Gel for preserving stem cells as well as preparation method and application of gel

Abstract

The invention provides gel for preserving stem cells. The gel comprises sodium alginate, gelatin, hyaluronic acid, dimethyl sulfoxide and human albumin. The gel provided by the invention can be used for preserving the stem cells at-80 DEG C for a long time. The invention further discloses a preparation method and application of the gel.

Description

Technical Field

[0001] The present invention relates to a gel for preserving stem cells, in particular to a gel capable of preserving stem cells for a long time at -80°C. The present invention also provides a preparation method and application of the gel. Background Art

[0002] Cell cryopreservation is one of the main methods of cell preservation. When the temperature drops to -70°C, the metabolic activities and enzyme activities in the cells almost stop. When the temperature is below -130°C, the cells can achieve a better survival rate. Liquid nitrogen (-196°C) can achieve long-term storage of cells. In order to protect cells from damage during freezing and thawing, very complex components are often required to prepare cryopreservation solutions.

[0003] Stem cells have the potential for self-renewal and multidirectional differentiation, and can differentiate into specific tissue cells under regulation. At the same time, because stem cells can continuously secrete various cytokines that promote wound healing, they can promote tissue repair and regeneration. Mesenchymal stem cells currently used in clinical or beauty industries need to be stored and transported at ultra-low temperatures (such as -196°C), and temperature fluctuations should be avoided as much as possible.

[0004] Gel preparations containing stem cells have demonstrated their potential for repair and treatment in many fields, including wound closure, neural tissue repair and regeneration, but how to stably preserve stem cells in gels and maintain their survival rate and proliferation ability as much as possible remains to be studied. Summary of the invention

[0005] In one aspect, the present invention provides a gel for preserving stem cells, comprising sodium alginate, gelatin, hyaluronic acid, dimethyl sulfoxide and human albumin.

[0006] In some embodiments, the gel comprises the following components: 2.0%-3.0% sodium alginate, 2.0-4.0% gelatin, 0.5%-0.8% hyaluronic acid, 3.0%-6.0% dimethyl sulfoxide, 5.0%-10.0% human albumin, and the balance is normal saline.

[0007] In some embodiments, the gel comprises the following components:

[0008] 1) 2.0% sodium alginate, 2.5% gelatin, 0.5% hyaluronic acid, 5.0% dimethyl sulfoxide, 5.0% human albumin, and the balance is normal saline;

[0009] 2) 3.0% sodium alginate, 2.0% gelatin, 0.8% hyaluronic acid, 3.0% dimethyl sulfoxide, 8.0% human albumin, and the balance is normal saline; or

[0010] 3) 2.5% sodium alginate, 4.0% gelatin, 0.6% hyaluronic acid, 6.0% dimethyl sulfoxide, 10.0% human albumin, and the balance is normal saline.

[0011] In some embodiments, the gel further comprises GM-CSF.

[0012] In some embodiments, the gel further comprises IL-8.

[0013] In some embodiments, the gel includes 0.2 μg / g GM-CSF and 0.1 μg / g IL-8.

[0014] In another aspect, the present invention provides a method for preparing the above-mentioned gel, comprising uniformly mixing the components contained in the above-mentioned gel.

[0015] In another aspect, the present invention provides use of the above gel in preserving stem cells.

[0016] In some embodiments, the storage is performed at -80°C.

[0017] In some embodiments, the stem cells are umbilical cord mesenchymal stem cells. DETAILED DESCRIPTION

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0019] Herein, the term "comprising" generally means including the elements explicitly specified but not excluding other elements, and also includes the case consisting of these elements.

[0020] As used herein, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0021] In this document, the term "and / or" should be understood to mean either one or both of the alternatives.

[0022] Unless otherwise indicated, percentages and ratios herein are by weight.

[0023] The term "gel", which may also be called "hydrogel" or "gel solution", refers to a dispersion system formed by dissolving a hydrophilic polymer material (such as gelatin, hyaluronic acid, etc.) in water. In the dispersion system, the hydrophilic polymer material may be one or more, and other non-polymer materials may also be dispersed therein. The gel may have good cell affinity, which is conducive to the preservation or proliferation of cells therein.

[0024] The term "stem cell" refers to an undifferentiated or underdifferentiated cell that can self-renew, that is, produce more cells identical to itself, and can differentiate into two or more mature cell types. According to the source of stem cells, stem cells can be divided into embryonic stem cells (ES cells) and adult stem cells. Mesenchymal stem cells, as adult stem cells, can be derived from a variety of tissues, such as bone marrow, adipose tissue, umbilical cord, etc. Mesenchymal stem cells have the functions of self-renewal and multidirectional differentiation, can differentiate into osteoblasts, chondrocytes, adipocytes, neurons, etc., and also have the ability to promote tissue regeneration and repair.

[0025] The present invention provides a gel for preserving stem cells, which can preserve stem cells for a long period of time (e.g., up to 8 months) at -80°C. The inventors have also found that adding GM-CSF (and IL-8) to the gel can enhance the proliferation ability after resuscitation.

[0026] The present invention is described below by means of specific examples.

[0027] Test reagents used

[0028] Sodium alginate, Macklin, S875554; gelatin, Aladdin, G108396; hyaluronic acid, Macklin, H909936; dimethyl sulfoxide, Merck, 900645; human albumin, Merck, 106331; stem cell freezing solution LiveCyte, LC1602; granulocyte macrophage colony stimulating factor (GM-CSF), Bioengineering, C610017; interleukin-8 (IL-8), Bioengineering, C620008.

[0029] Example 1

[0030] The gel used to store stem cells includes the following components:

[0031] 2.0% sodium alginate, 2.5% gelatin, 0.5% hyaluronic acid, 5.0% dimethyl sulfoxide, 5.0% human albumin, and the balance is normal saline.

[0032] Mix the components in the above proportions and stir evenly, then store in a refrigerator at 4°C for later use.

[0033] Example 2

[0034] The gel used to store stem cells includes the following components:

[0035] 3.0% sodium alginate, 2.0% gelatin, 0.8% hyaluronic acid, 3.0% dimethyl sulfoxide, 8.0% human albumin, and the balance is normal saline.

[0036] Mix the components in the above proportions and stir evenly, then store in a refrigerator at 4°C for later use.

[0037] Example 3

[0038] The gel used to store stem cells includes the following components:

[0039] 2.5% sodium alginate, 4.0% gelatin, 0.6% hyaluronic acid, 6.0% dimethyl sulfoxide, 10.0% human albumin, and the balance is normal saline.

[0040] Mix the components in the above proportions and stir evenly, then store in a refrigerator at 4°C for later use.

[0041] Example 4

[0042] The 4th generation umbilical cord mesenchymal stem cells (UC-MSC, purchased from ATCC) were revived and cultured in mesenchymal stem cell medium (Sciencell, 7501) supplemented with 5% fetal bovine serum, 1% MSCGS (Sciencell, 7552) and double antibody (P / S) (hereinafter referred to as complete medium) until about 85% confluence, and then digested with 0.25% trypsin. The digested cell suspension was centrifuged, and the cell pellet was resuspended with physiological saline and adjusted to a stem cell concentration of about 2.0×10 7 Pieces / mL.

[0043] Stem cell cryopreservation grouping: dilute the above-mentioned stem cell physiological saline suspension 10 times with the gel or stem cell cryopreservation solution prepared in Examples 1-3, and each group has 8 cryopreservation tubes. The cryopreservation tubes using the gel prepared in Examples 1-3 are stored at -80°C, and the cryopreservation tubes using the stem cell cryopreservation solution are stored at -80°C overnight and then transferred to -196°C liquid nitrogen for storage (no programmed cooling is required during the cryopreservation process). On the 60th day (D60), 120th day (D120), 180th day (D180) and 240th day (D240) of storage, two cryopreservation tubes are taken out from each group, thawed and revived, centrifuged, and cultured for 12 hours with a complete medium twice the volume of the gel or stem cell cryopreservation solution used during cryopreservation, and the cell survival rate is detected by trypan blue staining. Cell survival rate = (total number of cells - number of blue cells) / total number of cells × 100%.

[0044] The mean results of cell viability in two cryopreserved tubes are shown in Table 1.

[0045] Table 1 Cell viability test results (average of two cryopreserved tubes)

[0046] D60 D120 D180 D240 Example 1 Gel 95.3 91.7 90.6 82.7 Example 2 Gel 94.1 92.6 89.6 84.2 Example 3 Gel 92.3 89.3 88.5 83.4 Stem cell cryopreservation medium 96.6 94.2 91.7 89.5

[0047] From the results in Table 1, it can be seen that the gel for preserving stem cells prepared by the present invention can maintain the survival rate of stem cells for a long time. After 240 days of cryopreservation, the cell survival rate is still above 80%, and there is no need to adopt a programmed cooling process during cryopreservation.

[0048] Example 5

[0049] In later studies, we found that when the gel prepared by the present invention is used to cryopreserve stem cells, although there is no significant difference in cell survival rate after recovery from stem cells preserved in stem cell cryopreservation solution, the proliferation ability of stem cells after recovery is significantly lower than that of stem cells preserved in stem cell cryopreservation solution. The live cell count result (number of non-blue cells microscopically examined after trypan blue staining) after 72 hours of culture in complete medium is equivalent to about 60% of the live cell count corresponding to the stem cell cryopreservation solution, and the time required to grow to 85% confluence is extended by about 2 days. This suggests that although the cryopreservation process using the gel prepared by the present invention does not affect the cell survival rate, it affects the proliferation ability of stem cells after recovery relative to the stem cell cryopreservation solution.

[0050] In order to improve the proliferation ability of stem cells after cryopreservation, we studied the possible effects of various factors, including the control of the cooling program during cryopreservation, adding some antioxidants (such as ascorbic acid, vitamin E, etc.) to the gel, changing the cryoprotectant (such as using hydroxyethyl starch in combination with dimethyl sulfoxide), adding some cytokines (such as PDGF, IFN-γ, IL-8, G-CSF), etc. The results showed that the best effect was achieved when granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 8 (IL-8) were simultaneously supplemented to the gel prepared by the present invention.

[0051] Experimental groups (all frozen for 120 days):

[0052] 1#: Gel was prepared according to Example 1, and stem cells were cryopreserved according to the gel set of Example 4, except that the cryopreservation method was changed to cooling to -80°C at 2°C / min, and 10 tubes were cryopreserved;

[0053] 2#: Gel was prepared according to Example 1, except that 0.5% ascorbic acid was added, and stem cells were cryopreserved according to the gel set of Example 4, and 10 tubes were cryopreserved;

[0054] #3: Gel prepared in Example 1, except that 1.0% hydroxyethyl starch was added, and stem cells were cryopreserved according to the gel group in Example 4, with 10 tubes cryopreserved;

[0055] 4#: Gel was prepared as in Example 1, except that 0.2 μg / g GM-CSF was added, and stem cells were cryopreserved as in the gel group in Example 4, with 10 tubes cryopreserved;

[0056] 5#: Gel was prepared as in Example 1, except that 0.1 μg / g IL-8 was supplemented, and stem cells were cryopreserved as in the gel group in Example 4, with 10 tubes cryopreserved;

[0057] #6: Gel was prepared as in Example 1, except that 0.2 μg / g GM-CSF and 0.1 μg / g IL-8 were added, and stem cells were cryopreserved as in the gel group in Example 4, with 10 tubes cryopreserved;

[0058] 7#: using stem cell freezing solution, cryopreserving stem cells according to the stem cell freezing solution set of Example 4, freezing 10 tubes;

[0059] 8#: using stem cell freezing solution, supplemented with 0.2 μg / g GM-CSF and 0.1 μg / g IL-8, and freezing the stem cells according to the stem cell freezing solution set of Example 4, freezing 10 tubes;

[0060] 9#: Prepare gel according to Example 1, and cryopreserve stem cells according to the gel set of Example 4, and cryopreserve 10 tubes.

[0061] Table 2 shows the cell viability and viable cell counts detected at different times after recovery after various modifications (the viable cell count detected by the 7# stem cell cryopreservation solution was 100%). Five tubes were taken for testing at 24h and 72h, and the average value was taken.

[0062] Table 2 Cell viability and viable cell number test results

[0063]

[0064] *The letter superscript after the value indicates the significance of the difference between the same columns; the same letters indicate no significant difference (p>0.05), and different letters indicate significant difference (p<0.05)

[0065] From the results in Table 2, it can be seen that the addition of GM-CSF alone to the gel (4#) can improve the proliferation ability of stem cells after cryopreservation. The addition of IL-8 alone (5#) has little effect on the proliferation ability, but the addition of IL-8 can further improve the enhancement (or recovery) of the proliferation ability caused by GM-CSF (6#). The addition of these two cytokines basically does not affect the cryopreservation effect of the stem cell cryopreservation solution (8#). Changes in other factors (1#, 2# and 3#) have little effect on the proliferation ability.

[0066] Since GM-CSF and IL-8 are cytokines secreted by mesenchymal stem cell fluid, we also studied the effects of other cytokines secreted by mesenchymal stem cell fluid, such as G-CSF, HGF, IL-6, IL-11, etc., and have not found that their use alone or in combination can improve the proliferation ability of stem cells after cryopreservation.

[0067] In addition, the phenotype of cells cultured in complete culture medium for 24 hours after recovery was identified by flow cytometry. Compared with the stem cell freezing solution, the gel of the present invention was not found to affect the expression of stem cell markers such as CD73, CD90 and CD105, and the ratio of cells expressing CD45 and CD34 was not increased. This shows that the gel of the present invention better maintains the stemness of stem cells.

Claims

1. Gels used to preserve stem cells, including sodium alginate, gelatin, hyaluronic acid, dimethyl sulfoxide and human albumin.

2. The gel as claimed in claim 1, comprising the following components: 2.0%-3.0% sodium alginate, 2.0-4.0% gelatin, 0.5%-0.8% hyaluronic acid, 3.0%-6.0% dimethyl sulfoxide, 5.0%-10.0% human albumin, and the balance being normal saline.

3. The gel according to claim 1, comprising the following components: 1) 2.0% sodium alginate, 2.5% gelatin, 0.5% hyaluronic acid, 5.0% dimethyl sulfoxide, 5.0% human albumin, and the balance is normal saline; 2) 3.0% sodium alginate, 2.0% gelatin, 0.8% hyaluronic acid, 3.0% dimethyl sulfoxide, 8.0% human albumin, and the balance is normal saline; or 3) 2.5% sodium alginate, 4.0% gelatin, 0.6% hyaluronic acid, 6.0% dimethyl sulfoxide, 10.0% human albumin, and the balance is normal saline.

4. The gel according to any one of claims 1 to 3, further comprising GM-CSF.

5. The gel according to any one of claims 4, further comprising IL-8.

6. The gel according to claim 5, comprising 0.2 μg / g GM-CSF and 0.1 μg / g IL-8.

7. A method for preparing the gel according to any one of claims 1 to 6, comprising uniformly mixing the components contained in the gel according to any one of claims 1 to 6.

8. Use of the gel according to any one of claims 1 to 6 in preserving stem cells.

9. The use according to claim 8, wherein the storage is carried out at -80°C.

10. The use according to claim 8 or 9, wherein the stem cells are umbilical cord mesenchymal stem cells.

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