4
L-Proline: 32-48
L-Serine: 20-30
L-Threonine: 38.4-57.6
L-Tryprophan: 8-12
L-Tyrosine (disodium salt): 41.6-62.4
L-Valine: 36.8-55.2
L-Ascorbic Acid: 40-60
Biotin: 0.08-0.12
D-Ca Pantothenate: 0.8-1.2
Choline Chloride: 0.8-1.2
Folic Acid: 0.8-1.2
i-Inositol: 1.6-2.4
Niacinamide: 0.8-1.2
Pyridoxal HCl: 0.8-1.2
Riboflavin: 0.08-0.12
Thiamine HCl: 0.8-1.2
Vitamin B ₁₂ : 1.12-1.68
Adenosine: 8-12
Cytidine: 8-12
Guanosine: 8-12
Uridine: 8-12
2′Deoxyadenosine: 8-12
2′Deoxycytidine HCl: 8.8-13.2
2′Deoxyguanosine: 8-12
Thymidine: 8-12 In the step (iii), the 0.05% trypsin / 0.53 mM EDTA solution or an enzyme such as protease having the same action is used as a cell dissociating agent, and the reaction is performed in the culture vessel for 1 to 10 minutes. The method according to claim 1, wherein leaf stem cells are selectively detached. The cell concentration of the mesenchymal stem cells separated and purified after detachment in step (iii) is adjusted to 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL, seeded on a scaffold, and 8 to 12 mM β-Glycerophosphate. ,
16-24 μg / mL Vitamin C and 80-120 nM dexamethasone were added,
The method according to any one of claims 1 to 5, which is cultured in human serum-containing α-MEM medium for 7 to 28 days and differentiated into osteoblasts or progenitor cells thereof. The subcultured cells are treated with trypsin or a cell dissociating agent such as protease having the same action, and the cell concentration is adjusted to 1 × 10 ⁶ to 1 × 10 ⁸ cells / mL. The method according to any one of claims 1 to 6, wherein the scaffold is sown and cultured for 1 to 24 hours. The method according to claim 6 or 7, wherein the scaffold is any one of porous hydroxyapatite, calcium phosphate ceramics such as porous calcium triphosphate, porous calcium carbonate, and alumina having a surface porous structure. . The scaffold is made of a biological metal material, or a material made by coating a surface of a biological metal mainly composed of titanium with a bioactive glass or an inorganic material mainly composed of calcium phosphate such as hydroxyapatite. The method according to claim 6 or 7. The method according to any one of claims 1 to 9, wherein the human serum is the same human serum as bone marrow fluid, that is, autoserum.

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