PRODUCTION METHOD FOR SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS AND PRODUCTION METHOD FOR CELL PREPARATION FOR JOINT MEDICAL TREATMENT

Abstract

An object of the present invention is to provide a production method for a synovium-derived mesenchymal stem cell, by which proliferation efficiency is improved so that a sufficient amount of synovium-derived mesenchymal stem cells can be obtained from a synovial tissue, a production method for a cell preparation for joint medical treatment using the production method for a synovium-derived mesenchymal stem cell, synovium-derived mesenchymal stem cell, and a cell preparation for a joint medical treatment. According to the present invention, there is provided a production method for a synovium-derived mesenchymal stem cell, which is a method of producing a synovium-derived mesenchymal stem cell from a synovial tissue, where the production method includes treating the synovial tissue with a mixed enzyme including one or more kinds of collagenases and one or more kinds of neutral proteases for 2 hours or more without causing the synovial tissue to undergo a disinfection step.

Claims

1. A method of producing a synovium-derived mesenchymal stem cell, which is a production method for a synovium-derived mesenchymal stem cell from a synovial tissue, the method comprising: treating the synovial tissue with a mixed enzyme including one or more kinds of collagenases and one or more kinds of neutral proteases for 2 hours or more without causing the synovial tissue to undergo a disinfection step.

2. The method according to claim 1, wherein the mixed enzyme is Liberase (registered trade name).

3. The method according to claim 1, wherein an enzyme concentration in the enzyme treatment is 0.01 mg/ml to 10 mg/ml.

4. The method according to claim 1, wherein the synovial tissue is derived from a human.

5. The method according to claim 1, wherein the synovial tissue is a synovial tissue derived from a single donor.

6. The method according to claim 1, wherein a subject from which synovium is collected and a subject to which the synovium-derived mesenchymal stem cell is transplanted are the same subject.

7. The method according to claim 1, wherein a mass ratio of the synovial tissue to the enzyme is 1,000:1 to 10:1.

8. The method according to claim 1, further comprising: culturing, for 10 days or more, the synovium-derived mesenchymal stem cell after the enzyme treatment to proliferate the synovium-derived mesenchymal stem cell.

9. The method according to claim 8, wherein the culturing for 10 days or more is carried out without culture medium exchange.

10. The method according to claim 8, wherein the culturing for 10 days or more is carried out in a culture medium containing an autologous serum.

11. The method according to claim 8, wherein the synovium-derived mesenchymal stem cell after the enzyme treatment is seeded at a cell number of 100 cells/cm.sup.2 or more and 5,000 cells/cm.sup.2 or less and cultured for 10 days or more.

12. The method according to claim 8, wherein a proliferation ratio in the culturing for 10 days or more is 3 times or more and 50 times or less.

13. The method according to claim 1, wherein the synovium-derived mesenchymal stem cell is produced without being co-cultured with a cell other than the synovium-derived mesenchymal stem cell.

14. A production method for a cell preparation for a joint medical treatment, the production method comprising: producing a synovium-derived mesenchymal stem cell by the method according to claim 1; and producing a cell preparation for a joint medical treatment using the synovium-derived mesenchymal stem cell.

15. The production method for a cell preparation for a joint medical treatment according to claim 14, wherein the joint medical treatment is a medical treatment for a meniscus injury.

16. Synovium-derived mesenchymal stem cell produced by the method according to claim 1.

17. A cell preparation for a joint medical treatment, produced by the method according to claim 14.

Description

EXAMPLES

Comparative Example 1

[0105] Production of Synovium-Derived Mesenchymal Stem Cell According to General Production Step (With Tissue Disinfection, Enzyme Treatment Time: 1 Hour and 1 Minute)

[0106] Using a human specimen, synovium-derived mesenchymal stem cells were prepared from a synovial tissue according to a general production method. Specifically, an M1 specimen (synovial tissue: 0.62 g) was subjected to disinfection with a 0.1% Isodine solution for 1 minute as a tissue disinfection step. Then, the above-described tissue was shredded with scissors and immersed in 5.0 mL of a Liberase aqueous solution. It is noted that as the Liberase aqueous solution, a solution obtained by dissolving 5.0 mg of Liberase MNP-S (manufactured by F. Hoffmann-La Roche, Ltd.) in 5.0 mL of water for injection containing human autologous serum of a final concentration of 20% was used. The enzymatic reaction was carried out at room temperature of 25° C. for 1 hour and 1 minute. Then, the digested tissue solution was transferred to a 50 mL centrifuge tube through a cell strainer and centrifuged at 400 g for 5 minutes. The supernatant was removed, the obtained cell-concentrated suspension was suspended in a culture medium, and the entire amount thereof was seeded in a flask (1,000 cells/cm.sup.2). It is noted that as the culture medium, a culture medium obtained by dissolving, in αMEM, human autologous serum to a final concentration of 10% was used. The culture was carried out in a CO.sub.2 incubator (37° C., 5% CO.sub.2), and after 2 weeks, the cells were recovered from the flask to produce synovium-derived mesenchymal stem cells. It is noted that a feature of the present cell production is that the culture medium is not exchanged during the culture period. The cell quantity finally obtained in this way was 1.5×10.sup.7 cells for Ml. In addition, the proliferation ratio calculated as the ratio of the number of cells after culturing for 2 weeks to the number of cells at the time of seeding was 2.0 times. A summary of the results is shown in Table 1.

Comparative Example 2

[0107] Production of Synovium-Derived Mesenchymal Stem Cell According to Production Step in Which Enzyme Treatment Time is Extended (With Tissue Disinfection, Enzyme Treatment Time: 1.5 Hours)

[0108] Using a human specimen, synovium-derived mesenchymal stem cells were prepared from a synovial tissue under the condition in which the enzyme treatment time was extended by 30 minutes as compared with the general production method. Specifically, an M2 specimen (synovial tissue: 0.85 g) was subjected to disinfection with a 0.1% Isodine solution for 1 minute as a tissue disinfection step. Then, the above-described tissue was shredded with scissors and immersed in 5.0 mL of a Liberase aqueous solution. It is noted that as the Liberase aqueous solution, a solution obtained by dissolving 5.0 mg of Liberase MNP-S (manufactured by F. Hoffmann-La Roche, Ltd.) in 5.0 mL of water for injection containing human autologous serum of a final concentration of 20% was used. The enzymatic reaction was carried out at room temperature of 25° C. for 1.5 hours. Then, the digested tissue solution was transferred to a 50 mL centrifuge tube through a cell strainer and centrifuged at 400 g for 5 minutes. The supernatant was removed, the obtained cell-concentrated suspension was suspended in a culture medium, and the entire amount thereof was seeded in a flask (1,880 cells/cm.sup.2). It is noted that as the culture medium, a culture medium obtained by dissolving, in αMEM, human autologous serum to a final concentration of 10% was used. The culture was carried out in a CO.sub.2 incubator (37° C., 5% CO.sub.2), and after 2 weeks, the cells were recovered from the flask to produce synovium-derived mesenchymal stem cells. It is noted that a feature of the present cell production is that the culture medium is not exchanged during the culture period. The cell quantity finally obtained in this way was 0.8×10.sup.7 cells as M2. This was less than 1.0×10.sup.7 cells considered to be necessary for the medical treatment, which was at a level in which a cell amount required for the medical treatment could not be provided. In addition, the proliferation ratio calculated as the ratio of the number of cells after culturing for 2 weeks to the number of cells at the time of seeding was 0.6 times. A summary of the results is shown in Table 1.

Example 1

[0109] Production of Synovium-Derived Mesenchymal Stem Cell According to Production Step in Which Tissue Disinfection Step is Eliminated and Enzyme Treatment Time is Extended (Without Tissue Disinfection, Enzyme Treatment Time: 3 Hours)

[0110] Using a human specimen, synovium-derived mesenchymal stem cells were prepared from a synovial tissue under the condition in which the tissue disinfection step was eliminated and the enzyme treatment time was extended by 2 hours, unlike the general production method. Specifically, tissues of an N1 specimen (synovial tissue: 0.42 g), an N2 specimen (synovial tissue: 0.60 g), an N3 specimen (synovial tissue: 0.86 g), and an N4 specimen (synovial tissue: 0.33 g) were each shredded with scissors and immersed in 5.0 mL of a Liberase aqueous solution without causing them to undergo the tissue disinfection step. It is noted that as the Liberase aqueous solution, a solution obtained by dissolving 5.0 mg of Liberase MNP-S (manufactured by F. Hoffmann-La Roche, Ltd.) in 5.0 mL of water for injection containing human autologous serum of a final concentration of 20% was used. The enzymatic reaction was carried out at room temperature of 25° C. for 3 hours. Then, the digested tissue solution was transferred to a 50 mL centrifuge tube through a cell strainer and centrifuged at 400 g for 5 minutes. The supernatant was removed, the collected cell-concentrated suspension was suspended in a culture medium, and the entire amount was seeded in a flask (690 cells/cm.sup.2 for N1, 1,059 cells/cm.sup.2 for N2, 1,028 cells/cm.sup.2 for N3, and 787 cells/cm.sup.2 for N4). It is noted that as the culture medium, a culture medium obtained by dissolving, in αMEM, human autologous serum to a final concentration of 10% was used. The culture was carried out in a CO.sub.2 incubator (37° C., 5% CO.sub.2), and after 2 weeks, the cells were recovered from the flask to produce synovium-derived mesenchymal stem cells. It is noted that a feature of the present cell production is that the culture medium is not exchanged during the culture period. The cell quantities finally obtained in this way were 4.9×10.sup.7 cells for N1, 7.6×10.sup.7 cells for N2, 7.0×10.sup.7 cells for N3, and 6.5×10.sup.7 cells for N4. This significantly exceeded 1.0×10.sup.7 cells considered to be necessary for the medical treatment, which was at a level in which a cell amount required for the medical treatment could be sufficiently and stably provided. In addition, the proliferation ratios calculated as the ratio of the number of cells after culturing for 2 weeks to the number of cells at the time of seeding were respectively 9.5 times, 9.6 times, 9.1 times, and 11.0 times, and thus the effect of improving the proliferation ratio was recognized. A summary of the results is shown in Table 1.

Example 2

Checking Test of Mesenchymal Stem Cell Marker

[0111] The synovium-derived mesenchymal stem cells prepared in Comparative Examples 1 and 2 and Example 1 were subjected to a checking test for the expression of the mesenchymal stem cell markers. As a result, it was confirmed that all of the cells exhibit CD90 positivity, CD45 negativity, and cartilage differentiation potency and thus are synovium -derived mesenchymal stem cells.

TABLE-US-00001 TABLE 1 Comparative Example Example Tissue code M1 M2 N1 N2 N3 N4 Tissue weight (g) 0.62 0.85 0.42 0.60 0.86 0.33 Presence or absence of tissue Present Present Absent Absent Absent Absent disinfection Enzyme concentration(mg/mL) 1 1 1 1 1 1 Mass ratio of synovial tissue to 124:1 170:1 84:1 120:1 172:1 66:1 enzyme Enzyme treatment time 1:01 1:30 3:00 3:00 3:00 3:00 (hour, minute) Seeding density (cells/cm.sup.2) 1000 1880 690 1059 1028 787 Number of seeded cells (×10.sup.6 7.7 14.4 5.3 8.1 7.9 6.0 cells) Number of total cells recovered 15.0 8.0 49.0 76.0 70.0 65.0 (×10.sup.6 cells) Proliferation ratio 2.0 0.6 9.5 9.6 9.1 11.0

[0112] <Interpretation of Results>

[0113] From the results shown in Table 1, it was revealed that the weight of the synovial tissue is not the dominant parameter with respect to the cell quantity, and two factors, that is, the extension of the enzyme treatment time and the elimination of the disinfection step, are indispensable for proliferation ratio. In addition, as it was conceived that extending the enzyme treatment time should be avoided since it causes injury to the synovial tissue, it was found that in M2 in which the enzyme treatment time was extended by 30 minutes with respect to Ml, the cell quantity is actually reduced significantly, and the proliferation ratio is also decreased. On the other hand, it was conceived that not undergoing the tissue disinfection step should be avoided since the possibility that impurities in the tissue derived from the production step, particularly bacteria, microorganisms, and the like increase during the enzyme treatment time and the culture period increased. However, it was revealed that in a case of deliberately eliminating the tissue disinfection step and significantly extending the enzyme treatment time, a positive effect in terms of the proliferation ratio is obtained unexpectedly. In addition, the cell quantity obtained from the production according to the present invention as a result of achieving the above-described proliferation ratio was significantly exceeded 1.0×10.sup.7 cells considered to be necessary for the medical treatment, and this significantly exceeded the cell amount (2.0×10.sup.7 cells) capable of exhibiting the effect even in a case of allogeneic cells. The combination that achieves the present proliferation ratio was conceived to have remarkable value in a method of producing a synovium-derived mesenchymal stem cell assuming autologous cell therapy.

Example 3

[0114] The synovium-derived mesenchymal stem cells produced as N1 to N4 were actually produced from specimens of meniscus injury patients themselves and, after the production, respectively administered to the meniscus injury patients as autologous cell therapy. As a result, favorable symptom relief was observed until 52 weeks after the administration in all the meniscus injury patients to which the synovium-derived mesenchymal stem cells produced as N1 to N4 were respectively administered. An improvement of 22 points or more was confirmed in terms of the Lysholm score (see Table 2 below), which is known as a symptom score, until 52 weeks after the administration compared with that before the treatment, and the reposition was also confirmed in the meniscus checking by magnetic resonance imaging (MRI), which indicated the medical treatment results were favorable.

TABLE-US-00002 TABLE 2 Lysholm score: Evaluation carried out by person in charge of evaluation Evaluation item Limping (foot Absent 5 limping) Seldom or often 3 (5 points) Always severe 0 Walking Walking is possible without problem 30 stability Knees rarely collapse during sports or other 25 (30 points) vigorous exercise Knees frequently collapse during sports or other 15 vigorous exercise Unstable at times in everyday life 10 Unstable frequently in everyday life 5 Always unstable when walking 0 Supporting Fully loadable 5 ability Stick or crutch is necessary 3 (5 points) Loading application is impossible 0 Swelling Absent 10 (10 points) Associated with Knee collapse 7 Due to vigorous exercise 5 With exercise in everyday life 2 Always 0 Going up and No problem 5 down stairs Some problems 3 (5 points) Going up and down is possible only step by step 2 Impossible 0 Pain Absent 30 (30 points) Occasionally slight during vigorous exercise 25 At time when knees collapse 20 During vigorous exercise 15 During or after walking 2 km or more 10 During or after walking less than 2 km 5 Always 0 Bending and No problem 5 stretching Some problems 3 of knee Up to 90 degrees 2 (5 points) Impossible 0 Femur atrophy Absent 5 (5 points) Atrophy of 1 to 2 cm 3 Atrophy of 2 cm 0