Stem cell cryopreservation method

Abstract

The invention discloses a stem cell cryopreservation method which comprises the following steps: incubating stem cells in a first culture medium for 0.5-6 hours, removing a biocompatible osmotic pressure regulator, collecting the stem cells, resuspending the stem cells by using a second culture medium to obtain a cell suspension, dropwise adding the cell suspension into a GG / Gel freeze-dried scaffold, and performing cryopreservation after the GG / Gel freeze-dried scaffold completely absorbs the cell suspension, the first culture medium comprises a biocompatible osmotic pressure regulator and a third culture medium, and the concentration of the biocompatible osmotic pressure regulator in the first culture medium is 1-6 wt%; the second culture medium comprises polyvinylpyrrolidone and a third culture medium, and the concentration of the polyvinylpyrrolidone in the second culture medium is 5-15 wt%; the third culture medium comprises a basic culture medium, serum and antibiotics. According to the stem cell cryopreservation method disclosed by the invention, the formation of ice crystals inside and outside the cells is inhibited through a combined pre-dehydration and cell encapsulation strategy, and efficient cryopreservation of the stem cells is realized.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a stem cell cryopreservation method. Background Art

[0002] Stem cell tissue engineering is an advanced medical method for repairing or replacing damaged tissues, and plays an increasingly important role in the repair and regeneration of tissues such as skin, cartilage and nerves. Efficient and safe cell preservation methods are the key to the application of stem cell tissue engineering, which can provide sufficient and stable stem cell resources to ensure the preparation of stem cell tissue engineering products. Ultra-low temperature cryopreservation is a common technology for long-term preservation of stem cells. However, during the process of cooling and freezing and warming and thawing, the formation and growth of ice crystals inside and outside the cells will cause irreversible damage to stem cells, affecting the survival and therapeutic efficacy of stem cells. At present, dimethyl sulfoxide (DMSO, 5-10wt%) is the most commonly used cryoprotectant for stem cells, which can effectively reduce the cryopreservation damage caused by ice crystals to cells. However, DMSO is cytotoxic and can easily cause uncontrollable differentiation of stem cells and cause cell apoptosis. At the same time, DMSO is difficult to completely remove from the cells. Many studies have confirmed that stem cells frozen with DMSO can cause serious clinical side effects such as nausea, allergies and kidney damage when infused into the human body.

[0003] Biocompatible scaffold materials, as stem cell carriers, can reduce the immune rejection of the recipient to the cells after implantation, while providing a suitable growth environment for stem cells and promoting their retention in damaged areas. Based on this, the organic combination of stem cell preservation technology and biocompatible scaffold materials is expected to be used to prepare off-the-shelf stem cell tissue engineering products and promote their clinical transformation and application. However, recent research reports have shown that although the method of encapsulating cells for cryopreservation using biocompatible hydrogel scaffold materials (such as calcium alginate hydrogel) can reduce the concentration of DMSO toxic protective agents, it is still impossible to completely abandon the use of DMSO, which limits the convenient application of stem cell tissue engineering products. Therefore, the development of a new DMSO-free stem cell cryopreservation system using biocompatible scaffold materials is a key challenge that needs to be urgently addressed in the field of stem cell tissue engineering. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a stem cell cryopreservation method, which combines a stem cell pre-dehydration strategy induced by a biocompatible osmotic pressure regulator and a cell encapsulation strategy based on a biocompatible antifreeze scaffold to achieve efficient DMSO-free stem cell cryopreservation and its convenient application in the field of tissue engineering.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] A method for cryopreservation of stem cells, comprising: incubating stem cells in a first culture medium for 0.5 to 6 hours, removing a biocompatible osmotic pressure regulator, collecting stem cells, resuspending the stem cells in a second culture medium to obtain a cell suspension, dropping the cell suspension onto a GG / Gel freeze-dried scaffold, and cryopreserving the cell suspension after the GG / Gel freeze-dried scaffold completely absorbs the cell suspension, wherein:

[0007] The first culture medium comprises: a biocompatible osmotic pressure regulator and a third culture medium, wherein the concentration of the biocompatible osmotic pressure regulator in the first culture medium is 1 to 6 wt %;

[0008] The second culture medium comprises: polyvinyl pyrrolidone and a third culture medium, wherein the concentration of polyvinyl pyrrolidone in the second culture medium is 5 to 15 wt %;

[0009] The third culture medium includes: basal culture medium, serum and antibiotics.

[0010] In the above technical solution, the concentration of the biocompatible osmotic pressure regulator in the first culture medium is 2-6 wt %.

[0011] In the above technical solution, the GG / Gel freeze-dried scaffold completely absorbs the cell suspension after 1 to 10 minutes.

[0012] In the above technical solution, the stem cells are incubated in the first culture medium for 0.5 to 4 hours.

[0013] In the above technical solution, the biocompatible osmotic pressure regulator is one or a mixture of proline (Pro), taurine, trehalose, sorbitol, betaine and mannitol.

[0014] In the above technical scheme, the cells were incubated at 37° C. and 5% CO 2 .

[0015] In the above technical solution, the content of serum in the third culture medium is 5-20% by volume, and the content of antibiotics in the third culture medium is 0.5-1%.

[0016] In the above technical solution, the antibiotics include: penicillin and streptomycin.

[0017] In the above technical solution, the ratio of the number of stem cells in the cell suspension to the mass fraction of the GG / Gel freeze-dried scaffold is (0.5×10 6 ~10×10 6 ): (0.005~0.1), the unit of mass fraction is g.

[0018] In the above technical scheme, the method for obtaining the GG / Gel freeze-dried scaffold includes: mixing the gellan gum solution and the gelatin solution evenly to obtain a first mixture, adding a calcium chloride aqueous solution to the first mixture for cross-linking for 5 to 20 minutes to obtain a second mixture, first storing it at -90 to -70°C for 0.5 to 24 hours, and then vacuum freeze-drying to obtain a GG / Gel freeze-dried scaffold.

[0019] In the above technical scheme, the gellan gum solution is a mixture of gellan gum (GG) and water, and the concentration of gellan gum (GG) in the gellan gum (GG) solution is 0.5-2wt%; the gelatin solution is a mixture of gelatin (Gel) and water, and the concentration of gelatin (Gel) in the gelatin (Gel) solution is 0.2-2wt%.

[0020] In the above technical solution, the ratio of gellan gum solution to gelatin solution is (1-10):(1-10) by volume.

[0021] In the above technical solution, the ratio of the first mixture to the calcium chloride aqueous solution is (10-50):1 by volume, and the concentration of calcium chloride in the calcium chloride aqueous solution is 2-10 wt %.

[0022] In the above technical solution, the slow freezing method is used for cryopreservation: cooling to -90 to -70°C at a cooling rate of -10 to -1°C / min and cryopreservation for 12 to 24 hours, and then cryopreservation in liquid nitrogen.

[0023] The invention discloses a biocompatible osmotic pressure regulator for promoting cell dehydration. The biocompatible osmotic pressure regulator is one or a mixture of proline (Pro), taurine, trehalose, sorbitol, betaine and mannitol.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The biocompatible osmotic pressure regulator, polyvinyl pyrrolidone (PVP) and GG / Gel freeze-dried scaffold used in the stem cell cryopreservation method of the present invention all have good biocompatibility, and the use of the toxic cryoprotectant DMSO is abandoned. Therefore, after thawing and recovery, the cells do not need to undergo an additional cryoprotectant removal process.

[0026] 2. The stem cell cryopreservation method of the present invention achieves efficient cryopreservation of stem cells by combining pre-dehydration and cell encapsulation strategies to inhibit the formation of ice crystals inside and outside the cells.

[0027] 3. The stem cell cryopreservation method of the present invention can realize the convenient application of stem cells in the field of tissue engineering and promote the healing process of mouse skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 (a) the control group, the proline group and the PGG group in Example 1-1 4:1 The cell survival rate of the proline experimental group and PGG in Example 1-2 4:1 Hemolysis rate of the experimental group, (c) the main organs (heart, liver, spleen, lung, kidney) and PGG of the mice in Examples 1-3 4:1 H&E staining of muscle tissue under the material;

[0029] Figure 2 (a) Diameter statistics and (b) diameter size schematic diagram of cells in Example 2;

[0030] Figure 3 (a) heat flow diagrams of the blank control group, PVP group and PGG group in Example 3, (b) freezing points of the blank control group, PVP group and PGG group in Example 3, (c) non-freezing water content of the blank control group, PVP group and PGG group in Example 3;

[0031] Figure 4 is (a) the cell survival rate in Example 4-1, (b) the cell survival rate in Example 4-2, (c) the cell survival rate in Example 4-3, (d) the cell survival rate in Example 4-4;

[0032] Figure 5 (a) wound pictures of the mouse control group and the mouse experimental group in Example 5, (b) wound healing rates of the mouse control group and the mouse experimental group in Example 5;

[0033] Figure 6 (a) the cell survival rate in Example 6-1, (b) the cell survival rate in Example 6-2, and (c) the cell survival rate in Example 6-3. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0035] Proline was purchased from Beijing J&K Scientific Co., Ltd. (Batch: LR90W38;

[0036] Serum and DMEM / F-12 medium were purchased from Viacell;

[0037] Antibiotics were purchased from Beijing Solebow Technology Co., Ltd., Lot No. 20150626. Antibiotics include penicillin and streptomycin, the concentration of penicillin in the antibiotics is 10000U / mL, and the concentration of streptomycin in the antibiotics is 10mg / mL.

[0038] In the following embodiments, the third culture medium comprises: Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 culture medium (DMEM / F-12 culture medium), serum and antibiotics, wherein the content of serum in the third culture medium is 10% (v / v), and the content of antibiotics is 1% (v / v).

[0039] In the following examples, the second culture medium comprises: polyvinyl pyrrolidone and a third culture medium, wherein the concentration of polyvinyl pyrrolidone in the second culture medium is 5 wt %.

[0040] In the following embodiments, the first culture medium includes: a biocompatible osmotic pressure regulator and a third culture medium.

[0041] In the following embodiments, the method for obtaining the GG / Gel freeze-dried scaffold includes: mixing a gellan gum (GG) solution and a gelatin (Gel) solution uniformly to obtain a first mixture, adding a calcium chloride aqueous solution to the first mixture for cross-linking for 10 minutes to obtain a second mixture, freezing and storing at -80°C for 24 hours, and vacuum freeze-drying for 72 hours to obtain a GG / Gel freeze-dried scaffold, wherein the gellan gum (GG) solution is a mixture of gellan gum (GG) and water, and the concentration of gellan gum (GG) in the gellan gum (GG) solution is 1wt%, the gelatin (Gel) solution is a mixture of gelatin (Gel) and water, and the gelatin (Gel) in the gelatin (Gel) solution is 0.5wt%, the concentration of calcium chloride in the calcium chloride aqueous solution is 5wt%, the volume ratio of the gellan gum (GG) solution to the gelatin (Gel) solution is W, the volume ratio of the first mixture to the calcium chloride aqueous solution is 50:1, and W and its number of the GG / Gel freeze-dried scaffold are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] In the following embodiments, the method for obtaining the PGG material includes: using a pipette to absorb the second culture medium, and dripping it onto the surface of the GG / Gel freeze-dried scaffold so that the second culture medium is loaded on the GG / Gel freeze-dried scaffold, and after 5 minutes, the GG / Gel freeze-dried scaffold completely absorbs the second culture medium to obtain the PGG material, wherein the ratio of the mass fraction of the GG / Gel freeze-dried scaffold to the volume fraction of the second culture medium is 0.005:100, the mass fraction unit is g, and the volume fraction unit is uL. The numbers of the GG / Gel freeze-dried scaffold and the PGG material used in the PGG material are shown in Table 2.

[0046] Table 2

[0047]

[0048] In the following examples, the 10 wt % trichloroacetaldehyde solution is a mixture of trichloroacetaldehyde and water, and the concentration of trichloroacetaldehyde in the 10 wt % trichloroacetaldehyde solution is 10 wt %.

[0049] Example 1

[0050] The proline and PGG used in the present invention were detected by cell live / dead fluorescence staining experiment, hemolysis experiment and mouse subcutaneous embedding experiment. 4:1 Cell compatibility, blood compatibility and tissue compatibility of materials.

[0051] Example 1-1

[0052] Control group: Mouse bone marrow-derived mesenchymal stem cells (BMSCs) were cultured at 3×10 3 The cells were inoculated into a 96-well plate at a density of cells / well and cultured in the third culture medium. After culturing for 24 hours at 37°C and 5% CO2, the third culture medium was removed and the cells were continued to be cultured with a new third culture medium for Th, where Th = 24h, 48h or 72h. After the culture was completed, the cells were stained with a live / dead fluorescent dye to observe the survival of the cells and to quantitatively calculate the cell survival rate. The cell survival rate was as follows: Figure 1 As shown in the "control" in (a);

[0053] Proline group: basically the same as the control group, the only difference is that after removing the third culture medium, the cells are continued to be cultured in the first culture medium, wherein the biocompatible osmotic pressure regulator in the first culture medium is proline, the proline concentration in the first culture medium is 4wt%, and the cell survival rate is Figure 1 As shown in "proline" in (a);

[0054] PGG 4:1 Group: basically the same as the control group, the only difference is that after removing the third culture medium, PGG 4:1 The cells were cultured with the extract to obtain PGG 4:1 The extraction method comprises: soaking 0.1 g PGG in 2 mL of the third culture medium 4:1 Material 24h, remove PGG 4:1 Materials, get PGG 4:1 Extract, cell viability Figure 1 (a) "PGG 4:1 ” as shown.

[0055] like Figure 1 As shown in (a), the proline group and PGG 4:1The cell survival rates of BMSCs in the two groups were higher than 95% when they were cultured for 24h, 48h and 72h, respectively, and there was no significant difference between them and the control group, indicating that proline and PGG 4:1 The material has good cell compatibility.

[0056] Example 1-2

[0057] Whole blood was obtained from Kunming mice (Kunming mice were purchased from Scobes) by cardiac blood sampling.

[0058] Positive control group: Take 20 μL of whole blood and add 1 mL of deionized water, incubate at 37°C for 30 min, centrifuge at 2000 r / min for 5 min after incubation to obtain the supernatant, measure the absorbance of the supernatant at 540 nm, and record the absorbance of the positive control group as A p ;

[0059] Negative control group: basically the same as the positive control group, the only difference is that 20 μL of whole blood was added to 1 mL of normal saline for incubation. The absorbance of the negative control group is recorded as A n ;

[0060] Proline experimental group: basically the same as the positive control group, the only difference is that 20 μL of whole blood was added to 1 mL of physiological saline containing 4 wt% proline for incubation. The absorbance of the proline experimental group was recorded as A h1 ;

[0061] PGG 4:1 Experimental group: basically the same as the positive control group, the only difference is that 20 μL of whole blood was added with 1 mL of 0.1 g PGG 4:1 Materials were incubated in saline, PGG 4:1 The absorbance of the experimental group is recorded as A h2 .

[0062] A h1 or A h2 As A h , according to A h , A n and A p Calculation of proline experimental group and PGG 4:1 The hemolysis rate of the experimental group was calculated as follows:

[0063]

[0064] The hemolysis rate of the proline experimental group was Figure 1 (b) "Proline" shows that PGG 4:1 The hemolysis rate of the experimental group was Figure 1 (b) "PGG 4:1 "As shown by Figure 1(b) shows that the proline experimental group and PGG 4:1 The hemolysis rates of the experimental groups were significantly lower than 5%, which was in line with the international hemolysis standards. 4:1 The material has good blood compatibility.

[0065] Examples 1-3

[0066] Control group: Three Kunming mice were anesthetized with 10 wt% chloral solution, and their main organs (heart, liver, spleen, lung, kidney) and muscle tissues were taken for H&E staining to observe the inflammatory response and visceral organ toxicity. Figure 1 as shown in "control" in (c);

[0067] Experimental 3-day group: 3 Kunming mice were anesthetized with 10wt% chloral solution, a 1cm wound was cut on the back of the mice, and 0.1g PGG 4:1 The material was implanted subcutaneously into the back of Kunming mice through the wound, the wound was sutured, and the mice were raised normally for 3 days. The mice were randomly selected and killed. The main organs (heart, liver, spleen, lung, kidney) and PGG were taken. 4:1 The muscle tissue under the material was stained with H&E to observe the inflammatory response and visceral organ toxicity. The main organs and muscle tissues of Kunming mice in the 3-day experimental group were as follows: Figure 1 As shown in “3 days” in (c);

[0068] The 7-day experimental group was basically the same as the 3-day experimental group, except that the mice were randomly selected and killed after being fed normally for 7 days. The main organs and muscle tissues of the Kunming mice in the 7-day experimental group were as follows: Figure 1 As shown in “7 days” in (c).

[0069] Depend on Figure 1 (c) shows that implantation of PGG 4:1 Materials The main organs (heart, liver, spleen, lung and kidney) and PGG of Kunming mice on the 3rd and 7th days of feeding 4:1 The muscle tissue under the material was not significantly different from that of the normally raised Kunming mice, indicating that PGG 4:1 The material did not cause significant acute or chronic damage to mice and had good tissue compatibility.

[0070] Example 2

[0071] BMSCs were resuspended in a first culture medium containing 4 wt % proline (i.e., the biocompatible osmotic pressure regulator was proline) and incubated at 37°C, 5% CO2 for 15 min, 30 min, 1 h, 2 h, or 4 h. After incubation, changes in cell diameter were observed under an optical microscope, and Image J was used to quantitatively count the cell diameter to analyze cell dehydration.

[0072] like Figure 2 (a) and Figure 2 (b) shows that compared with normal BMSCs ( Figure 2 Compared with the "0min" in the first culture medium containing 4wt% proline, after incubation for 15min in the first culture medium containing 4wt% proline, the cell diameter of BMSCs decreased from 21.31μm to 9.91μm. As the incubation time increased, the cell diameter of BMSCs gradually increased, but was smaller than the cell diameter of normal BMSCs. After incubation for 30min, the cell diameter was 11.59μm; after incubation for 1h, the cell diameter was 14.98μm; after incubation for 2h, the cell diameter was 17.61μm; after incubation for 4h, the cell diameter was 18.17μm. The above results show that proline can promote cell dehydration, reduce intracellular water content, and thus help reduce the formation of ice crystals in cells. At the same time, by changing the incubation time, the degree of cell dehydration can also be regulated.

[0073] Example 3

[0074] Both PVP and PGG materials are non-permeable macromolecular materials, and their ability to inhibit ice crystal formation outside cells was characterized by differential scanning calorimetry experiments.

[0075] Blank control group: 10-30 mg of the third culture medium was added to a sealed crucible, and a differential scanning calorimeter (Netzsch DSC 200F3) was used to monitor the heat flow changes during the cooling and heating processes. The temperature change program was set to cool from 20°C to -40°C at a cooling rate of -10°C / min, keep at -40°C for 5 minutes, and then heat to 10°C at a heating rate of 2°C / min. The freezing point and non-freezing water content were further calculated. The experimental results are shown in Figure 3 As shown in the “Control” in;

[0076] PVP group: basically the same as the blank control group, the only difference is the third culture medium, the third culture medium in the blank control group was replaced with the second culture medium containing 5wt% PVP. The experimental results are as follows Figure 3 As shown in "PVP" in;

[0077] PGG group: basically the same as the blank control group, the only difference is the third culture medium, the third culture medium in the blank control group was replaced with PGG material, PGG material is PGG 2:1 Materials, PGG 3:1 Materials and PGG 4:1 One of the materials.

[0078] like Figure 3 As shown in (a), the melting peaks of the PVP group and the PGG group shifted to the left, and the peak areas were smaller than those of the blank control group, indicating that the second culture medium containing 5wt% PVP and the PGG material can inhibit the formation of ice crystals. At the same time, the higher the gellan gum (GG) content of the GG / Gel freeze-dried scaffold in the PGG material, the lower the peak apex, the more obvious the peak shift to the left, and the smaller the melting peak area, indicating that the PGG material has a stronger inhibitory effect on ice crystals. The freezing points of the blank control group, the PVP group, and the PGG group are shown in Figure 3 As shown in (b), the non-freezing water content is Figure 3 As shown in (c), the results show that compared with the blank control group, the second culture medium containing 5wt% PVP and the PGG material can reduce the freezing point and the frozen water content. In addition, as the gellan gum content of the GG / Gel freeze-dried scaffold in the PGG material increases, the ability of the PGG material to reduce the freezing point and the frozen water content is stronger.

[0079] Example 4

[0080] Example 4-1

[0081] BMSCs (5×10 5 cells / mL) were resuspended in a first culture medium containing proline, wherein the concentration of proline in the first culture medium was 2, 4 and 6 wt%, respectively, and incubated at 37°C, 5% CO2 to achieve pre-dehydration, and the incubation time was 0 min, 15 min, 30 min, 1 h, 2 h and 4 h, respectively. After the incubation was completed, the cells were centrifuged at 1500 r / min for 5 min, the supernatant was removed, and the stem cells were collected. The stem cells were resuspended in a second culture medium containing 5 wt% PVP to obtain a cell suspension, and the cell suspension (5×10 5 cells / mL) were placed in a cryopreservation tube and cooled to -80°C at a rate of -1°C / min for 12 hours, and then placed in liquid nitrogen for one day. The cell suspension was taken out and placed in a 37°C water bath to thaw the cells.

[0082] Live / dead staining was used to characterize the survival rate of cells after recovery. Figure 4From (a), we can see that when BMSCs were directly cryopreserved using the second culture medium containing 5wt% PVP, the survival rate of the cells after recovery was 55%. When BMSCs were co-incubated with the first culture medium containing proline and then cryopreserved using the second culture medium containing 5wt% PVP, the survival rate of BMSCs after recovery was significantly improved. Among them, when the first culture medium with a proline concentration of 4wt% was incubated for 1h and then cryopreserved using the second culture medium containing 5wt% PVP, the cell recovery survival rate reached 85%, indicating that pre-dehydration treatment can reduce the formation of ice crystals in cells and provide protection to cells.

[0083] Example 4-2

[0084] A method for cryopreservation of stem cells, wherein BMSCs (5×10 5 cells / mL) in a first culture medium containing 4 wt% proline, incubated at 37°C, 5% CO2 for 1 h to achieve pre-dehydration, centrifuged at 1500 r / min for 5 min after incubation, removed the supernatant, collected the stem cells, and resuspended the stem cells in a second culture medium containing 5 wt% PVP to obtain a cell suspension (cell density of 5×10 6 / mL), 500 μL of cell suspension was taken with a pipette and dropped onto the surface of 0.02g GG / Gel freeze-dried scaffold for cell encapsulation, so that the cell suspension was loaded in the GG / Gel freeze-dried scaffold. 2:1 Freeze-dried scaffold, GG 3:1 Freeze-dried scaffold, GG 4:1 Freeze-dried scaffold, GG 3:2 Freeze-dried scaffolds and GG 2:3 One of the freeze-dried scaffolds. After 5 minutes, the GG / Gel freeze-dried scaffold completely absorbed the cell suspension to obtain BMSCs@PGG. BMSCs@PGG was placed in a cryopreservation tube, cooled to -80°C at a cooling rate of -1°C / min for 12 hours, and then placed in liquid nitrogen for one day. The BMSCs@PGG was taken out and placed in a 37°C water bath to revive the cells. After revival, the cell survival rate was characterized by live / dead staining. The cell survival rate was as follows: Figure 4 As shown in (b), when the GG / Gel freeze-dried scaffold is GG 2:1 Freeze-dried scaffold, GG 3:1 Freeze-dried scaffold, GG 4:1 Freeze-dried scaffold, GG 3:2 Freeze-dried scaffolds and GG 2:3 When freeze-dried scaffolds were used, the survival rates obtained were as follows Figure 4 (b) PGG 2:1 PGG 3:1 PGG 4:1 PGG 3:2and PGG 2:3 .Depend on Figure 4 (b) shows that the GG / Gel freeze-dried scaffold and PVP form the PGG material. After being encapsulated by the PGG material, the survival rate of the revived cells is further improved, and the survival rate is as high as 95%. The above results show that the efficient cryopreservation of stem cells is achieved by combining the pre-dehydration and cell encapsulation strategies, and when the volume ratio of 1wt% GG solution and 0.5wt% Gel solution in the GG / Gel freeze-dried scaffold used by the PGG material is 2:1, 3:1, 4:1, 3:2 and 2:3, there is no difference in the survival rate of the cells after cell freezing and resuscitation.

[0085] Example 4-3

[0086] The BMSCs@PGG (GG / Gel freeze-dried scaffold in Example 4-2 was GG 4:1 The freeze-dried scaffolds were placed in cryopreservation tubes, cooled to -80°C at a cooling rate of -1°C / min for 12 hours, and then placed in liquid nitrogen for cryopreservation. The cryopreservation time was 1 week, 1 month, 2 months, and 4 months. The BMSCs@PGG were taken out and placed in a 37°C water bath to revive the cells. After revival, the cell survival rate was characterized by live / dead staining to verify the effectiveness of the combined pre-dehydration and cell encapsulation strategy for long-term cryopreservation of stem cells. Figure 4 As shown in (c), the combined pre-dehydration and cell encapsulation strategy can provide stable protection for cells during cryopreservation for up to 4 months. When the cryopreservation time is 1 week, 1 month, 2 months and 4 months, there is no significant difference in the survival rate of cells after recovery, and the cell survival rate is all above 90%, which shows that the combined pre-dehydration and cell encapsulation strategy can provide effective long-term preservation for stem cells.

[0087] Example 4-4

[0088] A stem cell cryopreservation method is basically the same as the stem cell cryopreservation method of Example 4-2, except that BMSCs are replaced by human umbilical cord mesenchymal stem cells or rat bone marrow mesenchymal stem cells, and the GG / Gel freeze-dried scaffold is GG 4:1 Freeze-dried scaffolds.

[0089] The viability of the revived cells was characterized by live / dead staining to verify the wide applicability of the combined pre-dehydration and cell encapsulation strategy for cryopreservation of stem cells of different species. The viability of human umbilical cord mesenchymal stem cells was Figure 4 As shown in (d) "UC-MSCs", the survival rate of rat bone marrow mesenchymal stem cells is as follows Figure 4 As shown in (d) of "Rat-BMSCs", Figure 4(d) shows that the combined pre-dehydration and cell encapsulation strategy can also provide cryopreservation for human umbilical cord mesenchymal stem cells and rat bone marrow mesenchymal stem cells, and the survival rate of cells after recovery is above 92%. The above results show that the stem cell cryopreservation method of the present invention not only abandons the use of DMSO and does not require the traditional protective agent removal process, but also has a highly efficient cryopreservation protection effect on stem cells.

[0090] Example 5

[0091] A method for cryopreservation of stem cells, wherein BMSCs (cell density of 5×10 5 cells / mL) in a first culture medium containing 4 wt% proline, incubated at 37°C, 5% CO2 for 1 h to achieve pre-dehydration, centrifuged at 1500 r / min for 5 min after incubation, removed the supernatant, collected the stem cells, and resuspended the stem cells in a second culture medium containing 5 wt% PVP to obtain a cell suspension (cell density of 5×10 6 / mL), 100 μL of cell suspension was taken with a pipette and added to the surface of 0.005g GG / Gel freeze-dried scaffold for cell encapsulation, so that the cell suspension was loaded in the GG / Gel freeze-dried scaffold. 4:1 Freeze-dried scaffolds. After 5 minutes, the GG / Gel freeze-dried scaffolds completely absorbed the cell suspension to obtain BMSCs@PGG 4:1 , BMSCs@PGG 4:1 Place in a cryopreservation tube, cool to -80°C at a rate of -1°C / min and freeze for 12 hours, then freeze in liquid nitrogen for one day, and take out the BMSCs@PGG 4:1 Place in a 37°C water bath to thaw cells.

[0092] Mouse experimental group: Male Kunming mice weighing 25-35g and aged 5-6 weeks were randomly selected and anesthetized with 10wt% chloral solution. After anesthesia, a full-thickness acute cortical injury model with a diameter of 8mm was established on the back of the mice. After the full-thickness acute cortical injury model was established, the frozen-thawed BMSCs@PGG 4:1 Attach directly to the wound for treatment.

[0093] Mouse control group: basically the same as the mouse experimental group, except that no treatment was performed after the full-thickness acute cortical injury model was established.

[0094] The wound sites of the mice were photographed and recorded on days 0, 3, 7, 11, and 14 in the experimental and control groups, and the wound area was calculated using ImageJ software to evaluate the wound healing. Figure 5The experimental results of the mouse experimental group are shown in Figure 5 The “BMSCs@PGG 4:1 "As shown by Figure 5 (a) and Figure 5 (b) shows that the wound healing rates of the mouse experimental group on the 7th and 14th days were 71.48% and 99.11%, respectively, while those of the mouse control group were only 36.29% and 80.54%. Compared with the mouse control group, the mouse experimental group can significantly accelerate the wound healing speed. Therefore, the stem cell cryopreservation method of the present invention combines the GG / Gel freeze-dried scaffold to make the BMSCs@PGG 4:1 It can be directly used as a tissue engineering construct to promote the repair of mouse skin wounds, and has the advantage of convenient application.

[0095] Example 6

[0096] Example 6-1

[0097] BMSCs (5×10 5 cells / mL) in a first culture medium containing 4 wt% proline, incubated at 37°C, 5% CO2 for 1 h to achieve pre-dehydration, centrifuged at 1500 r / min for 5 min after incubation, removed the supernatant, collected the stem cells, and resuspended the stem cells in a third culture medium to obtain a cell suspension (cell density of 5×10 6 / mL), 500 μL of cell suspension was taken with a pipette and dropped onto the surface of 0.02g GG / Gel freeze-dried scaffold for cell encapsulation, so that the cell suspension was loaded in the GG / Gel freeze-dried scaffold. 4:1 After 5 minutes of freeze-drying the scaffold, the GG / Gel freeze-dried scaffold completely absorbed the cell suspension to obtain BMSCs@GG 4:1 . 4:1 Place in a cryopreservation tube, cool to -80°C at a rate of -1°C / min and freeze for 12 hours, then freeze in liquid nitrogen for one day, and take out the BMSCs@GG 4:1 The cells were placed in a 37°C water bath to thaw, and the cell survival rate was characterized by live / dead staining after thaw. Figure 6 When BMSCs were only encapsulated by GG / Gel freeze-dried scaffolds, the survival rate of cells after recovery was only about 62%. Figure 4 As shown in (b), when BMSCs are encapsulated by PGG material, the survival rate of cells after recovery can reach more than 90%, indicating that the PVP component in PGG material has a significant beneficial effect on cell cryopreservation.

[0098] Example 6-2

[0099] 500 μL of the cell suspension of Example 6-1 was pipetted and added to the surface of 0.02 g of PGG freeze-dried material (PGG freeze-dried material is obtained by freezing and preserving PGG material at -80°C for 24 hours and then freeze-drying it in vacuum for 72 hours) for cell encapsulation, so that the cell suspension is loaded in the PGG freeze-dried material. The PGG material is PGG 4:1 After 5 minutes, the PGG freeze-dried material completely absorbed the cell suspension to obtain BMSCs@P-GG 4:1 . 4:1 Place in a cryopreservation tube, cool to -80°C at a rate of -1°C / min and freeze for 12 hours, then freeze in liquid nitrogen for one day, and take out the BMSCs@P-GG 4:1 The cells were placed in a 37°C water bath to thaw, and the cell survival rate was characterized by live / dead staining after thaw. Figure 6 As shown in (b), Figure 6 As shown in (b), after encapsulation with PGG freeze-dried material, the survival rate of cells after recovery was only 61.37%.

[0100] Example 6-3

[0101] BMSCs (5×10 6 cells / mL) were resuspended in the third culture medium to obtain a cell suspension, and 500 μL of the cell suspension was pipetted and added dropwise to the surface of 0.02 g of PGG freeze-dried material (PGG freeze-dried material is obtained by freezing and preserving PGG material at -80°C for 24 hours and then freeze-drying in vacuum for 72 hours) for cell encapsulation, so that the cell suspension is loaded in the PGG freeze-dried material. The PGG material is PGG 4:1 After 5 minutes, the PGG freeze-dried material completely absorbed the cell suspension to obtain BMSCs-PGG 4:1 BMSCs-PGG 4:1 Place in a cryopreservation tube, cool to -80°C at a rate of -1°C / min and freeze for 12 hours, then freeze in liquid nitrogen for one day, and take out the BMSCs-PGG 4:1 The cells were placed in a 37°C water bath to thaw, and the cell survival rate was characterized by live / dead staining after thaw. Figure 6 As shown in (c), Figure 6 As shown in (c), when only PGG freeze-dried material was used for encapsulation, the survival rate of cells after recovery was only 8.05%.

[0102] Therefore, compared with directly resuspending the cells in the third culture medium, resuspending the cells in the third culture medium containing PVP after incubation with proline may be more conducive to maintaining the cells in a dehydrated state, so the cell survival rate after freezing is higher.

[0103] When the biocompatible osmotic pressure regulator is one or a mixture of taurine, trehalose, sorbitol, betaine and mannitol, the stem cell cryopreservation method can obtain the same technical effect as when the biocompatible osmotic pressure regulator is proline (Pro).

[0104] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for cryopreservation of stem cells, characterized in that: include: The stem cells are incubated in the first culture medium for 0.5 to 6 hours, the biocompatible osmotic pressure regulator is removed, the stem cells are collected, the stem cells are resuspended in the second culture medium to obtain a cell suspension, the cell suspension is added dropwise to the GG / Gel freeze-dried scaffold, and the GG / Gel freeze-dried scaffold is cryopreserved after the cell suspension is completely absorbed, wherein: The first culture medium comprises: a biocompatible osmotic pressure regulator and a third culture medium, wherein the concentration of the biocompatible osmotic pressure regulator in the first culture medium is 1 to 6 wt %, and the biocompatible osmotic pressure regulator is one or a mixture of proline, taurine, trehalose, sorbitol, betaine and mannitol; The second culture medium comprises: polyvinyl pyrrolidone and a third culture medium, wherein the concentration of polyvinyl pyrrolidone in the second culture medium is 5 to 15 wt %; The third culture medium includes: basal culture medium, serum and antibiotics.

2. The stem cell cryopreservation method according to claim 1, characterized in that: The ratio of the number of stem cells in the cell suspension to the mass fraction of the GG / Gel freeze-dried scaffold is (0.5×10 6 ~10×10 6 ): (0.005~0.1), the unit of mass fraction is g.

3. The stem cell cryopreservation method according to claim 1 or 2, characterized in that: The method for obtaining the GG / Gel freeze-dried scaffold comprises: uniformly mixing a gellan gum solution and a gelatin solution to obtain a first mixture, adding a calcium chloride aqueous solution to the first mixture for cross-linking for 5 to 20 minutes to obtain a second mixture, first storing the mixture at -90 to -70°C for 0.5 to 24 hours, and then vacuum freeze-drying to obtain the GG / Gel freeze-dried scaffold.

4. The stem cell cryopreservation method according to claim 3, characterized in that: The gellan gum solution is a mixture of gellan gum and water, and the concentration of gellan gum in the gellan gum solution is 0.5-2wt%; the gelatin solution is a mixture of gelatin and water, and the gelatin in the gelatin solution is 0.2-2wt%.

5. The stem cell cryopreservation method according to claim 4, characterized in that: The ratio of the gellan gum solution to the gelatin solution is (1-10):(1-10) by volume.

6. The method for cryopreservation of stem cells according to claim 5, characterized in that: The ratio of the first mixture to the calcium chloride aqueous solution is (10-50):1 by volume, and the concentration of calcium chloride in the calcium chloride aqueous solution is 2-10 wt %.

7. The method for cryopreservation of stem cells according to claim 1, characterized in that: The cryopreservation adopts a slow freezing method: cooling to -90 to -70°C at a cooling rate of -10 to -1°C / min and storing for 12 to 24 hours, and then storing in liquid nitrogen.

8. The stem cell cryopreservation method according to claim 1, characterized in that: The stem cells were incubated in the first culture medium for 0.5 to 4 h.

9. The method for cryopreservation of stem cells according to claim 1, characterized in that: After 1 to 10 minutes, the GG / Gel freeze-dried scaffold completely absorbs the cell suspension.

10. Use of a biocompatible osmotic pressure regulator in promoting cell dehydration, characterized in that: The biocompatible osmotic pressure regulator is one or a mixture of proline, taurine, trehalose, sorbitol, betaine and mannitol.

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