A gel for preserving stem cells and its preparation method and application

Abstract

The present invention provides a gel for preserving stem cells, comprising sodium alginate, gelatin, hyaluronic acid, dimethyl sulfoxide, and human albumin. The gel can be used for long-term preservation of stem cells at -80°C. The present invention also provides a preparation method and uses 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 at -80°C for a long time. The present invention also provides a preparation method and application of the gel. Background Art

[0002] Cryopreservation is one of the primary methods of cell preservation. At temperatures as low as -70°C, metabolic and enzyme activity within cells almost ceases. Cells achieve optimal survival rates below -130°C. Liquid nitrogen (-196°C) enables long-term cell preservation. To protect cells from damage during freezing and thawing, cryopreservation solutions often require the use of complex components.

[0003] Stem cells possess the potential for self-renewal and multidirectional differentiation, allowing them to differentiate into specific tissue cells under control. Furthermore, because they continuously secrete various cytokines that promote wound healing, they can promote tissue repair and regeneration. Currently, mesenchymal stem cells used in clinical practice or the cosmetic industry must be stored and transported at ultra-low temperatures (e.g., -196°C), and temperature fluctuations must be minimized.

[0004] Gel preparations containing stem cells have demonstrated their potential for repair and treatment in multiple 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 comprises 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 gel, comprising uniformly mixing the components of the gel.

[0015] In another aspect, the present invention provides use of the aforementioned 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 meaning 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 where it consists of these elements.

[0020] As used herein, the term "about" generally refers to a variation 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 of the alternatives or both of the alternatives.

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

[0023] The term "gel," also known as "hydrogel" or "gel solution," refers herein to a dispersion of a hydrophilic polymer (such as gelatin or hyaluronic acid) dissolved in water. This dispersion can contain one or more hydrophilic polymers, and other non-polymeric materials may also be dispersed therein. The gel exhibits good cell affinity, facilitating the preservation and proliferation of cells within it.

[0024] The term "stem cell" refers to undifferentiated or underdifferentiated cells that are capable of self-renewal, that is, producing more cells identical to themselves, and on the other hand, are capable of differentiating into two or more mature cell types. Depending on 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, for example, bone marrow, adipose tissue, umbilical cord, etc. Mesenchymal stem cells have the function 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 discovered that adding GM-CSF (and IL-8) to the gel can enhance the proliferation capacity 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 cryopreservation medium LiveCyte, LC1602; granulocyte macrophage colony-stimulating factor (GM-CSF), Sangon, C610017; interleukin-8 (IL-8), Sangon, C620008.

[0029] Example 1

[0030] The gel used to preserve 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, stir evenly, and store in a refrigerator at 4°C until use.

[0033] Example 2

[0034] The gel used to preserve 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, stir evenly, and store in a refrigerator at 4°C until use.

[0037] Example 3

[0038] The gel used to preserve 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, stir evenly, and store in a refrigerator at 4°C until 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 culture medium (Sciencell, 7501) supplemented with 5% fetal bovine serum, 1% MSCGS (Sciencell, 7552) and double antibody (P / S) (hereinafter referred to as complete culture medium) until about 85% confluence. The cells were then digested with 0.25% trypsin, the digested cell suspension was centrifuged, and the cell pellet was resuspended in physiological saline to adjust the stem cell concentration to about 2.0×10 7 pieces / mL.

[0043] Stem cell cryopreservation grouping: The above-mentioned stem cell saline suspension was diluted 10-fold with the gel or stem cell cryopreservation solution prepared in Examples 1-3, with 8 cryopreservation tubes per group. Cryopreservation tubes using the gel prepared in Examples 1-3 were stored at -80°C, while cryopreservation tubes using the stem cell cryopreservation solution were stored at -80°C overnight and then transferred to liquid nitrogen at -196°C for storage (no programmed cooling was required during the cryopreservation process). On days 60 (D60), 120 (D120), 180 (D180), and 240 (D240) of storage, two cryopreservation tubes were removed from each group, thawed, and centrifuged. The tubes were then cultured for 12 hours with complete medium containing 2 times the volume of the gel or stem cell cryopreservation solution used during cryopreservation. Cell viability was determined by trypan blue staining. Cell viability = (total cell number - number of blue cells) / total cell number × 100%.

[0044] The mean cell viability results of the 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] As can be seen from the results in Table 1, 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 no programmed cooling process is required during cryopreservation.

[0048] Example 5

[0049] In later studies, we found that when stem cells were cryopreserved using the gel prepared by the present invention, although there was no significant difference in cell survival rate after thawing compared to stem cells stored in stem cell cryopreservation solution, the proliferation capacity of the stem cells after thawing was significantly lower than that of stem cells stored in stem cell cryopreservation solution. The viable cell count (number of non-blue cells after microscopic examination after trypan blue staining) after 72 hours of culture in complete medium was approximately 60% of the corresponding viable cell count of the stem cell cryopreservation solution, and the time required to grow to 85% confluence was extended by approximately 2 days. This suggests that although the cryopreservation process using the gel prepared by the present invention did not affect cell survival rate, it did affect the proliferation capacity of stem cells after thawing compared to stem cell cryopreservation solution.

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

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

[0052] 1#: Gel was prepared as in Example 1, and stem cells were cryopreserved as in Example 4, except that the freezing method was changed to cooling to -80°C at 2°C / min, and 10 tubes were frozen;

[0053] 2#: Gel was prepared as in Example 1, except that 0.5% ascorbic acid was added. Stem cells were cryopreserved as in the gel set in Example 4, with 10 tubes frozen.

[0054] 3#: Gel prepared as in Example 1, except that 1.0% hydroxyethyl starch was added. Stem cells were cryopreserved as in the gel set in Example 4, with 10 tubes frozen.

[0055] 4#: Gel was prepared as in Example 1, except that 0.2 μg / g GM-CSF was supplemented. Stem cells were cryopreserved as in the gel set 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. Stem cells were cryopreserved as in the gel set 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 supplemented. Stem cells were cryopreserved as in the gel set in Example 4, with 10 vials cryopreserved.

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

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

[0060] 9#: Gel was prepared according to Example 1, and stem cells were cryopreserved according to the gel set of Example 4, with 10 tubes cryopreserved.

[0061] Table 2 shows the cell viability and viable cell counts at different times after recovery after various modifications (the viable cell count detected by freezing solution in group 7 was considered 100%). Five tubes were tested at 24h and 72h, and the average values ​​were taken.

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

[0063]

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

[0065] As shown in Table 2, the addition of GM-CSF alone to the gel (4#) improved the proliferation of stem cells after cryopreservation. The addition of IL-8 alone (5#) had little effect on proliferation, but the addition of IL-8 further improved (or restored) the GM-CSF-induced proliferation (6#). The addition of these two cytokines had little effect on the cryopreservation performance of the stem cell cryopreservation solution (8#). Changes in other factors (1#, 2#, and 3#) had little effect on proliferation.

[0066] Since GM-CSF and IL-8 happen to be 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, nor was the ratio of cells expressing CD45 and CD34 increased. This shows that the gel of the present invention better maintains the stemness of stem cells.

Claims

1. A gel for preserving stem cells, 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, 0.2 μg / g GM-CSF, and 0.1 μg / g IL-8, with the remainder being normal saline.

2. 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.

3. A method for preparing the gel according to claim 1 or 2, comprising uniformly mixing the components contained in the gel according to claim 1 or 2.

4. Use of the gel according to claim 1 or 2 in preserving stem cells. The use according to claim 4 , wherein the storage is carried out at -80° C. The use according to claim 4 or 5, wherein the stem cells are umbilical cord mesenchymal stem cells.

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