Mesenchymal stem cell cryopreservation solution, injection and cryopreservation method

Abstract

The application provides a mesenchymal stem cell cryopreservation solution which can be directly transfused into human body, the cryopreservation solution comprises dimethyl sulfoxide with a mass concentration greater than 0% and less than 5%, carboxymethyl cellulose salt with a mass concentration greater than 0% and less than or equal to 0.8%, and compound electrolyte injection solution. The mesenchymal stem cell cryopreservation solution of the application adopts clinical grade reagent, does not contain serum and animal-derived components, and the cell injection solution after cell cryopreservation and recovery can be directly transfused into human body, so that cell damage and loss caused by the washing process are significantly reduced, and the application is convenient. The carboxymethyl cellulose salt in the cryopreservation solution effectively replaces the protein protective agent, significantly reduces the dosage of dimethyl sulfoxide, has very low toxic side effects and irritability, and can ensure excellent 0-hour and 24-hour cell survival rate and recovery rate, and the cells after recovery can be placed at room temperature for a long time, and have good room temperature stability; meanwhile, the cryopreservation cost of the mesenchymal stem cells is greatly reduced, and the application is suitable for large-scale clinical application.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation technology, specifically to a cryopreservation solution, injection solution, and cryopreservation method for mesenchymal stem cells that can be directly infused into the human body. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent cells capable of self-renewal and differentiation into various cell types, such as osteoblasts, chondrocytes, adipose tissue, and neurons. Due to their self-renewal and multi-lineage differentiation potential, MSCs are widely used in the treatment of tissue regeneration and immune diseases, including bone / cartilage defects, cardiovascular diseases, inflammatory diseases, and autoimmune diseases. Numerous studies have demonstrated that MSC therapy is safe and effective.

[0003] Currently, the cryopreservation solutions used in MSC therapeutic formulations all contain serum, animal-derived components, or excipients that do not meet clinical standards, making them unsuitable for direct human use. Conventional methods involve washing and resuspending cells after thawing to remove harmful substances, finally resuspending them in clinical-grade excipients to prepare a fresh injection solution. However, this freshly prepared MSC injection solution has a short shelf life, making it unsuitable for long-term transportation, and cell loss can occur during the washing process. If cryopreservation formulations are to be used, clinical-grade cryoprotectants suitable for direct human injection must be employed. This ensures that cells are not easily damaged during cryopreservation, guarantees cell viability and recovery rates, and facilitates normal cell function after infusion into the body. However, some existing cryopreservation agents for mesenchymal stem cells, while exhibiting good cryopreservation effects, have strong toxic side effects, such as dimethyl sulfoxide (DMSO) and glycerol. Others, such as human serum albumin (HSA), while having fewer toxic side effects, cause significant cell damage after cryopreservation, resulting in low survival and recovery rates. These agents do not adequately meet clinical application needs, especially in the treatment of conditions such as knee osteoarthritis, where the knee joint is highly sensitive to the toxicity or irritation of cryopreservation agents. To better meet clinical needs, attempts have been made to develop cryopreservation solutions containing dimethyl sulfoxide and human serum albumin (HSA) to meet the clinical requirement of excellent cryopreservation effects with low toxicity while ensuring high survival and recovery rates. However, the DMSO content in existing cryopreservation solutions containing HSA cannot be further reduced to achieve the required low toxicity; otherwise, high survival and recovery rates cannot be guaranteed. Therefore, there is an urgent need to develop a mesenchymal stem cell cryopreservation solution that is directly infused into the human body and has very low toxicity or irritation while ensuring excellent cell survival and recovery rates. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a mesenchymal stem cell cryopreservation solution that can be directly infused into the human body. This cryopreservation solution has very low toxicity or irritation while ensuring excellent cell viability and recovery rates. The mesenchymal stem cell cryopreservation solution comprises dimethyl sulfoxide at a mass concentration greater than 0% and less than 5%, carboxymethyl cellulose salt at a mass concentration greater than 0% and less than or equal to 0.8%, and a compound electrolyte injection solution.

[0005] In some embodiments, the carboxymethyl cellulose salt is at least one of sodium carboxymethyl cellulose and potassium carboxymethyl cellulose.

[0006] In some embodiments, the mesenchymal stem cell cryopreservation solution further contains glucose at a mass concentration greater than 0% and less than 1.0%.

[0007] In some embodiments, the mesenchymal stem cell cryopreservation solution further contains 0.1-2 mg / mL of compound amino acids and has a pH of 7.0-7.5.

[0008] In some embodiments, the mass concentration of the carboxymethyl cellulose salt is greater than 0% and less than or equal to 0.3%.

[0009] In some embodiments, the mass concentration of the carboxymethyl cellulose salt is greater than 0% and less than or equal to 0.15%.

[0010] In some embodiments, the mass concentration of the dimethyl sulfoxide is greater than 1.5% and less than 5%.

[0011] In some embodiments, the glucose concentration is greater than 0.4% and less than 1.0% by mass.

[0012] The present invention provides a mesenchymal stem cell injection solution, wherein the mesenchymal stem cell injection solution comprises the mesenchymal stem cell cryopreservation solution and mesenchymal stem cells described in the present invention.

[0013] This invention provides a method for cryopreserving mesenchymal stem cells, comprising the following steps: cryopreserving mesenchymal stem cells using the mesenchymal stem cell cryopreservation solution described in this invention.

[0014] In some embodiments, the cryopreservation density of mesenchymal stem cells in the mesenchymal stem cell cryopreservation solution is 2 × 10⁻⁶. 6 N25×10 6 Cells / mL.

[0015] In some embodiments, the cryopreservation density of mesenchymal stem cells in the mesenchymal stem cell cryopreservation solution is 10 × 10⁻⁶. 6 N15×10 6 Cells / nL

[0016] Beneficial effects

[0017] The mesenchymal stem cell cryopreservation solutions of this invention all use clinical-grade reagents and are free of serum and animal-derived components, eliminating concerns about allergens or animal infectious diseases. The cryopreserved and thawed cell injection solution can be directly infused into the human body, significantly reducing cell damage and loss during the washing process, making it convenient to use. The carboxymethyl cellulose salt in the cryopreservation solution effectively replaces protein-based cryoprotectants such as HSA, significantly reducing the amount of dimethyl sulfoxide used, exhibiting very low toxicity or irritation, while ensuring excellent 0-hour and 24-hour cell viability and recovery rates. Furthermore, the cells can be stored at room temperature for extended periods after thawing, demonstrating good room temperature stability, suitable for long-term storage of mesenchymal stem cells, thus expanding the clinical application range of mesenchymal stem cells. Simultaneously, the use of clinical-grade carboxymethyl cellulose salt in the mesenchymal stem cell cryopreservation solution of this invention effectively replaces protein-based cryoprotectants such as HSA, greatly reducing the cost of mesenchymal stem cell cryopreservation and making it suitable for large-scale clinical applications. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments. In the accompanying drawings:

[0019] Figure 1 The effects of the addition of glucose to the cryopreservation solution and different concentrations of sodium carboxymethyl cellulose on the recovery rate and cell viability of iMSCs after cryopreservation and thawing at 0 hours were shown.

[0020] Figure 2 The effects of the addition of glucose to the cryopreservation solution and different concentrations of sodium carboxymethyl cellulose on the recovery rate and cell viability of iMSCs after 24 hours of culture following cryopreservation and thawing were shown.

[0021] Figure 3 The effects of different mass concentrations of glucose and sodium carboxymethyl cellulose in the cryopreservation solution on the recovery rate and cell viability of iMSCs at 0 hours after cryopreservation and thawing were shown.

[0022] Figure 4 The effects of different mass concentrations of glucose and sodium carboxymethyl cellulose in the cryopreservation solution on the recovery rate and cell viability of iMSCs after 24 hours of culture following cryopreservation and thawing were shown.

[0023] Figure 5 The image shows the cell morphology of iMSCs that were cryopreserved in cryopreservation solutions containing different mass concentrations of glucose and different mass concentrations of sodium carboxymethyl cellulose after thawing and inoculation for 24 hours.

[0024] Figure 6 The effects of different mass concentrations of dimethyl sulfoxide in the cryopreservation solution on the recovery rate and cell viability of iMSCs at 0 hours after cryopreservation and thawing were shown.

[0025] Figure 7 This study showed the effect of different mass concentrations of dimethyl sulfoxide in the cryopreservation solution on the recovery rate and cell viability of iMSCs after 24 hours of culture following cryopreservation and thawing.

[0026] Figure 8 The image shows the cell morphology of iMSCs that were cryopreserved in cryopreservation solutions containing different mass concentrations of dimethyl sulfoxide and then cultured for 24 hours after thawing.

[0027] Figure 9 The effect of cryopreservation solutions containing compound amino acids at different mass concentrations of sodium carboxymethyl cellulose on the recovery rate and cell viability of iMSCs at 0 hours after cryopreservation and thawing was shown.

[0028] Figure 10 The effect of cryopreservation solutions containing compound amino acids at different mass concentrations of sodium carboxymethyl cellulose on the recovery rate and cell viability of iMSCs after cryopreservation and thawing for 24 hours was shown.

[0029] Figure 11 The image shows the cell morphology of iMSCs that were cryopreserved in a compound amino acid-containing cryopreservation solution at different mass concentrations of sodium carboxymethyl cellulose after thawing and inoculation for 24 hours.

[0030] Figure 12 This study demonstrates the effect of using a medical injection solution different from the compound electrolyte injection solution on the recovery rate and cell viability of iMSCs at 0 hours after cryopreservation and thawing.

[0031] Figure 13 This study demonstrates the effect of using a medical injection solution different from the compound electrolyte injection solution on the recovery rate and cell viability of iMSCs after cryopreservation and thawing for 24 hours.

[0032] Figure 14 The image shows the cell morphology of iMSCs that were cryopreserved in a medical injection solution different from the compound electrolyte injection solution, after being thawed and cultured for 24 hours.

[0033] Figure 15 The effects of different cryopreservation densities on the recovery rate and cell viability of iMSCs after thawing and placing them at room temperature for different times after 0 hours were shown.

[0034] Figure 16 The study showed the effect of different cryopreservation densities on the recovery rate and cell viability of iMSCs after thawing at room temperature for different times after 24 hours of culture.

[0035] Figure 17A The image shows the cell morphology of iMSCs that were frozen in cryopreservation solution at different cryopreservation densities, after being thawed and placed at room temperature for 0 hours and then cultured for 24 hours.

[0036] Figure 17B The image shows the morphology of iMSCs that were thawed after being placed at room temperature for 2 hours and then cultured for 24 hours after being frozen in cryopreservation solution at different cryopreservation densities.

[0037] Figure 17C The image shows the cell morphology of iMSCs that were frozen in cryopreservation solution at different cryopreservation densities, after which they were thawed and placed at room temperature for 4 hours, and then inoculated and cultured for 24 hours. Detailed Implementation

[0038] The mesenchymal stem cell cryopreservation solution provided by this invention is free of serum and animal-derived components and can be directly infused into the human body. It contains dimethyl sulfoxide at a mass concentration greater than 0% and less than 5%, carboxymethyl cellulose salt at a mass concentration greater than 0% and less than or equal to 0.8%, and a compound electrolyte injection solution. This mesenchymal stem cell cryopreservation solution has very low toxicity or irritation, while ensuring excellent cell viability and recovery rates at 0 hours and 24 hours.

[0039] Furthermore, the mesenchymal stem cell cryopreservation solution of the present invention uses compound electrolyte injection as the base solution because other injection solutions cannot simultaneously ensure excellent cell viability and recovery rate at 0 hours and 24 hours, while the 24-hour cell recovery rate is important for clinical applications.

[0040] The addition of DMSO can improve the cell viability and live cell recovery rate of cryopreserved cells at 0 hours and 24 hours. However, excessively high DMSO concentrations can lead to significant toxicity and irritation, making it unsuitable for specific clinical needs such as knee osteoarthritis. In this invention, the mass concentration of DMSO in the cryopreservation solution is greater than 0% and less than 5%, and a mass concentration of dimethyl sulfoxide greater than 1.5% and less than 5% is more conducive to a high 24-hour live cell recovery rate.

[0041] According to the present invention, although the concentration of DMSO in the cryopreservation solution is low, the addition of carboxymethyl cellulose salt allows the cryopreservation solution to simultaneously ensure high cell viability and cell recovery rates at both 0 hours and 24 hours. The mass concentration of carboxymethyl cellulose salt in the cryopreservation solution of the present invention is greater than 0% and less than or equal to 0.8%. When the mass concentration exceeds 0.8%, carboxymethyl cellulose salt is not easily dissolved in the cryopreservation solution and cannot achieve a good cryopreservation effect. Furthermore, according to the present invention, a low concentration of carboxymethyl cellulose salt is more beneficial to cell recovery rates at both 0 hours and 24 hours compared to a high concentration. Compared to higher concentrations of carboxymethyl cellulose salt, a mass concentration of carboxymethyl cellulose salt greater than 0% and less than or equal to 0.3% not only benefits the cell recovery rate at 0 hours but also significantly improves the cell recovery rate at 24 hours. Furthermore, a mass concentration of carboxymethyl cellulose salt greater than 0% and less than or equal to 0.15% can further significantly improve both the cell recovery rate at 0 hours and 24 hours, especially the latter.

[0042] The carboxymethyl cellulose salt is sodium carboxymethyl cellulose, potassium carboxymethyl cellulose, or other carboxymethyl cellulose salts such as magnesium carboxymethyl cellulose or calcium carboxymethyl cellulose, or a mixture thereof. Preferably, the carboxymethyl cellulose salt is selected from at least one of sodium carboxymethyl cellulose and potassium carboxymethyl cellulose.

[0043] The mesenchymal stem cell cryopreservation solution of the present invention can also be composed of dimethyl sulfoxide with a mass concentration greater than 0% and less than 5%, sodium carboxymethyl cellulose with a mass concentration greater than 0% and less than or equal to 0.8%, and the balance being a compound electrolyte injection solution.

[0044] The mesenchymal stem cell cryopreservation solution of the present invention may further contain glucose at a mass concentration greater than 0% and less than 1.0%. The addition of glucose to the cryopreservation solution of the present invention can improve the 0-hour viable cell recovery rate. More advantageously, the glucose mass concentration is greater than 0.4% and less than 1.0%, and according to this embodiment, the 0-hour viable cell recovery rate can be significantly improved.

[0045] The mesenchymal stem cell cryopreservation solution of the present invention may also be composed of dimethyl sulfoxide with a mass concentration greater than 0% and less than 5%, carboxymethyl cellulose salt with a mass concentration greater than 0% and less than or equal to 0.8%, glucose with a mass concentration greater than 0% and less than 1.0%, and the balance being a compound electrolyte injection solution.

[0046] The mesenchymal stem cell cryopreservation solution of the present invention may further contain 0.1-2 mg / mL of compound amino acids. Preferably, the mass concentration of the compound amino acids is 0.1-1 mg / mL. Further, depending on the specific circumstances, the mesenchymal stem cell cryopreservation solution of the present invention also contains a pH adjuster, used to adjust the pH of the cryopreservation solution to 7.0-7.5 after the addition of the compound amino acids. Specifically, when the concentration of the compound amino acids is low, the pH adjuster may not be added, while when the concentration of the compound amino acids is high, the pH adjuster may be added to adjust the pH to 7.0-7.5. The pH adjuster is a commonly used pH adjuster such as sodium hydroxide or sodium bicarbonate.

[0047] The mesenchymal stem cell cryopreservation solution of the present invention may also be composed of dimethyl sulfoxide with a mass concentration greater than 0% and less than 5%, sodium carboxymethyl cellulose with a mass concentration greater than 0% and less than or equal to 0.8%, glucose with a mass concentration greater than 0% and less than 1.0%, compound amino acids with a mass concentration of 0.1-2 mg / mL, and the balance being compound electrolyte injection solution, and has a pH value of 7.0-7.5.

[0048] The present invention provides a mesenchymal stem cell injection solution, wherein the mesenchymal stem cell injection solution comprises the mesenchymal stem cell cryopreservation solution and mesenchymal stem cells described in the present invention.

[0049] The mesenchymal stem cells described in this invention include primary mesenchymal stem cells derived from natural tissues and mesenchymal stem cells derived from human induced pluripotent stem cells. Furthermore, the mesenchymal stem cells described in this invention also include genetically modified mesenchymal stem cells. Primary mesenchymal stem cells, induced mesenchymal stem cells, or genetically modified mesenchymal stem cells can all be obtained using conventional techniques in the art or commercially available.

[0050] This invention provides a method for cryopreserving mesenchymal stem cells, comprising the following steps: cryopreserving mesenchymal stem cells using the mesenchymal stem cell cryopreservation solution described in this invention.

[0051] More specifically, the cryopreservation method includes: first, using a programmed cooling method to freeze the mesenchymal stem cells at a first temperature, and then transferring them to a second temperature, such as below -196°C, for further freezing. For example, the mesenchymal stem cell cryopreservation solution can be mixed with the mesenchymal stem cells and filled into a cryopreservation container, the mesenchymal stem cell injection solution can be frozen using a programmed cooling method, and then transferred to a liquid nitrogen tank below -196°C for storage.

[0052] The programmed cooling method may include cooling from room temperature to -100°C at a certain cooling rate.

[0053] The aforementioned programmable cooling method may also include:

[0054] S1: Set the cooling start temperature to 25℃;

[0055] S2: Cools down to 4℃ at a rate of -10℃ / minute;

[0056] S3: Maintain at 4℃ for 15 minutes;

[0057] S4: Cools down to -80℃ at a rate of -1℃ / minute;

[0058] S5: Cooling down to -100℃ at a rate of -10℃ / minute;

[0059] The mesenchymal stem cell cryopreservation formulation of this invention adjusts the cryopreservation dosage of cells according to the requirements of clinical application, with a cryopreservation density of 2×10⁻⁶ cells / year. 6 N25×10 6 Cells / mL, specifically 2 × 10⁻⁶ 6 cells / mL, 5×10 6 8 × 10⁸ cells / mL 6 cells / mL, 10×10 6 cells / mL, 12 × 10 6 cells / mL, 15 × 10 6 cells / mL, 20×10 6 cells / mL, 25×10 6 Cells / mL. The preferred cryopreservation density of mesenchymal stem cells is 10 × 10⁻⁶ cells / mL. 6 Cells / mL - 15 × 10 6 Cells / mL. According to this implementation scheme, cells frozen at this low cryopreservation density have better stability after thawing (24-hour live cell recovery stability) compared to high cryopreservation density.

[0060] Example

[0061] The present invention will be described in detail below with reference to the embodiments, but the embodiments should not be construed as limiting the scope of protection of the present invention.

[0062] The sources of reagents and raw materials in the following embodiments are described below. It should be noted that the present invention does not limit the source of the reagents and materials used.

[0063] Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) were obtained by inducing iPSC differentiation at Anhui Zhong Sheng Su Yuan Biotechnology Co., Ltd. (CN201910884482.4); sodium carboxymethyl cellulose (CMC-Na) was purchased from Anhui Shanhe Pharmaceutical Excipients Co., Ltd. (SH-SJJ-1000); dimethyl sulfoxide (DMSO) was purchased from OirGen. Biomedical (CP-70); glucose was purchased from Chenxin Pharmaceutical Co., Ltd. (National Drug Approval Number H37021755); compound amino acid injection was purchased from Hubei Yibantian Pharmaceutical Co., Ltd. (National Drug Approval Number H20023293); compound electrolyte injection was purchased from Shanxi Zhendong Pharmaceutical Co., Ltd. (National Drug Approval Number H20113035); 0.9% sodium chloride injection was purchased from Anhui Shuanghe Pharmaceutical Co., Ltd. (National Drug Approval Number H34023608); dextran 40 sodium chloride injection was purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd. (National Drug Approval Number H13022493); serum-free culture medium for human mesenchymal stem cells was from Anhui Shouning Biotechnology Co., Ltd. (SN-02-1010); DPBS was purchased from Thermo Fisher Scientific (C14190500BT); TrypLE TM SelectEnzyme (10×) was purchased from Thermo Fisher (A1217702).

[0064] Example 1

[0065] 1. Preparation of cryopreservation solution for mesenchymal stem cells

[0066] The mesenchymal stem cell cryopreservation solution was prepared according to the formula shown in Table 1. The preparation method of the cryopreservation solution includes the following steps:

[0067] (1) Weigh an appropriate amount of sodium carboxymethyl cellulose powder and add it to a 50mL centrifuge tube;

[0068] (2) Take a clean 100mL Erlenmeyer shaker, add an appropriate amount of medical injection solution, and place a magnetic stirrer rotor inside.

[0069] (3) Turn on the stirring machine at 800 rpm to stir the solution, and then slowly add sodium carboxymethyl cellulose powder to the solution while stirring to mix.

[0070] (4) After adding sodium carboxymethyl cellulose powder, continue stirring for 3-4 hours, and then autoclave at 115°C for 30 minutes.

[0071] (5) After sterilization, allow the mixture to return to room temperature and filter it using a 0.45μm filter for later use;

[0072] (6) Calculate the amount of each component in the cryopreservation solution based on the volume of the cryopreservation solution prepared, and mix the corresponding amounts of compound electrolyte injection, CMC-Na solution and glucose solution thoroughly.

[0073] (7) Take the corresponding amount of dimethyl sulfoxide and add it to the above mixed solution, and mix thoroughly;

[0074] (8) Store the prepared mesenchymal stem cell cryopreservation solution at 2-8℃.

[0075] Table 1. Mesenchymal stem cell cryopreservation solution ratio

[0076]

[0077] 2. Cryopreservation of mesenchymal stem cells

[0078] (1) Culture of induced pluripotent stem cell-derived mesenchymal-like stem cells (iMSCs)

[0079] 1) Take one iMSCP1 generation cell from the cell bank, place it in a cryogenic transport container, and transport it to the intercellular space;

[0080] 2) During the cell resuscitation stage, following the standard operating procedure for iMSC resuscitation, mesenchymal stem cells were seeded into three T150 cell culture flasks using serum-free mesenchymal stem cell culture medium.

[0081] 3) iMSC cells P2, change the medium on the second day, passage to P3 on the third day, and then seed into 5 T150 cell culture flasks;

[0082] 4) iMSC cells were passaged to P4 on the second day after the medium was changed, and then seeded into a ten-layer cell factory on the third day.

[0083] 5) iMSC cells P4, change the medium on the second day, and the cells can be used on the third day of culture, at which time the cell confluence is 80% to 95%.

[0084] (2) Cell collection

[0085] 1) Remove one 10-layer cell factory from the 37℃ incubator and discard the supernatant;

[0086] 2) Wash the cells 1N2 times with an appropriate amount of DPBS, then discard the DPBS;

[0087] 3) Add an appropriate amount of 0.5×TrypLE TM Add enough Select Enzyme cell digestion solution to cover the bottom of the container, then close the lid.

[0088] 4) Transfer the ten-layer cell factory to a 37°C incubator and digest for 12 minutes;

[0089] 5) After digestion, transfer the cells to a biosafety cabinet, gently shake the cell factory to allow the iMSCs to fully detach from the substrate, and then transfer them to three 50mL centrifuge tubes and centrifuge at 250×g for 5 minutes.

[0090] 6) After centrifugation, transfer the 50mL centrifuge tube to the biosafety cabinet, discard the supernatant, and collect the cell pellet.

[0091] (3) Adding mesenchymal stem cell cryopreservation solution to prepare cryopreservation formulation

[0092] 1) Add an appropriate amount of the cryopreservation solution shown in Table 1 to the cell pellet collected in the centrifuge tube in step (2) and resuspend the cell pellet;

[0093] 2) Take an appropriate amount of cell suspension, dilute it 10 times with culture medium, and then count the cells using a Vi-CELLXR cell counter and record the cell volume;

[0094] 3) Based on the counting results, replenish the cryopreservation solution for each group. The replenishment should be done slowly, dropwise while stirring. After replenishment, count again using a Vi-CELLXR cell counter to confirm the cell density. At this point, the final cell density should be adjusted to approximately 10 × 10⁻⁶. 6 A resuspension of mesenchymal stem cells can be obtained by measuring approximately 10 cells / mL.

[0095] (4) Filling cells into cryopreservation containers

[0096] The cell suspension obtained in step (3) is dispensed into 1 mL / tube, and each group is dispensed into 3 tubes.

[0097] (5) Cell cryopreservation

[0098] Each group of cells was frozen to below -100°C using a programmed freezing device and finally transferred to a gas phase liquid nitrogen tank for storage.

[0099] The cooling program of the programmed cooling device is as follows:

[0100] S1: Set the cooling start temperature to 25℃;

[0101] S2: Cooling down to 4℃ at a rate of -10℃ / minute;

[0102] S3: Maintain at 4℃ for 15 minutes;

[0103] S4: Cooling down to -80℃ at a rate of -1℃ / minute;

[0104] S5: Cools down to -100℃ at a rate of -10℃ / minute.

[0105] 3. Thawing of mesenchymal stem cell cryopreservation preparations and detection of cell viability and live cell recovery rate at 0 hours.

[0106] (1) Take out the frozen mesenchymal stem cells from the gas phase liquid nitrogen tank, and place each group in a 37°C water bath. Gently shake during thawing until completely thawed.

[0107] (2) After thawing, transfer the cells to a 50ml centrifuge tube, add 9ml of MSC amplification complete medium and mix well. Take an appropriate amount of cell suspension and count the cells using a Vi-CELLXR cell counter. Record the cell viability and viable cell density at 0 hours after thawing, and calculate the viable cell recovery rate at 0 hours. Viable cell recovery rate at 0 hours = [(viable cell density × 10mL) / (total number of viable cells before cryopreservation)] × 100%.

[0108] 4. Detection of 24-hour cell viability and live cell recovery rate of mesenchymal stem cells

[0109] (1) Add an appropriate amount of culture medium to the T25 cell culture flask, taking 1×10⁶ cells per group. 6 ~2×10 6 A total of [number] cells were seeded into T25 cell culture flasks and then placed in a carbon dioxide incubator at 37°C, 5% CO2 concentration, and saturated humidity for 24 hours for static culture.

[0110] (2) Take iMSC cells from each group that have been cultured for 24 hours and photograph their adherence status under a microscope.

[0111] (3) Transfer the T25 cell culture flasks of each group into the biosafety cabinet;

[0112] (4) Discard the supernatant, wash 1-2 times with DBPS, and discard DPBS;

[0113] (5) Add 1 mL of 0.5×TrypLE to each T25 cell culture flask TM Select Enzyme, digest at 37°C for 6 minutes;

[0114] (6) After digestion, use a 1mL pipette tip to blow the cells 3-5 times, take out an appropriate amount of cell suspension, count the cells with a Vi-CELL XR cell counter, obtain the viable cell density and cell viability of each group, and calculate the 24-hour cell recovery rate; 24-hour viable cell recovery rate = [(viable cell density × 1mL) / (total number of viable cells inoculated)] × 100%.

[0115] The results of cell viability and live cell recovery rate of iMSCs at 0 hours after resuscitation showed that... Figure 1 In the study, the cell viability and live cell recovery rate of iMSCs after 24 hours of culture following resuscitation were shown to be... Figure 2 middle.

[0116] like Figure 1As shown, the cell viability of iMSCs cryopreserved in each group was over 85% at 0 hours after thawing, and the cell recovery rate at 0 hours was also high, all exceeding 70%. Figure 2 As shown, the cell viability of iMSCs cryopreserved in each group remained above 95% after thawing and culturing for 24 hours, and the 24-hour viable cell recovery rate reached over 70%. In particular, when the glucose concentration was 0.5%, the 0-hour viable cell recovery rate significantly increased from 70.5% to 90.6% compared to cryopreservation solutions without added glucose. This indicates that mesenchymal stem cells cryopreserved using the cryopreservation solution of this embodiment not only have excellent 0-hour and 24-hour cell viability but also excellent 0-hour and 24-hour viable cell recovery rates, demonstrating high cell proliferation capacity.

[0117] Example 2

[0118] 1. Preparation of cryopreservation solution for mesenchymal stem cells

[0119] The mesenchymal stem cell cryopreservation solution was prepared according to the formula shown in Table 2. The preparation method of the cryopreservation solution is the same as in Example 1.

[0120] Table 2. Mesenchymal stem cell cryopreservation solution ratio

[0121]

[0122] 2. Cryopreservation of mesenchymal stem cells

[0123] Mesenchymal stem cells (iMSCs) were cryopreserved using the cryopreservation solution with the formulation shown in Table 2. Specific steps are described in Example 1.

[0124] 3. Resuscitation of mesenchymal stem cells and detection of cell viability and live cell recovery rate at 0 hours and 24 hours: The frozen mesenchymal stem cells were resuscitated and relevant tests were performed. The specific steps are as described in Example 1.

[0125] The results of cell viability and live cell recovery rate of iMSCs at 0 hours after resuscitation showed that... Figure 3 In the study, the cell viability and live cell recovery rate of iMSCs after 24 hours of culture following resuscitation were shown to be... Figure 4 The image shows the cell state of iMSCs after 24 hours of adherent culture following resuscitation. Figure 5 middle.

[0126] like Figure 3 As shown, the cell viability of iMSCs cryopreserved in each group was over 85% at 0 hours after thawing, and the cell recovery rate at 0 hours was also high, all exceeding 70%. Figure 4As shown, the cell viability of iMSCs thawed and cultured for 24 hours after freezing in each group of cell cryopreservation solutions remained above 90%, and the 24-hour viable cell recovery rate was similar to that at 0 hours, reaching approximately 68% or more. This indicates that mesenchymal stem cells cryopreserved using the cryopreservation solution of this invention not only have excellent 0-hour and 24-hour cell viability but also excellent 0-hour and 24-hour viable cell recovery rates, demonstrating high cell proliferation capacity. In particular, when the glucose concentration increased from 0.1% to 0.5%, the 0-hour cell recovery rate significantly increased from approximately 70% to over 80%. Furthermore, when maintaining a glucose concentration of 0.5 w / v% and reducing the CMC-Na concentration from 0.5% to 0.2%, not only was the 0-hour viable cell recovery rate improved, but the 24-hour viable cell recovery rate also significantly increased from approximately 68% to over 80%, indicating that a low concentration of CMC-Na is more advantageous for both 0-hour and 24-hour viable cell recovery rates (especially the latter). Figure 5 As shown, after thawing and adhering to the culture medium, each group of iMSCs in the cryopreservation solution exhibited a considerable number of adherent cells, with group 3 showing a significantly higher number of adherent cells than the other groups. This indicates that the proliferative capacity of cells in each group was effectively maintained after cryopreservation and thawing.

[0127] Example 3

[0128] 1. Preparation of cryopreservation solution for mesenchymal stem cells

[0129] The mesenchymal stem cell cryopreservation solution was prepared according to the formula shown in Table 3, and the preparation method of the cryopreservation solution was the same as in Example 1. Group 1 served as the control group and did not add DMSO.

[0130] Table 3 shows the composition ratios of cryopreservation solutions containing different mass concentrations of dimethyl sulfoxide.

[0131]

[0132] 2. Cryopreservation of mesenchymal stem cells

[0133] Mesenchymal stem cells (iMSCs) were cryopreserved using the cryopreservation solution with the formulation shown in Table 3. Specific steps are described in Example 1.

[0134] 3. Resuscitation of mesenchymal stem cells and detection of cell viability and live cell recovery rate at 0 hours and 24 hours: The frozen mesenchymal stem cells were resuscitated and subjected to relevant tests. The specific steps are as described in Example 1.

[0135] like Figure 6 and 7As shown, except for Group 1 which does not contain DMSO, the cryopreservation solutions containing different concentrations of DMSO in the other groups have little effect on the cell viability and cell recovery rate at 0 hours, which can all reach more than 80%, and also have little effect on the cell viability at 24 hours, which can all reach more than 95%, but have a greater impact on the cell recovery rate at 24 hours. Specifically, as the concentration of DMSO decreases, the cell recovery rate at 24 hours decreases.

[0136] like Figure 8 As shown, after the iMSC cells were thawed and cultured in different concentrations of dimethyl sulfoxide (DMSO) cryopreservation solution, the cells in groups 2-4 adhered to the wall for 24 hours. The results showed that the number of cells in groups 2-4 was relatively large, with groups 3 and 4 showing similar performance. However, the number of cells in group 1, which did not contain DMSO, was very small.

[0137] Example 4

[0138] 1. Preparation of cryopreservation solution for mesenchymal stem cells

[0139] The mesenchymal stem cell cryopreservation solution was prepared according to the formula shown in Table 4. The preparation method of the cryopreservation solution includes the following steps:

[0140] The preparation steps for the sodium carboxymethyl cellulose solution in steps (1)-(5) are the same as in Example 1;

[0141] (6) Based on the volume of the cryopreservation solution, calculate the amount of each component in the cryopreservation solution, add the corresponding amounts of compound electrolyte injection, compound amino acid injection and glucose injection into a 0.22μm filter bottle, filter under vacuum and mix well.

[0142] (7) Take the corresponding volume of dimethyl sulfoxide, add it to the filtered solution, mix thoroughly, and take the corresponding volume of sodium carboxymethyl cellulose solution and mix well.

[0143] (8) Adjust the pH of the cell cryopreservation solution in each group to 7.2–7.4 using 1 mol / L NaOH;

[0144] (9) Store the prepared mesenchymal stem cell cryopreservation solution at 2-8℃.

[0145] Table 4. Partition ratios of cryopreservation solutions containing different mass concentrations of sodium carboxymethyl cellulose

[0146]

[0147] 2. Cryopreservation of mesenchymal stem cells

[0148] Mesenchymal stem cells (iMSCs) were cryopreserved using cryopreservation solution with the formulation shown in Table 4. Specific steps are described in Example 1.

[0149] 3. Resuscitation of mesenchymal stem cells and detection of cell viability and live cell recovery rate at 0 hours and 24 hours: The frozen mesenchymal stem cells were resuscitated and subjected to relevant tests. The specific steps are as described in Example 1.

[0150] The results of cell viability and live cell recovery rate of iMSCs at 0 hours after resuscitation showed that... Figure 9 In the study, the cell viability and live cell recovery rate of iMSCs after 24 hours of culture following resuscitation were shown to be... Figure 10 The image shows the cell state of iMSCs after 24 hours of adherent culture following resuscitation. Figure 11 middle.

[0151] like Figure 9 As shown, the cell viability of iMSCs cryopreserved in each group was over 90% at 0 hours after thawing, and the cell recovery rate at 0 hours was also high, all exceeding 80%. Figure 10 As shown, the cell viability of iMSCs cryopreserved in each group of cryopreservation solutions remained above 95% after 24 hours of inoculation following thawing, and the viable cell recovery rate was above 85%. Specifically, the cell viability and viable cell recovery rate of iMSCs cryopreserved in groups 1 and 2 (CMC-Na concentrations of 0.05% and 0.1%, respectively) at 0 hours after thawing were both above 90%, higher than that of group 3 (CMC-Na concentration of 0.2%). This indicates that lower CMC-Na concentrations are more beneficial for viable cell recovery at 0 hours. In particular, the 24-hour viable cell recovery rate of iMSCs cryopreserved in groups 1 and 2 was above 100%, significantly higher than that of iMSCs cryopreserved in group 3, indicating that lower CMC-Na concentrations are also more beneficial for viable cell recovery at 24 hours. Figure 11 As shown, iMSCs cryopreserved in different concentrations of CMC-Na in each group showed a considerable number of adherent cells after thawing.

[0152] Comparative Example

[0153] 1. Preparation of cryopreservation solution for mesenchymal stem cells

[0154] The mesenchymal stem cell cryopreservation solution was prepared according to the formula shown in Table 5. The preparation method of the cryopreservation solution is the same as in Example 1. The traditional Chinese medicine injection solutions for the cryopreservation solutions of Group 1 and Group 2 are sodium chloride injection and dextran 40 sodium chloride injection, respectively.

[0155] Table 5. Composition ratios of cryopreservation solutions containing different medical injection solutions

[0156]

[0157] 2. Cryopreservation of mesenchymal stem cells

[0158] Mesenchymal stem cells (iMSCs) were cryopreserved using the cryopreservation solution with the formulation shown in Table 5. Specific steps are described in Example 1.

[0159] 3. Resuscitation of mesenchymal stem cells and detection of cell viability and live cell recovery rate at 0 hours and 24 hours: The frozen mesenchymal stem cells were resuscitated and subjected to relevant tests. The specific steps are as described in Example 1.

[0160] Cell viability and viable cell recovery rate of iMSCs thawed in cryopreserved solutions at 0 hours post-thaw were shown in the following figures. Figure 12 In the study, the cell viability and live cell recovery rate of iMSCs after 24 hours of culture following resuscitation were shown to be... Figure 13 The image shows the cell state of iMSCs after 24 hours of adherent culture following resuscitation. Figure 14 middle.

[0161] like Figure 12 As shown, the cell viability of iMSCs cryopreserved in both groups was over 80% at 0 hours after thawing, and the cell recovery rate was over 70%, but lower than the 80.89% 0-hour recovery rate of the compound electrolyte injection solution used under the same conditions (Group 3 in Example 3) (see...). Figure 6 ).like Figure 13 As shown, the cell viability of iMSCs thawed and cultured for 24 hours after freezing in both groups was over 90%, but the 24-hour viable cell recovery rate was only about 30-40%, significantly lower than the 86.17% 24-hour viable cell recovery rate of group 3 in Example 3 when using compound electrolyte injection under the same conditions (see [reference]). Figure 7 ).like Figure 14 As shown, the number of iMSCs that adhered to the culture vessel after thawing and culturing for 24 hours in both groups of cryopreservation solutions was significantly lower than the number of cells that adhered under the same conditions when using compound electrolyte injection solution (Group 3 in Example 3) (see...). Figure 8 ).

[0162] Example 5

[0163] 1. Preparation of cryopreservation solution for mesenchymal stem cells

[0164] The mesenchymal stem cell cryopreservation solution was prepared according to the formula shown in Table 6. The preparation method of the cryopreservation solution is the same as in Example 4.

[0165] Table 6. Distribution ratio of cryopreservation solutions

[0166]

[0167] 2. Cryopreservation of mesenchymal stem cells

[0168] Mesenchymal stem cells (iMSCs) were cryopreserved using the cryopreservation solution with the formulation shown in Table 6. Specific steps are described in Example 1.

[0169] 3. Stability test of iMSC injection:

[0170] The cryopreserved mesenchymal stem cells were thawed, following the specific steps outlined in Example 1. After thawing, the cell suspensions from each group were divided into three portions and placed at room temperature for 0h, 2h, and 4h, respectively. A suitable amount of cell suspension was then collected, and the cells were counted using a Vi-CELLXR cell counter. The cell viability and viable cell density at room temperature for 0h, 2h, and 4h after thawing were recorded. The cell recovery rate at 0 hours for each group was calculated, following the specific detection and calculation steps outlined in Example 1.

[0171] Add an appropriate amount of culture medium to a T25 cell culture flask, and seed each group at room temperature for 0h, 2h, and 4h, respectively, with a total cell count of 1×10⁻⁶. 6 ~2×10 6 The cells were transferred to T25 cell culture flasks and then placed in a carbon dioxide incubator at 37°C, 5% CO2 concentration, and saturated humidity for 24 hours. The iMSC cells of each group were taken after 24 hours of adherent culture, and the cell adhesion status was photographed under a microscope. The live cell recovery rate of each group after 24 hours was also detected.

[0172] The specific stability test conditions for iMSC injection are shown in Table 7 below.

[0173] Table 7. Stability testing conditions for iMSC injection.

[0174]

[0175] The results of iMSC cell viability and viable cell recovery rate at 0h, 2h, and 4h after resuscitation and incubation at room temperature were shown in... Figure 15 In the study, the cell viability and viable cell recovery rate of iMSCs after resuscitation and subsequent inoculation for 24 hours were shown to be... Figure 16 The cell status images of iMSCs after resuscitation (0h, 2h, and 4h) followed by 24 hours of adherence culture are shown in the figure. Figure 17A -C

[0176] like Figure 15 As shown, the cell viability of iMSCs cryopreserved in each group was over 80% at 0h, 2h, and 4h after thawing and being placed at room temperature, and the viable cell recovery rate was also high (divided by 10×10). 6 Except for the case where the cryopreservation density was 1 cell / mL and the cells were stored for 0 hours after thawing (approximately 87%), the recovery rate was over 90%. Moreover, the recovery rate of viable cells after 0 hours actually increased with the extension of storage time. Figure 16As shown, the cell viability of iMSCs cryopreserved in the cryopreservation solutions after 0h, 2h, and 4h at room temperature remained above 95% after 24 hours of inoculation. The viable cell recovery rate after 24 hours was above 145% for all cryopreservation densities after 0h at room temperature. The viable cell recovery rate decreased after 24 hours for all cryopreservation densities after 2h at room temperature, but still remained above 85%, even exceeding 100%. As the inoculation time at room temperature increased to 4h, the viable cell recovery rate decreased slightly, but still remained above 60%, especially for the 10⁻¹⁵ × 10⁻¹⁰ group. 6 Even at a low-density cryopreservation density of cells / mL, the viable cell recovery rate remained above approximately 80% after 24 hours. This indicates that the cryopreservation solution of the present invention provides good cell stability, allowing for extended storage at room temperature and suitability for various clinical applications. Figure 17A and 17B As shown, after cell resuscitation, cells in each group were placed at room temperature for 0 and 2 hours, and the cell adhesion was normal, with no significant differences between groups; Figure 17C As shown, after being placed at room temperature for 4 hours, the number of cells adhering to the wall in groups 3 and 4 with high cryopreservation density was reduced compared to groups 1 and 2 with low cryopreservation density, but a considerable number of cells still remained in a normal adherent state.

[0177] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A mesenchymal stem cell cryopreservation solution directly transfusable into a human body, the mesenchymal stem cell cryopreservation solution consisting of dimethyl sulfoxide having a mass concentration greater than 1.5% and less than 5%, a carboxymethyl cellulose salt having a mass concentration greater than 0% and less than or equal to 0.8%, glucose having a mass concentration greater than 0% and less than 1.0%, and a compound electrolyte injection.

2. The mesenchymal stem cell cryopreserved solution according to claim 1, characterized in that, The carboxymethyl cellulose salt is at least one selected from the group consisting of sodium carboxymethyl cellulose and potassium carboxymethyl cellulose.

3. The mesenchymal stem cell cryopreserved solution according to any one of claims 1-2, characterized in that, The carboxymethyl cellulose salt has a mass concentration greater than 0% and less than or equal to 0.3%.

4. The mesenchymal stem cell cryopreserved solution according to claim 3, characterized in that, The carboxymethyl cellulose salt has a mass concentration greater than 0% and less than or equal to 0.15%.

5. The mesenchymal stem cell cryopreserved solution according to any one of claims 1-2, characterized in that, The glucose has a mass concentration greater than 0.4% and less than 1.0%.

6. A mesenchymal stem cell injection solution comprising the mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 5 and mesenchymal stem cells.

7. A method for cryopreservation of mesenchymal stem cells, comprising the steps of: cryopreserving mesenchymal stem cells using the mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 5.

8. The cryopreservation method of claim 7, wherein, The density of the mesenchymal stem cells in the mesenchymal stem cell freezing solution is 2 x 10 6 ~ 25 x 10 6 cells / mL.

9. The cryopreservation method of claim 8, wherein, The density of the mesenchymal stem cells in the mesenchymal stem cell freezing solution is 10 x 10 6 -15 x 10 6 cells / mL.

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