A mesenchymal stem cell cryopreservation protective agent and its preparation method and application

Abstract

The present invention proposes a formula ratio of a mesenchymal stem cell cryopreservation protective agent, and its components include: 40-56.5mL of DMEM basal medium, 10-25mL of human albumin, 5-15mL of dextran (40000), 5-10mL of polyethylene glycol (400), 5-10mL of DMSO, and 1-2.5mL of platelet lysate. The present invention discloses a method for using the above-mentioned mesenchymal stem cell cryopreservation protective agent, comprising the following steps: mixing mesenchymal stem cells and the above-mentioned mesenchymal stem cell cryopreservation protective agent to obtain a cell suspension, transferring it into a sterile cryopreservation tube and then placing it in a cell program gradient cooling box, program cooling to -50--60°C, freezing for 5-15h, and then transferring it to liquid nitrogen for freezing.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cell preservation, and in particular to a mesenchymal stem cell cryoprotectant and a preparation method and a use method thereof, and a mesenchymal stem cell resuscitation method. Background Art

[0002] Mesenchymal stem cells (MSC) are an important member of the stem cell family and belong to adult stem cells. They originate from the mesoderm and ectoderm in the early stages of development. MSC was first discovered in Friedenstein's bone marrow in 1968 because of its multidirectional differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation and self-replication. For example, under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc., and they still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathology.

[0003] Cell cryopreservation is a technology that places cells in an ultra-low temperature environment (-198℃ liquid nitrogen) to almost stop the cell biological process, thereby maintaining cell activity and extending the cell preservation period. However, the cell cryopreservation process will significantly change the thermodynamic, chemical and physical environment of the cells, and there is a risk of causing biological damage. During the cryopreservation of cell suspensions, the formation of ice crystals is the main cause of cell death. Therefore, cell cryopreservation requires the use of cell cryopreservation protectants to protect cells during the cryopreservation process.

[0004] There are many types of mesenchymal stem cell cryopreservation protectants with various formulas, but they are usually prepared from basal culture medium, fetal bovine serum and DMSO. Basal culture medium is the main nutrient required to maintain cell growth and development, including amino acids, vitamins and inorganic salts. Fetal bovine serum is an important substance to promote cell growth, maintain cell physiological balance and assist in supplementing cell nutrition. DMSO can reduce the formation of ice crystals during cell cryopreservation, reduce the damage of free radicals to cells, and protect cells during cell cryopreservation.

[0005] At present, due to the complex composition of bovine serum and the presence of unknown components, the probability of infection by animal pathogens will increase, which will add uncertainty to the use of cells; in addition, the cryopreservation protectant prepared by simply using basal culture medium, fetal bovine serum and DMSO will significantly reduce the cell viability after thawing, and the cell number and viability cannot meet the standards for clinical application. Therefore, it is very promising to find a mesenchymal stem cell cryopreservation protectant with clear composition, high safety and convenient preparation. Summary of the invention

[0006] Based on the problems existing in the background technology, the present invention proposes a mesenchymal stem cell cryoprotectant and a preparation method and a use method thereof, as well as a mesenchymal stem cell resuscitation method.

[0007] A mesenchymal stem cell cryopreservation protective agent, the components of which include: 40-56.5 mL of DMEM basal culture medium, 10-25 mL of human serum albumin, 5-15 mL of dextran (40000), 5-10 mL of polyethylene glycol (400), 5-10 mL of DMSO, and 1-2.5 mL of platelet lysate.

[0008] The method for preparing the mesenchymal stem cell cryopreservation protective agent comprises the following steps: uniformly mixing the above components, filtering, and aseptically packaging.

[0009] The method for using the above-mentioned mesenchymal stem cell cryoprotectant comprises the following steps: uniformly mixing the mesenchymal stem cells with the mesenchymal stem cell cryoprotectant according to any one of claims 1 to 2 to obtain a cell suspension, transferring the suspension into a sterile cryopreservation tube and then placing the suspension into a cell programmed gradient cooling box, cooling the suspension to -50 to -70°C, freezing the suspension for 5 to 15 hours, and then transferring the suspension into a liquid nitrogen tank for freezing.

[0010] Preferably, the cell concentration in the cell suspension is 1×10 6 -2.5×10 7 Pieces / mL.

[0011] Preferably, umbilical cord mesenchymal stem cells are prepared by the following specific operation: take an umbilical cord, remove Wharton's jelly and cut into pieces of 1 to 3 mm 3 The cells were cultured and passaged in complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS after centrifugation, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells.

[0012] A method for resuscitating mesenchymal stem cells, characterized in that it comprises the following steps: placing the cryopreservation tube obtained by liquid nitrogen freezing in the method for using the mesenchymal stem cell cryoprotectant according to claim 4 or 5 in a constant temperature water bath at 36-40°C for 1-3 minutes, shaking until the cell suspension is completely melted, centrifuging, and washing with D-PBS for 2-6 times.

[0013] The technical effects of the present invention are as follows:

[0014] (1) The present invention uses human serum albumin, which is highly safe, does not contain animal serum, and has no possibility of infection by animal-borne pathogens.

[0015] (2) The present invention uses polyethylene glycol (400) to effectively reduce the addition ratio of DMSO, effectively inhibiting the formation of ice crystals and reducing the damage to the cell structure caused by the formation of ice crystals in cells, while greatly reducing the potential toxic side effects of DMSO on cells.

[0016] (3) The present invention uses dextran (40000), which can effectively avoid cell structure disorder after dehydration, promote rapid cell recovery, effectively enhance stability, and significantly improve the preservation effect of mesenchymal stem cells.

[0017] (4) The DMSO content of the present invention is not higher than 10%, the preparation is simple, the batch-to-batch difference is small, and it can be used directly. Moreover, on the basis of ensuring the rapid recovery of cell activity, its value-added ability is extremely superior, that is, it has an excellent protective effect on frozen cells and ensures a high cell viability after recovery. Even for large-scale cryopreservation of mesenchymal stem cells, the cell recovery rate is maintained at a high level, effectively ensuring the cell preservation cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Comparison of cell activity of umbilical cord mesenchymal stem cells before and after cryopreservation using the mesenchymal stem cell cryoprotectants obtained in Example 5 and Comparative Examples 1-2.

[0019] Figure 2-6 Comparison of the expression change rates of cell surface markers CD90+, CD73+, CD105+, and CD29+ before and after cryopreservation of umbilical cord mesenchymal stem cells using the mesenchymal stem cell cryopreservation protectants obtained in Example 5 and Comparative Examples 1-2.

[0020] Figure 7 After 60 days of cryopreservation of umbilical cord mesenchymal stem cells using the mesenchymal stem cell cryoprotectants obtained in Example 5 and Comparative Examples 1-2, the cells were revived, and the cell proliferation multiples of umbilical cord mesenchymal stem cells at 24, 48, and 72 hours were compared. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below through specific embodiments.

[0022] Example 1

[0023] A mesenchymal stem cell cryoprotectant, comprising: Components content DMEM basal medium 40mL Human serum albumin 10mL Dextran(40000) 5mL Polyethylene glycol (400) 5mL DMSO 5mL Platelet lysate 1mL

[0024] The method for using the above-mentioned mesenchymal stem cell cryoprotectant comprises the following steps:

[0025] Take an umbilical cord, remove Wharton's jelly and cut into pieces of 1-3 mm 3The cells were cultured and passaged in complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells;

[0026] The umbilical cord mesenchymal stem cells and the above mesenchymal stem cell cryopreservation protective agent were mixed evenly to obtain a cell suspension. The concentration of cells in the cell suspension was 1×10 6 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to cool to -50°C, frozen for 5 hours, and then transferred to a liquid nitrogen tank for freezing.

[0027] Example 2

[0028] A mesenchymal stem cell cryoprotectant, comprising:

[0029] The method for using the above-mentioned mesenchymal stem cell cryoprotectant comprises the following steps:

[0030] Take an umbilical cord, remove Wharton's jelly and cut into pieces of 1-3 mm 3 The cells were cultured and passaged in complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS, filtered with a 100 μm cell strainer, and centrifuged to obtain umbilical cord mesenchymal stem cells;

[0031] The umbilical cord mesenchymal stem cells and the above mesenchymal stem cell cryopreservation protective agent were mixed evenly to obtain a cell suspension. The concentration of cells in the cell suspension was 2.5×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -70°C, frozen for 15 hours, and then transferred to a liquid nitrogen tank for freezing.

[0032] Example 3

[0028] A mesenchymal stem cell cryoprotectant, comprising: Components content DMEM basal medium 45mL Human serum albumin 15mL Dextran(40000) 9mL Polyethylene glycol (400) 7mL DMSO 7mL Platelet lysate 1.5mL

[0029] The method for using the above-mentioned mesenchymal stem cell cryopreservation protective agent comprises the following steps: mixing umbilical cord mesenchymal stem cells and the above-mentioned mesenchymal stem cell cryopreservation protective agent evenly to obtain a cell suspension, wherein the concentration of cells in the cell suspension is 1.0×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -60°C, frozen for 12 hours, and then transferred to a liquid nitrogen tank for freezing.

[0030] Example 4

[0031] A mesenchymal stem cell cryoprotectant, comprising: Components content DMEM basal medium 55mL Human serum albumin 20mL Dextran(40000) 14mL Polyethylene glycol (400) 9mL DMSO 9mL Platelet lysate 2mL

[0032] The method for using the above-mentioned mesenchymal stem cell cryopreservation protective agent comprises the following steps: mixing umbilical cord mesenchymal stem cells and the above-mentioned mesenchymal stem cell cryopreservation protective agent evenly to obtain a cell suspension, wherein the concentration of cells in the cell suspension is 1.5×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -68°C, frozen for 8 hours, and then transferred to a liquid nitrogen tank for freezing.

[0033] Example 5

[0034] A mesenchymal stem cell cryoprotectant, comprising: Components content DMEM basal medium 44mL Human serum albumin 17mL Dextran(40000) 23mL Polyethylene glycol (400) 8.5mL DMSO 5.3mL Platelet lysate 2.2mL

[0035] The method for using the above-mentioned mesenchymal stem cell cryopreservation protective agent comprises the following steps: mixing umbilical cord mesenchymal stem cells and the above-mentioned mesenchymal stem cell cryopreservation protective agent evenly to obtain a cell suspension, wherein the concentration of cells in the cell suspension is 2.2×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -65°C, frozen for 10 hours, and then transferred to a liquid nitrogen tank for freezing.

[0036] Comparative Example 1

[0037] A mesenchymal stem cell cryoprotectant, comprising: Components content DMEM basal medium 44mL Polyethylene glycol (400) 8.5mL DMSO 5.3mL Platelet lysate 2.2mL

[0038] The method for using the above-mentioned mesenchymal stem cell cryopreservation protective agent comprises the following steps: mixing umbilical cord mesenchymal stem cells and the above-mentioned mesenchymal stem cell cryopreservation protective agent evenly to obtain a cell suspension, wherein the concentration of cells in the cell suspension is 2.2×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -65°C, frozen for 10 hours, and then transferred to a liquid nitrogen tank for freezing.

[0039] Comparative Example 2

[0040] A mesenchymal stem cell cryoprotectant, comprising: Components content DMEM basal medium 44mL Human serum albumin 17mL Dextran(40000) 23mL DMSO 5.3mL Platelet lysate 2.2mL

[0041] The method for using the above-mentioned mesenchymal stem cell cryopreservation protective agent comprises the following steps: mixing umbilical cord mesenchymal stem cells and the above-mentioned mesenchymal stem cell cryopreservation protective agent evenly to obtain a cell suspension, wherein the concentration of cells in the cell suspension is 2.2×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -65°C, frozen for 10 hours, and then transferred to a liquid nitrogen tank for freezing.

[0042] Verification experiment

[0043] Take an umbilical cord, remove Wharton's jelly and cut into pieces of 1-3 mm 3 The cells were cultured and passaged in a complete stem cell culture medium, and the digested cells were centrifuged, washed with D-PBS, filtered with a 100 μm cell filter, and centrifuged to obtain umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cells were mixed evenly with the mesenchymal stem cell cryopreservation protective agents obtained in Example 5 and Comparative Example 1-2 to obtain a cell suspension. The concentration of cells in the cell suspension was 2.0×10 7 The obtained cell suspension was sampled and the activity was calculated by trypan blue staining, and the surface markers of umbilical cord mesenchymal stem cells, CD90+, CD73+, CD105+, and CD29+, were detected by flow cytometry.

[0044] Add the remaining cell suspension into sterile cryopreservation tubes, 1 mL per tube; divide into three groups of 10 days, 30 days, and 60 days, 3 tubes of cells in each group, a total of 27 tubes of cells; then put into a cell programmed gradient cooling box, program the temperature to -68°C, freeze for 8 hours, and then transfer to a liquid nitrogen tank for freezing for the corresponding time.

[0045] The umbilical cord mesenchymal stem cells that have completed the corresponding cryopreservation time are taken out and revived (placed in a 37°C constant temperature water bath for 2 minutes, shaken until the cell suspension is completely thawed, centrifuged, and washed 4 times with D-PBS medium).

[0046] The activity of the revived umbilical cord mesenchymal stem cells was calculated by trypan blue staining, and the surface markers of umbilical cord mesenchymal stem cells, CD90+, CD73+, CD105+, and CD29+, were detected by flow cytometry. The average values ​​of the cell activity and surface markers after resuscitation were compared with the values ​​of the cells before cryopreservation. Figure 1 As shown in Figure 2, the expression rate of cell surface markers changes as Figure 2-6 As shown, when the mesenchymal stem cell cryoprotectant obtained in Example 5 is used to treat mesenchymal stem cells, the cell activity is maintained at the highest level, while the expression rate of cell surface markers changes at the lowest level.

[0046] Umbilical cord mesenchymal stem cells were mixed with the mesenchymal stem cell cryopreservation protective agents obtained in Example 5 and Comparative Example 1-2 to obtain a cell suspension. The concentration of cells in the cell suspension was 2.2×10 7 / mL; transferred into a sterile cryopreservation tube and then placed in a cell programmed gradient cooling box, programmed to -65℃, frozen for 10 hours, and then transferred to a liquid nitrogen tank for 60 days. After 60 days, the cells were taken out, revived, centrifuged, and resuspended in complete medium. After counting, they were inoculated into 96-well plates for continued culture. After 24, 48 and 72 hours, 12 wells of cells were digested respectively to further evaluate the proliferation ability of cells after freezing. Figure 7 As shown, the mesenchymal stem cell cryopreservation protectant obtained in Example 5 was used to cryopreserve umbilical cord mesenchymal stem cells for 60 days, and the cells after thawing had the strongest proliferation ability.

[0047] The present application believes that: this is because the present invention reduces the DMSO content and adds polyethylene glycol (400) to effectively inhibit and reduce the formation of ice crystals in cells, thereby reducing the damage of ice crystals to the cell structure and reducing cell damage. At the same time, adding dextran (40000) can effectively avoid structural disorder after cell dehydration, and can promote the rapid recovery of cell activity after cell recovery, effectively increasing the stability of the cells and improving the cryopreservation effect of mesenchymal stem cells.

[0048] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A mesenchymal stem cell cryoprotectant, characterized in that: Its components include:

2. The mesenchymal stem cell cryoprotectant according to claim 1, characterized in that: The mass ratio of DMEM basal culture medium: human albumin: dextran (40000): polyethylene glycol (400): DMSO: platelet lysate is 40-56.5:10-25:5-15:5-10:5-10:1-2.

5.

3. The method for preparing a mesenchymal stem cell cryoprotectant according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: mixing DMEM basic culture medium, human serum albumin, dextran, polyethylene glycol, DMSO and platelet lysate, filtering and packaging under nitrogen protection.

4. The method for using the mesenchymal stem cell cryoprotectant according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: uniformly mixing mesenchymal stem cells with the mesenchymal stem cell cryoprotectant according to any one of claims 1 to 2 to obtain a cell suspension, transferring the suspension into a sterile cryotube and then placing the suspension into a cell programmed gradient cooling box, cooling the suspension to -50 to -70°C, freezing the suspension for 5 to 15 hours, and then transferring the suspension into a liquid nitrogen tank for freezing.

5. The method for using the mesenchymal stem cell cryoprotectant according to claim 4, characterized in that: The cell concentration in the cell suspension was 1×10 6 -2.5×10 7 Pieces / mL.

6. A method for resuscitating mesenchymal stem cells, characterized in that: The method comprises the following steps: placing the cryopreservation tube obtained by liquid nitrogen freezing in the method for using the mesenchymal stem cell cryopreservation protectant according to claim 4 or 5 in a constant temperature water bath at 36-40°C for 1-3 minutes, shaking until the cell suspension is completely melted, centrifuging, and washing with D-PBS for 2-6 times.

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