CN117084231A - Optimized formula and preparation and cryopreservation method of a cell cryopreservation solution - Google Patents

Abstract

The invention provides a cell cryopreservation solution, which is characterized in that the raw materials for preparing the cell cryopreservation solution include pharmaceutical grade DMSO, 20% human albumin, and compound electrolyte injection; the content of the pharmaceutical grade DMSO In the cell cryopreservation solution: 0-10%; the cell cryopreservation solution has an optimized formula, and the preparation ratio of the optimized formula is: pharmaceutical grade DMSO: 20% human albumin: compound electrolyte injection It's 8%: 25%: 67%. The cryopreservation solution used in the present invention causes little damage to cells and has little toxic and side effects, is highly safe, and can be directly used for clinical reinfusion and local injection. The cryopreservation density spans a wide range of 2×10 ⁶ -2×10 ⁷ cells/ml. It can be frozen for a long time, and the viability rate after cell recovery reaches more than 95%.

Description

Optimized formula and preparation and cryopreservation method of a cell cryopreservation solution

Technical field

The present invention relates to the technical field of cell biology, and specifically, to an optimized formula and preparation and cryopreservation method of a cell cryopreservation solution.

Background technique

For the vast majority of cells, if cryoprotectants are not added and a suitable cryopreservation rate is not given during the cryopreservation process, almost all cells will die. During the freezing process, adding a cryoprotectant to the cell suspension and maintaining a relatively slow freezing rate can allow the water in the cells to be slowly discharged out of the cells under the osmotic pressure of the cryoprotectant. The cryoprotectant slowly enters the cells. When the concentration of cryoprotectant in the cells reaches the highest level, the cells are almost completely dehydrated and shrunk, thus reaching the optimal cryopreservation state.

Currently commonly used cryopreservation agents usually contain dimethyl sulfoxide (DMSO), fetal bovine serum, cell culture media, etc. At present, the common formula of universal cell cryopreservation solution is prepared with fetal bovine serum and DMSO in a certain proportion. The specific operation is: mix 10% DMSO and 90% fetal calf serum. However, the DMSO content of this universal cryopreservation solution is high, which will increase the risk of clinical use.

Contents of the invention

In view of the defects in the existing technology, in order to solve the technical problems of high cost of cell cryopreservation solution and high risk of clinical use. The object of the present invention is to provide an optimized formula of a cell cryopreservation solution, which includes pharmaceutical grade DMSO, human albumin and compound electrolyte injection.

Further, the human albumin is human albumin with a concentration of 20%.

Further, the contents of the pharmaceutical grade DMSO and the 20% human albumin in the cell cryopreservation solution range from 7.5% to 10% and from 5% to 50%.

Further, the pharmaceutical grade DMSO, the 20% human albumin and the compound electrolyte injection have an optimized ratio, and the optimized ratio is 8:25:67.

Further, the raw materials for preparing the compound electrolyte injection include: 2.63g of sodium chloride, 2.51g of sodium gluconate, 1.84g of sodium acetate, 0.185g of potassium chloride, and 0.15g of magnesium chloride per 500ml.

Further, the freezing density of the cell cryopreservation solution is 2×10 ⁶ -2×10 ⁷ cells/ml.

Further, the cell concentration is 1×10 ⁷ cells/ml.

Further steps include:

Step 1: Add pharmaceutical grade DMSO to the compound electrolyte injection, mix well and let stand to dissipate heat to obtain mixed solution a;

Step 2: Use compound electrolyte to resuspend the cells to a constant volume in the cell solution to obtain mixed solution b;

Step 3: Add 20% human albumin to the mixed solution b to obtain the mixed solution c;

Step 4: Mix the mixed solution a and the mixed solution c thoroughly to obtain the cell cryopreservation solution.

Further, the cryopreservation method of the cell cryopreservation solution includes the following steps:

Prepare cell cryopreservation solution;

The cell cryopreservation solution is sealed in a cryopreservation tube and placed in an isopropyl alcohol cryopreservation box that has been equilibrated at room temperature;

The cryopreservation box is placed in the freezer and transferred into a liquid nitrogen tank after a preset time is met, so as to realize the cryopreservation operation of the cell cryopreservation solution.

Further, the freezing temperature of the refrigerator in which the freezing box is stored is -80 degrees.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a cell cryopreservation solution with an optimized formula and a preparation and cryopreservation method. This technical solution has simple ingredients, extremely low cost, simple operation and preparation, and only uses pharmaceutical grade DMSO, human albumin, and compound electrolytes. Three reagents. And the content of 20% human serum albumin is only 25%, and the content of pharmaceutical grade DMSO is only 8%. The cryopreservation solution used in the present invention causes little damage to cells and has little toxic and side effects, is highly safe, and can be directly used for clinical reinfusion and local injection.

The cryopreservation density that the present invention can achieve is wide, ranging from 2×10 ⁶ to 2×10 ⁷ cells/ml, and can be cryopreserved for a long time; the viability of cells in the cell cryopreservation solution after cryopreservation using the technical solution of the present invention after recovery is Reaching more than 95%. The optimized ratio of the frozen storage solution is compound electrolyte injection (containing 2.63g sodium chloride, 2.51g sodium gluconate, 1.84g sodium acetate, 0.185g potassium chloride, 0.15g magnesium chloride per 500ml): DMSO: 20% Human albumin=67:8:25. When the DMSO content in the cryopreservation solution is less than 10%, the toxic side effects on cells and patients are small. The final concentration of 20% human serum albumin is 25%, which reduces the cost of the cryopreservation solution. It can be directly used for clinical reinfusion and local injection, and can also be used for cell bank and cryopreservation of injection solutions.

Description of the drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

Figure 1 is a flow chart of a cell cryopreservation solution preparation method provided by an embodiment of the present invention;

Figure 2 is a flow chart of a cell cryopreservation solution optimization test provided by an embodiment of the present invention;

Figure 3 is the cell viability (human albumin) after cell recovery provided by the embodiment of the present invention;

Figure 4 is the cell wall adhesion rate (human albumin) of cells after recovery provided by the embodiment of the present invention;

Figure 5 is the cell viability (DMSO) after cell recovery provided by the embodiment of the present invention;

Figure 6 shows the cell adhesion rate (DMSO) after cell recovery provided by the embodiment of the present invention;

Figure 7 is the kynurenine concentration increase multiple (DMSO) after cell recovery provided by the embodiment of the present invention;

Figure 8 shows the cell viability (cells) after resuscitation of cells provided by the embodiment of the present invention;

Figure 9 shows the cell adhesion rate (cells) after cell recovery according to the embodiment of the present invention;

Figure 10 shows the cell viability at various periods after cell recovery provided by the embodiment of the present invention.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

In one embodiment, an optimized formula and preparation and cryopreservation method of a cell cryopreservation solution are provided, wherein the optimized formula of the cell cryopreservation solution is pharmaceutical grade DMSO:human albumin:compound electrolyte injection in a ratio of 8:25: 67. Among them, the concentration of human albumin is 20% of human albumin. The raw materials for preparing the compound electrolyte injection also include: 2.63g of sodium chloride, 2.51g of sodium gluconate, 1.84g of sodium acetate, 0.185g of potassium chloride, and 0.15g of magnesium chloride per 500ml.

According to records in the Pharmacopoeia, pharmaceutical grade DMSO does no harm to the human body when the content in the cell cryopreservation solution is 0-10%. In the optimized formula in this embodiment, pharmaceutical grade DMSO accounts for 8% of the cell freezing solution, which obviously causes less harm and toxic side effects to the human body.

In one embodiment, in order to better apply the optimized formula of the above-mentioned cell cryopreservation solution, we adopted a special preparation method, as shown in Figure 1. Figure 1 shows the preparation of cell cryopreservation solution provided by the embodiment of the present invention. For the method flow chart, refer to Figure 1; in this preparation method, the total volume of the cell cryopreservation solution is set to 1 ml. The specific preparation steps are as follows: add 0.08 ml of pharmaceutical grade DMSO to 0.42 ml of compound electrolyte injection, mix Let it stand to dissipate heat to obtain mixed solution a; use compound electrolyte to resuspend the cells and adjust the volume to 0.25ml to obtain mixed solution b; add 0.25ml of 20% human albumin to the mixed solution b to obtain a mixed solution Liquid c; mix liquid a and liquid c thoroughly to obtain a cell cryopreservation liquid.

In order to verify that the above optimized formula is the optimal formula among different cell cryopreservation solutions, this technical plan designed the following three-stage experiment, as shown in Figure 2. Figure 2 is the cell cryopreservation solution optimization test provided by the embodiment of the present invention. For the flow chart, refer to Figure 2. The main process is divided into three stages. The first stage is to compare the cryopreservation solution formulas; the second stage is to optimize the cryopreservation solution formula; the third stage is to optimize the cell density of the cryopreservation solution. Optimization.

First, in order to select the optimal formula of the cell cryopreservation solution among the cell cryopreservation solutions with various cell concentration ratios, we designed the following experiments. In one embodiment, optionally, umbilical cord mesenchymal stem cells were used. Formula optimization test:

In the first stage, the purpose of comparing various cryopreservation solution formulas is to select the optimal human serum albumin content in the cryopreservation solution formula; Table 1 is the grouping information table of cryopreservation solution formulas in the first stage, which is mainly used to analyze and compare different cryopreservation solution formulas. The storage solution formula corresponds to the cell viability and cell adhesion rate after resuscitation of cryopreserved cells when the human serum albumin content is different.

In one embodiment, the experimental steps can be simply divided into four steps. After the experiment is completed, the data in Table 1 above are obtained. We will use the data in Table 1 to analyze the data after the cryopreserved cells are recovered. The specific steps are as follows:

Experimental step one is to establish cell culture medium.

The pre-cultured and expanded cells were resuspended in physiological saline for injection, pipetted and evenly counted, divided into 10 groups according to number, with 3 cryopreservation tubes in each group (i.e. three replicates), and the cell content in each tube was approximately 1×10 ⁷ ; That is to say, there are 1×10 ⁷ viable cells in each cryopreservation tube in a 15ml centrifuge tube, and then centrifuged at a speed of 1500 rpm for 5 minutes; each cryopreservation tube corresponds to a centrifuge tube, and the number of the cryopreservation tube corresponds to the centrifuge tube. Tubes are numbered to facilitate experiments in distinguishing groups.

Experimental step two is to freeze the cell solution.

After centrifugation, the cell cryopreservation solution is left to settle in the centrifuge tube to allow the solutes to precipitate, and the supernatant in the centrifuge tube is discarded; cell cryopreservation solutions of different concentrations are prepared according to the proportions in Table 1, and 2.7 mL of the corresponding numbered cell cryopreservation solution is taken from each group. Resuspend the cells into the corresponding numbered centrifuge tubes, pipet evenly, and distribute evenly into three cryopreservation tubes. The specification of the cryopreservation tube is 1mL/tube. Write a label on the outside of each cryovial tube corresponding to the number of the centrifuge tube, put it into the isopropyl alcohol cryopreservation box, and place the isopropyl alcohol cryopreservation box containing the cryopreservation tube at temperature for -80℃ refrigerator.

Experimental step three is to resuscitate the cells in the freezing solution.

Place the isopropyl alcohol freezing box in the refrigerator for 24 hours, take it out, and put the freezing box into a liquid nitrogen tank and let it sit. After 3 days, take out the cryopreservation tube from the isopropyl alcohol freezing box and thaw it to recover the cells in the freezing solution.

Experimental step four: Calculate the cell viability and adhesion rate after recovery.

When cell recovery is complete, aspirate the recovered cells from the cryopreservation tube. Pour into a 15 ml centrifuge tube, add 5 ml of physiological saline for injection, resuspend, centrifuge at 1500 rpm for 5 min, and let stand. Discard the supernatant in the centrifuge tube and add 2 ml of physiological saline for injection to resuspend the cells.

All the above experimental steps are completed and the analysis phase begins. According to Table 1 and the specific experimental results, we analyzed and compared different cryopreservation solution formulas. Under the condition of different human albumin contents, the cell viability and adhesion rate corresponding to the recovery of cryopreserved cells were analyzed.

As shown in Figure 3, Figure 3 is the cell viability (human albumin) after cell recovery provided by the embodiment of the present invention. Referring to Figure 3, it can be concluded that the DMSO concentration is 5%-10%, and the human albumin concentration When the concentration was 1%-10% (the concentration of 20% human albumin was 5%-50%), they all showed good cell viability.

In one embodiment, the cell adhesion rate can also be calculated through the above steps. The calculation formula for the cell adhesion rate is: cell adhesion rate = total number of cells collected after 24 hours of adhesion/number of viable cells at the time of inoculation × 100% .

As shown in Figure 4, Figure 4 is the cell wall adhesion rate (human albumin) after cell recovery provided by the embodiment of the present invention. Referring to Figure 4, it can be concluded that 20% human albumin: DMSO = 25%: 10 % mixed solution group had a higher cell wall adhesion rate after recovery than other groups. The cell wall adhesion rate, also known as the cell seeding survival rate, is used to express the viability of cells and the vitality of the cell population.

The second stage of optimization of the cryopreservation solution formula is to select the optimal concentration of pharmaceutical grade DMSO in the cryopreservation solution formula; for the above purpose, we further refine the content of pharmaceutical grade DMSO in the cryopreservation solution, as shown in the table 1 experiment, we found that the high-quality range of pharmaceutical grade DMSO is 5%-10%, and the effect of 10% concentration DMSO is better than 5%. Combining the experimental results and clinical needs, the pharmaceutical grade DMSO concentration is 7.5-10%, 20 The range of % human albumin concentration of 25% was selected as the test range, and the optimal solution content of pharmaceutical grade DMSO and 20% human albumin was obtained through experiments. The content of the 20% human albumin solution was fixed at 25%. The cryopreservation solution formulas with different concentrations of pharmaceutical grade DMSO are shown in Table 2. The experiment was conducted according to the experimental design in Table 2.

Table 2 is the second stage cryopreservation solution formula optimization grouping information table

Prepare cryopreservation solutions of different concentrations according to the proportions in Table 2 above to complete this stage of the experiment. Experimental steps five to eight in this stage are repeated with experimental steps one to four in the first stage. However, due to different experimental subjects, they are described below.

Experimental step 5: Establish cell culture medium.

The pre-cultured and expanded cells were resuspended in physiological saline for injection, pipetted and evenly counted, divided into 5 groups according to number, with 3 cryopreservation tubes in each group (i.e. three replicates), and the cell content in each tube was approximately 1×10 ⁷ ; That is to say, there are 1×10 ⁷ viable cells in each cryopreservation tube in a 15ml centrifuge tube, and then centrifuged at a speed of 1500 rpm for 5 minutes; each cryopreservation tube corresponds to a centrifuge tube, and the number of the cryopreservation tube corresponds to the centrifuge tube. Tubes are numbered to facilitate experiments in distinguishing groups.

Experimental step six: freeze the cell solution.

After centrifugation, the cell cryopreservation solution is left to settle in the centrifuge tube to allow the solutes to precipitate, and the supernatant in the centrifuge tube is discarded; prepare cell cryopreservation solutions of different concentrations according to the proportions in Table 2, and take 2.7 mL of the corresponding numbered cell cryopreservation solution for each group. Resuspend the cells into the corresponding numbered centrifuge tubes, pipet evenly, and distribute evenly into three cryopreservation tubes. The specification of the cryopreservation tube is 1mL/tube. Write a label on the outside of each cryovial tube corresponding to the number of the centrifuge tube, put it into the isopropyl alcohol cryopreservation box, and place the isopropyl alcohol cryopreservation box containing the cryopreservation tube at temperature for -80℃ refrigerator.

Experimental step seven is to resuscitate the cells in the freezing solution.

Place the isopropyl alcohol freezing box in the refrigerator for 24 hours, take it out, and put the freezing box into a liquid nitrogen tank and let it sit. After 3 days, take out the cryopreservation tube from the isopropyl alcohol freezing box and thaw it to recover the cells in the freezing solution.

Experimental step 8: Calculate the cell viability and adhesion rate after recovery.

When cell recovery is complete, aspirate the recovered cells from the cryopreservation tube. Pour into a 15 ml centrifuge tube, add 5 ml of physiological saline for injection, resuspend, centrifuge at 1500 rpm for 5 min, and let stand. Discard the supernatant in the centrifuge tube and add 2 ml of physiological saline for injection to resuspend the cells.

All the above experimental steps are completed and the analysis phase begins. According to Table 2 and the specific experimental results, we analyzed and compared the cell viability of different cryopreservation solution formulas when the human serum albumin content is the same and the pharmaceutical grade DMSO content is different. wall ratio.

As shown in Figure 5, Figure 5 is the cell viability (DMSO) after cell recovery provided by the embodiment of the present invention. Referring to Figure 5, it can be concluded that a mixed solution of 20% human albumin: DMSO = 25%: 7.5% The group had a higher cell viability after recovery than the other groups.

In one embodiment, the cell adhesion rate can also be calculated through the above steps; the cell adhesion rate is also called the cell seeding survival rate, as shown in Figure 6. Figure 6 shows the cell adhesion after cell recovery provided by the embodiment of the present invention. Wall ratio (DMSO), referring to Figure 6, it can be concluded that the group with a mixture of 20% human albumin: DMSO = 25%: 8% has a higher cell seeding survival rate after recovery than other groups.

In one embodiment, the IDO1 immune activity can also be calculated through the above steps; as shown in Figure 7, Figure 7 is the kynurenine concentration increase multiple (DMSO) after cell recovery provided by the embodiment of the present invention, refer to Figure 7 It can be concluded that compared with other groups, the group with 20% human serum albumin and DMSO=25%:8% mixture has no significant difference in IDO1 activity after recovery; this data shows that the cell cryopreservation solution in this example does not It will cause damage to cellular immune activity and is safe and reliable.

The third stage, optimization of cell density, is to optimize and upgrade this technical solution in terms of cell density. Experimental steps 9 to 12 in this stage are repeated as experimental steps 1 to 4 in the first stage, but due to different experimental subjects, they are described below.

In one embodiment, the cryopreservation density of the optimized formula of cell cryopreservation solution is 2×10 ⁶ -2×10 ⁷ cells/ml. In this embodiment, we selected four sets of cell density values, corresponding to four sets of experimental data. To conduct the experiment, the concentration of pharmaceutical grade DMSO in this experiment was all 8%, the concentration of compound electrolyte was all 67%, and the concentration of 20% human serum albumin was all 25%.

Table 3 shows the optimal grouping of cells for cryopreservation density.

Group No Cell density DMSO concentration Compound electrolyte solution 20% human albumin 1 2×10 ⁶ cells/ml 8% 67% 25% 2 5×10 ⁶ cells/ml 8% 67% 25% 3 1×10 ⁷ cells/ml 8% 67% 25% 4 2×10 ⁷ cells/ml 8% 67% 25%

Experimental step 9: Establish cell culture medium;

The pre-cultured and expanded cells were resuspended in physiological saline for injection, pipetted and evenly counted, divided into 4 groups according to number, with 3 cryopreservation tubes in each group (i.e. three replicates), and the cell content in each tube was approximately 1×10 ⁷ ; That is to say, there are 1×10 ⁷ viable cells in each cryopreservation tube in a 15ml centrifuge tube, and then centrifuged at a speed of 1500 rpm for 5 minutes; each cryopreservation tube corresponds to a centrifuge tube, and the number of the cryopreservation tube corresponds to the centrifuge tube. Tubes are numbered to facilitate experiments in distinguishing groups.

Experimental step ten: freeze the cell solution;

After centrifugation, the cell cryopreservation solution is left to settle in the centrifuge tube to allow the solutes to precipitate, and the supernatant in the centrifuge tube is discarded; prepare cell cryopreservation solutions of different concentrations according to the proportions in Table 3, and take 2.7 mL of the corresponding numbered cell cryopreservation solution for each group. Resuspend the cells into the corresponding numbered centrifuge tubes, pipet evenly, and distribute evenly into three cryopreservation tubes. The specification of the cryopreservation tube is 1mL/tube. Write a label on the outside of each cryovial tube corresponding to the number of the centrifuge tube, put it into the isopropyl alcohol cryopreservation box, and place the isopropyl alcohol cryopreservation box containing the cryopreservation tube at temperature For -80℃ refrigerator.

Experimental step 11, resuscitate cells in frozen solution

Place the isopropyl alcohol freezing box in the refrigerator for 24 hours, take it out, and put the freezing box into a liquid nitrogen tank and let it sit. After 3 days, take out the cryopreservation tube from the isopropyl alcohol freezing box and thaw it to recover the cells in the freezing solution.

Experimental step 12: Calculate cell viability and adhesion rate after recovery

When cell recovery is complete, aspirate the recovered cells from the cryopreservation tube. Pour into a 15 ml centrifuge tube, add 5 ml of physiological saline for injection, resuspend, centrifuge at 1500 rpm for 5 min, and let stand. Discard the supernatant in the centrifuge tube and add 2 ml of physiological saline for injection to resuspend the cells.

All the above experimental steps are completed and the analysis phase begins. According to Table 3 and the specific experimental results, we analyzed and compared different cell densities, analyzed and compared the cell viability and cell adhesion rate corresponding to the recovery of cryopreserved cells under the same cryopreservation solution formula with different cell densities.

As shown in Figure 8, Figure 8 shows the cell viability (cells) after resuscitation of the cells provided by the embodiment of the present invention. Referring to Figure 8, it can be concluded that under the conditions of different cell densities in each group, the cell viability after resuscitation is very low. OK, no significant difference.

The cell adhesion rate is shown in Figure 9. Figure 9 is the cell adhesion rate (cells) after cell recovery provided by the embodiment of the present invention. Refer to Figure 9. DMSO: 20% human albumin = 8%: 25% Under concentration conditions, the test results of the adhesion rate of recovered cells at different cryopreservation densities showed that when the cryopreservation density was 1×10 ⁷ cells/ml, the adhesion rate of revived cells was better than that at the cryopreservation density of 2×10 ⁶ cells/ml. 5×10 ⁶ cells/ml and 2×10 ⁷ cells/ml groups. At the same time, the cryopreservation densities of 2×10 ⁶ cells/ml and 5×10 ⁶ cells/ml showed good conditions in terms of cryopreservation recovery rate and recovery adhesion rate, and can be used for cryopreservation of low-density cells.

After determining the optimized formula, we investigated the stability of the cryopreservation solution. The 2ml cryopreservation tube contained 5×10 ⁶ cells/ml, 1ml/tube, 3 tubes per group, at 0 months, 3 months, Cell viability, sterility, mycoplasma examination, bacterial endotoxin, cell morphology, tumorigenicity, cell surface antigen analysis, induction of differentiation ability and karyotype examination were tested at 6 months, 9 months and 12 months, as shown in Figure 10 As shown, Figure 10 shows the cell viability rate in various periods after cell recovery provided by the embodiment of the present invention. Referring to the results of Figure 10, the viability detection results at 0, 3, 6, 9, and 12 months were 93.14 respectively. %, 94.82%, 94.98%, 95.45%, 94.81%;

The sterility test results of the above five groups were all sterile growth; the mycoplasma test results were all negative; the bacterial endotoxin results were all less than 0.3EU/ml; the cell morphology test results were all adherent growth and spindle shape; tumorigenic-soft The results of the agar colony formation test were all no colony formation; the results of the cell surface antigen analysis were all positive rates of CD14, CD19, CD31, CD34, CD45, and HLA-DR ≤ 2%; the positive rates of CD73, CD90, and CD105 were ≥ 95%; The results of the induced differentiation ability test showed differentiation ability; the karyotype test results showed no abnormalities, and the test results were all qualified.

In this embodiment, using the cell cryopreservation solution and cryopreservation method of this embodiment, the cryopreservation solution can be directly used for clinical reinfusion and local injection after thawing and resuscitation.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above. Those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. The embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily without conflict.

Claims (10)

1. An optimized formula of a cell cryopreservation solution is characterized in that the cell cryopreservation solution comprises pharmaceutical grade DMSO, human serum albumin and a compound electrolyte injection.

2. The optimized formulation of cell cryopreservation solution according to claim 1, wherein the human serum albumin is 20% human serum albumin.

3. The optimized formulation of cell cryopreservation solution according to claim 2, wherein the pharmaceutical grade DMSO and 20% human serum albumin are present in the cell cryopreservation solution in the range of 7.5% -10% and 5% -50%.

4. The optimized formulation of cell cryopreservation solution according to claim 2, wherein the pharmaceutical grade DMSO, the 20% human serum albumin, and the compound electrolyte injection solution have an optimized ratio of 8:25:67.

5. The optimized formula of the cell freezing solution according to claim 1, wherein the preparation raw materials of the compound electrolyte injection comprise: every 500ml contains 2.63g of sodium chloride, 2.51g of sodium gluconate, 1.84g of sodium acetate, 0.185g of potassium chloride and 0.15g of magnesium chloride.

6. The optimized formulation of cell cryopreservation solution according to claim 1, wherein the cell cryopreservation solution has a cryopreservation density of 2 x 10 ⁶ -2×10 ⁷ cells/ml。

7. The optimized formulation of cell cryopreservation solution according to claim 1, wherein the cell concentration is 1 x 10 ⁷ cells/ml。

8. The preparation method of the cell freezing solution is characterized by comprising the following steps:

step 1, adding pharmaceutical grade DMSO into a compound electrolyte injection, uniformly mixing, standing and radiating to obtain a mixed solution a;

step 2, re-suspending the cells in the cell solution to a constant volume by using a compound electrolyte to obtain a mixed solution b;

step 3, adding 20% human serum albumin into the mixed solution b to obtain a mixed solution c;

and step 4, fully mixing and uniformly stirring the mixed solution a and the mixed solution c to obtain the cell frozen stock solution.

9. A method for freezing a cell freezing solution according to any one of claims 1 to 6, characterized in that the method for freezing a cell freezing solution comprises the steps of:

preparing cell frozen stock solution;

packaging the cell freezing solution in a freezing tube, and then placing the cell freezing solution in an isopropanol freezing box balanced at room temperature;

and after the freezing box is placed in the refrigerator, the freezing box is transferred into a liquid nitrogen tank after meeting the preset time, so that the freezing operation of the cell freezing liquid is realized.

10. The method for freezing and preserving cell freezing and preserving solution according to claim 9, wherein the freezing and preserving temperature of the freezing and preserving box for storing the refrigerator is-80 ℃.