Hematopoietic stem cell storage method

Abstract

The invention provides a hematopoietic stem cell storage method. According to the technical scheme, a cell cryopreservation system with brand new components is firstly constructed, and a sectional type cryopreservation scheme is designed on the basis of the cell cryopreservation system. Specifically, the fetal calf serum is added to serve as a basic nutrient under the condition that dimethyl sulfoxide is reserved, meanwhile, sodium cromoglycate and chrysin are added to serve as protective components, and the freezing protection effect is achieved through the hydrophilicity of sodium polystyrenesulfonate and glycerol; in addition, the components such as the compound amino acid, the tea polyphenol and the arachidonic acid are added, so that the cell cryopreservation time is prolonged, and the cell activity is kept. In the cryopreservation process, the three-stage pre-cryopreservation process is firstly carried out under the conditions of low concentration and high temperature, and a protective agent is added, so that the damage of the cells in the pre-cryopreservation process is relieved; compared with direct cooling to-80 DEG C, the method has the advantages that the survival rate of the pre-cryopreserved cells is higher, the influence on the cell activity is smaller, and the differentiation capacity of the recovered cells is fully ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and specifically relates to a hematopoietic stem cell storage method. Background Art

[0002] Hematopoietic stem cells are the most primitive hematopoietic cells differentiated from yolk sac totipotent mesenchymal cells. They are the precursors of all blood cells in the body. They are a type of self-renewal cells that can differentiate into various blood cell precursor cells, and eventually generate various blood cells including red blood cells, white blood cells and platelets. They can also differentiate into other cells. Therefore, they are widely used in the treatment of malignant blood diseases, severe aplastic anemia, certain solid tumors, certain abnormal immune diseases, certain genetic diseases, metabolic diseases and extremely severe acute bone marrow radiation sickness. At the same time, hematopoietic stem cell transplantation is also an effective means to restore normal hematopoietic and immune functions for cancer patients.

[0003] Hematopoietic stem cells are widely used in clinical practice. Hematopoietic stem cell cryopreservation is one of the key technologies to ensure the success of hematopoietic stem cell transplantation, so the cryopreservation method is particularly important. At present, clinical hematopoietic stem cell cryopreservation includes -196℃ liquid nitrogen programmed cooling storage and -80℃ non-programmed cooling storage. The former can be stored for a long time with less cell damage, generally 23 to 25 years, but the operation is complicated, the equipment is expensive, and there is cell agglutination after thawing. The latter can preserve stem cells for 1 to 2 years. The operation is relatively simple and the cost is low, but the cell preservation time and cell activity are limited. Exploring a stem cell cryopreservation method that is simple to operate, fast, low-cost and has a longer storage time is of great significance for the storage, application and successful hematopoietic stem cell transplantation of clinical hematopoietic stem cells. In the cryopreservation of hematopoietic stem cells, the cryopreservation agent and method used are very important. The protective agents currently used for -80°C cryopreservation of stem cells include dimethyl sulfoxide (DMSO), CP-1, hydroxyethyl starch, RPMI1640 culture medium, human serum albumin, etc. The formulations and usage methods of different protective agents vary greatly, and the preservation effects and preservation times on stem cells also vary. The survival rate of most preserved cells is only about 80%. Summary of the invention

[0004] The present invention aims to provide a hematopoietic stem cell storage method in view of the technical defects of the prior art, so as to solve the technical problems of the conventional storage method such as the short preservation time and low survival rate of hematopoietic stem cells.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: A method for storing hematopoietic stem cells, comprising: 1) Prepare a cryopreservation solution containing the following ingredients: arachidonic acid 0.04-0.08 mg / mL, tea polyphenols 0.2-0.6 mg / mL, hesperidin 0.02-0.05 mg / mL, sodium cromoglycate 0.02-0.06 mg / mL, monoolein 0.05-0.09 mg / mL, sodium polystyrene sulfonate 6-10 μg / mL, glycerol 0.2-0.5 mg / mL, DMSO 12-18 mg / mL, fetal bovine serum 15-18 mg / mL, citrulline 0.01-0.03 mg / mL, arginine 0.08-0.12 mg / mL, isoleucine 0.04-0.06 mg / mL, chrysin 0.2-0.3 mg / mL; 2) diluting the cryopreservation solution in step 1) to 4-6 times the volume, dispersing the hematopoietic stem cells therein, cooling the solution to 8-12° C. under shaking conditions, maintaining the temperature for 6-8 hours, and then returning the solution to room temperature and recovering the cells; 3) diluting the freezing solution of step 1) to 2-3 times the volume, dispersing the cells recovered in step 2) therein, cooling to 2-6°C under shaking conditions, adding transferrin at a final concentration of 0.02-0.06 mg / mL, L-cysteine ​​at 0.08-0.1 mg / mL, and dimethyl fumarate at 0.01-0.03 mg / mL, maintaining for 2-3 hours, and then cooling to -12--8°C, maintaining for 6-8 hours, and then returning to room temperature and recovering the cells; 4) Add the freezing solution in step 1) into a clean centrifuge tube, disperse the cells recovered in step 3) into the tube, shake to mix the cells and the freezing solution evenly, and then transfer to a -80°C refrigerator for storage.

[0006] Preferably, the freezing solution in step 1) contains the following ingredients: arachidonic acid 0.05 mg / mL, tea polyphenols 0.3 mg / mL, hesperidin 0.03 mg / mL, sodium cromoglycate 0.03 mg / mL, monoolein 0.06 mg / mL, sodium polystyrene sulfonate 7 μg / mL, glycerol 0.3 mg / mL, DMSO 14 mg / mL, fetal bovine serum 16 mg / mL, citrulline 0.02 mg / mL, arginine 0.09 mg / mL, isoleucine 0.05 mg / mL, and chrysin 0.25 mg / mL.

[0007] Preferably, the freezing solution in step 1) contains the following ingredients: arachidonic acid 0.06 mg / mL, tea polyphenols 0.4 mg / mL, hesperetin 0.04 mg / mL, sodium cromoglycate 0.04 mg / mL, monoolein 0.07 mg / mL, sodium polystyrene sulfonate 8 μg / mL, glycerol 0.4 mg / mL, DMSO 15 mg / mL, fetal bovine serum 17 mg / mL, citrulline 0.015 mg / mL, arginine 0.1 mg / mL, isoleucine 0.055 mg / mL, and chrysin 0.28 mg / mL.

[0008] Preferably, the freezing solution in step 1) contains the following ingredients: arachidonic acid 0.07 mg / mL, tea polyphenols 0.5 mg / mL, hesperidin 0.035 mg / mL, sodium cromoglycate 0.05 mg / mL, monoolein 0.05-0.09 mg / mL, sodium polystyrene sulfonate 6-10 μg / mL, glycerol 0.2-0.5 mg / mL, DMSO 17 mg / mL, fetal bovine serum 17.5 mg / mL, citrulline 0.015 mg / mL, arginine 0.11 mg / mL, isoleucine 0.055 mg / mL, and chrysin 0.22 mg / mL.

[0009] Preferably, the freezing solution in step 1) further contains 0.02-0.08 mg / mL of myoglobin.

[0010] Preferably, the freezing solution in step 1) further contains 0.08-0.1 mg / mL of mandelic acid.

[0011] Preferably, the freezing solution in step 1) further contains 0.1-0.15 mg / mL 5-hydroxymethylfurfural.

[0012] Preferably, in step 3), the final concentration of transferrin is 0.04 mg / mL, the final concentration of L-cysteine ​​is 0.09 mg / mL, and the final concentration of dimethyl fumarate is 0.02 mg / mL.

[0013] Preferably, in step 2), the dilution multiple is 5 times the volume, the final temperature of the cooling is 10° C., and the holding time is 7 hours.

[0014] Preferably, the dilution multiple in step 3) is 2.5 times the volume. After the cells are dispersed therein, the temperature is lowered to 4°C under shaking conditions. After transferrin, L-cysteine ​​and dimethyl fumarate are added, the temperature is maintained for 2.5 hours, and then the temperature is lowered to -10°C and maintained for 7 hours.

[0015] The present invention provides a method for storing hematopoietic stem cells. The technical solution first constructs a cell freezing system with a completely new component, and designs a segmented freezing scheme on this basis. Specifically, the present invention adds fetal bovine serum as a basic nutrient while retaining dimethyl sulfoxide, and adds sodium cromoglycate and chrysin as protective components, and utilizes the hydrophilicity of sodium polystyrene sulfonate and glycerol to play a cryoprotective role; in addition, the present invention adds composite amino acids, tea polyphenols, arachidonic acid and other components, which are beneficial to prolong the cell freezing time and maintain cell activity. During the freezing process, the present invention first performs a three-stage pre-freezing process under low concentration and high temperature conditions, and adds a protective agent to alleviate the damage to the cells during the pre-freezing process; compared with directly cooling to -80°C, the survival rate of pre-frozen cells is higher, the effect on cell activity is smaller, and the differentiation ability of cells after recovery is fully guaranteed. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application. In addition, ordinary technicians in this field know that with the development of technology and the emergence of new scenarios, the technical solutions provided in the embodiments of the present application are also applicable to similar technical problems.

[0017] In the description of the present application, it should be understood that, unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as those commonly understood by those skilled in the art of the present application. In addition, any terminology used is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0018] In addition, in order to better illustrate the present application, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0019] Example 1 A method for storing hematopoietic stem cells, comprising: 1) Prepare a cryopreservation solution containing the following ingredients: arachidonic acid 0.06 mg / mL, tea polyphenols 0.4 mg / mL, hesperidin 0.03 mg / mL, sodium cromolyn 0.04 mg / mL, monoolein 0.07 mg / mL, sodium polystyrene sulfonate 8 μg / mL, glycerol 0.4 mg / mL, DMSO 15 mg / mL, fetal bovine serum 16 mg / mL, citrulline 0.02 mg / mL, arginine 0.1 mg / mL, isoleucine 0.05 mg / mL, chrysin 0.25 mg / mL; 2) diluting the cryopreservation solution in step 1) to 5 times the volume, dispersing the hematopoietic stem cells therein, cooling to 10° C. under shaking conditions, maintaining for 7 h, and then returning to room temperature and recovering the cells; 3) diluting the freezing solution of step 1) to 2.5 times the volume, dispersing the cells recovered in step 2) therein, cooling to 4°C under shaking conditions, adding transferrin at a final concentration of 0.04 mg / mL, L-cysteine ​​at 0.09 mg / mL, and dimethyl fumarate at 0.02 mg / mL, maintaining for 2.5 hours, and then cooling to -10°C, maintaining for 7 hours, and then returning to room temperature and recovering the cells; 4) Add the freezing solution in step 1) into a clean centrifuge tube, and then disperse the cells recovered in step 3) into it (the cell concentration does not exceed 10 7 / mL), shake to mix the cells and freezing solution evenly, and then transfer to a -80℃ refrigerator for storage.

[0020] Example 2 A method for storing hematopoietic stem cells, comprising: 1) Prepare a cryopreservation solution containing the following ingredients: arachidonic acid 0.04 mg / mL, tea polyphenols 0.2 mg / mL, hesperidin 0.02 mg / mL, sodium cromolyn 0.02 mg / mL, monoolein 0.05 mg / mL, sodium polystyrene sulfonate 6 μg / mL, glycerol 0.2 mg / mL, DMSO 12 mg / mL, fetal bovine serum 15 mg / mL, citrulline 0.01 mg / mL, arginine 0.08 mg / mL, isoleucine 0.04 mg / mL, chrysin 0.2 mg / mL; 2) diluting the cryopreservation solution in step 1) to 4 times the volume, dispersing the hematopoietic stem cells therein, cooling the solution to 8° C. under shaking conditions, maintaining the temperature for 6 h, and then returning the solution to room temperature and recovering the cells; 3) diluting the freezing solution of step 1) to 2 times the volume, dispersing the cells recovered in step 2) therein, cooling to 2°C under shaking conditions, adding transferrin at a final concentration of 0.02 mg / mL, L-cysteine ​​at 0.08 mg / mL, and dimethyl fumarate at 0.01 mg / mL, and maintaining for 2 hours, then cooling to -12°C, maintaining for 6 hours, and then returning to room temperature and recovering the cells; 4) Add the freezing solution in step 1) into a clean centrifuge tube, and then disperse the cells recovered in step 3) into it (the cell concentration does not exceed 10 7 / mL), shake to mix the cells and freezing solution evenly, and then transfer to a -80℃ refrigerator for storage.

[0021] Example 3 A method for storing hematopoietic stem cells, comprising: 1) Prepare a cryopreservation solution containing the following ingredients: arachidonic acid 0.08 mg / mL, tea polyphenols 0.6 mg / mL, hesperidin 0.05 mg / mL, sodium cromolyn 0.06 mg / mL, monoolein 0.09 mg / mL, sodium polystyrene sulfonate 10 μg / mL, glycerol 0.5 mg / mL, DMSO 18 mg / mL, fetal bovine serum 18 mg / mL, citrulline 0.03 mg / mL, arginine 0.12 mg / mL, isoleucine 0.06 mg / mL, chrysin 0.3 mg / mL; 2) diluting the cryopreservation solution in step 1) to 6 times the volume, dispersing the hematopoietic stem cells therein, cooling the solution to 12° C. under shaking conditions, maintaining the temperature for 8 h, and then returning the solution to room temperature and recovering the cells; 3) diluting the cryopreservation solution of step 1) to 3 times the volume, dispersing the cells recovered in step 2) therein, cooling to 6°C under shaking conditions, adding transferrin at a final concentration of 0.06 mg / mL, L-cysteine ​​at 0.1 mg / mL, and dimethyl fumarate at 0.03 mg / mL, and maintaining for 3 hours, then cooling to -8°C, maintaining for 8 hours, and then returning to room temperature and recovering the cells; 4) Add the freezing solution in step 1) into a clean centrifuge tube, and then disperse the cells recovered in step 3) into it (the cell concentration does not exceed 10 7 / mL), shake to mix the cells and freezing solution evenly, and then transfer to a -80℃ refrigerator for storage.

[0022] Example 4 A method for storing hematopoietic stem cells, comprising: 1) Prepare a cryopreservation solution containing the following ingredients: arachidonic acid 0.06 mg / mL, tea polyphenols 0.4 mg / mL, hesperidin 0.03 mg / mL, sodium cromoglycate 0.04 mg / mL, monoolein 0.07 mg / mL, sodium polystyrene sulfonate 8 μg / mL, glycerol 0.4 mg / mL, DMSO 15 mg / mL, fetal bovine serum 16 mg / mL, citrulline 0.02 mg / mL, arginine 0.1 mg / mL, isoleucine 0.05 mg / mL, chrysin 0.25 mg / mL, myoglobin 0.05 mg / mL, mandelic acid 0.09 mg / mL, 5-hydroxymethylfurfural 0.13 mg / mL; 2) diluting the cryopreservation solution in step 1) to 5 times the volume, dispersing the hematopoietic stem cells therein, cooling to 10° C. under shaking conditions, maintaining for 7 h, and then returning to room temperature and recovering the cells; 3) diluting the freezing solution of step 1) to 2.5 times the volume, dispersing the cells recovered in step 2) therein, cooling to 4°C under shaking conditions, adding transferrin at a final concentration of 0.04 mg / mL, L-cysteine ​​at 0.09 mg / mL, and dimethyl fumarate at 0.02 mg / mL, maintaining for 2.5 hours, and then cooling to -10°C, maintaining for 7 hours, and then returning to room temperature and recovering the cells; 4) Add the freezing solution in step 1) into a clean centrifuge tube, and then disperse the cells recovered in step 3) into it (the cell concentration does not exceed 10 7 / mL), shake to mix the cells and freezing solution evenly, and then transfer to a -80℃ refrigerator for storage.

[0023] Comparative Example 1 Mix DMSO, 1640 sterile culture medium, and blood preservation solution I in a volume ratio of 8:3:3.5. Mix them with the hematopoietic stem cells to be stored in a clean centrifuge tube (cell concentration should not exceed 10 7 / mL), shake to mix the cells and freezing solution evenly, and then transfer to a -80℃ refrigerator for storage.

[0024] Comparative Example 2 Add 5% w / v dimethyl sulfoxide, 0.5% w / v low-density lipoprotein, 1% w / v trehalose, 1% w / v lecithin, 1% w / v bFGF, 1% w / v vitamin E, and finally use DMEM medium and fetal bovine serum in a 1:1 ratio to make up to 100 ml. Mix it with the hematopoietic stem cells to be stored in a clean centrifuge tube (the cell concentration should not exceed 10 7 / mL), shake to mix the cells and freezing solution evenly, and then transfer to a -80℃ refrigerator for storage.

[0025] Example 5 This example is used to examine the cryopreservation effects of the above examples and comparative examples on hematopoietic stem cells.

[0026] After freezing for 2 weeks, 6 months, 12 months, 24 months, and 48 months, cells were revived: the cryopreserved tubes were taken out and quickly placed in a 37°C water bath, shaken until the cell suspension was completely thawed, then diluted with 5-fold DMEM solution, centrifuged at 1000r / min for 5 minutes, centrifuged to remove the supernatant, and repeated 3 times to calculate the cell survival rate. The results are shown in Table 1 below: Table 1 Cell survival rate statistics Cell survival rate after 2 weeks Cell survival rate after 6 months Cell survival rate after 12 months Cell survival rate after 24 months Cell survival rate after 48 months Example 1 99.7% 98.9% 98.0% 97.3% 96.5% Example 2 99.8% 99.2% 98.8% 97.9% 96.8% Example 3 99.9% 99.1% 98.7% 98.1% 97.0% Example 4 99.8% 99.5% 98.9% 98.4% 97.9% Comparative Example 1 90.2% 72.5% 45.7% 21.8% 3.2% Comparative Example 2 92.1% 75.9% 52.3% 23.5% 7.0% The hematopoietic stem cells of Examples 1 to 4 were cryopreserved for 48 months and cultured for cell differentiation. The results showed that the cells were able to differentiate normally, with differentiation rates of 98.6%, 98.8%, 98.5%, and 99.3%, respectively. In contrast, the hematopoietic stem cells of Comparative Examples 1 and 2 were cryopreserved for 6 months and cultured for cell differentiation, with differentiation rates of only 65.1% and 69.4%, respectively. The above experimental results show that the storage methods of Examples 1 to 4 have no effect on the cell differentiation ability, and the effect is significantly better than that of Comparative Examples 1 and 2.

[0027] In summary, after reading this detailed disclosure, it will be apparent to those skilled in the art that the aforementioned detailed disclosure may be presented only by way of example and may not be restrictive. Although not explicitly stated herein, it will be appreciated by those skilled in the art that this application is intended to encompass various reasonable changes, improvements and modifications to the embodiments. These changes, improvements and modifications are intended to be proposed by this application and are within the spirit and scope of the exemplary embodiments of this application.

[0028] It should be understood that in the foregoing description of the embodiments of the present application, in order to help understand a feature and to simplify the present application, the present application combines various features into a single embodiment or its description. However, this does not mean that the combination of these features is necessary, and it is entirely possible for those skilled in the art to extract some of the features and understand them as separate embodiments when reading the present application.

[0029] It should be understood that the embodiments disclosed herein are illustrations of the principles of the present application. Other modified embodiments are also within the scope of the present application. The embodiments disclosed herein are merely examples and not limitations, and the embodiments of the present application are not limited to the embodiments precisely described above.

Claims

1. A method for storing hematopoietic stem cells, characterized in that: include: 1) Prepare a cryopreservation solution containing the following ingredients: arachidonic acid 0.04-0.08 mg / mL, tea polyphenols 0.2-0.6 mg / mL, hesperidin 0.02-0.05 mg / mL, sodium cromoglycate 0.02-0.06 mg / mL, monoolein 0.05-0.09 mg / mL, sodium polystyrene sulfonate 6-10 μg / mL, glycerol 0.2-0.5 mg / mL, DMSO 12-18 mg / mL, fetal bovine serum 15-18 mg / mL, citrulline 0.01-0.03 mg / mL, arginine 0.08-0.12 mg / mL, isoleucine 0.04-0.06 mg / mL, chrysin 0.2-0.3 mg / mL; 2) diluting the cryopreservation solution in step 1) to 4-6 times the volume, dispersing the hematopoietic stem cells therein, cooling the solution to 8-12° C. under shaking conditions, maintaining the temperature for 6-8 hours, and then returning the solution to room temperature and recovering the cells; 3) diluting the freezing solution of step 1) to 2-3 times the volume, dispersing the cells recovered in step 2) therein, cooling to 2-6°C under shaking conditions, adding transferrin at a final concentration of 0.02-0.06 mg / mL, L-cysteine ​​at 0.08-0.1 mg / mL, and dimethyl fumarate at 0.01-0.03 mg / mL, maintaining for 2-3 hours, and then cooling to -12--8°C, maintaining for 6-8 hours, and then returning to room temperature and recovering the cells; 4) Add the freezing solution in step 1) into a clean centrifuge tube, disperse the cells recovered in step 3) into the tube, shake to mix the cells and the freezing solution evenly, and then transfer to a -80°C refrigerator for storage.

2. A method for storing hematopoietic stem cells according to claim 1, characterized in that: The freezing solution in step 1) contains the following ingredients: arachidonic acid 0.05 mg / mL, tea polyphenols 0.3 mg / mL, hesperidin 0.03 mg / mL, sodium cromoglycate 0.03 mg / mL, monoolein 0.06 mg / mL, sodium polystyrene sulfonate 7 μg / mL, glycerol 0.3 mg / mL, DMSO 14 mg / mL, fetal bovine serum 16 mg / mL, citrulline 0.02 mg / mL, arginine 0.09 mg / mL, isoleucine 0.05 mg / mL, and chrysin 0.25 mg / mL.

3. A method for storing hematopoietic stem cells according to claim 1, characterized in that: The freezing solution in step 1) contains the following ingredients: arachidonic acid 0.06 mg / mL, tea polyphenols 0.4 mg / mL, hesperidin 0.04 mg / mL, sodium cromoglycate 0.04 mg / mL, monoolein 0.07 mg / mL, sodium polystyrene sulfonate 8 μg / mL, glycerol 0.4 mg / mL, DMSO 15 mg / mL, fetal bovine serum 17 mg / mL, citrulline 0.015 mg / mL, arginine 0.1 mg / mL, isoleucine 0.055 mg / mL, and chrysin 0.28 mg / mL.

4. A method for storing hematopoietic stem cells according to claim 1, characterized in that: The freezing solution in step 1) contains the following ingredients: arachidonic acid 0.07 mg / mL, tea polyphenols 0.5 mg / mL, hesperidin 0.035 mg / mL, sodium cromoglycate 0.05 mg / mL, monoolein 0.05-0.09 mg / mL, sodium polystyrene sulfonate 6-10 μg / mL, glycerol 0.2-0.5 mg / mL, DMSO 17 mg / mL, fetal bovine serum 17.5 mg / mL, citrulline 0.015 mg / mL, arginine 0.11 mg / mL, isoleucine 0.055 mg / mL, and chrysin 0.22 mg / mL.

5. A method for storing hematopoietic stem cells according to claim 1, characterized in that: The freezing solution in step 1) also contains 0.02-0.08 mg / mL myoglobin.

6. A method for storing hematopoietic stem cells according to claim 1, characterized in that: The freezing solution in step 1) further contains 0.08-0.1 mg / mL of mandelic acid.

7. A method for storing hematopoietic stem cells according to claim 1, characterized in that: The freezing solution in step 1) further contains 0.1-0.15 mg / mL of 5-hydroxymethylfurfural.

8. A method for storing hematopoietic stem cells according to claim 1, characterized in that: In step 3), the final concentration of transferrin was 0.04 mg / mL, the final concentration of L-cysteine ​​was 0.09 mg / mL, and the final concentration of dimethyl fumarate was 0.02 mg / mL.

9. A method for storing hematopoietic stem cells according to claim 1, characterized in that: In step 2), the dilution multiple is 5 times the volume, the final temperature of the cooling is 10° C., and the holding time is 7 h.

10. A method for storing hematopoietic stem cells according to claim 1, characterized in that: In step 3), the dilution multiple is 2.5 times the volume. After the cells are dispersed therein, the temperature is lowered to 4°C under shaking conditions. After transferrin, L-cysteine ​​and dimethyl fumarate are added, the temperature is maintained for 2.5 hours, and then the temperature is lowered to -10°C and maintained for 7 hours.