A cryopreservation solution for adipose mesenchymal stem cells and its application

Abstract

This application relates to a cryopreservation solution for adipose mesenchymal stem cells and its application, belonging to the technical field of cell cryopreservation. The cryopreservation solution for adipose mesenchymal stem cells provided by this application is composed of the following components: human serum albumin solution 6-10% v / v, trehalose 2-4% w / v, poloxamer 188 0.1-0.6% w / v, resveratrol 1-2% w / v, glutathione 1-2% w / v, polyvinyl alcohol 4-6% w / v, and DMSO 3-4% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% in a ratio of 1:1. The cryopreservation solution provided by this application can maintain the cell viability of adipose mesenchymal stem cells, and has good regenerative ability after thawing, can maintain a high exosome secretion ability, and the expression level of cytokines is also high, having important industrial application value.

Description

Technical Field

[0001] The present application relates to a cryopreservation solution for adipose mesenchymal stem cells and its application, belonging to the technical field of cell cryopreservation. Background Art

[0002] Stem cells are a class of pluripotent cells with self-renewal ability and can differentiate into various functional cells under certain conditions. Mesenchymal stem cells (MSCs) are one of the members of the stem cell family and exist in various tissues such as bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc., and have multi-directional differentiation potential. Among them, adipose mesenchymal stem cells have unique cytokine secretion functions as well as immunomodulatory and anti-inflammatory effects, and are currently used in the treatment of various immune-related diseases. Since adipose mesenchymal stem cells can secrete exosomes, the research on preparing various products for biomedicine using the exosomes secreted by mesenchymal cells is also a current hotspot.

[0003] Adipose mesenchymal stem cells have differentiation ability and immunomodulatory characteristics and have great potential in the field of regenerative medicine. Cryopreservation, that is, storing cells at ultra-low temperature, is crucial for maintaining the supply of required adipose mesenchymal stem cells. The expanded adipose mesenchymal stem cells need to be cryopreserved to slow down the cell metabolic activities and maintain life in a low-temperature environment for future use in scientific research and clinical resuscitation at any time or for convenient transportation and storage.

[0004] Currently, cryoprotectants need to be added during the cryopreservation of mesenchymal stem cells. Cryoprotectants can be divided into two categories according to whether they can penetrate cell membranes: permeable and non-permeable. Permeable cryoprotectants are generally small-molecule substances such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, methanol, ethanol, formamide, etc. The main function is to prevent the formation of ice crystals inside cells, avoid the toxicity of high-concentration cryopreservants, and dehydrate cells within the tolerance range; non-permeable cryoprotectants are mainly some macromolecular substances such as trehalose, sucrose, glucose, mannitol, sorbitol, hydroxyethyl starch, and polyvinylpyrrolidone.

[0005] The biological activity of mesenchymal stem cells directly affects their cytokine secretion function, immunomodulatory and anti-inflammatory effects, and also affects the secretion efficiency of their exosomes. Therefore, it is a very important technical issue to ensure that mesenchymal stem cells have high biological activity while meeting long-term preservation and transportation requirements. Currently, common cryopreservation methods for mesenchymal stem cells mostly use a certain proportion of dimethyl sulfoxide, bovine serum, culture medium, etc. for cell cryopreservation. However, bovine serum is an animal-derived substance and cannot be applied to clinical products. DMSO also has certain toxicity to cells and can denature intracellular proteins at room temperature. Cryopreservation with serum may lead to the differentiation of stem cells.

[0006] During the freezing process, water in cells forms ice crystals that can pierce cell membranes and damage internal structures, leading to cell death. The removal of water during freezing causes changes in the concentration of solutes within cells, generating osmotic pressure that causes cells to shrink and damage cell components, thereby reducing cell viability. Cryoprotectants such as dimethyl sulfoxide, glycerol, ethylene glycol, or propylene glycol can reduce ice crystal formation and protect cell membranes. However, they are cytotoxic at high concentrations. Therefore, it is crucial to adjust the combination and dosage of each component to balance the cryoprotective effect and reduce cytotoxicity.

[0007] Normal saline or glucose solution as a preservation solution for mesenchymal stem cells leads to phenomena such as decreased cell viability, massive swelling and death, and increased cell aggregation rate during long-term preservation or transportation due to nutrient deficiency, isotonic or isosmotic force of the solution, etc. Preservation solutions containing fetal bovine serum or cell culture medium cause cells to endocytose fetal bovine serum when in contact with it, which can then cause changes in the expression characteristics of certain cell proteins. When mesenchymal stem cells are reinfused into patients, it may also cause allergic reactions. In addition, freezing and thawing may regulate gene expression, thereby affecting the ability of MSCs to self-renew and differentiate into specific cell types. For example, it is likely to result in the loss or weakening of cell stemness, or a decrease in the ability of mesenchymal stem cells to secrete exosomes.

[0008] Therefore, there is an urgent need for an adipose mesenchymal stem cell preservation solution with stable components, diverse nutrients, osmotic pressure close to that of blood, strong buffering capacity, long preservation time, and convenient transportation, which can maintain the activity of adipose mesenchymal stem cells after freezing and thawing and has a high exosome secretion ability. Summary of the Invention

[0009] To solve the above problems, an adipose mesenchymal stem cell cryopreservation solution and its application are provided. The cryopreservation solution provided in this application can maintain the cell viability of adipose mesenchymal stem cells, has good regenerative ability after thawing, can maintain a high exosome secretion ability, and has a high expression level of cytokines, having important industrial application value.

[0010] According to one aspect of the present application, an adipose mesenchymal stem cell cryopreservation solution is provided, which is composed of the following components: human serum albumin solution 6 - 10% v / v, trehalose 2 - 4% w / v, poloxamer 188 0.1 - 0.6% w / v, resveratrol 1 - 2% w / v, glutathione 1 - 2% w / v, polyvinyl alcohol 4 - 6% w / v, and DMSO 3 - 4% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% at a ratio of 1:1.

[0011] Optionally, the volume concentration of the human serum albumin solution is 20 ± 5%.

[0012] Optionally, the human serum albumin is recombinant human albumin fermented by Pichia pastoris.

[0013] Optionally, the Pichia pastoris is GS115, KM71, X-33 or SMD1168H or their mutants.

[0014] Optionally, the molecular weight of the polyvinyl alcohol is 12 kDa to 15 kDa. In previous studies, the researchers found that adding polyvinyl alcohol with a molecular weight of 12 kDa to 15 kDa can significantly improve the cell survival rate after cryopreserved cell recovery. It is speculated that polyvinyl alcohol with this molecular weight can cooperate with human serum albumin to increase the extracellular osmotic pressure and reduce the free water inside the cells, thereby reducing cell damage caused by ice crystals during cryopreservation. In addition, after adding polyvinyl alcohol with a molecular weight of 12 kDa to 15 kDa, the recovered cells also have better proliferation ability.

[0015] Optionally, the compound amino acid injection contains L-proline, L-serine, L-alanine, L-isoleucine, L-leucine, L-aspartic acid, L-tyrosine, L-glutamic acid, L-phenylalanine, L-arginine hydrochloride, L-lysine hydrochloride, L-valine, L-threonine, L-histidine hydrochloride, L-tryptophan, L-methionine, L-cystine, glycine, sorbitol and sodium bisulfite.

[0016] Optionally, per 1000 mL of the compound amino acid injection, it contains 1.00 g of L-proline, 1.00 g of L-serine, 2.00 g of L-alanine, 3.52 g of L-isoleucine, 4.90 g of L-leucine, 2.50 g of L-aspartic acid, 0.25 g of L-tyrosine, 0.75 g of L-glutamic acid, 5.33 g of L-phenylalanine, 5.00 g of L-arginine hydrochloride, 4.30 g of L-lysine hydrochloride, 3.60 g of L-valine, 2.50 g of L-threonine, 2.50 g of L-histidine hydrochloride, 0.90 g of L-tryptophan, 2.25 g of L-methionine, 0.10 g of L-cystine, 7.60 g of glycine, 50.00 g of sorbitol and 0.50 g of sodium bisulfite.

[0017] Optionally, the compound electrolyte solution contains sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

[0018] Optionally, per 1000 mL of the compound electrolyte solution, it contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride and 0.30 g of magnesium chloride.

[0019] Optionally, it consists of the following components: 8% v / v human serum albumin solution, 3% w / v trehalose, 0.4% w / v poloxamer 188, 1% w / v resveratrol, 1% w / v glutathione, 5% w / v polyvinyl alcohol, and 3% v / v DMSO. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% at a ratio of 1:1.

[0020] According to another aspect of the present application, there is provided the use of any of the above-mentioned adipose mesenchymal stem cell cryopreservation solutions in the cryopreservation of adipose mesenchymal stem cells.

[0021] The beneficial effects of the present application include but are not limited to:

[0022] 1. According to the adipose mesenchymal stem cell cryopreservation solution of the present application, it can enable adipose mesenchymal stem cells to maintain a cell survival rate of about 98%. Compared with conventional cryopreservation solutions, it can better protect the internal structure of cells, reduce the change in the intracellular solute concentration during the freeze-thaw process, and the cell survival rate is relatively high.

[0023] 2. According to the adipose mesenchymal stem cell cryopreservation solution of the present application, when the adipose mesenchymal stem cells after cryopreservation and resuscitation are proliferating, the latent period is shorter, and the growth effect during the logarithmic growth phase is better, and they still maintain good cell stemness.

[0024] 3. According to the adipose mesenchymal stem cell cryopreservation solution of the present application, the adipose mesenchymal stem cells after thawing and resuscitation can maintain a high exosome secretion ability. Through the coordination of each component and the adjustment of the dosage in the cryopreservation solution of the present application, not only the impact of freezing and thawing on adipose mesenchymal stem cells is minimized, but also the cryopreservation solution itself has relatively low cytotoxicity to adipose mesenchymal stem cells.

[0025] 4. According to the adipose mesenchymal stem cell cryopreservation solution of the present application, the secretion amounts of TGF-β, HGF, PGE2, and IL-6 cytokines of the adipose mesenchymal stem cells after thawing and resuscitation are higher than those of conventional cryopreservation solutions. It can be seen that they still maintain a high cytokine secretion ability and good cell stemness.

[0026] 5. According to the adipose mesenchymal stem cell cryopreservation solution of the present application, it can maintain the cell activity of adipose mesenchymal stem cells, and has good regeneration ability after thawing, can maintain a high exosome secretion ability, and the expression level of cytokines is also relatively high, which has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present application, and constitute a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation to the present application. In the drawings:

[0028] Figure 1 This is the growth curve of adipose mesenchymal stem cells after thawing involved in Test Example 2 of this application;

[0029] Figure 2 This is the growth state diagram of adipose mesenchymal stem cells at 1d, 2d, 4d, and 8d after thawing in Example 1 and Example 4 involved in Test Example 2 of this application;

[0030] Figure 3 This is the result diagram of the ability of adipose mesenchymal stem cells to secrete exosomes after thawing involved in Test Example 3 of this application;

[0031] Figure 4 This is the result diagram of the ability of adipose mesenchymal stem cells to secrete cytokines after thawing involved in Test Example 4 of this application;

[0032] Figure 5 This is the liquid phase detection result diagram of recombinant human albumin (ART101L-240126-01) involved in Experimental Example 1 of this application. Detailed implementation manners

[0033] The following describes this application in detail with reference to the examples. However, this application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of this application are purchased through commercial channels.

[0034] In this application, blood-derived human serum albumin refers to albumin isolated and purified from healthy human blood, plasma, or specific immune plasma; recombinant human albumin refers to albumin prepared using recombinant DNA technology for treatment and prevention. For example, the recombinant human albumin used in this application is obtained by fermentation of Pichia pastoris.

[0035] In this application, the compound amino acid injection used contains L-proline, L-serine, L-alanine, L-isoleucine, L-leucine, L-aspartic acid, L-tyrosine, L-glutamic acid, L-phenylalanine, L-arginine hydrochloride, L-lysine hydrochloride, L-valine, L-threonine, L-histidine hydrochloride, L-tryptophan, L-methionine, L-cystine, glycine, sorbitol and sodium bisulfite; wherein for every 1000 mL of the compound amino acid injection, it contains 1.00 g of L-proline, 1.00 g of L-serine, 2.00 g of L-alanine, 3.52 g of L-isoleucine, 4.90 g of L-leucine, 2.50 g of L-aspartic acid, 0.25 g of L-tyrosine, 0.75 g of L-glutamic acid, 5.33 g of L-phenylalanine, 5.00 g of L-arginine hydrochloride, 4.30 g of L-lysine hydrochloride, 3.60 g of L-valine, 2.50 g of L-threonine, 2.50 g of L-histidine hydrochloride, 0.90 g of L-tryptophan, 2.25 g of L-methionine, 0.10 g of L-cystine, 7.60 g of glycine, 50.00 g of sorbitol and 0.5 g of sodium bisulfite.

[0036] In this application, the compound electrolyte solution used contains sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride; wherein for every 1000 mL of the compound electrolyte solution, it contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride and 0.3 g of magnesium chloride.

[0037] In this application, the MTT and BCA protein concentration assay kits were purchased from Beyotime Biotechnology Company. In this application, the recombinant human albumin solutions used in Examples 1-3 and Comparative Examples 1-4 were fermented by Pichia pastoris, with a volume concentration of 20%, which were self-developed products of the applicant, from Tonghua Anruite Biopharmaceutical Co., Ltd., product number ART101L; Example 4 used a blood-derived human serum albumin solution with a volume concentration of 20%, purchased from Boyaa Biopharmaceutical Group Co., Ltd. The BI basal medium was MSC NutriStem® XF Medium, of Sartorius brand, product number 05-200-1A, which is serum-free, does not contain heterologous animal components and does not contain antibiotics.

[0038] Example 1

[0039] A cryopreservation solution for adipose mesenchymal stem cells consists of the following components: recombinant human albumin solution 8% v / v, trehalose 3% w / v, poloxamer 188 0.4% w / v, resveratrol 1% w / v, glutathione 1% w / v, polyvinyl alcohol 5% w / v, and DMSO 3% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% in a ratio of 1:1; among them, the molecular weight of polyvinyl alcohol is 12 kDa to 15 kDa.

[0040] Example 2

[0041] A cryopreservation solution for adipose mesenchymal stem cells consists of the following components: recombinant human albumin solution 6% v / v, trehalose 4% w / v, poloxamer 188 0.1% w / v, resveratrol 2% w / v, glutathione 2% w / v, polyvinyl alcohol 6% w / v, and DMSO 3% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% in a ratio of 1:1; among them, the molecular weight of polyvinyl alcohol is 12 kDa to 15 kDa.

[0042] Example 3

[0043] A cryopreservation solution for adipose mesenchymal stem cells consists of the following components: recombinant human albumin solution 10% v / v, trehalose 2% w / v, poloxamer 188 0.6% w / v, resveratrol 1% w / v, glutathione 1% w / v, polyvinyl alcohol 4% w / v, and DMSO 4% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% in a ratio of 1:1; among them, the molecular weight of polyvinyl alcohol is 12 kDa to 15 kDa.

[0044] Example 4

[0045] A cryopreservation solution for adipose mesenchymal stem cells consists of the following components: blood-derived human serum albumin solution 8% v / v, trehalose 3% w / v, poloxamer 188 0.4% w / v, resveratrol 1% w / v, glutathione 1% w / v, polyvinyl alcohol 5% w / v, and DMSO 3% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% in a ratio of 1:1; among them, the molecular weight of polyvinyl alcohol is 12 kDa to 15 kDa.

[0046] Comparative Example 1

[0047] A cryopreservation solution for adipose mesenchymal stem cells consists of the following components: recombinant human albumin solution 24% v / v, DMSO 5.7% v / v, compound amino acid injection 30.5% v / v, and finally supplemented to 100% with BI basal medium.

[0048] Comparative Example 2

[0049] A cryopreservation solution for adipose mesenchymal stem cells, which is composed of the following components: recombinant human albumin solution 40% v / v, DMSO 3% v / v, glycerol 7% v / v, compound electrolyte injection 25% v / v, and dextran 25% v / v.

[0050] Comparative Example 3

[0051] A cryopreservation solution for adipose mesenchymal stem cells, which is composed of the following components: recombinant human albumin solution 10% v / v, trehalose 2% w / v, glutathione 1% w / v, polyvinyl alcohol 4% w / v, and DMSO 4% v / v, and finally the compound electrolyte injection and compound amino acid injection are supplemented to 100% at a ratio of 1:1; among them, the molecular weight of polyvinyl alcohol is 12 kDa to 15 kDa.

[0052] Comparative Example 4

[0053] A cryopreservation solution for adipose mesenchymal stem cells, which is composed of the following components: recombinant human albumin solution 10% v / v, trehalose 2% w / v, poloxamer 188 0.6% w / v, glutathione 1% w / v, and DMSO 4% v / v, and finally the compound electrolyte injection and compound amino acid injection are supplemented to 100% at a ratio of 1:1.

[0054] The experimenters conducted experiments and detections on the cryopreservation effects of the cryopreservation solutions in the above Examples 1 to 4 and Comparative Examples 1 to 4 on adipose mesenchymal stem cells, and respectively tested the cell survival rate, the proliferation ability of cells after thawing, the exosome secretion ability, and the cytokine expression ability. The specific detection steps and results are shown in the following text.

[0055] Test Example 1 Detection of cell survival rate

[0056] Cell cryopreservation process: Digest the P5 generation adipose mesenchymal stem cells (purchased from Wuhan Punosai Life Science Co., Ltd., product number CP-H202) with 0.2% trypsin for 4 minutes, add 15 mL of culture medium (composed of BI basal medium and Elite serum substitute, and their volume ratio is 95:5), gently pipette to make the cells uniform, centrifuge at 4000 revolutions per minute, discard the supernatant, add 2 mL of cryopreservation solution and mix well, and place it in a sterile cryopreservation tube. Put the cryopreservation tube into a cooling program instrument for the cooling program. The cooling program is: wait at 4°C for 30 minutes, cool down to -7°C at a rate of 1°C / min, cool down to -40°C at a rate of 20°C / min, warm up to -18°C at a rate of 15°C / min, cool down to -50°C at a rate of 1°C / min, warm up to -80°C at a rate of 10°C / min, and after maintaining at -80°C for 20 minutes, transfer the cryopreservation solution into liquid nitrogen for storage for 6 months.

[0057] Cell resuscitation: Take out the cryopreservation tube and quickly place it in a 37°C water bath. Shake it until the cells are completely melted and suspended. Then dilute it with 5 times the culture medium, centrifuge at 1000 rpm for 5 minutes, remove the supernatant by centrifugation, repeat the experiment 3 times, calculate the cell survival rate, and take the average value. Among them, the trypan blue exclusion test is used to calculate the cell survival rate. After centrifugation, take 5 μL of the cell precipitate containing adipose mesenchymal stem cells and dilute it, mix it with 5 μL of 0.4% (volume fraction) trypan blue solution. After 1 minute, drop it into the cell counting chamber, observe and count it under the microscope with a CountStar cell counter. Dead cells are stained light blue, and live cells are not stained. Statistically calculate the cell survival rate (%), and the cell survival rate (%) = live cells / (number of live cells + number of dead cells) × 100%. The results are shown in Table 1 below.

[0058] Table 1 Detection results of cell survival rate

[0059]

[0060] According to the results in Table 1, it can be seen that the cryopreservation solutions in Examples 1 to 3 can keep the cell survival rate of adipose mesenchymal stem cells at about 98%, and Example 4 can keep the cell survival rate of adipose mesenchymal stem cells above 96%. The effects of Examples 1 to 3 are significantly better than those of Example 4; however, compared with the cryopreservation solutions in Comparative Examples 1 to 4, each example can better protect the internal structure of cells, reduce the change in the intracellular solute concentration during the freezing and thawing process, and the cell survival rate is relatively high.

[0061] It can be seen that the cryopreservation solution provided by the solution of the present application can maintain a good cell survival rate of adipose mesenchymal stem cells and has a good cryoprotective effect. However, since dimethyl sulfoxide and the like are used, which may be cytotoxic to adipose mesenchymal stem cells, it is necessary to verify the cytotoxicity of the cryopreservation solution.

[0062] Test Example 2 Growth curve plotting and detection

[0063] Prepare a 96-well plate, take the single-cell suspension of P5-generation adipose mesenchymal stem cells after thawing, and adjust the cell concentration to about 1×10 6 L -1 , and inoculate it into a 96-well plate (200 μL / well). Take one specimen from each group every day. 4 hours before terminating the culture, add 20 μL of 5 mg / L MTT, continue to culture for 4 hours, add 150 μL of DMSO, place it at room temperature for 15 - 20 minutes, shake the culture plate for 10 minutes to make the staining uniform, measure the absorbance value (A) of each well at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, calculate the average value of each group, and continuously measure for 8 days to plot the growth curve. The results are as shown in the appendix Figure 1 as follows.

[0064] Appendix Figure 1The results showed that, compared with Comparative Examples 1-4, the growth curves of adipose mesenchymal stem cells treated in Examples 1-4 had a shorter latency period and better growth effect during the logarithmic growth phase, with Examples 1-3 having a better effect. It can be seen that the adipose mesenchymal stem cells treated with the cryopreservation solution in Examples 1-4 of the present application had better proliferation ability after thawing, and the adipose mesenchymal stem cells still maintained good cell stemness.

[0065] Attached Figure 2 The results showed the growth of adipose mesenchymal stem cells in Examples 1 and 4 at 1d, 2d, 4d and 8d. According to the growth state of adipose mesenchymal stem cells in the figure, when the recombinant human albumin produced by Pichia pastoris was used in combination with other components in the cryopreservation solution, compared with the human serum albumin from blood-derived sources, the mesenchymal stem cells grew better after thawing, and the influence of freezing on adipose mesenchymal stem cells was smaller.

[0066] Test Example 3 Purification, collection and detection of exosomes

[0067] The thawed adipose mesenchymal stem cells at passage 5 were cultured in serum-free DMEM / F12 medium for 48 h. The supernatant was taken, and after centrifugation, the supernatant was taken and concentrated with a 100 kD ultrafiltration tube. EXOquick-TC exosome precipitation reagent was added at a ratio of 5:1 (v:v). After centrifugation, the precipitate was resuspended with PBS to obtain exosomes of adipose mesenchymal stem cells, and the protein concentration of exosomes expressed by adipose mesenchymal stem cells was detected by the BCA method. Using Comparative Example 1 as a control, the relative expression amount results of exosomes are as attached Figure 3 shown.

[0068] According to the attached Figure 3 results, it can be seen that the adipose mesenchymal stem cells frozen and thawed in Examples 1-4 of the present application can maintain a high exosome secretion ability, with Examples 1-3 having a better effect; it can be seen that the cryopreservation solution provided by the present application has weak cytotoxicity to adipose mesenchymal stem cells through the coordination of each component and the adjustment of the dosage.

[0069] Test Example 4 Detection of secreted factors

[0070] The thawed adipose mesenchymal stem cells at passage 5 were cultured in serum-free DMEM / F12 medium for 96 h. The culture supernatant was collected by centrifugation, and the contents of secreted factors TGF-β, HGF, PGE2, and IL-6 in the supernatant were detected by ELISA method. Using Comparative Example 1 as a control, the relative results of the secretion amounts of each secreted factor are as attached Figure 4 shown.

[0071] According to the attached Figure 4It can be seen from the results that the adipose mesenchymal stem cells treated with the cryopreservation solutions of Examples 1 to 4 still maintain a high cytokine secretion capacity, and the adipose mesenchymal stem cells still maintain good cell stemness. Among them, the effects of Examples 1 to 3 are better, indicating that in the cryopreservation solution provided in this application, the effect of using recombinant human albumin is better than that of blood-derived human serum albumin.

[0072] Experimental Example 1 Comparison between Recombinant Human Albumin and Blood-Derived Human Serum Albumin

[0073] The experimenters found that when recombinant human albumin is combined with other components in the solution of this application, it has a better effect as a cell cryopreservation solution for mesenchymal stem cells. Therefore, an exploration was conducted on the structural differences between recombinant human blood albumin and blood-derived human serum albumin. Specifically, the DTNB method was used to detect the free sulfhydryl group content of albumin, and the results are shown in Table 2 below.

[0074] Table 2 Free Sulfhydryl Group Content of Recombinant Human Albumin and Blood-Derived Human Serum Albumin

[0075]

[0076] The experimenters found that the content of free sulfhydryl groups in recombinant human albumin is higher than that in blood-derived human serum albumin. It is speculated that this may be one of the reasons for the better effect of the cell cryopreservation solution prepared from recombinant human albumin. Sulfhydryl groups have antioxidant properties and can cooperate with other components to better protect adipose mesenchymal stem cells from oxidative stress damage.

[0077] In addition, as shown in the appendix Figure 5 is the liquid phase diagram of recombinant human albumin. The homogeneity of recombinant human albumin is better and the monomer content is higher. Due to individual differences in humans, the proteins of blood-derived human serum albumin vary greatly. The experimenters speculate that this is also one of the reasons why the effect of using recombinant human albumin is better than that of blood-derived human serum albumin.

[0078] The above is only the embodiments of this application. The protection scope of this application is not limited by these specific embodiments, but is determined by the claims of this application. For those skilled in the art, various changes and modifications can be made to this application. Any modifications, equivalent replacements, improvements, etc. made within the technical idea and principle of this application should be included within the protection scope of this application.

Claims

1. Use of adipose mesenchymal stem cell cryopreservation solution in the preparation of exosomes from adipose mesenchymal stem cells, characterized in that, The adipose mesenchymal stem cell cryopreservation solution is used for cryopreserving adipose mesenchymal stem cells before exosome secretion; The adipose mesenchymal stem cell cryopreservation solution consists of the following components: human serum albumin solution 6 - 10% v / v, trehalose 2 - 4% w / v, poloxamer 188 0.1 - 0.6% w / v, resveratrol 1 - 2% w / v, glutathione 1 - 2% w / v, polyvinyl alcohol 4 - 6% w / v, and DMSO 3 - 4% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% at a ratio of 1:

1. The compound amino acid injection contains L - proline, L - serine, L - alanine, L - isoleucine, L - leucine, L - aspartic acid, L - tyrosine, L - glutamic acid, L - phenylalanine, L - arginine hydrochloride, L - lysine hydrochloride, L - valine, L - threonine, L - histidine hydrochloride, L - tryptophan, L - methionine, L - cystine, glycine, sorbitol, and sodium bisulfite; The compound electrolyte injection contains sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride; The volume concentration of the human serum albumin solution is 20 ± 5%, and the human serum albumin is recombinant human albumin fermented by Pichia pastoris; The molecular weight of the polyvinyl alcohol is 12 kDa - 15 kDa.

2. The application according to claim 1, wherein For every 1000 mL of the compound amino acid injection, it contains 1.00 g of L - proline, 1.00 g of L - serine, 2.00 g of L - alanine, 3.52 g of L - isoleucine, 4.90 g of L - leucine, 2.50 g of L - aspartic acid, 0.25 g of L - tyrosine, 0.75 g of L - glutamic acid, 5.33 g of L - phenylalanine, 5.00 g of L - arginine hydrochloride, 4.30 g of L - lysine hydrochloride, 3.60 g of L - valine, 2.50 g of L - threonine, 2.50 g of L - histidine hydrochloride, 0.90 g of L - tryptophan, 2.25 g of L - methionine, 0.10 g of L - cystine, 7.60 g of glycine, 50.00 g of sorbitol, and 0.50 g of sodium bisulfite.

3. The application according to claim 1, wherein For every 1000 mL of the compound electrolyte injection, it contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride, and 0.30 g of magnesium chloride.

4. The application according to claim 1, wherein The adipose mesenchymal stem cell cryopreservation solution consists of the following components: human serum albumin solution 8% v / v, trehalose 3% w / v, poloxamer 188 0.4% w / v, resveratrol 1% w / v, glutathione 1% w / v, polyvinyl alcohol 5% w / v, and DMSO 3% v / v. Finally, compound electrolyte injection and compound amino acid injection are supplemented to 100% at a ratio of 1:1.

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