CN107258766A - A kind of cell freezing method and cells frozen storing liquid - Google Patents

Abstract

The embodiment of the present invention provides a cell cryopreservation method and a cell cryopreservation solution, which relate to the field of biotechnology. Since the cell cryopreservation solution does not contain serum, the recovery rate of the cells can be improved, and the activity and performance of the cells after recovery are high, which can satisfy The need for long-term cryopreservation of a variety of different cells. The cell cryopreservation method specifically includes the following two steps: step 1, the cell cryopreservation solution mixed with cells is loaded into container 1, and then container 1 is loaded into container 2, and a vacuum is formed between container 1 and container 2; step 2, A. Use normal temperature air cooling to reduce the temperature of container 2 from normal temperature to 4°C, and the temperature drop rate is 3‑4°C per minute; B. Use a refrigerator to reduce the temperature of container 2 from 4°C to ‑20°C. The rate of decline is 1-2°C per minute; C. Use a refrigerator to drop the temperature of container 2 from -20°C to -80°C, and the rate of temperature drop is 2-3°C per minute; D. Put container 2 Freeze in a liquid nitrogen tank with a temperature of -196°C.

Description

A kind of cell cryopreservation method and cell cryopreservation solution

technical field

The invention relates to the field of biotechnology, in particular to a cell freezing method and a cell freezing solution.

Background technique

Ultra-low temperature cryopreservation is an effective method for long-term preservation of cells, which mainly plays a protective role by reducing the metabolic rate of cells. Preserving cells in liquid nitrogen (-196°C) is the most common way of long-term cell preservation. The effects of various enzymes that regulate and control cell growth and metabolism are greatly inhibited, making the biochemical reactions inside the cells very slow or even stopped, thus Avoiding changes in cytogenetic traits, cells and tissues do not lose their morphogenetic potential. However, frostbite is the biggest negative reaction of cryoprotection, which is caused by mechanical damage and penetrative damage caused by ice crystals. At present, cell cryopreservation is mainly rapid cryopreservation method, that is, vitrification method. However, if the appropriate cryopreservation method and appropriate cryoprotectant are not adopted, the cells are prone to frostbite during the cryopreservation process. Improving cryopreservation solution and cryopreservation method is an important way to improve the preservation of cells. At present, many studies are aimed at improving cryopreservation solution and cryopreservation method to protect cells from frostbite during cryopreservation.

At present, most of the cryopreserved liquids contain animal-derived serum components, and animal-derived serum has the risk of infecting viruses; due to different animals, different serum origins and batch numbers, the quality of each batch varies greatly, and its components cannot be kept consistent. Affect each batch of test or production results; the high cost of serum increases production costs. Therefore, serum-free cell cryopreservation is the trend of future development, and some units or institutions are researching serum-free cell cryopreservation. The patent application number CN201610322202.7 discloses a serum-free cell cryopreservation solution, which is used to solve the problems of inconvenient use, high cost and low cell survival rate of the cryopreservation solution. The patent application number CN201610124499.6 discloses a serum-free cryopreservation solution for human adipose stem cells and its preparation method.

The existing serum-free cryopreservation solution is thawed after long-term freezing, the cell recovery rate is low, the cell viability and performance decrease after recovery, and it cannot meet the needs of long-term cryopreservation of different cells.

At present, most cryopreserved liquids contain animal-derived serum components, and animal-derived serum has the risk of infecting viruses; due to different animals, serum origins and batch numbers, the quality of each batch varies greatly, and its components cannot be kept consistent, affecting Each batch of test or production results; the cost of product safety inspection in the later stage after serum is contained will be very high, which will increase the production cost. Although vitrification is currently an ideal cryopreservation method, it will still form ice crystals during vitrification to damage cells, and after recovery, the cell viability is low, the number of cell proliferation is insufficient, and cells are prone to aging.

Contents of the invention

The embodiment of the present invention provides a cell cryopreservation method and a cell cryopreservation solution. Since the cell cryopreservation solution does not contain serum, the recovery rate of the cells can be improved, the activity and performance of the cells after recovery are high, and it can meet the requirements of various cell types. The need for long-term freezing.

In order to achieve the above object, the embodiment of the present invention adopts the following technical solutions:

The embodiment of the present invention provides a cell cryopreservation method, the cell cryopreservation method includes the following two steps:

Step 1, filling the cell cryopreservation solution mixed with cells into container 1, and then loading container 1 into container 2, wherein a vacuum is formed between container 1 and container 2;

Step 2, perform the following four steps A-D on the container 2 obtained in step 1:

A. The temperature of the container 2 is dropped from normal temperature to 4°C by air cooling at normal temperature, wherein the temperature drop rate is 3-4°C per minute;

B. Use a refrigerator to lower the temperature of the container 2 from 4°C to -20°C, wherein the rate of temperature drop is 1-2°C per minute;

C. Use a refrigerator to lower the temperature of the container 2 from -20°C to -80°C, wherein the rate of temperature drop is 2-3°C per minute;

D. Put container 2 into a liquid nitrogen tank for freezing storage, wherein the temperature of the liquid nitrogen tank is -196°C.

Further, Step 2 specifically includes: performing the following four steps A-D on the container 2 obtained in Step 1:

A. The temperature of container 2 is lowered from normal temperature to 4°C by air cooling at normal temperature, wherein the temperature drop rate is 4°C per minute;

B. Use a refrigerator to lower the temperature of the container 2 from 4°C to -20°C, wherein the rate of temperature drop is 1°C per minute;

C. Use a refrigerator to lower the temperature of the container 2 from -20°C to -80°C, wherein the rate of temperature drop is 2°C per minute;

D. Put container 2 into a liquid nitrogen tank for freezing storage, wherein the temperature of the liquid nitrogen tank is -196°C.

Further, the cells are epidermal stem cells, fibroblasts, corneal cells, adipose stem cells, melanocytes, oral mucosal epithelial cells or oral gingival cells.

Further, the cells are cells of the 2nd to 7th passages.

The embodiment of the present invention also provides a cell cryopreservation solution, which is applied to the cell cryopreservation method according to any one of claims 1-4, and the cell cryopreservation solution includes: DMEM/F12, DMSO, glycerol and serum substitution;

Among them, the content of DMSO in the cell freezing solution is 2-10% of the volume of DMEM/F12; the content of glycerol in the cell freezing solution is 5-30% of the volume of DMEM/F12; The content in the stock solution is 20-60% of the volume of DMEM/F12;

Serum substitutes include hydroxyethyl starch, dextran, human albumin, recombinant human transferrin, human AB serum, epidermal growth factor, recombinant human insulin, progesterone, hydrocortisone, putrescine, sodium selenate and flavonoids.

Further, the concentration of hydroxyethyl starch is 0.1-10%W/V, the concentration of dextran is 0.1-10%W/V, the concentration of human serum albumin is 0.1-15%W/V, recombinant human The concentration of ferritin is 1-10%W/V, the concentration of human AB serum is 1-10%W/V, the concentration of epidermal growth factor is 0.01-10%W/V, and the concentration of recombinant human insulin is 1-10% %W/V, the concentration of progesterone is 0.01-5%W/V, the concentration of hydrocortisone is 0.01-5%W/V, the concentration of putrescine is 0.01-5%W/V, the concentration of sodium selenate The concentration is 0.1-5%W/V, and the concentration of flavonoids is 0.01-5%W/V.

Further, the content of glycerol in the cell cryopreservation solution is 10-25% of the volume of DMEM/F12. Preferably, the content of glycerol in the cell cryopreservation solution is 15-20% of the volume of DMEM/F12.

Further, the content of the serum substitute in the cell cryopreservation solution is 30-55% of the volume of DMEM/F12. Preferably, the content of the serum substitute in the cell cryopreservation solution is 35-50% of the volume of DMEM/F12.

Preferably, the content of DMSO in the cell cryopreservation solution is 5-7% of the volume of DMEM/F12.

Further, DMEM/F12 is a mixture of DMEM culture solution and F12 culture solution at a volume ratio of 3:1.

The embodiment of the present invention provides a cell cryopreservation method, the cell cryopreservation method includes the following two steps: step 1, put the cell cryopreservation solution mixed with the cells into the container 1, and then put the container 1 into the container 2, wherein the container Form a vacuum between 1 and container 2; step 2, perform the following four steps A-D on the container 2 obtained in step 1: A, use normal temperature air cooling to reduce the temperature of container 2 from normal temperature to 4 ° C, wherein the temperature drops The rate of decrease is 3-4°C per minute; B. Use the refrigerator to lower the temperature of container 2 from 4°C to -20°C, wherein the temperature drop rate is 1-2°C per minute; C. Use the refrigerator to lower the temperature of the container 2 from 4°C to -20°C. The temperature of 2 drops from -20°C to -80°C, wherein the rate of temperature drop is 2-3°C per minute; D. Put container 2 into a liquid nitrogen tank for freezing storage, wherein the temperature of the liquid nitrogen tank is -196°C. Based on the description of the above examples, since the cell cryopreservation solution does not contain serum, there is no risk of zoonotic virus infection, and it has a wide range of applications. After the long-term frozen cells of the present invention are thawed, the recovery rate of the cells is as high as over 90%, and the recovered cells have high activity and are not easy to age, which can meet the needs of long-term cryopreservation of various different cells.

Description of drawings

Figure 1 is a comparison diagram of cell morphology before and after recovery of cells frozen for 6 months in the cell freezing method provided by the embodiment of the present invention and cells frozen in the existing method for 6 months;

Figure 2 is a comparison chart of the recovery rate of cells frozen for 6 months in the cell freezing method provided by the embodiment of the present invention and cells frozen in 6 months using the existing method;

Fig. 3 is a comparison diagram of cell morphology before and after recovery of cells frozen in the cell freezing solution provided by the embodiment of the present invention for 6 months and cells frozen in the existing solution for 6 months;

Fig. 4 is a comparison chart of the recovery rate of cells frozen in the cell freezing solution provided by the embodiment of the present invention for 6 months and cells frozen in the existing solution for 6 months.

detailed description

The technical solutions in the embodiments of the present invention will be described in detail below with reference to the drawings in the embodiments of the present invention.

Example 1

An embodiment of the present invention provides a cell cryopreservation method, the cell cryopreservation method includes the following two steps:

Step 1, filling the cell cryopreservation solution mixed with the cells into the container 1, and then loading the container 1 into the container 2, wherein a vacuum is formed between the container 1 and the container 2;

Step 2, perform the following four steps of A-D on the container 2 obtained in step 1:

A. The temperature of the container 2 is dropped from normal temperature to 4°C by air cooling at normal temperature, wherein the temperature drop rate is 3-4°C per minute;

B. Use a refrigerator to lower the temperature of the container 2 from 4°C to -20°C, wherein the rate of temperature drop is 1-2°C per minute;

C. Use a refrigerator to drop the temperature of the container 2 from -20°C to -80°C, wherein the temperature drop rate is 2-3°C per minute;

D. Put the container 2 into a liquid nitrogen tank for freezing storage, wherein the temperature of the liquid nitrogen tank is -196°C.

In a possible implementation, the primary cells of epidermal stem cells, fibroblasts, adipose stem cells, melanocytes, and oral mucosal epithelial cells are mixed into the cryopreservation solution, put into 1ml centrifuge tubes and sealed; the cryopreservation solution is prepared by Composition of DMEM/F12, DMSO, glycerin and serum substitute consisting of hydroxyethyl starch, dextran, human serum albumin, recombinant human transferrin, human AB serum, epidermal growth factor, recombinant human insulin , progesterone, hydrocortisone, putrescine, sodium selenate, flavonoids, dexamethasone.

Then put the 1ml centrifuge tube containing the primary cells into a 15ml centrifuge tube, seal it tightly, and cool it to 4°C with cold air at a cooling rate of 3°C;

Put the 15ml centrifuge tube at 4°C in the refrigerator for 180 minutes, and cool down to -20°C at a cooling rate of 1°C;

Then quickly put the 15ml centrifuge tube at -20°C into a -80°C refrigerator for 8 hours;

Finally, it was directly transferred to liquid nitrogen for freezing at -196°C.

In another possible implementation, the primary cells of epidermal stem cells, fibroblasts, adipose stem cells, melanocytes, and oral mucosal epithelial cells are mixed into the cryopreservation solution, filled into 1ml centrifuge tubes and sealed; the cryopreservation solution Consists of DMEM/F12, DMSO, glycerin and serum substitute consisting of hydroxyethyl starch, dextran, human albumin, recombinant human transferrin, human AB serum, epidermal growth factor, recombinant human Insulin, progesterone, hydrocortisone, putrescine, sodium selenate, flavonoids, dexamethasone.

Then put the 1ml centrifuge tube containing the primary cells into a 15ml centrifuge tube, seal it tightly, and cool it to 4°C with cold air at a cooling rate of 4°C;

Put the 15ml centrifuge tube at 4°C in the refrigerator for 90 minutes, and cool down to -20°C at a cooling rate of 2°C;

Then quickly put the 15ml centrifuge tube at -20°C into the -80°C refrigerator for 12 small tests;

Finally, it was directly transferred to liquid nitrogen for freezing at -196°C.

It should be noted that the cells cryopreserved for 6 months by the cryopreservation method provided in the examples of the present invention were directly placed in water at 40°C for recovery, and the morphology of the cells before and after recovery was observed. Compared with the cells frozen for 6 months using the existing method, it can be seen from Figure 1 that the cells before freezing are very similar to the cells after 6 months of freezing using the freezing method of the present invention, and there is almost no changes, indicating that the cryopreservation method of the present invention can protect cells well during the cryopreservation process.

At the same time, the cells frozen for 6 months by the cryopreservation method provided in the embodiment of the present invention and the existing conventional cryopreservation method were directly placed in water at 42°C for 3 minutes, and the recovery rate of the cells after recovery was observed. For specific experimental results, see figure 2. It is found from Fig. 2 that the cell recovery rate of the cryopreservation method of the present invention is significantly better than that of the existing cryopreservation method.

The embodiment of the present invention provides a cell cryopreservation method, the cell cryopreservation method includes the following two steps: step 1, put the cell cryopreservation solution mixed with the cells into the container 1, and then put the container 1 into the container 2, wherein the container Form a vacuum between 1 and container 2; step 2, perform the following four steps A-D on the container 2 obtained in step 1: A, use normal temperature air cooling to reduce the temperature of container 2 from normal temperature to 4 ° C, wherein the temperature drops The rate of decrease is 3-4°C per minute; B. Use the refrigerator to lower the temperature of container 2 from 4°C to -20°C, wherein the temperature drop rate is 1-2°C per minute; C. Use the refrigerator to lower the temperature of the container 2 from 4°C to -20°C. The temperature of 2 drops from -20°C to -80°C, wherein the rate of temperature drop is 2-3°C per minute; D. Put container 2 into a liquid nitrogen tank for freezing storage, wherein the temperature of the liquid nitrogen tank is -196°C. Based on the description of the above examples, since the cell cryopreservation solution does not contain serum, there is no risk of zoonotic virus infection, and it has a wide range of applications. After the long-term frozen cells of the present invention are thawed, the recovery rate of the cells is as high as over 90%, and the recovered cells have high activity and are not easy to age, which can meet the needs of long-term cryopreservation of various different cells.

Example 2

An embodiment of the present invention provides a cell cryopreservation solution, which is applied to the cell cryopreservation method described in any one of Example 1, and the cell cryopreservation solution includes: DMEM/F12, DMSO, Glycerin and serum substitutes;

Wherein, the content of the DMSO in the cell cryopreservation solution is 2-10% of the volume of the DMEM/F12; the content of the glycerol in the cell cryopreservation solution is 5-30% of the volume of the DMEM/F12 The volume of DMEM/F12; the content of the serum substitute in the cell freezing solution is 20-60% of the volume of the DMEM/F12;

The serum substitutes include hydroxyethyl starch, dextran, human serum albumin, recombinant human transferrin, human AB serum, epidermal growth factor, recombinant human insulin, progesterone, hydrocortisone, putrescine, selenium sodium and flavonoids.

Further, the concentration of the hydroxyethyl starch is 0.1-10%W/V, the concentration of the dextran is 0.1-10%W/V, and the concentration of the human serum albumin is 0.1-15%W /V, the concentration of the recombinant human transferrin is 1-10%W/V, the concentration of the human AB serum is 1-10%W/V, the concentration of the epidermal growth factor is 0.01-10%W /V, the concentration of the recombinant human insulin is 1-10%W/V, the concentration of the progesterone is 0.01-5%W/V, and the concentration of the hydrocortisone is 0.01-5%W/V , the concentration of putrescine is 0.01-5%W/V, the concentration of sodium selenate is 0.1-5%W/V, and the concentration of flavone is 0.01-5%W/V.

In a possible implementation, the cell cryopreservation solution provided by the embodiment of the present invention is configured according to the following proportions: 10% DMEM/F12 volume of DMSO, 30% DMEM/F12 volume glycerin, 50% DMEM/F12 volume F12 volume serum substitute and DMEM/F12 with a volume ratio of 10%, wherein the composition ratio of the serum substitute is as follows:

10% (W/V) hydroxyethyl starch, 0.1% (W/V) dextran, 15% (W/V) human albumin, 5% (W/V) recombinant human transferrin, 10% (W/V) human AB serum, 0.01% (W/V) epidermal growth factor, 10% (W/V) recombinant human insulin, 5% (W/V) progesterone, 5% (W/V) hydrocortisone Composition of pine, 0.01% (W/V) putrescine, 5% (W/V) sodium selenate, 0.01% (W/V) flavonoids.

After mixing the same amount of epidermal stem cells, fibroblasts, adipose stem cells, and melanocytes into the cryopreservation solution prepared above, after freezing in liquid nitrogen for 6 months, they were all recovered in water at 42°C for 2 minutes, and the cells before and after freezing were observed. The form is shown in Figure 3. As can be seen from Figure 3, with the cryopreservation solution of the present invention, the cells before cryopreservation are very similar to the cell morphology after 6 months of cryopreservation, and there is almost no change, which shows that the cryopreservation solution of the present invention has played a role in the cryopreservation of cells. Protects well.

In another possible implementation, the cell cryopreservation solution provided by the embodiment of the present invention is configured according to the following ratio:

DMSO at 2% volume of DMEM/F12, glycerin at 30% volume of DMEM/F12, serum substitute at 20% volume of DMEM/F12, and DMEM/F12 at a volume ratio of 48%; the composition ratio of the serum substitute is as follows:

0.1% (W/V) hydroxyethyl starch, 10% (W/V) dextran, 10% (W/V) human albumin, 1% (W/V) recombinant human transferrin, 10% (W/V) human AB serum, 0.5% (W/V) epidermal growth factor, 10% (W/V) recombinant human insulin, 0.01% (W/V) progesterone, 0.01% (W/V) hydrocortisone Composition of pine, 0.5% (W/V) putrescine, 0.01% (W/V) sodium selenate, 5% (W/V) flavonoids.

After mixing epidermal stem cells, fibroblasts, adipose stem cells, melanocytes, keratocytes, and oral gingival cells into the cryopreservation solution prepared above and a commercial serum-free cryopreservation solution, freeze them in liquid nitrogen for 6 months, and use the same conventional recovery method After recovering at 40°C for 2 minutes, the results of cell recovery rate are shown in Figure 4.

What needs to be added is that after mixing epidermal stem cells, fibroblasts, adipose stem cells, and melanocytes into the above-prepared cryopreservation solution and commercial serum-free cryopreservation solution, they were frozen in liquid nitrogen for 6 months and revived with the same recovery method. Subculture was carried out, and the aging generation of the cells was observed. The specific results are shown in Table 1.

Table 1

this invention commercial serum-free freezing solution epidermal stem cells P15 P9 Fibroblasts P21 P10 fat stem cells P15 P12 Melanocytes P18 P9 Oral mucosa model P24 P20

From Table 1, it can be seen that the aging generation of cells frozen in the cryopreservation solution provided by the present invention is significantly greater than that of the commercial serum-free cryopreservation solution after resuscitation, indicating that the cells frozen in the cryopreservation solution provided by the present invention The activity or performance is better than that of commercial serum-free cryopreservation solution.

What needs to be added is that after 6 months of cryopreservation provided by the existing conventional cryopreservation and the example of the present invention, the cells are resuscitated by the resuscitation method mentioned in the example of the present invention, and then continuously used commercial serum-free DMEM/F-12 Subculture, change the liquid every day, and observe the days when the cells begin to age. The specific experimental results are shown in the table.

Table 2

this invention Existing cryopreservation methods epidermal stem cells day 10 day 6 Fibroblasts day 12 day 7 fat stem cells day 18 day 10 Melanocytes day 14 Day 9 Oral mucosa model day 24 day 21

From Table 2, it can be seen that the number of days for cells frozen by the cryopreservation method provided by the present invention to start aging after resuscitation is significantly later than that of the existing cryopreservation methods, which means that the cells frozen by the cryopreservation method provided by the present invention Cells with activity or performance superior to existing cryopreservation methods.

The above is only a specific implementation of the application, but the protection scope of the application is not limited thereto, and any changes or replacements within the technical scope disclosed in the application should be covered within the protection scope of the application . Therefore, the protection scope of the present application should be determined by the protection scope of the claims.

Claims (9)

1. a kind of cell freezing method, it is characterised in that the cell freezing method includes following two steps：

Step 1: the cells frozen storing liquid for being mixed into cell is loaded into container 1, then the container 1 is loaded into container 2, wherein, it is described to hold Vacuum is formed between device 1 and the container 2；

Step 2: performing following tetra- steps of A-D according to this to the container 2 obtained by step one：

A, using the air-cooled temperature for making the container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is every point Clock declines 3-4 DEG C；

B, using refrigerator the temperature of the container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop is under per minute 1-2 DEG C of drop；

C, using refrigerator the temperature of the container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop is per minute Decline 2-3 DEG C；

D, the container 2 is put into liquid nitrogen container frozen, wherein, the temperature of the liquid nitrogen container is -196 DEG C.

2. cell freezing method according to claim 1, it is characterised in that the step 2 is specifically included：To step one The container 2 of gained performs following tetra- steps of A-D according to this：

A, using the air-cooled temperature for making the container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is every point Clock declines 4 DEG C；

B, using refrigerator the temperature of the container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop is under per minute 1 DEG C of drop；

C, using refrigerator the temperature of the container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop is per minute Decline 2 DEG C；

D, the container 2 is put into liquid nitrogen container frozen, wherein, the temperature of the liquid nitrogen container is -196 DEG C.

3. cell freezing method according to claim 1 or 2, it is characterised in that the cell is epidermal stem cells, into fibre Tie up cell, keratocyte, fat stem cell, melanocyte, Buccal mucosa cell or oral cavity gingival cell.

4. cell freezing method according to claim 3, it is characterised in that the cell is the cell in the 2nd ~ 7 generation.

5. a kind of cells frozen storing liquid, it is characterised in that the cells frozen storing liquid is applied to such as any one institute in claim 1-4 The cell freezing method stated, the cells frozen storing liquid includes：DMEM/F12, DMSO, glycerine and serum substitute；

Wherein, contents of the DMSO in the cells frozen storing liquid is 2-10% DMEM/F12 volume；The glycerine The volume for the DMEM/F12 that content in the cells frozen storing liquid is 5-30%；The serum substitute is in the cell The volume for the DMEM/F12 that content in frozen stock solution is 20-60%；

The serum substitute includes HES, glucan, human serum albumin, restructuring human transferrin, people AB serum, table Skin growth factor, rh-insulin, progesterone, hydrocortisone, putrescine, sodium selenate and flavones.

6. cells frozen storing liquid according to claim 5, it is characterised in that the concentration of the HES is 0.1-10% W/V, the concentration of the glucan is 0.1-10%W/V, and the concentration of the human serum albumin is 0.1-15%W/V, the recombined human The concentration of transferrins is 1-10%W/V, and the concentration of the people AB serum is 1-10%W/V, the concentration of the EGF For 0.01-10%W/V, the concentration of the rh-insulin is 1-10%W/V, and the concentration of the progesterone is 0.01-5%W/V, institute The concentration for stating hydrocortisone is 0.01-5%W/V, and the concentration of the putrescine is 0.01-5%W/V, and the concentration of the sodium selenate is 0.1-5%W/V, the concentration of the flavones is 0.01-5%W/V.

7. the cells frozen storing liquid according to claim 5 or 6, it is characterised in that the glycerine is in the cells frozen storing liquid Content for 10-25% the DMEM/F12 volume.

8. the cells frozen storing liquid according to claim 5 or 6, it is characterised in that the serum substitute freezes in the cell The volume for the DMEM/F12 that content in liquid storage is 30-40%.

9. the cells frozen storing liquid according to claim 5 or 6, it is characterised in that the DMEM/F12 be DMEM nutrient solutions and F12 nutrient solutions are with volume ratio 3:1 ratio mixing.