Menstrual blood preserving fluid and menstrual blood source mesenchymal stem cell cryopreservation method

Abstract

The invention relates to the technical field of biological agents, in particular to a menstrual blood preserving fluid, which is characterized in that components such as modified hydroxyethyl starch, glucose, human serum albumin and the like are compounded to serve as components of the cryopreservation fluid according to a menstrual blood source mesenchymal stem cell cryopreservation method; the preparation method comprises the following steps: firstly, carrying out amination treatment on hydroxyethyl starch by adopting bromopropylamine hydrobromic acid to obtain aminated hydroxyethyl starch, and sequentially reacting the aminated hydroxyethyl starch with activated heparin sodium and proline so as to sequentially introduce the heparin sodium and the proline to obtain modified hydroxyethyl starch; according to the present invention, the modified hydroxyethyl starch is added, such that the hydration ability and the water binding ability of the modified hydroxyethyl starch can be significantly enhanced, and the binding water is difficult to form the large and sharp ice crystal during the freezing process so as to physically inhibit the growth of the ice crystal and reduce the damage of the ice crystal generated by the cryopreservation liquid during the freezing process to the cell membrane of the cell;

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a menstrual blood preservation solution and a method for cryopreserving mesenchymal stem cells derived from menstrual blood. Background Technology

[0002] Mesenchymal stem cells (MSCs) have shown great promise in regenerative medicine, tissue engineering, and immunotherapy due to their self-renewal, multi-lineage differentiation potential, and immunomodulatory properties. In recent years, menstrual blood-derived mesenchymal stem cells (MenSCs) have become a highly attractive new source of stem cells due to their relatively non-invasive sourcing, ease of periodic acquisition, strong proliferative capacity, low immunogenicity, and lack of ethical controversies.

[0003] However, effective storage of MenSCs is a crucial step towards their clinical translation and application. Currently, cell cryopreservation is the main method for long-term preservation of viable cells. During cryopreservation, cells undergo various physicochemical stresses, such as ice crystal formation, osmotic pressure changes, and low-temperature damage, leading to cell membrane damage, protein denaturation, and the accumulation of reactive oxygen species, ultimately resulting in decreased cell viability or even death. Therefore, highly efficient and low-toxicity cryoprotectants (cryopreservation solutions) are essential for maintaining high viability, good recovery status, and biological function of MenSCs after cryopreservation.

[0004] Current cryopreservation technology for menstrual blood-derived mesenchymal stem cells (MenSCs) faces significant challenges: traditional cryopreservation solutions rely on high concentrations of DMSO, which is cytotoxic, and often require the addition of serum, leading to risks of contamination and immunogenicity; existing protocols provide insufficient protection for MenSCs, resulting in poor cell viability, adhesion, and stemness maintenance after cryopreservation and thawing; furthermore, improper storage of menstrual blood during collection can easily lead to the inactivation of target cells; and the protective efficacy of conventional cryoprotectants is also limited. These problems severely restrict the clinical translation and application of MenSCs.

[0005] Therefore, developing a highly efficient, low-toxicity, and clearly defined method for the preservation and cryopreservation of MenSCs that can be applied throughout the entire process from collection to cryopreservation, and especially designing a novel cryoprotectant formulation that can significantly reduce DMSO usage, enhance cell protection, and is suitable for serum-free systems, is of paramount importance for promoting basic research and clinical applications of MenSCs. The technical solution of this invention addresses the key problems in the aforementioned existing technologies. Summary of the Invention

[0006] One object of the present invention is to provide a method for cryopreservation of mesenchymal stem cells derived from menstrual blood, comprising the following steps: S1. Collect menstrual blood, mix the menstrual blood with the preservation solution to obtain intermediate product 1; S2. Centrifuge intermediate product 1, remove the supernatant, add separation liquid, centrifuge again, remove the supernatant, aspirate the white film liquid into a centrifuge tube, add physiological saline, centrifuge, resuspend, add cryopreservation solution, and freeze. The cryopreservation solution comprises the following components by mass fraction: DMSO 2-7% glucose 4-6% Human serum albumin 9-13% Modified hydroxyethyl starch 3-5% Remaining basal culture medium; The modified hydroxyethyl starch is obtained by reacting aminated hydroxyethyl starch, sodium heparin, and proline.

[0007] Furthermore, the basal culture medium is selected from serum-free culture media.

[0008] Furthermore, the preparation method of the modified hydroxyethyl starch includes the following steps: L1. Aminated hydroxyethyl starch is mixed with sodium heparin, NHS, and EDC for the first reaction. Then proline, NHS, and EDC are added, and the reaction is repeated to obtain modified hydroxyethyl starch. L2. The modified hydroxyethyl starch is blended with other components to obtain the cryopreservation solution.

[0009] Furthermore, in step L1, the temperature of the first reaction is 30-35°C.

[0010] Furthermore, in step L1, the temperature of the second reaction is 25-35°C.

[0011] Furthermore, the centrifugation conditions are 700-900g and centrifugation for 15-45 minutes.

[0012] The present invention also provides a menstrual blood preservation solution, which is used for the preservation of menstrual blood in the method for cryopreservation of mesenchymal stem cells derived from menstrual blood. The menstrual blood preservation solution comprises the following mass fractions: Heparin sodium 50-100 U / mL Streptomycin 0.5-3% Penicillin 0.5-2% Remaining amount of physiological saline; Furthermore, the saline solution is 0.9% saline solution.

[0013] The present invention also provides the application of the menstrual blood preservation solution in the field of menstrual blood preservation.

[0014] The present invention has the following beneficial effects: The present invention discloses a method for cryopreserving mesenchymal stem cells derived from menstrual blood. The cryopreservation solution is composed of modified hydroxyethyl starch, glucose, and human serum albumin. First, hydroxyethyl starch is aminated with bromopropylamine hydrobromic acid to obtain aminated hydroxyethyl starch. Then, the aminated hydroxyethyl starch is reacted sequentially with activated sodium heparin and proline to introduce sodium heparin and proline in sequence, thereby obtaining modified hydroxyethyl starch. On the one hand, because sodium heparin contains hydrophilic groups such as sulfonic acid groups, which are similar to the components of cells, it can provide cells with a better survival environment and help maintain the morphology of cells during cryopreservation. Moreover, these hydrophilic groups can significantly enhance the hydration capacity and water-binding capacity of modified hydroxyethyl starch. During freezing, these bound waters are less likely to form large, sharp ice crystals, thereby physically inhibiting ice crystal growth and reducing the damage to cell membranes caused by ice crystals generated by the cryopreservation solution during freezing. On the other hand, the introduced proline is an excellent osmotic protectant and cryoprotectant. Introducing it into modified hydroxyethyl starch can effectively improve the osmotic regulation and cryoprotection capabilities of modified hydroxyethyl starch, and can more effectively balance the osmotic pressure inside and outside the cell, preventing cell damage caused by excessive dehydration during freezing / thawing, which can lead to excessive or insufficient osmotic pressure. Thus, together with heparin sodium, it can improve the cell survival rate. Detailed Implementation

[0015] To more clearly illustrate the technical solution of the present invention, the following embodiments are provided. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well known to those skilled in the art.

[0016] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0017] It should be understood that, except in any operational instance or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term "about". Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying with the desired performance to be obtained according to the invention.

[0018] In this embodiment, the menstrual blood preservation solution comprises the following mass fractions: Heparin sodium 100 U / mL Streptomycin 2% 2% penicillin Remaining amount of physiological saline; Weigh the raw materials according to the above mass fraction, dissolve them in 1L of 0.9% physiological saline, stir evenly at 450rpm, then sterilize under high temperature and high pressure, and store at 4℃.

[0019] The basal culture medium was Lonza UltraCULTURE serum-free medium. Hydroxyethyl starch nanoparticles (CasNo: 1306-06-5) were purchased from Beijing Innocare Technology Co., Ltd., brand name: A82708.

[0020] The cleaning solution is 0.9% physiological saline containing 100 U / mL penicillin, 100 U / mL streptomycin, and 10 mg / mL fluconazole; The menstrual blood was collected from menstrual blood samples of healthy women (20-40 years old). Example

[0021] A method for cryopreserving mesenchymal stem cells derived from menstrual blood includes the following steps: S1. Mix menstrual blood and preservation solution at a mass ratio of 1:10 to obtain intermediate product 1; S2-1. Centrifuge intermediate product 1 at 2500 rpm for 20 min and remove the supernatant. Add an equal volume of washing solution and centrifuge at 2500 rpm for 15 min. Remove the supernatant. Add an equal volume of 0.9% physiological saline and an equal volume of lymphocyte separation medium (TBD) as described above. Centrifuge at 2100 rpm for 15 min. Remove the uppermost separation layer. Transfer the liquid from the white membrane layer to a centrifuge tube. Dilute with an equal volume of 0.9% physiological saline and mix with the cell suspension. Centrifuge at 2000 rpm for 15 min. After centrifugation, remove the supernatant. Add an equal volume of 0.9% physiological saline to resuspend the cells. Take a sample and count the cells. Centrifuge at 1800 rpm for 10 min. After centrifugation, discard the supernatant to obtain menstrual blood mononuclear cells. S2-2, per 1×10 7 Add 1 mL of cryopreservation solution to each of the aforementioned nuclear cells, freeze at -80°C for 24 h, and then transfer to liquid nitrogen at -196°C for storage.

[0022] The cryopreservation solution comprises the following components by mass fraction: DMSO 3% 4% glucose Human serum albumin 10% Modified hydroxyethyl starch 3% Remaining basal culture medium; The preparation method of the modified hydroxyethyl starch includes the following steps: L1-1. Add heparin sodium at a concentration of 8 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activate for 30 min to obtain activation solution 1. L1-2. Proline was added at a concentration of 5 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activated for 30 min to obtain activation solution 2. L1-3. Add 1g of bromopropylamine hydrobromic acid and 1g of hydroxyethyl starch to 4mL of 30wt% sodium hydroxide solution and react at 4℃ for 20min. Add 37% concentrated hydrochloric acid to adjust the pH value to 7. Dialyze with deionized water. The molecular weight of the dialysis bag is 3500. After freeze-drying, aminoated hydroxyethyl starch is obtained. L1-4. Add 1g of amino-modified hydroxyethyl starch to 50mL of activation solution 1, react at 35℃ for 24h, filter, wash three times each with PBS buffer, 4M NaCl solution and deionized water, then add 50mL of activation solution 2, react at 30℃ for 24h, filter, wash three times each with PBS buffer, 4M NaCl solution and deionized water, freeze-dry to obtain modified hydroxyethyl starch.

[0023] The modified hydroxyethyl starch was blended with other components according to the above mass fraction, and the mixture was brought up to 100% using basal culture medium to obtain the cryopreservation solution. Example

[0024] A method for cryopreserving mesenchymal stem cells derived from menstrual blood includes the following steps: S1. Mix menstrual blood and preservation solution at a mass ratio of 1:10 to obtain intermediate product 1; S2-1. Centrifuge intermediate product 1 at 2500 rpm for 20 min and remove the supernatant. Add an equal volume of washing solution and centrifuge at 2500 rpm for 15 min. Remove the supernatant. Add an equal volume of 0.9% physiological saline and an equal volume of lymphocyte separation medium (TBD) as described above. Centrifuge at 2100 rpm for 15 min. Remove the uppermost separation layer. Transfer the liquid from the white membrane layer to a centrifuge tube. Dilute with an equal volume of 0.9% physiological saline and mix with the cell suspension. Centrifuge at 2000 rpm for 15 min. After centrifugation, remove the supernatant. Add an equal volume of 0.9% physiological saline to resuspend the cells. Take a sample and count the cells. Centrifuge at 1800 rpm for 10 min. After centrifugation, discard the supernatant to obtain menstrual blood mononuclear cells. S2-2, per 1×10 7 Add 1 mL of cryopreservation solution to each of the aforementioned nuclear cells, freeze at -80°C for 24 h, and then transfer to liquid nitrogen at -196°C for storage.

[0025] The cryopreservation solution comprises the following components by mass fraction: DMSO 5% 5% glucose Human serum albumin 11% 4% Modified hydroxyethyl starch Remaining basal culture medium; The preparation method of the modified hydroxyethyl starch includes the following steps: L1-1. Add heparin sodium at a concentration of 8 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activate for 30 min to obtain activation solution 1. L1-2. Proline was added at a concentration of 5 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activated for 30 min to obtain activation solution 2. L1-3. Add 1g of bromopropylamine hydrobromic acid and 1g of hydroxyethyl starch to 4mL of 30wt% sodium hydroxide solution and react at 4℃ for 20min. Add 37% concentrated hydrochloric acid to adjust the pH value to 7. Dialyze with deionized water. The molecular weight of the dialysis bag is 3500. After freeze-drying, aminoated hydroxyethyl starch is obtained. L1-4. Add 1g of amino-modified hydroxyethyl starch to 50mL of activation solution 1, react at 35℃ for 24h, filter, wash three times each with PBS buffer, 4M NaCl solution and deionized water, then add 50mL of activation solution 2, react at 30℃ for 24h, filter, wash three times each with PBS buffer, 4M NaCl solution and deionized water, freeze-dry to obtain modified hydroxyethyl starch. L2. According to the above mass fraction, the modified hydroxyethyl starch is blended with other components and supplemented to 100% with basal culture medium to obtain the cryopreservation solution. Example

[0026] A method for cryopreserving mesenchymal stem cells derived from menstrual blood includes the following steps: S1. Mix menstrual blood and preservation solution at a mass ratio of 1:10 to obtain intermediate product 1; S2-1. Centrifuge intermediate product 1 at 2500 rpm for 20 min and remove the supernatant. Add an equal volume of washing solution and centrifuge at 2500 rpm for 15 min. Remove the supernatant. Add an equal volume of 0.9% physiological saline and an equal volume of lymphocyte separation medium (TBD) as described above. Centrifuge at 2100 rpm for 15 min. Remove the uppermost separation layer. Transfer the liquid from the white membrane layer to a centrifuge tube. Dilute with an equal volume of 0.9% physiological saline and mix with the cell suspension. Centrifuge at 2000 rpm for 15 min. After centrifugation, remove the supernatant. Add an equal volume of 0.9% physiological saline to resuspend the cells. Take a sample and count the cells. Centrifuge at 1800 rpm for 10 min. After centrifugation, discard the supernatant to obtain menstrual blood mononuclear cells. S2-2, per 1×10 7 Add 1 mL of cryopreservation solution to each of the aforementioned nuclear cells, freeze at -80°C for 24 h, and then transfer to liquid nitrogen at -196°C for storage.

[0027] The cryopreservation solution comprises the following components by mass fraction: DMSO 7% 6% glucose Human serum albumin 12% Modified hydroxyethyl starch 5% Remaining basal culture medium; The preparation method of the modified hydroxyethyl starch includes the following steps: L1-1. Add heparin sodium at a concentration of 8 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activate for 30 min to obtain activation solution 1. L1-2. Proline was added at a concentration of 5 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activated for 30 min to obtain activation solution 2. L1-3. Add 1g of bromopropylamine hydrobromic acid and 1g of hydroxyethyl starch to 4mL of 30wt% sodium hydroxide solution and react at 4℃ for 20min. Add 37% concentrated hydrochloric acid to adjust the pH value to 7. Dialyze with deionized water. The molecular weight of the dialysis bag is 3500. After freeze-drying, aminoated hydroxyethyl starch is obtained. L1-4. Add 1g of amino-modified hydroxyethyl starch to 50mL of activation solution 1, react at 35℃ for 24h, filter, wash three times each with PBS buffer, 4M NaCl solution and deionized water, then add 50mL of activation solution 2, react at 30℃ for 24h, filter, wash three times each with PBS buffer, 4M NaCl solution and deionized water, freeze-dry to obtain modified hydroxyethyl starch. L2. According to the above mass fraction, the modified hydroxyethyl starch is blended with other components and supplemented to 100% with basal culture medium to obtain the cryopreservation solution.

[0028] Comparative Example 1 A method for cryopreserving mesenchymal stem cells derived from menstrual blood. The difference between this comparative example and Example 1 is that steps L1-2 are removed. In steps L1-4, an equal volume of activation solution 2 is replaced with activation solution 1. Other components and preparation methods are the same.

[0029] Comparative Example 2 A method for cryopreserving mesenchymal stem cells derived from menstrual blood. The difference between this comparative example and Example 1 is that step L1-1 is removed. In steps L1-4, arginine activation solution is used to replace an equal volume of activation solution 1. Other components and preparation methods are the same. The preparation method of the arginine activation solution includes the following steps: Arginine was added at a concentration of 5 mg / mL to 0.05 M MES buffer containing 25 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mM N-hydroxysuccinimide, and activated for 30 min to obtain arginine activation solution.

[0030] Comparative Example 3 A method for cryopreserving mesenchymal stem cells derived from menstrual blood. The difference between this comparative example and Example 1 is that steps L1-4 are removed. That is, activation solution 1, activation solution 2, and modified hydroxyethyl starch are physically mixed as components of the cryopreservation solution. Other components and preparation methods are the same.

[0031] Test Example 1 Cell counting and viability were performed on the cryopreservation methods of Examples 1-3 and Comparative Examples 1-3.

[0032] Cell counting and viability assay: Cells cryopreserved using the methods described in Examples 1-3 and Comparative Examples 1-3 were cryopreserved. After one month of cryopreservation, the cells were thawed in a 37°C water bath for 24 hours and diluted with culture medium to a concentration of 2×10⁻⁶. 6 For a concentration of / mL, take 10μL of cell suspension and 10μL of LAOPI reagent, mix thoroughly, and add to the sample slide for detection.

[0033] The CountStar cell counter was used to count the number of cells before and after cryopreservation and to calculate the survival rate.

[0034] The test results are shown in Table 1.

[0035] Table 1. Cell counting and viability test results of the cryopreservation methods in Examples 1-3 and Comparative Examples 1-3. Serial Number Cell count Cell viability (%) Example 1 1.83 x 10 6 ]] 96.2 Example 2 1.85 x 10 6 ]] 96.5 Example 3 1.86 x 10 6 ]] 95.8 Comparative Example 1 1.80 x 10 6 ]]> 92.7 Comparative Example 2 1.79 x 10 6 ]] 89.5 Comparative Example 3 1.77 x 10 6 ]]> 86.3 Table 1 shows that the cryopreservation methods in Examples 1-3 performed better in terms of cell count and viability. In Comparative Example 1, the modified hydroxyethyl starch exhibited decreased osmotic regulation and cryoprotection capabilities due to the lack of proline modification. In Comparative Example 2, the replacement of heparin sodium with arginine resulted in the loss of anticoagulation and anti-aggregation functions. Comparative Example 3, representing a physical mixture of components without modification, showed the lowest viability. This demonstrates that the technical solution of the present invention can more effectively protect cells from cryopreservation damage, and that the synergistic effect of heparin sodium and proline is crucial for maintaining cell viability.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0037] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for cryopreserving mesenchymal stem cells derived from menstrual blood, characterized in that, Includes the following steps: S1. Collect menstrual blood, mix the menstrual blood with the preservation solution to obtain intermediate product 1; S2. Centrifuge intermediate product 1, remove the supernatant, add separation liquid, centrifuge again, remove the supernatant, aspirate the white film layer liquid into a centrifuge tube, add physiological saline, centrifuge, resuspend, add cryopreservation solution, and freeze. The cryopreservation solution comprises the following components by mass fraction: DMSO 2-7% glucose 4-6% Human serum albumin 9-13% Modified hydroxyethyl starch 3-5% Remaining basal culture medium; The modified hydroxyethyl starch is obtained by reacting aminated hydroxyethyl starch, sodium heparin, and proline.

2. The method for cryopreserving mesenchymal stem cells derived from menstrual blood according to claim 1, characterized in that, The basal culture medium is selected from serum-free culture medium.

3. The method for cryopreservation of mesenchymal stem cells derived from menstrual blood according to claim 1, characterized in that, The preparation method of the modified hydroxyethyl starch includes the following steps: L1. Aminated hydroxyethyl starch is mixed with sodium heparin, NHS, and EDC for the first reaction. Then proline, NHS, and EDC are added, and the reaction is repeated to obtain modified hydroxyethyl starch. L2. The modified hydroxyethyl starch is blended with other components to obtain the cryopreservation solution.

4. The method for cryopreserving mesenchymal stem cells derived from menstrual blood according to claim 3, characterized in that, In step L1, the temperature of the first reaction is 30-35℃.

5. The method for cryopreserving mesenchymal stem cells derived from menstrual blood according to claim 3, characterized in that, In step L1, the temperature of the second reaction is 25-35°C.

6. The method for cryopreservation of mesenchymal stem cells derived from menstrual blood according to claim 1, characterized in that, The centrifugation conditions are 700-900g and centrifugation for 15-45 minutes.

7. A menstrual blood preservation solution, characterized in that, The menstrual blood preservation solution is used for the preservation of menstrual blood in the cryopreservation method for mesenchymal stem cells derived from menstrual blood according to any one of claims 1-6, and the menstrual blood preservation solution comprises the following mass fractions: Heparin sodium 50-100 U / mL Streptomycin 0.5-3% Penicillin 0.5-2% Remaining amount of physiological saline.

8. The menstrual blood preservation solution according to claim 7, characterized in that, The saline solution is 0.9% saline solution.

9. The application of the menstrual blood preservation solution according to claim 7 in the field of menstrual blood preservation.