CN105861415A - Serum-free medium used for suspension culture of insect cells, and application thereof - Google Patents
Serum-free medium used for suspension culture of insect cells, and application thereof Download PDFInfo
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Abstract
A serum-free medium used for suspension culture of insect cells includes 5-10g/L of carbohydrates, 1-5g/L of amino acids, 5-10g/L of an inorganic salt, 0.01-0.05g/L of vitamins, 5-10g/L of yeast extract and 10-20ml/L of a lipid emulsifier. Compared with serum component-containing media, the serum-free medium has the advantages of low cost, easy preparation, no animal pollution, and high batch stability; and compared with general commercial media, the serum-free medium has the advantages of realization of suspension culture of cells, improvement of the expression level, suitableness for large-scale production application, and facilitation of stable obtaining of recombinant proteins.
Description
Technical field
The present invention relates to culture medium field, relate to a kind of serum free medium and application thereof cultivated that suspend for insect cell.
Background technology
Cell is cultivated and is referred to be bred in vitro by cell and cultivate.The cell culture technology arising from for 20 beginnings of the century has become the important tool in medical science and biological study field, and increasing bio-pharmaceutical such as monoclonal antibody, vaccine and reactive protein etc. are all to utilize cell successful expression and production.In nearest 20 years, the invertebral zooblast with insect cell as representative cultivates the effect ever more important played in application biomedical sector.With baculoviral as carrier, insect cell expression system has been widely used in producing foreign protein, enjoys the favor of people.It addition, insect cell expression system is also considered to be the optimizer system expressing virus sample particle vaccines, the most successfully develops multiple people and use and animal vaccines.
Large-scale Cell culture invitro needs provide the favorable environment being suitable for cell growth, including nutritional labeling, temperature, pH, osmotic pressure, oxygen and blake bottle or specific cultivation interface etc..Good culture medium can provide nutritional labeling, suitable pH and the osmotic pressure needed for cell growth, is factor the most key during cell is cultivated.
Traditional commercialization cell culture medium needs to add substantial amounts of serum or seralbumin for assisting cell to grow.High price and the serum being difficult to obtain often become important limiting factor.From the perspective of producing, animal blood serum or seralbumin is used to be also problematic in that, owing to serum existing the most certified protein so that the purifying products in downstream more complicates the most in a large number.It addition, the animal virus polluted in serum is likely to result in serious safety problem.Serum batch between quality there are differences, scientist or production engineer need to be adjusted according to this otherness, thus increase workload.
Have been developed at present some for the serum free medium cultivating insect cell, but these culture mediums generally exist high cost or be not applied for suspend cultivate.CN101988047A as submitted on May 13rd, 2010 is not suitable for suspending and cultivates, and the cell density cultivated is low.For another example, the culture medium described in JP20060185008 contains looks for little molecule additive and recombinant protein more, has the highest cost.Culture medium described in Publication No. CN1498267A contains lactalbumin hydrolysate and the yeast extract of a large amount of import, significantly improves cost.The another drawback of the culture medium developed at present is to cannot be directly used to purify on post, needs to wait other just to can be used for purifying after processing step through concentration, is unfavorable for the subsequent treatment of expression product.
Summary of the invention
It is an object of the invention to provide the serum free medium of a kind of cultivation that suspends for insect cell, to overcome, the preparation of existing culture medium is complicated, prepare complexity, the problem that production cost is high.
To achieve these goals, the present invention adopts the following technical scheme that
A kind of serum free medium of the cultivation that suspends for insect cell, it is characterised in that: described serum free medium comprises the lipid emulsifying agent of 5-10g/L carbohydrate, 1-5g/L amino acid, 5-10g/L inorganic salts, 0.01 ~ 0.05g/L vitamin, 5 ~ 10g/L yeast extract and 10-20ml/L.
nullFurther，Amino acid used can include 150-250mg/L aspartic acid、20-50mg/L threonine、40-80mg/L serine、200-300mg glutamic acid、40-80mg/L glycine、50-100mg/L valine、100-200mg/L methionine、100-200mg/L isoleucine、30-50mg/L leucine、100-200mg phenylalanine、80-140mg/L lysine、20-50mg/L histidine、100-150mg/L arginine、50-100mg/L proline、180-300mg/L asparagine、1000-1200mg/L glutamine、10-30mg/L cysteine and 30-80mg/L tyrosine.
Further, vitamin used includes 0.1-0.3mg/L biotin, 5-10mg/L pantothenic acid, 10-15mg/L nicotinic acid, 0.1-0.5mg/L p-aminobenzoic acid, 0.5-0.8mg/L vitamin B1,1-3mg/L vitamin B6,0.2-0.5mg/L vitamin B12,1-3mg/L riboflavin, 0.1-0.4mg/L folic acid, 15-20mg/L Choline Chloride.
Further, inorganic salts used can include 200-800mg/L calcium chloride, 800-1200mg/L magnesium sulfate, 500-800mg/L potassium chloride, 400-1000mg/L sodium acid carbonate, 1500-3000mg/L sodium chloride, 800-1200mg/L sodium dihydrogen phosphate；It is contained in the 0.005-0.015mg/L cobalt chloride hexahydrate of 1000X concentrate, 0.01-0.05mg/L Copper dichloride dihydrate, 0.001-0.01mg/L tetra-chloride hydrate manganese, 0.05-0.20mg/L zinc chloride, 0.1-0.2mg/L green vitriol, 0.001-0.01mg/L seven Ammonium paramolybdate tetrahydrate.
The present invention also aims to provide a kind of serum free medium cultivated that suspends for insect cell in the application cultivated in insect cell that suspends.
For insect cell suspend cultivate serum free medium be mainly composed of carbohydrate, amino acid, inorganic salts, vitamin, yeast extract and lipid emulsifying agent.
Carbohydrate such as glucose, fructose and maltose are primary carbon source and the energy of major part insect cell.In most culture mediums, glucose can be utilized rapidly as carbon source, although after baculovirus infection, the growth of insect cell also can consume part sucrose, but it to be played a major role in the medium be regulation osmotic pressure.In the culture medium of the present invention, contained carbohydrate content is at 5-8g/L, and without control to osmotic pressure in sucrose and maltose, beneficially sweat.
Different types of insect cell has different requirements to amino acid, and wherein having 14 kinds is essential amino acid, and cell itself can not synthesize, it is necessary to is provided by culture medium.
nullThe amino acid that can be used for the present invention includes，150-250mg/L aspartic acid、20-50mg/L threonine、40-80mg/L serine、200-300mg glutamic acid、40-80mg/L glycine、50-100mg/L valine、100-200mg/L methionine、100-200mg/L isoleucine、30-50mg/L leucine、100-200mg phenylalanine、80-140mg/L lysine、20-50mg/L histidine、100-150mg/L arginine、50-100mg/L proline、180-300mg/L asparagine、1000-1200mg/L glutamine、10-30mg/L cysteine and 30-80mg/L tyrosine.
The vitamin that can be used for the present invention includes, 0.1-0.3mg/L biotin, 5-10mg/L pantothenic acid, 10-15mg/L nicotinic acid, 0.1-0.5mg/L p-aminobenzoic acid, 0.5-0.8mg/L vitamin B1,1-3mg/L vitamin B6,0.2-0.5mg/L vitamin B12,1-3mg/L riboflavin, 0.1-0.4mg/L folic acid, 15-20mg/L Choline Chloride.
Basal culture medium contains 5-10g/L inorganic salts.The inorganic salts that can be used for the present invention include, 200-800mg/L calcium chloride, 800-1200mg/L magnesium sulfate, 500-800mg/L potassium chloride, 400-1000mg/L sodium acid carbonate, 1500-3000mg/L sodium chloride, 800-1200mg/L sodium dihydrogen phosphate；It is contained in the 0.005-0.015mg/L cobalt chloride hexahydrate of 1000X concentrate, 0.01-0.05mg/L Copper dichloride dihydrate, 0.001-0.01mg/L tetra-chloride hydrate manganese, 0.05-0.20mg/L zinc chloride, 0.1-0.2mg/L green vitriol, 0.001-0.01mg/L seven Ammonium paramolybdate tetrahydrate.
Yeast extract (Yeastolate, YL) is the basis of insect cell medium, and it is the main source of vitamin and purine, can comprise in the present invention, such as, and the yeast extract of 5-10g/L.
The Serum-free complete medium of the present invention also comprises other composition, such as, the lipid emulsifying agent of 5-15ml/L and the anti-foaming agent Kolliphor K188 of 1g/L.
The preparation of culture medium is the most relatively simple, and compound method is the most unique.Such as, culture medium can be prepared by method described below, and first with respective suitable concn, all the components and additives are dissolved in water, and pH is to desired value in regulation, and then pressurization makes solution obtain the aseptic culture medium without mycoplasma by a membrane filter.
In one embodiment, the invention provides a kind of for the serum free medium cultivating insect cell that suspends, described serum free medium comprises the lipid emulsifying agent of 5-10g/L carbohydrate, 1-5g/L amino acid, 5-10g/L inorganic salts, 0.01 ~ 0.05g/L vitamin, 5 ~ 10g/L yeast extract and 10-20ml/L.
In another embodiment, carbohydrate of the present invention does not comprise the polysaccharide such as sucrose and maltose.
In another embodiment, the serum free medium of the present invention is without cystine, alanine and hydroxyproline.
In another embodiment, the serum free medium of the present invention contains the glutamine of 1.0-1.2g/L.
Culture medium ingredient kind involved in the present invention is few, and content is low, and preparation is simple, and can reach the effect identical with commercialization culture medium.All the components is reagent simple and easy to get, and it is convenient to buy, with low cost.Compared with the another kind of Zooblast culture medium (Publication No. CN101988047) similar with composition, the amino acid contained composition of culture medium of the present invention substantially reduces, the yeast extract that price is higher substantially reduces, liposome is self-control, the only composition of vitamin increased, therefore the production of culture medium and prepare convenient.All using as a example by Sigma, BD and chemical reagents corporation of traditional Chinese medicines group product by all the components, the culture medium cost of the present invention import like product that compares about reduces by more than 70%, has good Commercial Prospect.Compared with external classic products, basal culture medium can be directly used for purifying on post, it is not necessary to concentrates and waits other to process step, very convenient expression product subsequent treatment.
There is compared with traditional culture medium adding serum composition low cost, be prone to preparation, the features such as non-animal derived contact scar, batch are stable, compared with general commercialization culture medium, energy suspended culture cell, improve expression, it is more suitable for large-scale production and application, beneficially the stable acquisition of recombinant protein.
Accompanying drawing explanation
Fig. 1: homemade NB-SFM culture medium suspends with SF900 III culture medium and cultivates SF9 cell, contrasts culture effect figure
Fig. 2: homemade NB-SFM culture medium suspends with commercialization culture medium and cultivates High Five cell, contrasts culture effect figure.
Detailed description of the invention
Below for further describe the present invention in conjunction with specific embodiments, these embodiments are only exemplary, the scope of the present invention are not constituted any restriction.The details of technical scheme and form can be modified or replace lower without departing from the spirit and scope of the present invention; but these amendments and replacement all should be contained within protection scope of the present invention; embodiment of the present invention agents useful for same; such as non-specified otherwise; it is purchased from biochemical reagents supplier; experimental technique used, if not otherwise specified, is routine techniques.
The named NB-SFM of culture medium of the present invention.The following example, for illustrating the certain preferred embodiments of the present invention, should not be construed as the scope limiting it.In embodiment, amino acid is purchased from TCI, inorganic salts and vitamin and is purchased from Sigma and BD purchased from traditional Chinese medicines company and Sigma, other composition.Commercialization culture medium the most used is SF900III and Express Five, purchased from Life
Technologies company.Cell line SF9 and High
Five is purchased from ATCC.HISF cell is built by this laboratory.In cell culture experiments, cell density is counted by blood counting chamber, and cytoactive is determined by trypan blue staining.
Embodiment 1: the preparation of serum free medium of the present invention
(1) weigh
Raw material is accurately weighed according to table 1, table 2, table 3:
Table 1 amino acid
Table 2 vitamin
Table 3 inorganic salts
Table 4 lipid emulsifying agent
After table 4 ingredients listed weighs, with 1mL ethanol, mentioned component is dissolved, be slowly added dropwise 9mL water under conditions of then reheating (45-50 DEG C), be allowed to be slowly formed emulsion, before using, add culture medium.
Other composition of table 5
(2) dissolve
By raw material (table 1, table 2, table 3, table 5) it is put in a big beaker, taking 800mL ultra-pure water to be put in beaker, stirring is until dissolving completely, and the solution made by table 4 adds in beaker, with the NaOH regulation medium pH of 10M to 6.3, add ultra-pure water and make cumulative volume reach 1000mL.
Embodiment 2: culture medium of the present invention compares with import like product culture effect
Conventionally, with homemade NB-SFM culture medium and SF900
III culture medium suspends and cultivates SF9 cell, contrast culture effect (accompanying drawing 1).As can be seen from Figure 1, in same incubation time, two kinds of culture medium SF9 viable cell densities are consistent, do not have notable difference.
Also according to conventional method, cultivate High with homemade NB-SFM culture medium with the suspension of commercialization culture medium
Five cell, contrast culture effect (accompanying drawing 2).As can be seen from Figure 2, in same incubation time, two kinds of culture medium High Five viable cell densities are consistent, do not have notable difference.
Embodiment 3: self-made medium and the metainfective insect cell of commercialization medium culture
Conventionally, after the SF9 cell 96h after utilizing self-made medium and commercialization serum free medium to cultivate baculovirus infection, measure virus titer, compare the culture effect (table 6) of two kinds of culture mediums.As shown in Table 6, self-control serum free medium virus titer is apparently higher than commercialization culture medium.
Table 6 baculovirus infection cell cultivates the comparison of 96h restrovirus titre
Embodiment 4: self-made medium and commercialization medium culture expression of influenza hemagglutinin compare
Conventionally, utilize self-made medium and commercialization serum free medium to cultivate influenza hemagglutinin expressing protein cell, measure after collecting albumen and compare Hemagglutination titer.Two kinds of medium culture effects (table 7) are determined according to Hemagglutination titer.As shown in Table 7, self-control serum free medium Hemagglutination titer is apparently higher than commercialization culture medium.
The comparison of table 7 influenza virus hemagglutinin protein expression
| The culture medium used | Hemagglutination titer |
| Commercialization serum free medium | 128 |
| NB-SFM serum free medium | 256 |
Although some specific embodiments that schematically illustrate above illustrate and describe the present invention, but are not meant to present invention is limited only by various details therein.On the contrary, various amendment can be made in various details without departing from present invention spirit in the category being equivalent to claims and scope.
Claims (5)
1. the serum free medium cultivated for insect cell suspension, it is characterised in that described serum free medium comprises the lipid emulsifying agent of 5-10g/L carbohydrate, 1-5g/L amino acid, 5-10g/L inorganic salts, 0.01 ~ 0.05g/L vitamin, 5 ~ 10g/L yeast extract and 10-20ml/L.
nullThe serum free medium of the cultivation that suspends for insect cell the most according to claim 1，It is characterized in that，Amino acid used can include 150-250mg/L aspartic acid、20-50mg/L threonine、40-80mg/L serine、200-300mg glutamic acid、40-80mg/L glycine、50-100mg/L valine、100-200mg/L methionine、100-200mg/L isoleucine、30-50mg/L leucine、100-200mg phenylalanine、80-140mg/L lysine、20-50mg/L histidine、100-150mg/L arginine、50-100mg/L proline、180-300mg/L asparagine、1000-1200mg/L glutamine、10-30mg/L cysteine and 30-80mg/L tyrosine.
The serum free medium of the cultivation that suspends for insect cell the most according to claim 1, it is characterized in that, vitamin used includes 0.1-0.3mg/L biotin, 5-10mg/L pantothenic acid, 10-15mg/L nicotinic acid, 0.1-0.5mg/L p-aminobenzoic acid, 0.5-0.8mg/L vitamin B1,1-3mg/L vitamin B6,0.2-0.5mg/L vitamin B12,1-3mg/L riboflavin, 0.1-0.4mg/L folic acid, 15-20mg/L Choline Chloride.
The serum free medium of the cultivation that suspends for insect cell the most according to claim 1, it is characterized in that, inorganic salts used can include 200-800mg/L calcium chloride, 800-1200mg/L magnesium sulfate, 500-800mg/L potassium chloride, 400-1000mg/L sodium acid carbonate, 1500-3000mg/L sodium chloride, 800-1200mg/L sodium dihydrogen phosphate；It is contained in the 0.005-0.015mg/L cobalt chloride hexahydrate of 1000X concentrate, 0.01-0.05mg/L Copper dichloride dihydrate, 0.001-0.01mg/L tetra-chloride hydrate manganese, 0.05-0.20mg/L zinc chloride, 0.1-0.2mg/L green vitriol, 0.001-0.01mg/L seven Ammonium paramolybdate tetrahydrate.
5. the serum free medium cultivated that suspends for insect cell as described in claim 1-4 is in the application cultivated in insect cell that suspends.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834203A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of insect cell medium |
| CN107603941A (en) * | 2017-09-14 | 2018-01-19 | 丙申南京网络技术有限公司 | A kind of high-effect serum additive and preparation method thereof and application method |
| CN110564670A (en) * | 2019-09-04 | 2019-12-13 | 广州今成生物科技有限公司 | Insect cell serum-free culture medium and preparation process thereof |
| CN111304144A (en) * | 2019-11-30 | 2020-06-19 | 河南普诺易生物制品研究院有限公司 | Insect cell culture medium and preparation method thereof |
| CN114250191A (en) * | 2020-09-23 | 2022-03-29 | 上海倍谙基生物科技有限公司 | Serum-free culture medium suitable for high-density culture and high-expression of insect cells |
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| WO2001098517A2 (en) * | 2000-06-23 | 2001-12-27 | Basf Aktiengesellschaft | Cost effective media for large scale insect cell culture |
| CN101988047A (en) * | 2010-05-13 | 2011-03-23 | 维亚生物科技(上海)有限公司 | Insect cell serum-free medium with low cost |
| CN104726392A (en) * | 2013-12-18 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Method for preparing serum-free cultured suspension mammal cell line, prepared cell line thereof and application thereof |
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| WO2001098517A2 (en) * | 2000-06-23 | 2001-12-27 | Basf Aktiengesellschaft | Cost effective media for large scale insect cell culture |
| CN101988047A (en) * | 2010-05-13 | 2011-03-23 | 维亚生物科技(上海)有限公司 | Insect cell serum-free medium with low cost |
| CN104726392A (en) * | 2013-12-18 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Method for preparing serum-free cultured suspension mammal cell line, prepared cell line thereof and application thereof |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834203A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of insect cell medium |
| CN107603941A (en) * | 2017-09-14 | 2018-01-19 | 丙申南京网络技术有限公司 | A kind of high-effect serum additive and preparation method thereof and application method |
| CN110564670A (en) * | 2019-09-04 | 2019-12-13 | 广州今成生物科技有限公司 | Insect cell serum-free culture medium and preparation process thereof |
| CN111304144A (en) * | 2019-11-30 | 2020-06-19 | 河南普诺易生物制品研究院有限公司 | Insect cell culture medium and preparation method thereof |
| CN111304144B (en) * | 2019-11-30 | 2023-04-07 | 河南普诺易生物制品研究院有限公司 | Insect cell culture medium and preparation method thereof |
| CN114250191A (en) * | 2020-09-23 | 2022-03-29 | 上海倍谙基生物科技有限公司 | Serum-free culture medium suitable for high-density culture and high-expression of insect cells |
| CN114250191B (en) * | 2020-09-23 | 2023-03-24 | 上海倍谙基生物科技有限公司 | Serum-free culture medium suitable for high-density culture and high-expression of insect cells |
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