CN102827804B - Culture medium and method suitable for the amplification culture of Vero cell microcarrier suspension - Google Patents
Culture medium and method suitable for the amplification culture of Vero cell microcarrier suspension Download PDFInfo
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Abstract
The invention discloses a kind of culture mediums made for the amplification culture of Vero cell microcarrier suspension, including amino acid, inorganic salts, vitamin, protolysate, lipid, buffer composition and additive.Through the above way, the present invention can be not only suitable for Vero square vase stationary culture and roller bottle culture in the case where adding trace serum profiles, also microcarrier suspension culture and the amplification culture of reactor microcarrier are supported, serum-free virus maintaining liquid is also used as and realizes serum-free virus production.
Description
Technical field
The present invention relates to cell engineering fields, more particularly to one kind for supporting Vero cell adherent growth and branch
The culture medium and application method for holding virus multiplication, it is outstanding more particularly for Vero cell microcarrier suspension culture and microcarrier
The culture medium of float gauge mould amplification culture and the method for realizing pilot scale culture using the feeding base.
Background technique
Vero cell origin is clear, and history understands, is the World Health Organization (WHO) and I without oncogenicity in certain generation
State's biological products regulation ratifies the cell line that can be used for the mankind and beasts production of vaccine, is that viral vaccine production at this stage is main
Cellular matrix.Vero cell is sensitive to a variety of viruses, and virus multiplication titre is high.The country is various at present is made a living with Vero cell
The biological products industry for producing medium flourishes, and more and more enterprises are reached the requirement commercially produced, this is just needed
Largely for the cell culture medium of production, while also needing the in vitro culture of certain scale.
However, most of conventional mediums need to add 10% or so cow's serum, serum purchase and storage it is at high cost,
Importantly, the serum of addition high concentration considerably increases the pressure of downstream product purifying.The complexity of serum component and its
It is middle there are the potential risk of the microbiological contaminations such as virus, increase the unstability and control of product quality of production
Difficulty.It undeniably, the use of serum free medium is the preferable approach for avoiding the above risk, external also there are many commercializations
The listing of Vero cell non-serum culture medium, such as the EX-CELLTMVERO of JRH company, the VP-SFM of Invitrogen company and
OptiPro SFM etc..But serum free medium price is high, and Lead Time is long, and great amount of cost is increased for enterprise, reduces it
The market competitiveness；In addition, cell by serum-containing media is transferred to serum free medium, there are adaptation process, need according to cell strain
Characteristic attempt different serum-free production mediums, cell non-serum meets the needs of the time in 4~5 generations, under normal circumstances carefully
After born of the same parents cultivated for 8 generations in serum free medium, growth can be slack-off.The foundation in serum-free cell library is also a problem.
When being related to the scale amplification of reactor micro-carrier system, will be faced using the production line of serum free medium bigger
The problem of.The amplification of anchorage-dependent cells needs the digestion of trypsase, eliminates serum, how to neutralize the pancreas in culture medium
Protease is that technical staff will urgently solve.Existing frequently-used method is to add the trypsin inhibitor of plant origin, such as
Soybean trypsin inhibitor.But trypsin inhibitor is easy inactivation, condition of storage is harsh；More importantly largely make
Do not allowed in trypsin inhibitor cost.In addition, the growth of cell can be inhibited using pancreatin inhibitor for a long time.
The serum addition for reducing cell growth phase, is preferably down to trace (1% (v/v) is below) for serum-concentration and answers
This is feasible method.The present invention solves existing technical problem by providing a kind of inexpensive cell culture medium.That is Vero
Cell growth phase adds trace serum, and Vero can be supported to grow in square vase, roller bottle, and can support that Vero cell microcarrier is outstanding
Floating culture and the amplification culture of microcarrier suspension scale.In addition basal culture medium can be used as viral maintaining liquid, realize viral serum-free
Production.
Process scale based on micro-carriers cell culture production vaccine has reached 1000L or more [1-5].Scaling -up arrives
The basic transfer mode of next stage reactor has digestion transfer and ball to turn ball.
Ball turns new microcarrier to be added by a certain percentage, and take after ball refers to cell confluency on microcarrier at single layer
Certain measures (such as slow-speed of revolution stirring, intermittent stirring) promote cell to migrate from old microcarrier to new microcarrier and realize amplification.
Ball turns ball amplification process can directly add new microcarrier in former reactor, increase microcarrier concentration；It can also will be with
The old ball of cell is squeezed into next stage reactor, is added after new microcarrier and is diluted microcarrier to original concentration with fresh culture.
Although existing scholar reports the successful case [6] that ball turns ball amplification, require the transfer ability of cell strong, and on old ball
Cell is easy to grow old, therefore limits its extensive use.
Digestion transfer refers to is digested cell with trypsase from microcarrier, and cell or cell and micro- load
Body accesses in next stage reactor, adds the micro-carriers cell culture of a beginning new round after new microcarrier and fresh culture.From
Digestion reactor is added between two-stage reactor as long as saying on operating level, digestion transfer is suitable for most microcarriers and puts
Big technique.However more and more scholars find in practice microcarrier culture serial passage be it is sufficiently complex, with biography
Algebra increases, and growth rate of the cell on microcarrier sharply declines.Van etc. reports a kind of MK cells and uploads in microcarrier
For when cell most high-density be gradually reduced, when reaching the 6th generation or seven generations cell stop proliferation [7].Hu etc. also reports similar
The phenomenon that, FS-4 cell is successfully digested from microcarrier with trypsase and is inoculated on new microcarrier by they, knot
Fruit discovery cell has doubled 2.2 times in the first generation, and only doubles 1.5 times [8] in the third generation.It cannot be maintained in succeeding generations steady
Fixed cell proliferation rate has become the big obstacle that microcarrier is applied to mass cell culture.
Two-phase method production biological products are widely used in the country: the basal mediums such as cell growth phase MEM, M199 add
Add 10%NBCS, to cell confluency at single layer, washs removal serum through PBS；Production phase is added appropriate white with basal medium
The production of albumen or a small amount of serum as cell maintenance medium, for product.
Firstly, since being added to a large amount of serum in cell growth phase, it is residual to might have excessive serum when being washed with PBS
It stays and causes product unqualified, be very cumbersome and tired to the washing of upper kilolitre cultivating system especially in large-scale production process
Difficult.
Secondly, an important factor for serum differences between batches are very much difference between causing biological products batch greatly, 10%NBCS's adds
Add unstable between undoubtedly considerably increasing batch.
Again, the production phase removes or after serum-concentration is greatly lowered, and is difficult to keep good cell state for a long time,
To significantly affect the yield and quality of product.
Complete serum-free production: self-developing or directly purchase commercialization serum free medium grow into production from cell
Product production all uses serum free medium.
Firstly, serum free medium development difficulty is big, development cost are high.
Secondly, commercialization serum free medium price is high, Lead Time is long, and cell strain universality is poor.
Again, attached cell needs trypsin digestion that could pass on, and loses the neutralization of serum, trypsase meeting
To cell damage, the activity of pancreatin can be inhibited using pancreatin inhibitor (plant polypeptide), but pancreatin inhibitor itself
There is certain toxic action to cell, is difficult to realize the amplification of micro-carrier system culture scale.
The starting point of most conventional cell culture medium is a kind of basic basal medium, generally comprise amino acid, carbohydrate,
Vitamin, inorganic salts, microelement, buffer.For the culture medium of most conventional, by animal blood serum appropriate or blood
Pure albumen and lipid components are added in the basic basal medium and are made and grow and breed, again for sertoli cell
Complete medium necessary to histone expression and virus replication or viral product are expressed.In view of the safety and batch of serum
Between stability problem, in the prior art with other egg white substitutes replace serum composition in terms of made some attempts.
For example, Chinese patent 200710187466 discloses a kind of serum free medium, it is by 1) conventional basal medium, 2) lipid
Mixture, 3) recombinant protein and growth factor composition, wherein it is raw to be added to liposoluble vitamin, transferrins, insulin, epidermis
Recombinant proteins, polypeptide and the hormones such as the long factor, prostaglandin, triiodo thyroid hormone.Although may be implemented in the culture medium
Vero cell single-cell suspension culture, but maximum cell density is cultivated only up to 2.1 × 106 cell numbers/milliliter with basis
Cell density is consistent when base adds 10% cow's serum adhere-wall culture, illustrates that complete serum free suspension culture cannot be supported carefully well
Intracellular growth.Although realizing Vero cell non-serum culture, wherein adding ingredient is very expensive, considerably increases culture medium
Cost adds the cost of 10% cow's serum considerably beyond basal medium, and be unable to sertoli cell grows to sufficiently high level
For economic production.Therefore, at the performance of culture medium and two aspect of totle drilling cost, there is still a need for improvement.Illustrate to get rid of completely at this stage
Good Vero cell culture effect can not be obtained by abandoning serum.
Chinese patent 201110231805.3 describes Vero cell non-serum microcarrier suspension culture method, uses
Culture medium is commercialization culture medium.Although realizing Vero cell non-serum microcarrier suspension culture, 5 liters are only realized
Total volume bioreactor culture is cultivated without reference to amplification, and it is only realized in 2 grams per liter cytodex1 microcarriers
1.2 × 106 cell numbers of maximum cell density/milliliter.
Summary of the invention
Amplify culture suitable for Vero cell microcarrier suspension the invention mainly solves the technical problem of providing a kind of
Culture medium and method can be not only suitable for Vero square vase stationary culture and roller bottle culture in the case where adding trace serum profiles, also prop up
Microcarrier suspension culture and the amplification culture of reactor microcarrier are held, serum-free virus maintaining liquid is also used as and realizes serum-free disease
Poison production.
In order to solve the above technical problems, one technical scheme adopted by the invention is that: it provides a kind of suitable for Vero cell
The culture medium of microcarrier suspension amplification culture, it is characterised in that: including amino acid, inorganic salts, vitamin, protolysate, rouge
Class, buffer composition and additive (ratio or overall ratio range that several substances please be provide).
Preferably, the content of the amino acid is calculated as with mg/litre:
Preferably, the content of the vitamin is calculated as with mg/litre:
Preferably, the content of the inorganic salts is calculated as with mg/litre:
Preferably, the hydrolysate content is calculated as with mg/litre:
Soy hydrolyzate 500~6000
Preferably, the content of the lipid is calculated as with mg/litre:
Linoleic acid 0.01~0.1
Blocked polyethers F68 500~1000.
Preferably, the content of the buffer is calculated as with mg/litre:
Sodium bicarbonate 2400~2800
Hydroxyethyl piperazineethanesulfonic acid 2000~3800.
Preferably, the content of the additive is calculated as with mg/litre:
Preferably, the culture medium can also contain other water soluble ingredients, including insulin, for that can reinforce carbohydrate
Absorption；Transferrin, for transporting iron；Trace element such as selenium, as peroxidization protective agent；Ethanol amine, as lipid
Precursor；Steroid hormone such as cortisol；Thyroid hormone such as triiodo thryonine；Nucleic acid precurser such as hypoxanthine, thymus gland is phonetic
Pyridine deoxyribonucleoside, desoxyadenossine and deoxycytidine, and in the conventional culture medium for cell culture supplemented with serum or
Other nutritional ingredients contained in serum free medium.
It is a kind of for Vero bioreactor microcarrier culture and to realize the Bioreactor scaleup works of various ways
Skill amplifies the culture medium cultivated adhere-wall culture Vero T-25 tissue culture flasks using suitable for Vero cell microcarrier suspension
Cell will be used in T-25 tissue culture flasks with the Vero cell of the MEM culture medium culture containing 10% (v/v) newborn bovine serum
The trypsin digestion of 0.25% (w/v) 8 minutes；
Contain the low blood serum medium of 1% (v/v) newborn bovine serum listed by following table 1 respectively and contains 10% (v/v) newly
Cell is resuspended in the MEM culture medium of raw cow's serum, and adjustment cell density is 2.5 × 105Cell number/milliliter.It is inoculated in by 5ml/ bottles
T-25 tissue culture flasks, 37 DEG C, 5%CO2Static gas wave refrigerator is observed cellular morphology with inverted microscope and is photographed to record.Culture 72
After hour, Vero cell contains the low blood serum medium of 1% (v/v) newborn bovine serum in the present invention and contains 10% (v/v) new born bovine
Monolayer is merged into the MEM culture medium of serum；
Table 1
| Ingredient | Concentration |
| Calcium chloride | 116 mg/litres |
| 50 × main salt | 20 ml ls |
| 10000 × secondary salt | 0.1 ml l |
| 200 × vitamin and other substances | 5 ml ls |
| Glucose | 4500 mg/litres |
| Glutamine | 484 mg/litres |
| 10 × amino acid | 100 ml ls |
| 10%Pluronic F-68 | 10 ml ls |
| Sodium bicarbonate | 2600 mg/litres |
| Soy hydrolyzate | 2000 mg/litres |
| Recombinant human epidermal growth factor | 0.005 mg/litre |
It preferably, further include that 5 liters of bioreactor digestion amplifications of micro-carrier system are cultivated to 25 liters of bioreactors,
Microcarrier suspension culture Vero cell in 5 liters of reactors was cultivated to 72 hours, cells trypsinised on microcarrier,
New microcarrier is added, squeezes into 25 liters of bioreactors and cultivates after the cell digested and microcarrier are mixed, working volume
It is 18 liters；25 liters of reactor reaction device parameter settings: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturated air, stirring
Speed is 35 revs/min；By once digesting amplifying operation, Vero cell grows normally in 5 liters of reactors, cultivates 96 hours
Cell still forms fine and close single layer, and cell density reaches 6.5 × 10 when cultivating 144 hours6Cell number/milliliter, most high-density
Reduction may be to digest microcarrier loss in transfer operation to cause.
It preferably, further include that 25 liters of bioreactor balls of micro-carrier system turn ball technique, the microcarrier in 5 liters of reactors
Suspension Cultivation of Vero was cultivated to 60 hours, and cell will grow up to single layer, with the old and new's microcarrier ratio 3: 1 into culture medium
New microcarrier is added, is mixed with the old microcarrier for covering with cell, volume of culture is constant；Initial stage stir within intermittent stirring 30 minutes
3 minutes, speed of agitator was 30 revs/min, was cultivated 6 hours, the attaching situation of sampling observation cell, subsequent to be continuously stirred；
Reactor parameter setting when continuously stirring: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturated air, mixing speed is
60 revs/min；Vero cell can be migrated from new microcarrier to old microcarrier, and culture in 5 liters of reactors is transferred to 25 liters of lifes
In object reactor and culture solution is added to 11 liters, and entire incubation continues 200 hours, and highest cell density reaches 6.5 × 106
Cell number/milliliter.
A kind of serum free medium, including amino acid, inorganic salts, vitamin, carbohydrate etc., wherein joined protolysate
Ingredient, lipid components and protective ingredient, these substances together provide basic necessary to sertoli cell life, growth and breeding
Nutritional ingredient.
Preferably, described also includes protolysate ingredient, which not only compensates for amino in culture medium
The content of acid, additionally provides the substitute to serum or albumin.
Preferably, the protolysate is commercially available, including lactalbumin hydrolysate, soy hydrolyzate,
Casein hydrolysate, any one of tryptone phosphoric acid meat soup and plant hydrolyzed object.
It is produced the beneficial effects of the present invention are: conventional preparation method can be used in the present invention, application method is also
Conventional method；Of the invention is suitable for Vero cell square vase, roller bottle culture, microcarrier suspension culture and the amplification of microcarrier suspension scale
The low-cost culture medium of culture, add trace serum after can be with cell under the conditions of the static and microcarrier suspension culture of sertoli cell
Growth, and can support virus production under serum-free environment.Later period cell maintenance medium is free of serum, and component is clear, is conducive to
Product isolates and purifies, and improves product quality, not only can avoid various unfavorable factors of serum free culture system, but also can eliminate roller bottle patch
Wall culture is difficult to the problem of scale amplification；Vero cell long-period secondary culture is supported, without long-term and complex adaptation process；Energy
Good support Vero cell microcarrier suspension culture and the amplification culture of microcarrier suspension scale；It prepares to use and be easy, and cost
It is cheap, it is suitable for powder culture medium and prepares, and utilized for the large-scale production of biological products, improves the production of Vero cell culture
Efficiency has good application value and huge market prospects.
Detailed description of the invention
Fig. 1 is the result of the present invention with the prior art serum free medium GIBCO VP-SFM and OptiPRO comparison culture
Schematic diagram；
Fig. 2 is schematic diagram of the present invention using the result of culture medium culture under commercialization the same terms；
Fig. 3 A is the cell schematic diagram that Vero cell of the present invention is cultivated 72 hours in MEM+10%NBCS；
Fig. 3 B is the cell schematic diagram that Vero cell of the present invention is cultivated 72 hours in low blood serum medium of the invention；
Fig. 4 is that the cell that Vero cell is cultivated 96 hours on 1 microcarrier of Cytodex in 25 liters of L reactors of the invention shows
It is intended to.
Specific embodiment
The preferred embodiments of the present invention will be described in detail with reference to the accompanying drawing, so that advantages and features of the invention energy
It is easier to be readily appreciated by one skilled in the art, so as to make a clearer definition of the protection scope of the present invention.
Low blood serum medium of the invention includes that inorganic salts, amino acid, vitamin, microelement, carbohydrate etc. pass
The substance usually added in system culture medium, also contains the uncommon microelement such as selenium and a small amount of epidermal growth factor (EGF).
In addition, the present invention has adjusted the mixed proportion of common inorganic salts, amino acid, vitamin.This is because low blood serum medium is opened
Hair usually requires to consider following factor:
(1) inorganic salts anchorage-dependent cell needs to be attached to support surface and could be proliferated after extending, calcium and magnesium ion
It is the co-factor of many adhesion proteins, is played an important role during cell attachment.Increasing calcium and magnesium ion concentration can promote
It is adherent into cell, but excessive concentration cell is easy conglomeration, is unfavorable for cell adherent growth instead.Developing low blood serum medium
In the process, the optimization of calcium and magnesium ion concentration is very important.
(2) amino acid animal cell to amino acid be in great demand and also each cell strain is to the preferences of amino acid classes
Difference, wherein the consumption of several essential amino acids is generally all larger, such as lysine, leucine, isoleucine, methionine.It fills
Amino acid supply that is sufficient and balancing can alleviate the generation [9] of lactic acid and ammonia.Glutamine with the adherent related substances of cell
Play a significant role in synthesis [10], the adherent ability of cell declines to a great extent under low serum condition, therefore the appropriate of glutamine adds
Adding is necessary.In addition, the reduction of serum will increase demand [11] of the cell to amino acid, so low blood serum medium
Development process in amino acid there is very important status.
(3) growth metabolism of vitamin cell is substantially a series of chemical reactions regulated and controled by various enzymes, and enzyme, which plays, to be made
With too busy to get away coenzyme, and vitamin is the important component of coenzyme, therefore vitamin is that cell eubolism is indispensable.
Vitamin C makes an addition in culture medium [12] generally as antioxidant.The inoxidizability of vitamin E is also stronger than vitamin C, can have
Effect removal free radical, is protected cell [13].
(4) the low blood serum medium of some cell lines of microelement also needs supplement V, Cr, Mo, Mn, Cu, the micro member such as Zn
Element, cell are different to the preference of these elements, generally require to carry out big flux screening test.Cell itself can not synthesize micro member
Element, it is necessary to be provided by the external world.Although the additive amount of microelement in the medium is seldom, the adjusting of cellular physiological environment is not from
Open the prothetic group [14,15] that microelement or even some trace elements are also used as certain enzymes.
(5) reduction of growth-stimulating factor serum means that the reduction of growth-stimulating factor, cell division can accordingly slow down,
This just needs to add the substances such as EGF in right amount and promotes cell division, maintains cellular morphology.
(6) ability for buffering and protecting system serum that there is very strong pH buffer capacity and repair cell to damage, thus it is low
Need to add HEPES, bovine serum albumin(BSA) (BSA), methylcellulose, F68 etc. in blood serum medium, these substances, which have, to be improved
The effect [16] of culture medium buffer capacity and protection cell.
The purpose of the invention will be achieved through the following technical solutions:
One kind being suitable for Vero cell square vase and roller bottle culture, microcarrier suspension culture and the amplification culture of microcarrier suspension scale
Culture medium, it is characterised in that: including amino acid, inorganic salts, vitamin, protolysate, lipid, buffer composition and addition
Object；The amino acid is L-Trp, L-cysteine, l-Isoleucine, L-Leu, Valine, L-arginine hydrochloric acid
Salt, L-Histidine hydrochloride, L-lysine, L-Methionine, Pidolidone, L-phenylalanine, L- asparagus fern acyl, glycine, L- dried meat
Propylhomoserin, l-Alanine, L-Aspartic acid, Serine, L-threonine, L-Glutamine, l-cysteine hydrochloride and L- junket
Propylhomoserin disodium salt；The inorganic salts are magnesium chloride hexahydrate, anhydrous magnesium sulfate, anhydrous calcium chloride, potassium chloride, sodium chloride, a water phosphorus
Acid dihydride sodium, disodium hydrogen phosphate, white vitriol, Fe(NO3)39H2O, sodium selenite and cupric sulfate pentahydrate；The vitamin
For inositol, vitamin C, pyridoxine phosphate, phosphopyridoxal pyridoxal phosphate, cobalamin, niacinamide, riboflavin, thiamine hydrochloride, D-VB5
Calcium, folic acid, biotin (Biotin) and choline chloride；The protolysate be milk protein hydrolysate, yeast hydrolyate, or
Any one of plant hydrolyzed object of person；The lipid is linoleic acid and blocked polyethers F68 (PluronicF-68)；The buffering
Ingredient is sodium bicarbonate and hydroxyethyl piperazineethanesulfonic acid (HEPES)；The additive is D-Glucose, Sodium Pyruvate, reduced form
Glutathione, hypoxanthine sodium salt, putrescine and lipoic acid, rhGM-CSF (rhEGF).
The content of the amino acid is calculated as with mg/litre:
The content of the vitamin is calculated as with mg/litre:
The content of the inorganic salts is calculated as with mg/litre:
The hydrolysate content is calculated as with mg/litre:
Soy hydrolyzate 500~6000
The content of the lipid is calculated as with mg/litre:
Linoleic acid 0.01~0.1
Blocked polyethers F68 500~1000.
The content of the buffer is calculated as with mg/litre:
Sodium bicarbonate 2400~2800
Hydroxyethyl piperazineethanesulfonic acid 2000~3800.
The content of the additive is calculated as with mg/litre:
The content of its each component is calculated as with mg/litre:
Recombinant human epidermal growth factor 0.005.
In the present invention by adjusting the content of certain amino acid, the protein content in culture medium is eliminated, and not shadow
Ring the performance of culture medium.By using trace serum in cell growth phase, cultivated in square vase using culture medium of the invention
Vero cell has obtained the maximum cell density that 1.5 times of Chinese patents 200710187466 are reported, and can also support
Viral infection and proliferation under serum-free environment.Therefore culture medium of the invention provided in the case where add trace serum and
Serum-free composition described in the prior is compared the saving of extra cost, while providing fabulous effect.
Chinese patent 201110231805.3 describes Vero cell non-serum microcarrier suspension culture method, uses
Culture medium is commercialization culture medium.Although realizing Vero cell non-serum microcarrier suspension culture, 5 liters are only realized
Total volume bioreactor culture is cultivated without reference to amplification, and it is only realized in 2 grams per liter cytodex1 microcarriers
Maximum cell density 1.2 × 106Cell number/milliliter.By using trace serum in cell growth phase, training of the invention is used
It supports 3 grams per liter cytodex-1 microcarrier suspension culture Vero cell of base and realizes 7.1 × 106 cell numbers of maximum cell density/milliliter.
The preferred embodiment of serum free medium in the present invention includes amino acid, inorganic salts, vitamin, carbohydrate etc., wherein
It joined protolysate ingredient, lipid components and protective ingredient.These substances together provide sertoli cell life, growth and
Basic nutrition ingredient necessary to breeding.
Serum-free complete medium of the invention further includes protolysate ingredient, which does not compensate only for
The content of amino acid, additionally provides the substitute to serum or albumin in culture medium.Term " protolysate " refers to water
The protein product of solution refers generally to the egg that native protein arrives 30KD about 5 through acid or the resulting average molecular weight of enzyme partial hydrolysis
White matter cleavage product mixtures.Selectively, for combine prepare protolysate ingredient independent peptone component parts will
It purifies, removes in any remaining protease, endotoxin or other potential interference culture mediums in advance through ultrafiltration or similar step
The product of Vero cell growth and expression recombinant protein product or the high component of virus.In culture medium the concentration of protolysate according to
Rely several factors, such as used specific protolysate, the feature of the cell line to be cultivated, given peptone to thin
Intracellular growth generates toxicity or the concentration of inhibiting effect etc., and the optium concentration of protolysate is all determined with experience.In general,
In Serum-free complete medium of the present invention the total amount of protolysate be 0.1 grams per liter to about 15 grams per liters in the range of, it is more unobstructed
Ground is 0.5 grams per liter to 10 grams per liters.It is commercially available for being suitable for the invention protolysate, including the milky white egg of hydrolysis
It is white, soy hydrolyzate, casein hydrolysate, any one of tryptone phosphoric acid meat soup and plant hydrolyzed object.
Culture medium of the invention can also contain other water soluble ingredients, such as insulin (absorption that can reinforce carbohydrate),
Transferrin (Transshipment Permitted iron), trace element such as selenium, catalase (as peroxidization protective agent), ethanol amine (can be made
For lipid precursor), steroid hormone such as cortisol, thyroid hormone such as triiodo thryonine, Nucleic acid precurser such as hypoxanthine,
Thymidine, desoxyadenossine and deoxycytidine, and in the conventional training supplemented with serum for cell culture
Support other nutritional ingredients contained in base or serum free medium.
The method for preparing culture medium is not critical.For example, culture medium can be made by method as described below, i.e.,
All components and additives are first dissolved in water with respective suitable concentration, then pressurization makes solution pass through a membrane filter mistake
Filter obtains sterile culture medium.As mentioned above, when in order to express recombinant protein, antibody or virus and Cultivation of Vero
When, preferentially prepurification so that later purifying is easier, such as is passed through filtering by peptone component in culture medium, is then used
Film with the small molecular weight boundary of any albumen or viral product than that need to collect carries out ultrafiltration.
It is also not critical with the method for culture medium culture cell of the invention.With about the same with conventional medium
Condition culture medium culture cell of the invention.In general, the cell grown in culture medium of the present invention is in certain temperature range
And selected specific cell line is cultivated under the conditions of suitable.For example, Vero cell, is in about 36~37.5 DEG C of temperature models
Interior culture is enclosed, wherein the pH value of culture medium is preferably held in the range of about 7.0~7.5, more preferably 7.0~7.2
In range.Cell culture is advantageous in good environment of ventilating.
In general, Vero cell can successfully use a large amount of serum, albumin or other protein liposomals to carry wherein
It is grown in the culture medium of body, then the cell carries out normally in the case where can adding trace serum in the medium of the present invention
Passage growth.And the culture medium of the invention for being added to trace serum can support Vero cell microcarrier suspension culture and micro-
The amplification culture of carrier suspension scale.Culture medium of the invention is applicable not only to the growth of cell, and is also applied for producing useful
Physiological activity substance such as recombinant protein, the factor or antibody, can be also used for production viral vaccine.It benefits from the disclosure
Those skilled in the art of appearance will recognize that can be thin by this according to the present invention many other recombinant protein or virus
Born of the same parents' strain is made.Further, it is possible to which the Vero cell grown in the medium of the present invention can be different subcloned cells strains.?
The preferred Vero cell line cultivated in culture medium of the invention is suitable for the expression of many recombinant proteins, and be suitable for rabies viruses and
A variety of virus amplifications such as influenza virus, bursal disease virus.
Embodiment 1
One kind of the present embodiment is suitable for Vero cell square vase, roller bottle culture, microcarrier suspension culture and microcarrier suspension rule
The low-cost culture medium of mould amplification culture, it includes 21 kinds of amino acid, 11 kinds of inorganic salts, 12 kinds of vitamins, a kind of proteolysis
Object, 2 kinds of lipids, 2 kinds of buffer compositions, 7 kinds of additives；Materials described below is purchased from Sigma.
The specific embodiment of the present embodiment serum free medium, mark are prepared for using ingredient and amount listed in table 1
It is denoted as V-SFM.
Table 1
| Ingredient | Concentration |
| Calcium chloride | 116 mg/litres |
| 50 × main salt | 20 ml ls |
| 10000 × secondary salt | 0.1 ml l |
| 200 × vitamin and other substances | 5 ml ls |
| Glucose | 4500 mg/litres |
| Glutamine | 484 mg/litres |
| 10 × amino acid | 100 ml ls |
| 10%Pluronic F-68 | 10 ml ls |
| Sodium bicarbonate | 2600 mg/litres |
| Soy hydrolyzate | 2000 mg/litres |
| Recombinant human epidermal growth factor | 0.005 mg/litre |
Table 2
Table 3
Table 4
Table 5:
Table 6
Liquid concentrate is made with ingredient and amount listed by table 2 to table 5.Powdered concentrate is according to listed by upper table 1
Ingredient and amount are made.Concentrate and powder are added in 800 milliliters of water by sequence listed by table 1.By the pH value tune of final mixture
To 7.2~7.3 and volume is added to 1 liter.Amino acid in addition to glutamine is made according to table 5.
As shown in table 6, by Vero cell with 0.3 × 106Cell number/milliliter density is inoculated in 5 liters of stirring-type biological respinses
In device, cytodex-1 microcarrier concentration is the maximum cell density that 3 grams per liter cells can achieve, and gives and is grown in three not
With the obtained maximum cell density of vero cell in the V-SFM culture medium of batch.With prior art serum free medium
The results are shown in attached figure 1 for (GIBCO VP-SFM and OptiPRO) comparison culture.Reactor parameter setting: temperature is 37 DEG C, pH value is
7.2, dissolved oxygen is 40% saturated air, mixing speed is 60 revs/min.Fresh medium is replaced according to remaining sugar concentration, it is entire to train
Feeding process continues 144 hours.Cellular morphology is observed every the inverted microscope of sampling in 24 hours, with crystal violet method living cell counting
Density.From the above it is clear that, the cell being grown in the culture medium of the addition trace serum of low cost of the invention
Can with preferable cell density.In addition basal culture medium can also support that herpes simplex virus is in culture medium under absence of serum
It is replicated on the Vero cell of middle culture.
Although invention above-mentioned has quite been retouched in detail for the purpose of clarification and understanding by explanation and embodiment
It states, it is obvious that still having certain change and alter within the scope of the appended claims.For example, being proposed in table 1~5
The relative quantity of separate constituent can be repaired by those skilled in the art according to the specific needs of interested specific cell line
Change, the needs are known to those skilled in the art and are easy to utilize.In general, the specific quantity listed in table 1~5
It can be changed in the range of about 50%.
Embodiment 2
One kind of the present embodiment is suitable for Vero cell square vase, roller bottle culture, microcarrier suspension culture and microcarrier suspension rule
The low-cost culture medium of mould amplification culture, it includes 21 kinds of amino acid, 11 kinds of inorganic salts, 12 kinds of vitamins, a kind of proteolysis
Object, 2 kinds of lipids, 2 kinds of buffer compositions, 7 kinds of additives；Materials described below is purchased from Sigma.
Ingredient is as follows, is calculated as with mg/litre:
Said components are dissolved in no heat source ultrapure water and are prepared, can suitable for Vero cell square vase, roller bottle culture,
Microcarrier suspension culture and the low-cost culture medium of microcarrier suspension scale amplification culture.It is added using above-mentioned V-SFM culture medium
Trace serum Cultivation of Vero in domestic 25 liters of stirring-type biological respinses, reaches 3.5 × 10 in cell density6Cell number/
After milliliter, it is changed to serum-free V-SFM using PBS washing microcarrier, infects herpes simplex virus, maximum virus titer is 1.2
×108Pfu/ milliliters, reach domestically leading level.Fig. 2 is seen using the result of commercialization culture medium culture under the same terms.Ben Pei
Supporting effect, still cost but only has every liter of dozens of yuan, and the import well below several hundred every liter of members is commercialized culture medium, can apply
Animal vaccine manufacturing enterprise or people's medication manufacturing enterprise at home.
Still there are many embodiment, all technical sides formed using equivalents or equivalent transformation by the present invention
Case is within the scope of the present invention.
Embodiment 3
Using the culture medium in embodiment 1 in T25 tissue culture flasks adhere-wall culture Vero cell
It will be used in T25 tissue culture flasks with the Vero cell of the MEM culture medium culture containing 10% (v/v) newborn bovine serum
The trypsin digestion of 0.25% (w/v) 8 minutes.The low serum listed by table 1 containing 1% (v/v) newborn bovine serum is trained respectively
It supports base and cell is resuspended in the MEM culture medium containing 10% (v/v) newborn bovine serum, adjustment cell density is 2.5 × 105Cell number/
Milliliter.T25 tissue culture flasks are inoculated in by 5 milliliters/bottle, 37 DEG C, 5%CO2 static gas wave refrigerator observe cell shape with inverted microscope
State simultaneously photographs to record.After culture 72 hours, Vero cell contains the low blood serum medium of 1% (v/v) newborn bovine serum in the present invention
Monolayer is merged into in the MEM culture medium containing 10% (v/v) newborn bovine serum, as shown in Figure 3A and Figure 3B.
Embodiment 4
5 liters of bioreactor digestion amplifications of micro-carrier system are cultivated to 25 liters of bioreactors
By described in embodiment 1, the microcarrier suspension culture Vero cell in 5 liters of reactors.Culture was to 72 hours, microcarrier
On it is cells trypsinised, add new microcarrier, will the cell that digested and microcarrier mix after squeeze into 25 liters of lifes
Culture in object reactor, working volume are 18 liters.25 liters of reactor reaction device parameter settings: temperature is 37 DEG C, pH value 7.2,
Dissolved oxygen is 40% saturated air, mixing speed is 35 revs/min.By once digesting amplifying operation, Vero cell is reacted at 5 liters
It is grown in device normally, 96 hour cells of culture still form fine and close single layer as shown in figure 4,144 hour cell density of culture reach
6.5×106Cell number/milliliter (reduction of most high-density may be to digest microcarrier loss in transfer operation to cause).
Embodiment 5
25 liters of bioreactor balls of micro-carrier system turn ball technique
By described in embodiment 1, the microcarrier suspension culture Vero cell in 5 liters of reactors, culture to 60 hours, cell is
Single layer will be grown up to, new microcarrier is added into culture medium with the old and new's microcarrier ratio 3: 1, mixed with the old microcarrier for covering with cell
It closes, volume of culture is constant.Initial stage carries out intermittent stirring and (stirs 3 minutes within 30 minutes, speed of agitator is 30 revs/min) culture 6h,
The attaching situation of sampling observation cell, it is subsequent to be continuously stirred.Reactor parameter setting when continuously stirring: temperature 37
DEG C, pH value 7.2, dissolved oxygen be 40% saturated air, mixing speed is 60 revs/min.Vero cell can be migrated from new microcarrier
To old microcarrier, culture in 5 liters of reactors is transferred in 25 liters of bioreactors and adds culture solution to 11 liters, is entirely trained
The process of supporting continues 200h, and highest cell density reaches 6.5 × 106Cell number/milliliter.
Technical solution of the present invention bring beneficial effect
(1) serum content in Vero cell growth phase culture medium can be lower than to 1% (v/v), cell growth is vigorous, carefully
Born of the same parents' form and density are superior to containing the growth in 10% serum conventional medium, and the ingredient of culture medium is simple, at low cost
It is honest and clean.After being prepared into powder culture medium, cost is also well below commercialized culture, and its serum content is very low, can reduce
The influence that serum batch stability grows cell.It can support square vase, roller bottle Cultivation of Vero.
(2) V-SFM of the invention can support microcarrier suspension culture and microcarrier in the case where containing trace serum
The amplification culture of suspension scale, can help production enterprise to realize bioreactor large-scale culture.And the training can be used
Base is supported as serum-free virus maintaining liquid and supports virus production.
(3) V-SFM of the invention can support microcarrier suspension culture to pass through digestion in the case where containing trace serum
Transfer (as described in example 4 above) and ball turn ball (as described in Example 5) two ways and realize that microcarrier suspension culture scale is put
Greatly.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright specification and accompanying drawing content is applied directly or indirectly in other relevant skills
Art field, is included within the scope of the present invention.
Claims (1)
- The application of the low-cost culture medium of 1.Vero microcarrier suspension scale amplification culture, it is characterised in that:The low-cost culture medium includes 21 kinds of amino acid, 11 kinds of inorganic salts, 12 kinds of vitamins, a kind of protolysate, 2 kinds of rouge Class, 2 kinds of buffer compositions, 7 kinds of additives；The ingredient of the low-cost culture medium is as follows:Described 50 × main salt:Described 10000 × secondary salt:200 × the vitamin and other are as follows:10 × the amino acid are as follows:The applying step are as follows: by Vero cell with 0.3 × 106Cell number/milliliter density is inoculated in 5 liters of stirring-type biological respinses In device, the microcarrier suspension culture Vero cell in 5 liters of reactors cultivates the cell tryptose to 72 hours, on microcarrier New microcarrier is added in enzymic digestion, is squeezed into 25 liters of bioreactors and is cultivated after the cell digested and microcarrier are mixed, work Making volume is 18 liters；25 liters of reactor reaction device parameter settings: temperature is 37 DEG C, pH value 7.2, dissolved oxygen are 40% saturation sky Gas, mixing speed are 35 revs/min, and by once digesting amplifying operation, Vero cell is grown normally in 5 liters of reactors, training It supports 96 hour cells and still forms fine and close single layer, 144 hour cell density of culture reach 6.5 × 106Cell number/milliliter.
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| EP4071237A4 (en) * | 2019-12-24 | 2023-03-08 | Lg Chem, Ltd. | Low-serum medium composition for culturing vero cells and use thereof |
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| CN104130971B (en) * | 2014-08-12 | 2015-03-11 | 山西威奇达光明制药有限公司 | Serum-free medium of recombinant human erythropoietin, applications of serum-free medium and method for preparing recombinant human erythropoietin |
| CN105176916A (en) * | 2015-10-15 | 2015-12-23 | 南京三生生物技术有限公司 | Low-serum protein-free culture medium applicable to Vero cell growth and preparation method thereof |
| CN106754634A (en) * | 2016-12-31 | 2017-05-31 | 山东金周生物科技有限公司 | A kind of serum free medium and preparation method thereof |
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| CN110616183A (en) * | 2019-09-23 | 2019-12-27 | 山东甲骨文生物科技有限公司 | Low-serum culture medium for Vero cell culture and corresponding virus production |
| CN110628697A (en) * | 2019-09-23 | 2019-12-31 | 山东甲骨文生物科技有限公司 | Serum-free culture medium for VERO serum-free cell culture and corresponding virus production |
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| CN115896035B (en) * | 2022-12-22 | 2024-04-16 | 苏州沃美生物有限公司 | Vero cell strain applied to virus amplification, construction method and application thereof |
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| EP4071237A4 (en) * | 2019-12-24 | 2023-03-08 | Lg Chem, Ltd. | Low-serum medium composition for culturing vero cells and use thereof |
| CN112094802A (en) * | 2020-09-28 | 2020-12-18 | 成都柏奥特克生物科技股份有限公司 | Serum-free culture medium for culturing Vero cells |
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