CN107151649A - A kind of method of Vero cell expansions culture - Google Patents

A kind of method of Vero cell expansions culture Download PDF

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CN107151649A
CN107151649A CN201710524381.7A CN201710524381A CN107151649A CN 107151649 A CN107151649 A CN 107151649A CN 201710524381 A CN201710524381 A CN 201710524381A CN 107151649 A CN107151649 A CN 107151649A
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parts
culture
vero cell
cell
liquid
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马荣华
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SUZHOU BEIKAI BIOCHEMICAL EQUIPMENT Co Ltd
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SUZHOU BEIKAI BIOCHEMICAL EQUIPMENT Co Ltd
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Abstract

The invention provides a kind of method of Vero cell expansions culture, Vero cells special culture media is exhausted with liquid-transfering gun, add 3 5ml PBS washings cell 23 times, 1 2ml 0.38% trypsase is added into Tissue Culture Dish, 2 3min are digested under the conditions of 37 DEG C, 2 3ml fresh Vero cell special culture medias are added, Tissue Culture Dish bottom is blown and beaten 40 50 times using liquid-transfering gun；Liquid is moved in cell centrifuge tube, 3 5min are centrifuged under the conditions of 1000 1200g/min, supernatant is removed with liquid-transfering gun, the fresh Vero cell special culture medias of 2 3ml are added, piping and druming cell 40 50 times above and below liquid-transfering gun are used；Averagely it is added in 38 Tissue Culture Dish, is enlarged culture, rationally, using conveniently, the Vero cell growths speed of culture is fast, and cell state is good for its method.

Description

A kind of method of Vero cell expansions culture
Technical field
The invention belongs to biological technology application, in particular it relates to a kind of method of Vero cell expansions culture.
Background technology
Cell culture is a biological important technology with healthy Related Research Domain.With the rapid hair of life science Exhibition, current cell culture turns into the important base of the disciplinary studies such as cell biology, molecular biology, science of heredity and immunology Plinth.In order to further investigated cell vegetative activity rule, have the pathology of related disorders and the pharmacology of medicine（Toxicity）Mechanism, exploitation tool There are different targetedly cell culture processes significant.
Vero cells are that Japanese scholars are built for 1962 from cercopithecus aethiops (cercopithecus aethiops monkey) kidney Vertical MK cells system.The origin of its title is taken from Esperanto two before VERDE the first two of " green " word letter and " kidney " word Individual letter is combined, but does not allow VERE to exist in Esperanto, and need to be ended up with " O ", therefore is named as " Vero ".Its 93rd generation The U.S. NIH allergy and tropic virus research department of Infectious Disease Research Institute are brought to, the 113rd generation was submitted to ATCC, and numbering is ATCC NO.CCL-81。
In addition to conventional scientific research needs, identify that Vero cells have karyogy stable, do not have by comprehensively research Exogenous factor pollutes the advantage with oncogenicity, complies fully with codes of the WHO in 1987 on the passage cell for biological products It is required that, and can be used as the matrix of production of vaccine by WHO approvals.Then, Vero cells polio inactivated vaccine, polio Live vaccine, Vero cell rabies are developed and granted production in France in succession.Therefore, the application of Vero cells Prospect and demand are very big.
The content of the invention
Goal of the invention：The invention provides a kind of method of Vero cell expansions culture.
Technical scheme：The invention provides a kind of method of Vero cell expansions culture, comprise the following steps：Use liquid-transfering gun Vero cells special culture media is exhausted, 3-5ml PBS washings cell is added 2-3 times, 1-2ml is added into Tissue Culture Dish 0.38% trypsase, digests 2-3min under the conditions of 37 DEG C, adds 2-3ml fresh Vero cell special culture medias, makes Tissue Culture Dish bottom is blown and beaten with liquid-transfering gun 40-50 times；Liquid is moved in cell centrifuge tube, under the conditions of 1000-1200g/min 3-5min is centrifuged, supernatant is removed with liquid-transfering gun, the fresh Vero cell special culture medias of 2-3ml are added, using on liquid-transfering gun It is lower to blow and beat cell 40-50 times；Averagely it is added in 3-8 Tissue Culture Dish, is enlarged culture.Vero of the present invention is thin The method that born of the same parents expand culture, rationally, using conveniently, the Vero cell growths speed of culture is fast, and cell state is good for method.
Further, also comprising 0.05% in the method for above-mentioned Vero cell expansion cultures, the trypsase EDTA.It can be engaged with trypsase, attached cell is departed from culture dish rapidly, reduce damage of the trypsase to cell.
Further, the method for above-mentioned Vero cell expansion cultures, the Vero cells special culture media includes 70- 90% basal medium and 10-30% hyclone.Hyclone can be further the cells with nutrient thing of culture Matter, while the digestion of trypsase can also be terminated.
Further, the method for above-mentioned Vero cell expansion cultures, the basal medium by weight part, including Following components：80-90 parts of glucose, 10-20 parts of sodium acid carbonate, 8-18 parts of Sodium Pyruvate, 2-10 parts of human serum albumin, chlorination 30-50 parts of potassium, 5-12 parts of anhydrous magnesium sulfate, 60-80 parts of sodium chloride, 8-16 parts of AMSP, PHA- plant blood cells 0.2-1 parts of agglutinin, 0.2-0.6 parts of folic acid, 0.5-1.5 parts of inositol, 0.2-0.8 parts of niacinamide, 20-40 parts of anhydrous calcium chloride, 0.1-0.5 parts of ferric nitrate, 5-10 parts of succinic acid, 9-18 parts of sodium succinate, 0.2-0.8 parts of D-VB5 calcium, choline tartrate 0.5-1 parts, 0.1-0.3 parts of riboflavin, 0.1-0.5 parts of thiamine hydrochloride, 0.1-0.6 parts of pyridoxine hydrochloride, reduced glutathione 0.1-0.8 parts, 1-5 parts of cholesterol, 0.5-1.2 parts of ascorbic acid phosphoric acid esters, 0.3-1.5 parts of makrolon, phenol red sodium 0.8-1.5 100 parts of part and deionized water.
Further, the method for above-mentioned Vero cell expansion cultures, the basal medium also includes 3-8 parts of amino Acid, the amino acid is by weight part, composed of the following components：5-10 parts of L- R-genes, L- hydrochloric acid cystines 3-8 Part, 3-6 parts of Serine, 1-5 parts of glycine, 4-6 parts of L- histidine monohydrochlorides, 8-20 parts of ILE, L-Leu 7-18 Part, 10-20 parts of LYS, 1-6 parts of METHIONINE, 2-8 parts of L-phenylalanine, 5-15 parts of L-threonine, L- color ammonia 8-12 parts of sour 1-3 parts, 5-9 parts of TYR and Valine.
Further, the method for above-mentioned Vero cell expansion cultures, the basal medium also include 2-5 part aid in because Son, the confactor is by weight part, composed of the following components：Recombination human basic fibroblast growth factor 2-10 Part, 3-8 parts of interleukin-22,3-6 parts of IL-4,1-5 parts of interleukin-11 4,3-6 parts of IFN-γ and 1-4 parts of neuroleukin. Basal medium component is rationally, nutritious, can provide more nutrients for Vero cell culture.
Further, the method for above-mentioned Vero cell expansion cultures, the basal medium by weight part, is also wrapped Include 0.006 part of penicillin and 0.01 part of streptomysin.Culture cell can be effectively prevented to be contaminated.
Further, the method for above-mentioned Vero cell expansion cultures, the penicillin is selected from 10000U/ml, the chain Mycin is selected from 10000 μ g/ml.Penicillin and streptomysin activity are good, and effect is good.
Further, the method for above-mentioned Vero cell expansion cultures, the Tissue Culture Dish is 10cm Tissue Culture Dish.
Beneficial effect：The method of Vero cell expansions culture of the present invention, method is reasonable, using convenience, culture Vero cell growths speed is fast, and cell state is good.
Embodiment
Below will be by several specific embodiments, the present invention is furture elucidated, these embodiments simply to illustrate that problem, It is not a kind of limitation.
Embodiment 1
A kind of method of Vero cell expansions culture, comprises the following steps：Vero cells special culture media is exhausted with liquid-transfering gun, Add 3ml PBS washings cell 3 times, 1ml 0.38% trypsase is added into 10cm Tissue Culture Dish, under the conditions of 37 DEG C 3min is digested, 2ml fresh Vero cell special culture medias are added, Tissue Culture Dish bottom is blown and beaten 40 times using liquid-transfering gun；Will Liquid is moved in cell centrifuge tube, and 5min is centrifuged under the conditions of 1000g/min, and supernatant is removed with liquid-transfering gun, adds 2ml fresh Vero cell special culture medias, use and cell 40 times blown and beaten above and below liquid-transfering gun；Averagely it is added in 3 Tissue Culture Dish, enters Row expands culture.
Wherein, the Vero cells special culture media includes 70% basal medium and 30% hyclone.It is described Basal medium by weight part, including following components：80 parts of glucose, 10 parts of sodium acid carbonate, 8 parts of Sodium Pyruvate, people 2 parts of blood albumin, 30 parts of potassium chloride, 5 parts of anhydrous magnesium sulfate, 60 parts of sodium chloride, 8 parts of AMSP, PHA- plants 0.2 part of hemagglutinin, 0.2 part of folic acid, 0.5 part of inositol, 0.2 part of niacinamide, 20 parts of anhydrous calcium chloride, 0.1 part of ferric nitrate, 5 parts of succinic acid, 9 parts of sodium succinate, 0.2 part of D-VB5 calcium, 0.5 part of choline tartrate, 0.1 part of riboflavin, thiamine hydrochloride 0.1 Part, 0.1 part of pyridoxine hydrochloride, 0.1 part of reduced glutathione, 1 part of cholesterol, 0.5 part of ascorbic acid phosphoric acid esters, makrolon 100 parts of 0.3 part, 0.8 part of phenol red sodium and deionized water.
In addition, the basal medium also includes 3 parts of amino acid, the amino acid by weight part, by following components group Into：5 parts of L- R-genes, 3 parts of L- hydrochloric acid cystine, 3 parts of Serine, 1 part of glycine, 4 parts of L- histidine monohydrochlorides, 8 parts of ILE, 7 parts of L-Leu, 10 parts of LYS, 1 part of METHIONINE, 2 parts of L-phenylalanine, L- Soviet Unions ammonia 8 parts of 5 parts of acid, 1 part of L-Trp, 5 parts of TYR and Valine.
Again, the basal medium also includes 2 parts of confactors, the confactor by weight part, by with the following group It is grouped into：2 parts of recombination human basic fibroblast growth factor, 3 parts of interleukin-22,3 parts of IL-4,41 parts of interleukin-11, 1 part of 3 parts of IFN-γ and neuroleukin.
Further, the basal medium by weight part, in addition to 0.006 part of penicillin and 0.01 part of strepto- Element.Also, the penicillin is selected from 10000U/ml, the streptomysin is selected from 10000 μ g/ml.In addition, in the trypsase Also include 0.05% EDTA.
Embodiment 2
A kind of method of Vero cell expansions culture, comprises the following steps：Vero cells special culture media is exhausted with liquid-transfering gun, Add 5ml PBS washings cell 2 times, 2ml 0.38% trypsase is added into 10cm Tissue Culture Dish, under the conditions of 37 DEG C 2min is digested, 3ml fresh Vero cell special culture medias are added, Tissue Culture Dish bottom is blown and beaten 50 times using liquid-transfering gun；Will Liquid is moved in cell centrifuge tube, and 3min is centrifuged under the conditions of 1200g/min, and supernatant is removed with liquid-transfering gun, adds 3ml fresh Vero cell special culture medias, use and cell 50 times blown and beaten above and below liquid-transfering gun；Averagely it is added in 8 Tissue Culture Dish, enters Row expands culture.
Wherein, the Vero cells special culture media includes 90% basal medium and 10% hyclone.It is described Basal medium by weight part, including following components：90 parts of glucose, 20 parts of sodium acid carbonate, 18 parts of Sodium Pyruvate, people 10 parts of blood albumin, 50 parts of potassium chloride, 12 parts of anhydrous magnesium sulfate, 80 parts of sodium chloride, 16 parts of AMSP, PHA- plant 1 part of thing hemagglutinin, 0.6 part of folic acid, 1.5 parts of inositol, 0.8 part of niacinamide, 40 parts of anhydrous calcium chloride, 0.5 part of ferric nitrate, 10 parts of succinic acid, 18 parts of sodium succinate, 0.8 part of D-VB5 calcium, 1 part of choline tartrate, 0.3 part of riboflavin, thiamine hydrochloride 0.5 Part, 0.6 part of pyridoxine hydrochloride, 0.8 part of reduced glutathione, 5 parts of cholesterol, 1.2 parts of ascorbic acid phosphoric acid esters, makrolon 100 parts of 1.5 parts, 1.5 parts of phenol red sodium and deionized water.
In addition, the basal medium also includes 8 parts of amino acid, the amino acid by weight part, by following components group Into：10 parts of L- R-genes, 8 parts of L- hydrochloric acid cystine, 6 parts of Serine, 5 parts of glycine, 6 parts of L- histidine monohydrochlorides, 20 parts of ILE, 18 parts of L-Leu, 20 parts of LYS, 6 parts of METHIONINE, 8 parts of L-phenylalanine, L- Soviet Unions 12 parts of 15 parts of propylhomoserin, 3 parts of L-Trp, 9 parts of TYR and Valine.
Again, the basal medium also includes 5 parts of confactors, the confactor by weight part, by with the following group It is grouped into：10 parts of recombination human basic fibroblast growth factor, 8 parts of interleukin-22,6 parts of IL-4,45 parts of interleukin-11, 4 parts of 6 parts of IFN-γ and neuroleukin.
Further, the basal medium by weight part, in addition to 0.006 part of penicillin and 0.01 part of strepto- Element.Also, the penicillin is selected from 10000U/ml, the streptomysin is selected from 10000 μ g/ml.In addition, in the trypsase Also include 0.05% EDTA.
Embodiment 3
A kind of method of Vero cell expansions culture, comprises the following steps：Vero cells special culture media is exhausted with liquid-transfering gun, Add 4ml PBS washings cell 2-3 times, 1ml 0.38% trypsase is added into 10cm Tissue Culture Dish, in 37 DEG C of conditions Lower digestion 3min, adds 3ml fresh Vero cell special culture medias, and Tissue Culture Dish bottom is blown and beaten 45 times using liquid-transfering gun； Liquid is moved in cell centrifuge tube, 4min is centrifuged under the conditions of 1100g/min, supernatant is removed with liquid-transfering gun, 3ml is added new Fresh Vero cell special culture medias, use piping and druming cell 45 times above and below liquid-transfering gun；Averagely it is added in 6 Tissue Culture Dish, It is enlarged culture.
Wherein, the Vero cells special culture media includes 80% basal medium and 20% hyclone.It is described Basal medium by weight part, including following components：86 parts of glucose, 18 parts of sodium acid carbonate, 12 parts of Sodium Pyruvate, people 6 parts of blood albumin, 40 parts of potassium chloride, 8 parts of anhydrous magnesium sulfate, 75 parts of sodium chloride, 12 parts of AMSP, PHA- plant blood 0.5 part of ball agglutinin, 0.4 part of folic acid, 1 part of inositol, 0.5 part of niacinamide, 30 parts of anhydrous calcium chloride, 0.3 part of ferric nitrate, fourth two Acid 7 parts, 14 parts of sodium succinate, 0.5 part of D-VB5 calcium, 0.7 part of choline tartrate, 0.2 part of riboflavin, 0.3 part of thiamine hydrochloride, 0.4 part of pyridoxine hydrochloride, 0.5 part of reduced glutathione, 3 parts of cholesterol, 0.8 part of ascorbic acid phosphoric acid esters, makrolon 0.8 100 parts of part, 1.2 parts of phenol red sodium and deionized water.
In addition, the basal medium also includes 4 parts of amino acid, the amino acid by weight part, by following components group Into：8 parts of L- R-genes, 6 parts of L- hydrochloric acid cystine, 5 parts of Serine, 3 parts of glycine, 5 parts of L- histidine monohydrochlorides, 12 parts of ILE, 10 parts of L-Leu, 15 parts of LYS, 3 parts of METHIONINE, 4 parts of L-phenylalanine, L- Soviet Unions 9 parts of 9 parts of propylhomoserin, 2 parts of L-Trp, 6 parts of TYR and Valine.
Again, the basal medium also includes 3 parts of confactors, the confactor by weight part, by with the following group It is grouped into：8 parts of recombination human basic fibroblast growth factor, 6 parts of interleukin-22,4 parts of IL-4,43 parts of interleukin-11, 2 parts of 5 parts of IFN-γ and neuroleukin.
Further, the basal medium by weight part, in addition to 0.006 part of penicillin and 0.01 part of strepto- Element.Also, the penicillin is selected from 10000U/ml, the streptomysin is selected from 10000 μ g/ml.In addition, in the trypsase Also include 0.05% EDTA.
Embodiment 4
A kind of method of Vero cell expansions culture, comprises the following steps：Vero cells special culture media is exhausted with liquid-transfering gun, Add 4ml PBS washings cell 2-3 times, 2ml 0.38% trypsase is added into 10cm Tissue Culture Dish, in 37 DEG C of conditions Lower digestion 2-3min, adds 3ml fresh Vero cell special culture medias, and Tissue Culture Dish bottom 50 is blown and beaten using liquid-transfering gun It is secondary；Liquid is moved in cell centrifuge tube, 3min is centrifuged under the conditions of 1200g/min, supernatant is removed with liquid-transfering gun, is added Vero cell special culture medias fresh 3ml, use piping and druming cell 40 times above and below liquid-transfering gun；Averagely it is added to 5 cell culture In ware, culture is enlarged.
Wherein, the Vero cells special culture media includes 90% basal medium and 10% hyclone.It is described Basal medium by weight part, including following components：88 parts of glucose, 12 parts of sodium acid carbonate, 12 parts of Sodium Pyruvate, people 6 parts of blood albumin, 35 parts of potassium chloride, 10 parts of anhydrous magnesium sulfate, 70 parts of sodium chloride, 14 parts of AMSP, PHA- plants 0.4 part of hemagglutinin, 0.4 part of folic acid, 0.9 part of inositol, 0.5 part of niacinamide, 28 parts of anhydrous calcium chloride, 0.3 part of ferric nitrate, 7 parts of succinic acid, 14 parts of sodium succinate, 0.5 part of D-VB5 calcium, 0.7 part of choline tartrate, 0.2 part of riboflavin, thiamine hydrochloride 0.2 Part, 0.5 part of pyridoxine hydrochloride, 0.6 part of reduced glutathione, 3 parts of cholesterol, 0.8 part of ascorbic acid phosphoric acid esters, makrolon 100 parts of 0.4 part, 1.3 parts of phenol red sodium and deionized water.
In addition, the basal medium also includes 3 parts of amino acid, the amino acid by weight part, by following components group Into：5 parts of L- R-genes, 3 parts of L- hydrochloric acid cystine, 5 parts of Serine, 3 parts of glycine, 4 parts of L- histidine monohydrochlorides, 20 parts of ILE, 7 parts of L-Leu, 20 parts of LYS, 4 parts of METHIONINE, 5 parts of L-phenylalanine, L- Soviet Unions 12 parts of 8 parts of propylhomoserin, 2 parts of L-Trp, 5 parts of TYR and Valine.
Again, the basal medium also includes 4 parts of confactors, the confactor by weight part, by with the following group It is grouped into：2 parts of recombination human basic fibroblast growth factor, 3 parts of interleukin-22,5 parts of IL-4,43 parts of interleukin-11, 4 parts of 6 parts of IFN-γ and neuroleukin.
Further, the basal medium by weight part, in addition to 0.006 part of penicillin and 0.01 part of strepto- Element.Also, the penicillin is selected from 10000U/ml, the streptomysin is selected from 10000 μ g/ml.In addition, in the trypsase Also include 0.05% EDTA.
Described above is only several embodiments of invention, it is noted that for those skilled in the art For, on the premise of inventive principle is not departed from, some improvement can also be made, these improvement also should be regarded as the protection of the present invention Scope.

Claims (9)

1. a kind of method of Vero cell expansions culture, it is characterised in that：Comprise the following steps：It is with liquid-transfering gun that Vero cells is special Exhausted with culture medium, add 3-5ml PBS washings cell 2-3 times, 1-2ml 0.38% tryptose is added into Tissue Culture Dish Enzyme, digests 2-3min under the conditions of 37 DEG C, adds 2-3ml fresh Vero cell special culture medias, is blown and beaten using liquid-transfering gun Tissue Culture Dish bottom 40-50 times；Liquid is moved in cell centrifuge tube, 3-5min is centrifuged under the conditions of 1000-1200g/min, Supernatant is removed with liquid-transfering gun, the fresh Vero cell special culture medias of 2-3ml are added, using blowing and beating cell above and below liquid-transfering gun 40-50 times；Averagely it is added in 3-8 Tissue Culture Dish, is enlarged culture.
2. the method for Vero cell expansions culture according to claim 1, it is characterised in that：Also wrapped in the trypsase Containing 0.05% EDTA.
3. the method for Vero cell expansions culture according to claim 1 or 2, it is characterised in that：The Vero cells are special Include 70-90% basal medium and 10-30% hyclone with culture medium.
4. the method for Vero cell expansions culture according to claim 3, it is characterised in that：The basal medium is with weight Measure component meter, including following components：80-90 parts of glucose, 10-20 parts of sodium acid carbonate, 8-18 parts of Sodium Pyruvate, the white egg of human blood White 2-10 parts, 30-50 parts of potassium chloride, 5-12 parts of anhydrous magnesium sulfate, 60-80 parts of sodium chloride, AMSP 8-16 Part, 0.2-1 parts of PHA- phytohemagglutinins, 0.2-0.6 parts of folic acid, 0.5-1.5 parts of inositol, 0.2-0.8 parts of niacinamide, nothing 20-40 parts of water calcium chloride, 0.1-0.5 parts of ferric nitrate, 5-10 parts of succinic acid, 9-18 parts of sodium succinate, D-VB5 calcium 0.2-0.8 Part, 0.5-1 parts of choline tartrate, 0.1-0.3 parts of riboflavin, 0.1-0.5 parts of thiamine hydrochloride, 0.1-0.6 parts of pyridoxine hydrochloride, 0.1-0.8 parts of reduced glutathione, 1-5 parts of cholesterol, 0.5-1.2 parts of ascorbic acid phosphoric acid esters, 0.3-1.5 parts of makrolon, Phenol red sodium 0.8-1.5 parts and 100 parts of deionized water.
5. the method for Vero cell expansions culture according to claim 4, it is characterised in that：The basal medium is also wrapped 3-8 parts of amino acid are included, the amino acid is by weight part, composed of the following components：5-10 parts of L- R-genes, L- salt Sour cystine 3-8 parts, 3-6 parts of Serine, 1-5 parts of glycine, 4-6 parts of L- histidine monohydrochlorides, 8-20 parts of ILE, 7-18 parts of L-Leu, 10-20 parts of LYS, 1-6 parts of METHIONINE, 2-8 parts of L-phenylalanine, L-threonine 5- 8-12 parts of 15 parts, 1-3 parts of L-Trp, 5-9 parts of TYR and Valine.
6. the method for Vero cell expansions culture according to claim 5, it is characterised in that：The basal medium is also wrapped 2-5 parts of confactors are included, the confactor is by weight part, composed of the following components：Recombination human basic fibroblast cell - 10 parts of growth factor-2,3-8 parts of interleukin-22,3-6 parts of IL-4,1-5 parts of interleukin-11 4,3-6 parts of IFN-γ and nerve are white 1-4 parts of cytokine.
7. the method for Vero cell expansions culture according to claim 6, it is characterised in that：By weight part, it is described Basal medium also includes 0.006 part of penicillin and 0.01 part of streptomysin.
8. the method for Vero cell expansions culture according to claim 7, it is characterised in that：The penicillin is selected from 10000U/ml, the streptomysin is selected from 10000 μ g/ml.
9. the method for Vero cell expansions culture according to claim 1, it is characterised in that：The Tissue Culture Dish is 10cm Tissue Culture Dish.
CN201710524381.7A 2017-06-30 2017-06-30 A kind of method of Vero cell expansions culture Pending CN107151649A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019056217A1 (en) * 2017-09-20 2019-03-28 苏州北开生化设备有限公司 Method for cryopreserving vero cells with high viability

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