WO2019056217A1

WO2019056217A1 - Method for cryopreserving vero cells with high viability

Info

Publication number: WO2019056217A1
Application number: PCT/CN2017/102485
Authority: WO
Inventors: 马荣华
Original assignee: 苏州北开生化设备有限公司
Priority date: 2017-09-20
Filing date: 2017-09-20
Publication date: 2019-03-28

Abstract

Provided is a method for cryopreserving Vero cells with high viability, the method comprising the steps of: sucking up a culture medium with a pipette, adding PBS to wash the Vero cells twice or three times, adding 0.38% trypsin to a cell culture dish, digesting for 3 to 5 min at 37ºC, adding 2 to 3 ml of a fresh culture medium, and pipetting the bottom of the cell culture dish using the pipette 40 to 50 times; transferring the liquid to a cell centrifuge tube, centrifuging for 3 to 10 min under the condition of 1000-1500 g/min, removing the supernatant using the pipette, adding a cryopreservation liquid, vertically pipetting the cells 40 to 50 times using the pipette, adding the cells into a cell cryopreservation tube, tightening the cap of the cryopreservation tube, and sealing the cryopreservation tube with a sealing film; and preserving for 10 to 30 min at 2 to 7ºC, and for another 40 to 60 min at -15 to -20ºC, and then transferring same into an environment of ≤-80ºC for cryopreservation.

Description

Cryopreservation method of high survival rate Vero cells

Technical field

The invention belongs to the field of biotechnology applications, and in particular relates to a method for cryopreservation of high survival Vero cells.

Background technique

Vero cells are monkey kidney cell lines established by Japanese scholars in 1962 from the kidneys of the cercopithecus aethiops monkey. The origin of the name is the first two letters of VERDE taken from the word "green" in Esperanto and the first two letters of the word "kidney". However, in Esperanto, VERE is not allowed to exist, but it needs to end with "O", so it is named. It is "Vero". The 93rd generation was taken to the Tropical Virus Research Laboratory of the Institute of Allergy and Infectious Diseases at NIH, USA, and the 113th generation was submitted to the ATCC under the number ATCC NO.CCL-81.

In addition to routine research needs, Vero cells have been cytokine-stable, with no exogenous factor contamination and tumorigenicity, and are fully compliant with WHO's 1987 guidelines for passage cells for biological products. And has been approved by the WHO as a substrate for vaccine production. Thus, the Vero cell polio inactivated vaccine, the polio live vaccine, and the Vero cell rabies vaccine were successively developed and approved for production in France. Therefore, the application prospects and demand of Vero cells are enormous. As more and more tissue cell culture and proliferation techniques are used in the research process, Vero cells are stored at low temperatures for several years, decades or even hundreds of years. Once needed, the preserved cells can be resuscitated and restored at any time. Its complete morphological structure and biological characteristics for life science research experiments.

Summary of the invention

OBJECTS OF THE INVENTION: The present invention provides a method for cryopreservation of high viability Vero cells dedicated to the culture of Vero cells.

Technical Solution: The present invention provides a method for cryopreservation of Vero cells with high survival rate, comprising the steps of: absorbing the culture medium with a pipette, and washing the Vero cells 2-3 times with 3-5 ml of PBS to culture the cells. Add 1-2ml of 0.38% trypsin to the dish, digest for 3-5min at 37 °C, add 2-3ml of fresh Vero cell-specific medium, and use a pipette to blow the bottom of the cell culture dish 40-50 times; Transfer the liquid to the cell centrifuge tube, centrifuge at 1000-1500g/min for 3-10min, remove the supernatant with a pipette, add Vero cell-specific cryopreservation solution, and use a pipette to blow Vero cells up and down 40-50 Once, add to the cell cryotube, tighten the cryotube cap, seal the cryotube with a sealing film; store at 2-7 ° C for 10-30 min, then store at -15--20 ° C for 40-60 min, then move in Cell cryopreservation to an environment of ≤-80 ° C; wherein the Vero cell-specific cryopreservation solution comprises, by weight component, the following components: by weight component, including the following components: dimethyl sulfoxide 5-15 Parts, 3-8 parts of hydroxyethyl starch, 5-15 parts of glucose, 1-5 parts of vitamin E, 20% mannitol 2-10 parts, propylene glycol 1-2 parts, (S)-(-)-1-(4-fluoroisoquinolin-5-yl)sulfonyl-2-methyl-1,4-di 0.8-2 parts of nitrogen cycloheptane and 80-90 parts of basal medium. The high survival rate Vero cell cryopreservation method of the invention has good safety and stability, and the survival rate after cell recovery after cryopreservation is high. Among them, Vero cell special cryopreservation solution is reasonable in composition and rich in nutrients. 20% mannitol is a osmotic protective solution, and (S)-(-)-1-(4-fluoroisoquinolin-5-yl)sulfonyl group -2-Methyl-1,4-diazacycloheptane inhibits cell activity and gene expression, and vitamin E has good antioxidant activity, which can significantly reduce cell damage and improve cell damage while ensuring the nutrition of dormant Vero cells. The survival rate of the frozen cells is also maintained in a good state in terms of proliferation.

Further, in the above method for cryopreservation of high-viable Vero cells, the frozen Vero cells are frozen overnight at ≤-80 ° C, and placed in a liquid nitrogen tank for the next day in liquid nitrogen immersed in liquid nitrogen. The cells were frozen. Preservation in liquid nitrogen can further prolong the storage time of Vero cells, and can maintain good cell viability and cell viability after several years and decades.

Further, in the above method for cryopreservation of high-viable Vero cells, the Vero cell cryopreservation density in each cryotube is 10 ⁵ -10 ⁶ cells. The cell density is reasonable, which can effectively ensure the cell survival rate and cell activity.

Further, in the above method for cryopreservation of high survival rate Vero cells, the trypsin further comprises 0.5% EDTA. It can be combined with trypsin to rapidly separate the adherent cells from the culture dish and reduce the damage of trypsin to the cells.

Further, in the above method for cryopreservation of high survival rate Vero cells, the Vero cell-specific medium comprises 70-90% of basal medium and 10-30% of fetal bovine serum. Fetal bovine serum can further provide nutrients to the cultured cells while also terminating the digestion of trypsin.

Further, in the above method for cryopreservation of high-viable Vero cells, the basal medium comprises, by weight components, the following components: glucose 82-88 parts, sodium hydrogencarbonate 12-25 parts, sodium pyruvate 10 -16 parts, 5-9 parts of human albumin, 35-45 parts of potassium chloride, 6-10 parts of anhydrous magnesium sulfate, 70-78 parts of sodium chloride, 10-14 parts of anhydrous sodium dihydrogen phosphate, PHA - phytohemagglutinin 0.4-0.8 parts, folic acid 0.3-0.5 parts, inositol 0.8-1.2 parts, nicotinamide 0.3-0.6 parts, anhydrous calcium chloride 25-35 parts, ferric nitrate 0.2-0.4 parts, succinic acid 6-8 parts, 12-16 parts of sodium succinate, 0.3-0.6 parts of D-pantothenate, 0.6-0.8 parts of choline tartrate, 0.1-0.3 parts of riboflavin, 0.2-0.4 parts of thiamine hydrochloride, pyridinium hydrochloride 0.3-0.5 parts of sim, 0.2-0.6 parts of reduced glutathione, 2-4 parts of cholesterol, 0.6-1 part of ascorbyl phosphate, 0.4-0.9 parts of polycarbonate, 1-1.3 parts of sodium phenol red and 100 parts of deionized water Share.

Further, in the above method for cryopreservation of high-viable Vero cells, the basal medium further comprises 3-8 parts of amino acids, and the amino acids are composed by weight of components: L-arginine hydrochloride 5-10 parts, L-HCl cystine 3-8 parts, L-serine 3-6 parts, glycine 1-5 parts, L-histidine 4-6 parts, L-isoleucine 8-20 , L-leucine 7-18 parts, L-Lysine hydrochloride 10-20 parts, L-methionine 1-6 parts, L-phenylalanine 2-8 parts, L-threonine 5-15 parts, 1-3 parts of L-tryptophan, 5-9 parts of L-tyrosine, and 8-12 parts of L-valine.

Further, in the above method for cryopreservation of high survival rate Vero cells, the basal medium further comprises 2-5 parts of cofactors, which are composed of the following components in terms of weight components: recombinant human alkaline 2-10 parts of fibroblast growth factor, 3-8 parts of interleukin 2, 4-6 parts of interleukin 4, 1-5 parts of interleukin 14 , 3-6 parts of IFN-γ and 1-4 parts of neuroleukocytidine. The basic medium has reasonable components and is rich in nutrients, which can improve the nutrients during cell cryopreservation and ensure the activity of frozen cells.

Further, in the above method for cryopreservation of high-viable Vero cells, the basal medium further comprises 0.006 parts of penicillin and 0.01 part of streptomycin, by weight component. It can effectively prevent frozen cells from being contaminated.

Further, in the above method for cryopreservation of high survival rate Vero cells, the penicillin is selected from the group consisting of 10000 U/ml, and the streptomycin is selected from the group consisting of 10000 μg/ml. Penicillin and streptomycin have good activity and good effect.

Advantageous Effects: The method for cryopreservation of high-survival Vero cells according to the present invention has a reasonable method, convenient application, good safety and stability, high survival rate after resuscitation of cells after cryopreservation, and also maintains very high in proliferation. Good state.

DRAWINGS

Figure 1 is a graph showing the measurement of cell viability of Examples 1-4.

Detailed ways

The invention will be further clarified by the following specific examples, which are merely illustrative and not limiting.

Example 1

A method for cryopreservation of high survival Vero cells, comprising the steps of: draining the medium with a pipette, washing Vero cells 3 times with 3 ml of PBS, and adding 1 ml of 0.38% trypsin to the cell culture dish. Digest for 5 min at 37 °C, add 2 ml of fresh Vero cell-specific medium, and pipette the bottom of the cell culture dish 40 times with a pipette; transfer the liquid to the cell centrifuge tube and centrifuge at 1000g/min for 10min. Remove the supernatant from the liquid gun, add Vero cell-specific cryopreservation solution, use a pipette to blow Vero cells up and down 40 times, and add to the cell cryotube. The frozen density of Vero cells in each cryotube is 10 ⁵ cells. . Tighten the frozen tube cap and seal the cryotube with a sealing film; store at 2 ° C for 10 min, then at -15 ° C for 60 min, then transfer to ≤ -80 ° C for cell cryopreservation. The frozen Vero cells were frozen overnight at ≤-80 ° C, and placed in a liquid nitrogen tank the next day and immersed in liquid nitrogen for liquid cryopreservation.

Wherein, the Vero cell-specific medium comprises 70% basal medium and 30% fetal calf serum. Further, the Vero cell-specific cryopreservation solution comprises, by weight component, the following components: by weight component, including the following components: 5 parts of dimethyl sulfoxide, 3 parts of hydroxyethyl starch, and 5 parts of glucose. , vitamin E1 part, 20% mannitol 2 parts, propylene glycol 1 part, (S)-(-)-1-(4-fluoroisoquinolin-5-yl)sulfonyl-2-methyl-1,4- 0.8 parts of diazepane and basic medium 80 Share.

Wherein, the basal medium comprises, by weight components, the following components: 82 parts of glucose, 12 parts of sodium hydrogencarbonate, 10 parts of sodium pyruvate, 5 parts of human serum albumin, 35 parts of potassium chloride, anhydrous sulfuric acid. 6 parts of magnesium, 70 parts of sodium chloride, 10 parts of anhydrous sodium dihydrogen phosphate, 0.4 parts of PHA-phytohemagglutinin, 0.3 parts of folic acid, 0.8 parts of inositol, 0.3 parts of nicotinamide, 25 parts of anhydrous calcium chloride, 0.2 parts of ferric nitrate, 6 parts of succinic acid, 12 parts of sodium succinate, 0.3 parts of D-pantothenate, 0.6 parts of choline tartrate, 0.1 parts of riboflavin, 0.2 parts of thiamine hydrochloride, 0.3 parts of pyridoxine hydrochloride, 0.2 parts of glutathione, 2 parts of cholesterol, 0.6 parts of ascorbyl phosphate, 0.4 parts of polycarbonate, 1 part of sodium phenol red, and 100 parts of deionized water were reduced.

In addition, the basal medium further comprises 3 parts of amino acids, which are composed by weight of L-HCl arginine 5 parts, L-HCl cystine 3 parts, L-serine 3 1 part, 1 part of glycine, 4 parts of L-histidine hydrochloride, 8 parts of L-isoleucine, 7 parts of L-leucine, 10 parts of L-lysine lysine, 1 part of L-methionine, 2 parts of L-phenylalanine, 5 parts of L-threonine, 1 part of L-tryptophan, 5 parts of L-tyrosine and 8 parts of L-valine.

Further, the basal medium further comprises 2 parts of cofactors, which are composed of the following components in terms of weight components: recombinant human basic fibroblast growth factor 2 parts, interleukin 2 3 parts, and interleukin 4 3 Parts, 14 parts of interleukin, 3 parts of IFN-γ and 1 part of neuroleukocytidine.

Further, the basal medium further comprises 0.006 parts of penicillin and 0.01 part of streptomycin, by weight component. And, the penicillin is selected from the group consisting of 10000 U/ml, and the streptomycin is selected from the group consisting of 10000 μg/ml. In addition, 0.5% EDTA is also included in the trypsin.

Example 2

A method for cryopreservation of high-viable Vero cells, comprising the steps of: pipetting the medium with a pipette, washing Vero cells twice with 5 ml of PBS, and adding 2 ml of 0.38% trypsin to the cell culture dish. Digest for 3 min at 37 °C, add 3 ml of fresh Vero cell-specific medium, and pipette the bottom of the cell culture dish 50 times with a pipette; transfer the liquid to the cell centrifuge tube and centrifuge at 1500 g/min for 3 min. Remove the supernatant from the liquid gun, add Vero cell-specific cryopreservation solution, use a pipette to blow Vero cells up and down 50 times, and add to the cell cryotube. The frozen density of Vero cells in each cryotube is 10 ⁶ cells. . Tighten the cryotube cover and seal the cryotube with a sealing film; store at 7 ° C for 30 min, then store at -20 ° C for 40 min, then transfer to ≤ -80 ° C for cell cryopreservation. The frozen Vero cells were frozen overnight at ≤-80 ° C, and placed in a liquid nitrogen tank the next day and immersed in liquid nitrogen for liquid cryopreservation.

Wherein, the Vero cell-specific medium comprises 90% basal medium and 10% fetal calf serum. In addition, the Vero cell-specific cryopreservation solution comprises, by weight component, the following components: in terms of weight components, including the following groups Points: 15 parts of dimethyl sulfoxide, 8 parts of hydroxyethyl starch, 15 parts of glucose, 5 parts of vitamin E, 10 parts of 20% mannitol, 2 parts of propylene glycol, (S)-(-)-1-(4-fluoro 2 parts of isoquinolin-5-yl)sulfonyl-2-methyl-1,4-diazacycloheptane and 90 parts of basal medium.

Wherein, the basal medium comprises, by weight components, the following components: 88 parts of glucose, 25 parts of sodium hydrogencarbonate, 16 parts of sodium pyruvate, 9 parts of human serum albumin, 45 parts of potassium chloride, anhydrous sulfuric acid. 10 parts of magnesium, 78 parts of sodium chloride, 14 parts of anhydrous sodium dihydrogen phosphate, 0.8 parts of PHA-phytohemagglutinin, 0.5 part of folic acid, 1.2 parts of inositol, 0.6 parts of nicotinamide, 35 parts of anhydrous calcium chloride, 0.4 parts of ferric nitrate, 8 parts of succinic acid, 16 parts of sodium succinate, 0.6 parts of D-pantothenate, 0.8 parts of choline tartrate, 0.3 parts of riboflavin, 0.4 parts of thiamine hydrochloride, 0.5 parts of pyridoxine hydrochloride, 0.6 parts of glutathione, 4 parts of cholesterol, 1 part of ascorbyl phosphate, 0.9 parts of polycarbonate, 1.3 parts of sodium phenol red, and 100 parts of deionized water were reduced.

In addition, the basal medium further comprises 8 parts of amino acids, which are composed of the following components in terms of weight components: L-arginine hydrochloride 10 parts, L-HCl cystine 8 parts, L-serine 6 5 parts, 5 parts of glycine, 6 parts of L-histidine hydrochloride, 20 parts of L-isoleucine, 18 parts of L-leucine, 20 parts of L-lysine lysine, 6 parts of L-methionine, 8 parts of L-phenylalanine, 15 parts of L-threonine, 3 parts of L-tryptophan, 9 parts of L-tyrosine, and 12 parts of L-valine.

Further, the basal medium further comprises 5 parts of cofactors, which are composed of the following components in terms of weight components: recombinant human basic fibroblast growth factor 10 parts, interleukin 28 parts, and interleukin 4 6 Parts, 14 parts of interleukin, 6 parts of IFN-γ and 4 parts of neuroleukocytidine.

Example 3

A method for cryopreservation of high-viable Vero cells, comprising the steps of: draining the medium with a pipette, washing Vero cells 3 times with 4 ml of PBS, and adding 2 ml of 0.38% trypsin to the cell culture dish. Digestion at 37 ° C for 4 min, add 3 ml of fresh Vero cell-specific medium, pipette the bottom of the cell culture dish 48 times with a pipette; transfer the liquid to the cell centrifuge tube, centrifuge at 1200g / min for 5min, use The supernatant was removed by liquid gun, Vero cell-specific cryopreservation solution was added, and Vero cells were blown up and down 45 times using a pipette, and added to the cell cryopreservation tube. The Vero cell cryopreservation density in each cryotube was 4×10 ⁵ Cells. Tighten the cryotube cover and seal the cryotube with a sealing film; store at 5 °C for 20 min, then store at -18 °C for 50 min, then transfer to ≤-80 °C for cell cryopreservation. The frozen Vero cells were frozen overnight at ≤-80 ° C, and placed in a liquid nitrogen tank the next day and immersed in liquid nitrogen for liquid cryopreservation.

Wherein, the Vero cell-specific medium comprises 80% basal medium and 20% fetal calf serum. Further, the Vero cell-specific cryopreservation solution comprises, by weight component, the following components: by weight component, including the following components: 12 parts of dimethyl sulfoxide, 5 parts of hydroxyethyl starch, 12 parts of glucose , vitamin E3 parts, 20% mannitol 8 parts, propylene glycol 1.5 parts, (S)-(-)-1-(4-fluoroisoquinolin-5-yl)sulfonyl-2-methyl-1,4- 1.2 parts of diazepane and 88 parts of basal medium.

Wherein, the basal medium comprises, by weight components, the following components: 86 parts of glucose, 18 parts of sodium hydrogencarbonate, 12 parts of sodium pyruvate, 6 parts of human serum albumin, 40 parts of potassium chloride, anhydrous sulfuric acid. 8 parts of magnesium, 75 parts of sodium chloride, 12 parts of anhydrous sodium dihydrogen phosphate, 0.5 parts of PHA-phytohemagglutinin, 0.4 parts of folic acid, 1 part of inositol, 0.5 parts of nicotinamide, 30 parts of anhydrous calcium chloride, 0.3 parts of ferric nitrate, 7 parts of succinic acid, 14 parts of sodium succinate, 0.5 parts of D-pantothenate, 0.7 parts of choline tartrate, 0.2 parts of riboflavin, 0.3 parts of thiamine hydrochloride, 0.4 parts of pyridoxine hydrochloride, 0.5 parts of glutathione, 3 parts of cholesterol, 0.8 parts of ascorbyl phosphate, 0.8 parts of polycarbonate, 1.2 parts of sodium phenol red, and 100 parts of deionized water were reduced.

In addition, the basal medium further comprises 4 parts of amino acids, which are composed of the following components in terms of weight components: 8 parts of L-arginine hydrochloride, 6 parts of L-cystine hydrochloride, L-serine 5 3 parts, 3 parts of glycine, 5 parts of L-histidine hydrochloride, 12 parts of L-isoleucine, 10 parts of L-leucine, 15 parts of L-Lysine hydrochloride, 3 parts of L-methionine, 4 parts of L-phenylalanine, 9 parts of L-threonine, 2 parts of L-tryptophan, 6 parts of L-tyrosine and 9 parts of L-valine.

Further, the basal medium further comprises 3 parts of cofactors, which are composed of the following components in terms of weight components: recombinant human basic fibroblast growth factor 8 parts, interleukin 26 parts, and interleukin 4 4 Serving, 14 parts of interleukin, 5 parts of IFN-γ and 2 parts of neuroleukocytidine.

Example 4

A method for cryopreservation of high-viable Vero cells, comprising the steps of: pipetting the medium with a pipette, washing Vero cells twice with 5 ml of PBS, and adding 2 ml of 0.38% trypsin to the cell culture dish. Digestion at 37 ° C for 4 min, add 3 ml of fresh Vero cell-specific medium, pipette the bottom of the cell culture dish 45 times with a pipette; transfer the liquid to the cell centrifuge tube, centrifuge at 1200g / min for 8min, use Remove the supernatant from the liquid gun, add Vero cell-specific cryopreservation solution, use a pipette to blow Vero cells up and down 40 times, and add to the cell cryotube. The frozen density of Vero cells in each cryotube is 10 ⁶ cells. . Tighten the frozen tube cover and seal the frozen tube with a sealing film; store at 4 ° C for 20 min, then at -20 ° C for 45 min, then transfer to ≤ -80 ° C for cell cryopreservation. The frozen Vero cells were frozen overnight at ≤-80 ° C, and placed in a liquid nitrogen tank the next day and immersed in liquid nitrogen for liquid cryopreservation.

Wherein, the Vero cell-specific medium comprises 90% basal medium and 10% fetal calf serum. Further, the Vero cell-specific cryopreservation solution comprises, by weight component, of the following components: by weight component, including the following components: 5 parts of dimethyl sulfoxide, 8 parts of hydroxyethyl starch, and 9 parts of glucose. , vitamin E5 parts, 20% mannitol 2 parts, propylene glycol 1 part, (S)-(-)-1-(4-fluoroisoquinolin-5-yl)sulfonyl-2-methyl-1,4- 1.6 parts of diazepane and 85 parts of basal medium.

Wherein, the basal medium comprises, by weight components, the following components: 88 parts of glucose, 12 parts of sodium hydrogencarbonate, 12 parts of sodium pyruvate, 6 parts of human serum albumin, 35 parts of potassium chloride, anhydrous sulfuric acid. 10 parts of magnesium, 70 parts of sodium chloride, 14 parts of anhydrous sodium dihydrogen phosphate, 0.4 parts of PHA-phytohemagglutinin, 0.4 parts of folic acid, 0.9 parts of inositol, 0.5 parts of nicotinamide, 28 parts of anhydrous calcium chloride, 0.3 parts of ferric nitrate, 7 parts of succinic acid, 14 parts of sodium succinate, 0.5 parts of D-pantothenate, 0.7 parts of choline tartrate, 0.2 parts of riboflavin, 0.2 parts of thiamine hydrochloride, 0.5 parts of pyridoxine hydrochloride, 0.6 parts of glutathione, 3 parts of cholesterol, 0.8 parts of ascorbyl phosphate, 0.4 parts of polycarbonate, 1.3 parts of sodium phenol red, and 100 parts of deionized water were reduced.

In addition, the basal medium further comprises 3 parts of amino acids, which are composed of the following components in terms of weight components: L-arginine hydrochloride 5 parts, L-HCl cystine 3 parts, L-serine 5 3 parts, 3 parts of glycine, 4 parts of L-histidine hydrochloride, 20 parts of L-isoleucine, 7 parts of L-leucine, 20 parts of L-lysine lysine, 4 parts of L-methionine, 5 parts of L-phenylalanine, 8 parts of L-threonine, 2 parts of L-tryptophan, 5 parts of L-tyrosine and 12 parts of L-valine.

Further, the basal medium further comprises 4 parts of cofactors, which are composed of the following components in terms of weight components: recombinant human basic fibroblast growth factor 2 parts, interleukin 2 3 parts, and interleukin 4 5 Serving, 14 parts of interleukin, 6 parts of IFN-γ and 4 parts of neuroleukocytidine.

The cryopreserved cells of Example 1-4 were resuscitated at 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months after cryopreservation, and the cell survival rate was obtained. For the measurement, the cell survival rate was above 88%.

The above description is only a few embodiments of the invention, and it should be noted that those skilled in the art can also make several improvements without departing from the principle of the invention. The scope of protection.

Claims

A method for cryopreservation of Vero cells with high survival rate, comprising the steps of: absorbing the medium with a pipette, washing Vero cells 2-3 times with 3-5 ml of PBS, and adding to the cell culture dish. Digestion of 1-2 ml of 0.38% trypsin at 37 ° C for 3-5 min, add 2-3 ml of fresh Vero cell-specific medium, and pipette the bottom of the cell culture dish 40-50 times with a pipette; move the liquid to Centrifuge in a cell centrifuge tube at 1000-1500 g/min for 3-10 min, remove the supernatant with a pipette, add Vero cell-specific cryopreservation solution, and use a pipette to blow Vero cells up and down 40-50 times. In the cell cryotube, tighten the cryotube cap and seal the cryotube with a sealing film; store at 2-7 °C for 10-30 min, then store at -15--20 °C for 40-60 min, then transfer to ≤-80 Cell cryopreservation in a °C environment; wherein the Vero cell-specific cryopreservation solution comprises, by weight component, the following components: by weight component, including the following components: dimethyl sulfoxide 5-15 parts, hydroxyethyl 3-8 parts of starch, 5-15 parts of glucose, 1-5 parts of vitamin E, 2-10 parts of 20% mannitol, 1-2 parts of propylene glycol, (S)-(-)-1-(4 -Fluoroisoquinolin-5-yl)sulfonyl-2-methyl-1,4-diazacycloheptane 0.8-2 parts and basal medium 80-90 parts.
The method for cryopreservation of high-survival Vero cells according to claim 1, characterized in that the frozen Vero cells are frozen in an environment of ≤ -80 ° C overnight, and placed in a liquid nitrogen tank in liquid nitrogen for the next day. The cells were cryopreserved by immersion in liquid nitrogen.
The method for cryopreservation of high-viable Vero cells according to claim 1, characterized in that the Vero cell cryopreservation density in each of the cryotubes is from 10 5 to 10 6 cells.
The method for cryopreservation of high survival Vero cells according to claim 1, characterized in that the trypsin further comprises 0.5% EDTA.
The method for cryopreservation of high survival Vero cells according to claim 1, wherein the Vero cell-specific medium comprises 70-90% of basal medium and 10-30% of fetal bovine serum.
The method for cryopreservation of high-viable Vero cells according to claim 1 or 5, wherein the basal medium comprises, by weight components, the following components: glucose 82-88 parts, sodium hydrogencarbonate 12 -25 parts, 10-16 parts of sodium pyruvate, 5-9 parts of human albumin, 35-45 parts of potassium chloride, 6-10 parts of anhydrous magnesium sulfate, 70-78 parts of sodium chloride, and anhydrous phosphoric acid 10-14 parts of sodium hydrogen, 0.4-0.8 parts of PHA-phytohemagglutinin, 0.3-0.5 parts of folic acid, 0.8-1.2 parts of inositol, 0.3-0.6 parts of nicotinamide, 25-35 parts of anhydrous calcium chloride, iron nitrate 0.2-0.4 parts, 6-8 parts of succinic acid, 12-16 parts of sodium succinate, 0.3-0.6 parts of D-pantothenate, 0.6-0.8 parts of choline tartrate, 0.1-0.3 parts of riboflavin, thiamine hydrochloride 0.2-0.4 parts, 0.3-0.5 parts of pyridoxine hydrochloride, 0.2-0.6 parts of reduced glutathione, 2-4 parts of cholesterol, 0.6-1 part of ascorbyl phosphate, 0.4-0.9 parts of polycarbonate, and sodium phenol red 1 - 1.3 parts and 100 parts of deionized water.
The method for cryopreservation of high viability Vero cells according to claim 6, wherein the basal medium further comprises 3-8 parts of amino acids, and the amino acids are composed of the following components in terms of weight components: 5-10 parts of L-arginine hydrochloride, 3-8 parts of L-cystine hydrochloride, 3-6 parts of L-serine, 1-5 parts of glycine, 4-6 parts of L-histidine hydrochloride, L-iso Bright 8-20 parts of amino acid, 7-18 parts of L-leucine, 10-20 parts of L-Lysine hydrochloride, 1-6 parts of L-methionine, 2-8 parts of L-phenylalanine, 5-15 parts of L-threonine, 1-3 parts of L-tryptophan, 5-9 parts of L-tyrosine, and 8-12 parts of L-valine.
The method for cryopreservation of high-survival Vero cells according to claim 7, wherein the basal medium further comprises 2-5 parts of cofactors, the cofactors are by weight components, and the following components Composition: recombinant human basic fibroblast growth factor 2-10 parts, interleukin 2 3-8 parts, interleukin 4 3-6 parts, interleukin 14 1-5 parts, IFN-γ 3-6 parts and neuroleukocyps 1-4 Share.
The method for cryopreservation of high-viable Vero cells according to claim 8, wherein the basal medium further comprises 0.006 parts of penicillin and 0.01 parts of streptomycin, by weight component.
The method for cryopreservation of high-viable Vero cells according to claim 9, wherein the penicillin is selected from the group consisting of 10000 U/ml and the streptomycin is selected from the group consisting of 10000 μg/ml.