Human umbilical cord mesenchymal stem cell injection and preparation method thereof

Abstract

The invention relates to the technical field of stem cell injections, in particular to a human umbilical cord mesenchymal stem cell injection and a preparation method thereof. The cell cryoprotectant comprises the following components: a cell cryoprotectant and P6-generation human umbilical cord mesenchymal stem cells. By selecting P6-generation human umbilical cord mesenchymal stem cells, the capacity of secreting hepatocyte growth factors (HGF) can be improved; the cell cryoprotectant contains 6-9wt% of dimethyl sulfoxide and a certain amount of human serum albumin, can better protect cells and maintain the motility rate and biological functions of the cells, and is more beneficial to preparation of cryopreserved human umbilical cord mesenchymal stem cell injection. The P6-generation human umbilical cord mesenchymal stem cells are obtained by culturing a culture medium containing fetal calf serum, and the ability of the injection to repair liver injury can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell injection solutions, and specifically, to a human umbilical cord mesenchymal stem cell injection solution and a preparation method thereof. Background Art

[0002] In the field of modern medicine, stem cells have attracted much attention due to their potential therapeutic potential. In particular, human umbilical cord mesenchymal stem cells (hUC-MSCs), as a cell population with high self-renewal ability and multi-directional differentiation potential, have become a research hotspot for treating liver-related diseases such as liver injury, liver cirrhosis, and liver failure. Research results in recent years have shown that umbilical cord mesenchymal stem cells can effectively promote the restoration of the balance between extracellular matrix degradation and generation in the liver, and reverse the development of liver fibrosis through paracrine cytokines, especially hepatic growth factor (HGF), by inhibiting the activation of hepatic stellate cells, degrading excessive extracellular matrix in the liver, promoting hepatocyte regeneration, and immunomodulation. Some clinical research results have also found that it can improve liver function, reduce the deposition of collagen fibers in the liver matrix, and promote hepatocyte regeneration, etc., and can effectively treat end-stage liver diseases such as liver cirrhosis and acute and chronic liver failure. With the continuous progress of stem cell technology, significant progress has also been made in the process research of human umbilical cord mesenchymal stem cell injection solutions and their preparation methods. Currently, cell injection solutions mainly exist in two forms: fresh preparations and cryopreserved preparations. Compared with the former, cryopreserved preparations are more conducive to long-distance transportation and long-term storage, and are suitable for diseases that require multiple treatments. China is a large country with a high incidence of liver diseases. Human umbilical cord mesenchymal stem cells show good clinical application prospects in the treatment of liver diseases due to their clear mechanism of action, good clinical safety, and efficacy.

[0003] The human umbilical cord mesenchymal stem cell injection solution developed by Tianjin Heze Stem Cell Technology Co., Ltd. has become the world's first stem cell product derived from human umbilical cord for the treatment of liver cirrhosis and liver failure. However, the cell injection solution of this company is a fresh preparation, which is not conducive to long-term storage. Summary of the Invention

[0004] In the first aspect of the present invention, a human umbilical cord mesenchymal stem cell injection solution is provided, and its components include: a cell cryoprotectant solution and P6-generation human umbilical cord mesenchymal stem cells.

[0005] The present invention has found through research that by selecting P6-generation human umbilical cord mesenchymal stem cells, the ability to secrete hepatocyte growth factor (HGF) can be improved. The biological characteristics of this generation of cells, such as proliferation ability, differentiation ability, and factor secretion ability, are relatively stable.

[0006] The cell cryoprotectant solution contains 6-9 wt% of dimethyl sulfoxide.

[0007] Optionally, the cell cryoprotectant contains 7-8 wt% dimethyl sulfoxide.

[0008] Optionally, the cell cryoprotectant contains dextran and human serum albumin.

[0009] The P6 passage of human umbilical cord mesenchymal stem cells is obtained by culturing in a medium containing fetal bovine serum.

[0010] The raw materials for producing the umbilical cord mesenchymal stem cell injection include normal saline, recombinant trypsin, human albumin, and compound electrolyte injection.

[0011] The second aspect of the present invention provides a method for preparing a human umbilical cord mesenchymal stem cell injection, comprising the following steps:

[0012] S1, washing the P6 passage of human umbilical cord mesenchymal stem cells with normal saline and adding recombinant trypsin;

[0013] S2, observing the state and confluence of the P6 passage of human umbilical cord mesenchymal stem cells under a microscope, and when it is observed that the cells become round and shed in flakes, adding human albumin to terminate digestion;

[0014] S3, adding compound electrolyte injection to the P6 passage of human umbilical cord mesenchymal stem cells to obtain a cell suspension, and performing cell counting;

[0015] S4, pre-cooling the cell suspension and then adding a cell cryoprotectant to obtain a cell injection, and after gradient cooling, performing cryopreservation.

[0016] The cell number of the cell injection is 0.5×10 7 ~5×10 7 cells / 20 mL.

[0017] Optionally, the cell number of the cell injection is 1×10 7 ~3×10 7 cells / 20 mL.

[0018] The gradient cooling includes: at a rate of 0.1-0.5 °C / min to -50 to -60 °C, and then at a rate of 0.8-1.5 °C / min to -80 °C.

[0019] Optionally, the gradient cooling includes: at a rate of 0.2-0.5 °C / min to -53 to -57 °C, and then at a rate of 0.8-1.2 °C / min to -80 °C.

[0020] The pre-cooling includes: maintaining at 2-8 °C for 5-20 min.

[0021] Optionally, the precooling includes: maintaining at 2-8°C for 8-15 minutes.

[0022] Beneficial effects

[0023] 1. By selecting passage 6 human umbilical cord mesenchymal stem cells, the ability to secrete hepatocyte growth factor (HGF) can be improved.

[0024] 2. The cryoprotectant solution for cells contains 6-9 wt% dimethyl sulfoxide and a certain amount of human serum albumin, which can better protect the cells and maintain the viability and biological functions of the cells, and is more conducive to the preparation of cryopreserved human umbilical cord mesenchymal stem cell injection.

[0025] 3. Passage 6 human umbilical cord mesenchymal stem cells are cultured from a medium containing fetal bovine serum, which can improve the ability of the injection to repair liver injury.

[0026] 4. The preparation method of the present human umbilical cord mesenchymal stem cell injection is simple, and the product performance is stable. Brief description of the drawings

[0027] Figure 1 The ability of hUC-MSC of different passages to secrete HGF, where the abscissa is P4, P5, and P6 corresponding to Comparative Example 1, Comparative Example 2, and Example 1 respectively.

[0028] Figure 2 Histopathological staining of the liver tissues of experimental rats in each group 4 weeks after cell treatment, where a: HE staining of the vehicle control group; b: HE staining of group A; c: HE staining of group B; d: HE staining of group C; e: Masson staining of the vehicle control group; f: Masson staining of group A; g: Masson staining of group B; h: Masson staining of group C.

[0029] Figure 3 Statistical analysis of the collagen area of liver tissue sections of experimental rats in each group after cell treatment.

[0030] Where Figure 1 and Figure 3 in *: P < 0.01; **: P < 0.0001. Detailed implementation manners

[0031] The component manufacturers and product numbers used in the examples and comparative examples are shown in Table 1, and the components not specified are not limited in source:

[0032] Table 1

[0033]

[0034] Example 1

[0035] A human umbilical cord mesenchymal stem cell injection, the components of which are: a cell cryoprotectant (trade name: Genuo, purchased from Nanjing Sansheng Biotechnology Co., Ltd., containing 7.5 wt% dimethyl sulfoxide) and P6 human umbilical cord mesenchymal stem cells (cultured from P0 human umbilical cord mesenchymal stem cells to P6 human umbilical cord mesenchymal stem cells using a culture medium, the composition of the culture medium: 50 mL fetal bovine serum, 448 mL DMEM / F-12 medium, 1 mL 25 μg / mL EGF, and 1 mL 5 μg / mL bFGF).

[0036] P0 human umbilical cord mesenchymal stem cells are primary cells isolated and cultured from umbilical cords by our company. When the cells of each passage reach 80 - 85% confluence, they are passaged, and 1 - 3×10 6 cells are inoculated into each T175 culture flask, and 15 - 20 ml of the culture medium is added for culture.

[0037] Among them, human umbilical cord mesenchymal stem cells are simply referred to as hUC-MSC.

[0038] A preparation method of a human umbilical cord mesenchymal stem cell injection, which comprises the following steps:

[0039] 1. Sterilize the clean bench with ultraviolet light for 30 min, and balance the reagents to room temperature.

[0040] 2. Observe the state and confluence of P6 hUC-MSC under an inverted microscope.

[0041] 3. Use a pipette to aspirate and discard the culture supernatant, wash the cells with 10 mL of normal saline per bottle twice, aspirate and discard the washing solution, and add 1 mL of recombinant trypsin to the culture flask.

[0042] 4. When it is observed that the cells (P6 hUC-MSC) become round and flake off, add 0.25 mL of human albumin to each culture flask with a pipette to terminate digestion; use a pipette to aspirate 10 mL of compound electrolyte injection to collect the cells in the culture flask into a centrifuge tube, mix well, and take 100 μL of the cell suspension as a counting sample; after adding 5 μL of AO / DAPI fluorescence staining solution to the counting sample and mixing well, take 10 μL and add it to a cell counting chamber, and then use NC-250 TM an automatic cell counter for cell counting.

[0043] 5. Place the cell suspension in a 2 - 8 °C pre-cooling for 10 min, and then centrifuge at 300 g for 5 min.

[0044] 6. Use a pipette to aspirate and discard the supernatant in the centrifuge tube. According to the cell counting result, add the corresponding volume of the therapeutic cell cryoprotectant to the centrifuge tube with a pipette, mix well, and prepare it into a specification of 2×10 7Human umbilical cord mesenchymal stem cell injection at a density of NC-250 TM cells / 20 mL; 100 μL of the cell suspension was taken as the counting sample. After adding 5 μL of AO / DAPI fluorescent staining solution to the counting sample and mixing well, 10 μL was taken and added to a cell counting chamber, and then an

[0045] automatic cell counter was used for cell counting and cell viability detection.

[0046] 7. The prepared P6-generation hUC-MSC injection was placed in a programmable freezer for gradient cooling at a rate of 0.3 °C / min to -55 °C, and then at a rate of 1.0 °C / min to -80 °C.

[0047] Comparative Example 1

[0048] The specific implementation method was the same as that of Example 1; the difference was that P4-generation human umbilical cord mesenchymal stem cells were used in Comparative Example 1 instead of P6-generation human umbilical cord mesenchymal stem cells.

[0049] Comparative Example 2

[0050] The specific implementation method was the same as that of Example 1; the difference was that P5-generation human umbilical cord mesenchymal stem cells were used in Comparative Example 2 instead of P6-generation human umbilical cord mesenchymal stem cells.

[0051] Comparative Example 3

[0052] The specific implementation method was the same as that of Example 1; the difference was that in Comparative Example 3, the cell cryoprotectant solution ( CS2, containing 2 wt% dimethyl sulfoxide).

[0053] Comparative Example 4

[0054] The specific implementation method was the same as that of Example 1; the difference was that in Comparative Example 4, the cell cryoprotectant solution ( CS5, containing 5 wt% dimethyl sulfoxide).

[0055] Comparative Example 5

[0056] The specific implementation method was the same as that of Example 1; the difference was that in Comparative Example 5, the cell cryoprotectant solution ( CS10, containing 10 wt% dimethyl sulfoxide).

[0057] Comparative Example 6

[0058] The specific implementation method was the same as that of Example 1; the difference was that in Comparative Example 6, the composition of the medium was: 25 mL of human platelet lysate and 475 mL of α-MEM basal medium.

[0059] Comparative Example 7

[0060] The specific implementation method is the same as that of Example 1; the difference is that the composition of the culture medium in Comparative Example 7 is: 505 mL of serum-free basal medium.

[0061] Performance test method

[0062] Perform performance tests on the human umbilical cord mesenchymal stem cell injection solutions prepared in the examples and comparative examples.

[0063] 1. Use the ELISA method to detect the ability of hUC-MSCs of different passages in Example 1 and Comparative Examples 1-2 to secrete HGF.

[0064] Prepare relevant reagents

[0065] 1) Take out the samples and the kit from the refrigerator 20 minutes in advance to equilibrate to room temperature;

[0066] 2) Dilute the 20× concentrated washing solution with ultrapure water to a 1× working solution. Put the unused part back and store it at 4°C;

[0067] 3) Preparation of standard products: Centrifuge the standard products (Human HGF ElisaKit, manufacturer: Xinbosheng, product number: EHC107b.96) at 6000 rpm for 5 s at room temperature. Add 1 mL of the standard product and the specimen universal diluent,

[0068] Let it stand for 15 minutes, and gently mix it after it is fully dissolved to prepare an HGF standard product with a concentration of 8000 pg / mL. Then perform serial dilutions in turn to prepare 6 different concentrations of HGF standard products of 4000 pg / mL, 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, and 125 pg / mL;

[0069] 4) 20 minutes before use, dilute the 30× concentrated biotinylated antibody with the biotinylated antibody diluent to a 1× working solution;

[0070] 5) 20 minutes before use, dilute the 30× concentrated enzyme conjugate with the enzyme conjugate diluent to a 1× working solution.

[0071] ELISA detection

[0072] 1) Respectively take 100 μL of the standard products and the test products of each group and add them to the corresponding wells. Add 100 μL of the standard product and the specimen universal diluent to the blank well. Set 2 parallel wells for the standard product and 3 parallel wells for each group of test products for detection. Seal the reaction wells with sealing tape and incubate at 37°C for 90 minutes;

[0073] 2) Prepare the biotinylated antibody working solution 20 minutes before use;

[0074] 3) Wash the plates using an automatic plate washer. Add 350 μL of washing solution to each well, with a 60 s interval between injection and aspiration. Repeat the plate washing 5 times;

[0075] 4) Add 100 μL of biotinylated antibody diluent to the blank well, and add 100 μL of biotinylated antibody working solution to the remaining wells. Incubate at 37 °C for 60 min;

[0076] 5) Prepare the enzyme conjugate working solution 20 min before use;

[0077] 6) Repeat step 3);

[0078] 7) Add 100 μL of enzyme conjugate diluent to the blank well, and add 100 μL of enzyme conjugate working solution to the remaining wells. Incubate at 37 °C for

[0079] 30 min;

[0080] 8) Turn on the microplate reader for preheating and set up the detection program;

[0081] 9) Repeat step 3);

[0082] 10) Add 100 μL of chromogenic substrate (TMB) to each well and incubate at 37 °C in the dark for 15 min;

[0083] 11) Add 100 μL of stop solution to each well, mix well, and immediately measure the OD450 value on the machine.

[0084] Evaluation index

[0085] The subgenerations of hUC-MSCs with greater advantages in treating liver injury were screened by comparing the abilities of P4, P5, and P6 subgenerations of hUC-MSCs to secrete HGF. The HGF secretion level of every 10 6 hUC-MSCs was evaluated.

[0086] Test results

[0087] The levels of HGF in the culture supernatants of P4, P5, and P6 subgenerations of hUC-MSCs from 5 different batches were detected by ELISA method. As Figure 1 shown, the secretion amount of P6 subgeneration per 10 6 hUC-MSCs was 2205.99 ± 426.43 pg / mL, which were 2.33 times (P < 0.01) and 1.82 times (P < 0.05) that of P4 subgeneration (948.72 ± 81.66 pg / mL) and P5 subgeneration (1213.13 ± 541.34 pg / mL) of hUC-MSCs, respectively. It shows that P6 subgeneration of hUC-MSCs has greater advantages in secreting HGF and is more suitable for preparing hUC-MSC injection for repairing liver injury.

[0088] 2. Comparative Test of Different Cryopreservation Protective Solutions for hUC-MSC Injection (Example 1, Comparative Examples 3-5)

[0089] Sterilize the clean bench with ultraviolet light for 30 min, and equilibrate the injection sample to room temperature for resuscitation; set the temperature of the Plasmatherm dry heat cell resuscitator (Barkey, 212.10043) to 37 °C; quickly take out each group of hUC-MSC injection from liquid nitrogen at -196 °C, and then place it in the resuscitator; resuscitate for 4-5 min until the injection is completely dissolved; use a disposable 50 mL sterile syringe to draw each group of cell injection into a 50 mL centrifuge tube, and then fully mix the cell suspension. Take 100 μL of the cell suspension from each group as the counting sample. Add 5 μL of AO / DAPI fluorescence staining solution to each counting sample, mix well, take 10 μL and add it to the cell counting chamber, and then use NC-250 TM an automatic cell counter to detect the cell viability.

[0090] Evaluation Index: Use NC-250 TM an automatic cell counter to detect the cell viability of human umbilical cord mesenchymal stem cell injections prepared from five different batches of four different cell cryopreservation solutions and the cell injections after resuscitation after 4 weeks of liquid nitrogen cryopreservation.

[0091] Among them, the cell injections corresponding to Group A, Group B, Group C, and Group D are Comparative Example 3, Comparative Example 4, Example 1, and Comparative Example 5, respectively.

[0092] Test results, as shown in Table 2 (Group A vs Group C: #P < 0.05; Group A vs Group B: **P < 0.01; Group A vs Group C: **P < 0.01). Among the human umbilical cord mesenchymal stem cell injections prepared from four different cryopreservation solutions, the cell viability of Group A (93.90 ± 1.98%) is the highest, and that of Group C is the lowest (81.46 ± 7.95%, P < 0.05). The cell viabilities of the remaining Group B and Group D are also significantly lower than that of Group A. After 4 weeks of liquid nitrogen cryopreservation and resuscitation, the cell viability of Group A (90.06 ± 2.63%) in the human umbilical cord mesenchymal stem cell injection still remains above 90%, while the other three groups show a significant decrease. Among them, Group B (73.54 ± 4.28%, P < 0.01) and Group C (73.92 ± 11.05%, P < 0.01) are the most obvious. It shows that the Sansheng Genuo cryoprotectant for therapeutic cells in Group A can better maintain the cell viability than the CS series cryopreservation solution and is more conducive to the preparation of cryopreserved human umbilical cord mesenchymal stem cell injections.

[0093] 3. Efficacy Study of hUC-MSC Injection in Treating Liver Cirrhosis Model Mice

[0094] The P6-generation hUC-MSCs cultured in three groups of culture media were respectively prepared into hUC-MSC injection solutions (Example 1, Comparative Examples 6-7), where Example 1, Comparative Example 6, and Comparative Example 7 corresponded to Group C, Group A, and Group B respectively. An additional solvent control group was set up: only containing a cell cryoprotectant (trade name: Genuo, purchased from Nanjing Sansheng Biotechnology Co., Ltd., containing 7.5 wt% dimethyl sulfoxide).

[0095] Establish a liver cirrhosis animal model

[0096] A rat liver cirrhosis model was prepared by intraperitoneal injection of CCl4-olive oil solution. The experimental animals were male Sprague Dawley rats with a body weight of 200±20 g. A 40% CCl4-olive oil solution was intraperitoneally injected once on the first and fourth days of each week, with an injection dose of 2.5 mL / Kg body weight, and the injection was carried out for a total of 10 weeks.

[0097] After the modeling was completed, all experimental rats were subjected to liver ultrasound examination. Experimental rats with a liver ultrasound density integral value greater than 90 were selected for the subsequent efficacy study of human umbilical cord mesenchymal stem cell injection solution in treating liver cirrhosis model rats.

[0098] Treatment plan

[0099] After 10 weeks of modeling, each group of experimental animals was intravenously injected with a human umbilical cord mesenchymal stem cell injection solution or a therapeutic cell cryoprotectant once through the tail vein for the treatment of liver cirrhosis. Each rat in the cell treatment group was injected with 5×10 6 cells, with a volume of 1 mL. The solvent control group was injected with an equal volume of the therapeutic cell cryoprotectant. After 4 weeks of treatment, the efficacy of each group of liver cirrhosis model rats was evaluated.

[0100] Evaluation indicators

[0101] 1) Liver ultrasound density: After 4 weeks of treatment, each group of experimental animals was subjected to liver ultrasound examination, and the improvement of liver fibrosis was evaluated by comparing the liver ultrasound density values.

[0102] 2) Pathological histology: After 4 weeks of treatment, the liver pathological sections of each group of experimental animals were subjected to HE staining and Masson staining, and the ability of different human umbilical cord mesenchymal stem cell injection solutions to repair liver injury was evaluated by observing the damage of hepatocytes, the degree of liver fibrosis, and statistically analyzing the collagen area.

[0103] Test results

[0104] After 4 weeks of treatment, each group of experimental animals was subjected to liver ultrasound examination, Figure 2The results showed that the liver ultrasound density in the cell therapy A group (58.90 ± 4.57) and the C group (42.5 ± 8.5) was significantly lower than that in the vehicle control group (76.07 ± 7.28). Among them, the decrease in the liver ultrasound density value in the cell therapy C group was the most obvious (P < 0.0001). There was no significant difference in the liver ultrasound density in the cell therapy B group (71.88 ± 7.51) compared with the vehicle control group, and it was significantly higher than that in the cell therapy A group and the C group.

[0105] As Figure 2 shown by HE staining and Masson staining of liver tissue pathology, 4 weeks after cell therapy, compared with the vehicle control group, the necrotic hepatocytes and collagen fibers in the livers of the cell therapy A group and the C group were reduced to varying degrees. In particular, the C group had the least hepatocyte death and the most obvious restoration of liver structure, with only a small amount of radioactive fibrous structure around the hepatic sinusoids. In the B group, more fibrous collagen deposits and pseudolobule structures were still visible. Further statistical analysis of the collagen area in the pathological sections ( Figure 3 ) showed that compared with the vehicle control group (36305.5 ± 14301.5), the collagen areas in the A group (24679.20 ± 4111.09) and the C group (18384.99 ± 5222.94) were significantly reduced. Among them, the reduction in the collagen area in the cell therapy C group was the most significant (P < 0.05). The collagen area in the B group increased slightly compared with the control group (40563.52 ± 8356.17), and it was significantly higher than that in the cell therapy A group and the C group (P < 0.01). The results of pathological detection were consistent with the results of liver ultrasound density, indicating that the degree of liver cirrhosis in the experimental rats decreased most significantly after cell therapy with the C group.

[0106] Performance test data

[0107] Table 2

[0108]

Claims

1. An injection of human umbilical cord mesenchymal stem cells, characterized in that, The components include: a cell cryoprotectant solution and passage 6 human umbilical cord mesenchymal stem cells.

2. The human umbilical cord mesenchymal stem cell injection according to claim 1, wherein The cell cryoprotectant solution contains 6-9 wt% dimethyl sulfoxide.

3. The human umbilical cord mesenchymal stem cell injection according to claim 2, characterized in that, The passage 6 human umbilical cord mesenchymal stem cells are obtained by culturing in a medium containing fetal bovine serum.

4. The human umbilical cord mesenchymal stem cell injection according to claim 3, characterized in that, The raw materials for producing the human umbilical cord mesenchymal stem cell injection include normal saline, recombinant trypsin, human albumin, and compound electrolyte injection.

5. A method for preparing the human umbilical cord mesenchymal stem cell injection according to claim 4, characterized in that, It includes the following steps: S1. Wash the passage 6 human umbilical cord mesenchymal stem cells with normal saline and add recombinant trypsin. S2. Observe the state and confluence of the passage 6 human umbilical cord mesenchymal stem cells under a microscope. When the cells become round and shed in flakes, add human albumin to terminate digestion. S3. Add compound electrolyte injection to the passage 6 human umbilical cord mesenchymal stem cells to obtain a cell suspension, perform cell counting, and calculate the cell concentration. S4. Pre-cool the cell suspension and then add the cell cryoprotectant solution to obtain a cell injection. After gradient cooling, perform cryopreservation.

6. The preparation method of the human umbilical cord mesenchymal stem cell injection according to claim 5, wherein, The number of cells in the cell injection is 0.5×10 7 ~5×10 7 cells / 20 mL.

7. The preparation method of the human umbilical cord mesenchymal stem cell injection according to claim 6, characterized in that, The number of cells in the cell injection is 1×10 7 ~3×10 7 cells / 20 mL.

8. The preparation method of the human umbilical cord mesenchymal stem cell injection according to claim 5, wherein, The gradient cooling includes: cooling at a rate of 0.1-0.5 °C / min to -50 to -60 °C, and then cooling at a rate of 0.8-1.5 °C / min to -80 °C.

9. The preparation method of the human umbilical cord mesenchymal stem cell injection according to claim 8, characterized in that, The steps of the gradient cooling include: cooling at a rate of 0.2-0.5 °C / min to -53 to -57 °C, and then cooling at a rate of 0.8-1.2 °C / min to -80 °C.

10. The preparation method of the human umbilical cord mesenchymal stem cell injection according to claim 5, characterized in that, The steps of pre-cooling include: maintaining at 2-8 °C for 5-20 min.

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