CN106148268A - A kind of serum-free insect cell culture medium and its preparation method and application - Google Patents
A kind of serum-free insect cell culture medium and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of serum-free insect cell culture medium and its preparation method and application.nullThis serum-free insect cell culture medium includes basic components、Protein component、Lipid composition and auxiliary agent，Wherein basic components includes the inorganic salt of 9～14 weight portions、The aminoacid of 3.3～5 weight portions or its salt、The water soluble vitamins of 0.17～0.26 weight portion、The hormone of 0.0016～0.0024 weight portion and the trace element of 0.0024～0.0036 weight portion，Protein component includes the animal proteinum hydrolysate of 1.6～2.4 weight portions、The vegetable protein hydrolyzate of 2.4～3.6 weight portions and the yeast extract of 2.4～3.6 weight portions，Lipid composition includes the fatty acid of 0.14～0.22 weight portion、The fatsoluble vitamin of 0.028～0.042 weight portion and the cholesterol of 0.24～0.36 weight portion，Auxiliary agent includes defoamer and the surfactant of 2.6～4 weight portions of 7.2～10.8 weight portions.This serum-free insect cell culture medium cost is low, and long term storage stability is good, and when to Insect cellculture, cell density is high, it is easy to accomplish large-scale production and application.
Description
Technical field
The present invention relates to a kind of insect cell medium, particularly relate to a kind of serum-free insect cell and cultivate
Base and its preparation method and application.
Background technology
Insect cellculture is the field developed rapidly in cell engineering in recent years, and it is widely used in
Medical science, agriculture and biological various aspects.Insect cell medium is the key of Insect cellculture,
It is that insect cell is rely growth, the key factor breeding, break up.Insect cell is to the requirement of nutrition relatively
Height, generally requires several amino acids, vitamin, coenzyme, nucleic acid, hormone, somatomedin etc..
Natural medium mainly takes from animal body fluid or from animal tissue's separation and Extraction, its rich in nutrition content,
Culture effect is good, common are lactoalbumin hydrolysate and new-born calf serum.Although the growth that serum is to cell
More effective, but there is shortcomings in its application: and high-quality animal serum limited source, cost are high,
Thus limit it and use in a large number；Animal serum complicated component, various biological sized molecules mix,
Some composition is the most unclear, to the separation of later stage cultured products, purify and detect cause certain tired
Difficult；Serum is also mycoplasma and the important sources of other virus pollution；The quality of every batch of serum is different, shadow
Ring cell growth and the stability replicated, blood serum medium the most also cannot be used to carry out on a large scale
Commercially produce and apply.Therefore, in the urgent need to a kind of low cost and can to realize large scale insect thin
The serum-free insect cell culture medium that born of the same parents cultivate.
Summary of the invention
The present invention provides a kind of serum-free insect cell culture medium and its preparation method and application, this serum-free
Insect cell medium advantage of lower cost, long term storage stability is good, is cultivating insect cell
Time cell density high, it is easy to accomplish large-scale production and application.
The serum-free insect cell culture medium that the present invention provides, including basic components, protein component, lipid
Component and auxiliary agent, wherein said basic components includes the inorganic salt of 9～14 weight portions, 3.3～5 weight portions
Aminoacid or its salt, 0.17～0.26 water soluble vitamins, 0.0016～0.0024 weight portion of weight portion
Hormone and the trace element of 0.0024～0.0036 weight portion, described protein component includes 1.6～2.4 weights
Amount the animal proteinum hydrolysate of part, 2.4～3.6 weight portion vegetable protein hydrolyzate and 2.4～3.6 weight
Part yeast extract, described lipid composition include the fatty acid of 0.14～0.22 weight portion, 0.028～
The fatsoluble vitamin of 0.042 weight portion and the cholesterol of 0.24～0.36 weight portion, described auxiliary agent includes
The defoamer of 7.2～10.8 weight portions and the surfactant of 2.6～4 weight portions.
In the serum-free insect cell culture medium of the present invention, described fatty acid includes the one-tenth of following weight portion
Point:
In the serum-free insect cell culture medium of the present invention, described fatsoluble vitamin includes following weight portion
Composition:
Vitamin A 0.008～0.012 weight portion
Vitamin D 0.004～0.006 weight portion
Vitamin E 0.016～0.024 weight portion.
In the serum-free insect cell culture medium of the present invention, described animal proteinum hydrolysate is selected from tryptone, meat
One or more in peptone and bone peptone；Described vegetable protein hydrolyzate is selected from soybean protein hydrolyate, Semen Tritici aestivi
One or more in protolysate, Potato protein concentrate hydrolysate and Semen Pisi sativi protein hydrolysate.
In the serum-free insect cell culture medium of the present invention, described inorganic salt can select this area routine to use
Inorganic salt in serum-free insect cell culture medium；Further, described inorganic salt includes following weight portion
Composition:
In the serum-free insect cell culture medium of the present invention, described aminoacid can select this area routine to use
In the aminoacid of insect cell medium, particularly selecting L-type aminoacid, described amino acid salts can be
Hydrochlorate；Further, described aminoacid or its salt include the composition of following weight portion:
In the serum-free insect cell culture medium of the present invention, described water soluble vitamins includes following weight portion
Composition:
In the serum-free insect cell culture medium of the present invention, described trace element includes the one-tenth of following weight portion
Point:
In the serum-free insect cell culture medium of the present invention, described hormone can be that this area is conventionally used for
The hormone of insect cell medium, such as insulin, it can strengthen the absorption of glucide；Described froth breaking
Agent can be the defoamer that this area is conventional, such as PLURONICS F87；Described surfactant can be
Nonionic surfactant, such as Tween 80.
The purity of each composition employed in serum-free insect cell culture medium of the present invention all >=98%, enters one
Step >=99%；Each composition all can be by common commercially available acquisition.Further, not serum-free elder brother to the present invention
Under the precondition that worm cell culture medium adversely affects when cultivating insect cell, this serum-free insect
Cell culture medium can also include other cellar culture based component, such as transferrins (its Transshipment Permitted
Ferrum), trace element-selenium, ethanolamine (it can be as lipid precursor) etc., these cellar culture based components
Consumption can be the conventional amount used of this area.This area conventional method can be used to prepare described serum-free
Insect cell medium.
Particularly, the present invention also provides for the preparation method of above-mentioned serum-free insect cell culture medium, including such as
Lower step:
After each composition in described lipid composition being mixed according to weight, add organic solvent, stir
Mix to being completely dissolved, prepare the first solution；
Excipient is soluble in water, prepare the second solution；
Under stirring, described first solution is slowly added in described second solution, forms mixing molten
Liquid, to described mixed solution vacuum lyophilization, prepares described lipid composition；
According to weight by other in described lipid composition and described serum-free insect cell culture medium
Composition mixes, and prepares described serum-free insect cell culture medium.
In the present invention, the weight proportion between each composition during described weight refers to each component；
Described excipient can be the excipient that this area is conventional, such as mannitol, potassium chloride, glucose, group ammonia
Acid, glycine etc., and can control excipient mass content in described lipid composition be 85～
95%；Described organic solvent can be to dissolve the solvent of each composition in described lipid composition, such as without
Water-ethanol etc..Lipid components is manufactured separately by this preparation method, and it is the most easily stored, and is difficult to
Rotten, thus advantageously ensure that the steady quality of culture medium product.
It is possible to further individually mix the trace element in basic components, mixed method is permissible
For: after each composition in trace element is made solution, it is mixed to form mixing according to weight molten
Liquid, after adding excipient, is dried, prepares trace element in described mixed solution.Particularly, in system
During standby trace element, can be prepared according to the weight portion expanded, so that the dispersion of each composition is more uniform.
It is possible to further individually the water soluble vitamins in basic components is mixed, mixed method
Can be: after each composition in water soluble vitamins is made solution, be mixed to form according to weight
Mixed solution, after adding excipient, is dried, prepares water soluble vitamins in described mixed solution.Special
Not, when preparing water soluble vitamins, can be prepared according to the weight portion expanded, so that each composition
Disperse more uniform.
When each composition is made solution, each composition can individually dissolve, it is possible to so that dissolution properties
Identical composition is dissolved in the one in water, acid solution, aqueous slkali according to weight portion simultaneously, in mixing
In the mixed solution formed, the weight portion of each composition is it suffices that the weight of each composition in each component
Each composition in requirement, and each component can be 5～15% at the gross mass content of mixed solution.
Specifically, dissolving the water that each composition used can be ultra-pure water；The mass concentration of acid solution can be
5%-30%, and acid solution used acid type be selected from the one in hydrochloric acid, sulphuric acid and nitric acid；
The mass concentration of aqueous slkali can be 10%-50%, and the type of alkali that aqueous slkali is used is selected from hydrogen
One in sodium oxide and potassium hydroxide.
Further, described lipid composition is being become with other in described serum-free insect cell culture medium
Carry out ball milling after point mixing, make the particle mean size of the serum-free insect cell culture medium of preparation be 120 mesh with
On；Ball-milling Time can be 2～4 hours.
The present invention also provides for any of the above-described described serum-free insect cell culture medium on Insect cellculture
Application.This serum-free insect cell culture medium is when for Insect cellculture, it is not necessary to add serum,
And insect cell growth can be made good and stable.
The present invention also provides for a kind of Insect cellculture method, by thin for any of the above-described described serum-free insect
After born of the same parents' culture medium makes culture fluid, insect cell is cultivated, wherein, control nothing in described culture fluid
The mass content of serum insect cell medium is 1～3%.Further, when insect cell is cultivated,
The inoculum density of insect cell can be conventional density, such as 0.1 × 106～1 × 106Individual cell/ml, enters one
Step can be 0.5 × 106Individual cell/ml.
Further, any of the above-described described serum-free insect cell culture medium is made culture fluid, including:
After any of the above-described described serum-free insect cell culture medium is dissolved in water, adds sodium bicarbonate, stir
Mix mixing, prepare culture fluid；Wherein it is possible to control the mass content of sodium bicarbonate in described culture fluid it is
2～3%.The pH value of the culture fluid prepared is 6.8～7.4.
Further, the insect cell cultivated includes but not limited to sf9 cell, sf21 cell, High
Five cell etc., the most preferably sf9 cell.
The enforcement of the present invention, at least has the advantage that
1, the serum-free insect cell culture medium of the present invention does not contains serum, and its composition is simple, each composition
By the most commercially available and be readily available, preparation cost is relatively low, thus is conducive to training at insect cell
Large-scale application in Yanging.
2, the preparation method of the serum-free insect cell culture medium of the present invention is simple to operate, easily controllable,
Particularly lipid composition is manufactured separately by it, the most easily stored, and is unlikely to deteriorate, the product prepared
Quality is stable, and long-time stability are good.
3, the suitability when application of the serum-free insect cell culture medium of the present invention is strong, can be used for major part
Insect cellculture, and the cell growth state cultivated is good and stable, and target product yield is high,
Can reach the conventional cultivation level containing blood serum medium, application prospect is extensive.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with the reality of the present invention
Execute example, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described
Embodiment be a part of embodiment of the present invention rather than whole embodiments.Based on the reality in the present invention
Executing example, it is every other that those of ordinary skill in the art are obtained under not making creative work premise
Embodiment, broadly falls into the scope of protection of the invention.
The prescription of the raw material that each embodiment is used and source be:
Each aminoacid or its salt, D-Glucose: pharmaceutical grade, purity >=99%, purchased from sigma company；
Each inorganic salt: injection stage, purity >=99%, purchased from sigma company；
Each vitamin: pharmaceutical grade, purity >=99%, purchased from sigma company；
Each trace element: SILVER REAGENT, purity >=98%, purchased from Pharmaceutical limited company of traditional Chinese medicines group；
Each composition in protein component: purchased from Kerry company；
Each fatty acid: SILVER REAGENT, purity >=98%, purchased from sigma company；
Choline, hormone: SILVER REAGENT, purity >=98%, purchased from Pharmaceutical limited company of traditional Chinese medicines group；
Each insect cell: from Nat'l Pharmaceutical & Biological Products Control Institute (examining institute in abbreviation).
Embodiment 1
The serum-free insect cell culture medium of the present embodiment, by basic components, protein component, lipid composition
Forming with auxiliary agent, wherein basic components is by inorganic salt, aminoacid or its salt, water soluble vitamins, trace
Element and 0.002 weight portion insulin (hormone) composition, protein component by the meat peptone of 2 weight portions, 3
The yeast extract composition of the soybean protein hydrolyate of weight portion and 3 weight portions, lipid composition by fatty acid,
The cholesterol composition of fatsoluble vitamin and 0.3 weight portion, auxiliary agent is by the PLURONICS F87 of 9 weight portions
Tween 80 composition (surfactant) of (defoamer) and 3.3 weight portions；Wherein:
Inorganic salt comprises the following components in parts by weight:
Aminoacid or its salt comprise the following components in parts by weight:
Water soluble vitamins comprises the following components in parts by weight:
Trace element comprises the following components in parts by weight:
Fatty acid comprises the following components in parts by weight:
Fatsoluble vitamin comprises the following components in parts by weight:
Vitamin A 0.01 weight portion
Vitamin D 0.005 weight portion
Vitamin E 0.02 weight portion.
This serum-free insect cell culture medium can be prepared via a method which to obtain:
1, lipid composition is prepared
Weigh each composition in lipid composition according to above-mentioned weight respectively, after mix homogeneously, add
Appropriate dehydrated alcohol, is slowly stirred to being completely dissolved, and prepares the first solution；Mannitol is dissolved in ultra-pure water
In, prepare the second solution；Under stirring, the first solution is slowly added in the second solution, is formed
Mixed solution, wherein controlling each composition gross mass content in mixed solution in lipid composition is 10%,
Mannitol mass content in mixed solution is 90%；Use freezer dryer that mixed solution is entered subsequently
Row vacuum lyophilization, prepares lipid composition, prepared lipid composition is placed in-20 DEG C and stores for future use.
2, serum-free insect cell culture medium is prepared
0 month, 6 months and the lipid composition of 12 months will be stored respectively according to above-mentioned weight
After mixing homogeneously with other composition of serum-free insect cell culture medium, use 3 hours left sides of ball mill ball milling
The right side, prepared particle mean size is the serum-free insect cell culture medium of more than 120 mesh；Wherein, employing is stored up
The lipid depositing 0 month (the most just preparing), 6 months, 12 months forms the serum-free insect cell made
Culture medium is denoted as culture medium 1, culture medium 2, culture medium 3 respectively.
Embodiment 2
The serum-free insect cell culture medium of the present embodiment, by basic components, protein component, lipid composition
Forming with auxiliary agent, wherein basic components is by inorganic salt, aminoacid or its salt, water soluble vitamins, trace
The insulin composition of element and 0.0016 weight portion, protein component is by the tryptone of 1.6 weight portions, 2.4 weight
Part wheat protein hydrolysate and 3.6 weight portions yeast extract composition, lipid composition by fatty acid,
The cholesterol composition of fatsoluble vitamin and 0.36 weight portion, auxiliary agent is by the PLURONICS F87 of 7.2 weight portions
Form with the Tween 80 of 4 weight portions；Wherein:
Inorganic salt comprises the following components in parts by weight:
Aminoacid or its salt comprise the following components in parts by weight:
Water soluble vitamins comprises the following components in parts by weight:
Trace element comprises the following components in parts by weight:
Fatty acid comprises the following components in parts by weight:
Fatsoluble vitamin comprises the following components in parts by weight:
Vitamin A 0.008 weight portion
Vitamin D 0.004 weight portion
Vitamin E 0.024 weight portion.
Above-mentioned serum-free insect cell culture medium is prepared, wherein by employing storage 0 with reference to embodiment 1 method
Individual month, 6 months, the serum-free insect cell culture medium made of the lipid composition of 12 months be denoted as training respectively
Support base 4, culture medium 5, culture medium 6.
Embodiment 3
The serum-free insect cell culture medium of the present embodiment, by basic components, protein component, lipid composition
Forming with auxiliary agent, wherein basic components is by inorganic salt, aminoacid or its salt, water soluble vitamins, trace
The insulin composition of element and 0.0024 weight portion, protein component is by the bone peptone of 2.4 weight portions, 3.6 weight
Part Potato protein concentrate hydrolysate and 2.4 weight portions yeast extract composition, lipid composition by fatty acid,
The cholesterol composition of fatsoluble vitamin and 0.24 weight portion, auxiliary agent is by the poloxamer of 10.8 weight portions
The Tween 80 composition of 188 and 2.6 weight portions；Wherein:
Inorganic salt comprises the following components in parts by weight:
Aminoacid or its salt comprise the following components in parts by weight:
Water soluble vitamins comprises the following components in parts by weight:
Trace element comprises the following components in parts by weight:
Fatty acid comprises the following components in parts by weight:
Fatsoluble vitamin comprises the following components in parts by weight:
Vitamin A 0.012 weight portion
Vitamin D 0.006 weight portion
Vitamin E 0.016 weight portion.
Above-mentioned serum-free insect cell culture medium is prepared, wherein by employing storage 0 with reference to embodiment 1 method
Individual month, 6 months, the serum-free insect cell culture medium made of the lipid of 12 months composition be denoted as training respectively
Support base 7, culture medium 8, culture medium 9.
Embodiment 4
Respectively culture medium 1 to culture medium 3 prepared by 2g embodiment 1 is dissolved in 100ml ultra-pure water, stirs
After mixing mixing, add 2g sodium bicarbonate, stirring and evenly mixing, prepare serum-free medium 1～serum-free culture
Liquid 3.
Meanwhile, 1640 cell culture mediums of 2g are dissolved in 100ml ultra-pure water, after stirring and evenly mixing, add
Enter 10g serum, prepare containing serum free culture system liquid 1.
Aseptically, with sterilizing pipettor respectively by serum-free medium 1～the serum-free of 50ml
Culture fluid 3 and transfer to, in 250ml Tissue Culture Flask, divide to each Tissue Culture Flask containing serum free culture system liquid 1
Not Jie Zhong sf9 cell suspension, inoculum density is 0.5 × 106Individual cell/ml, under 27 ± 2 DEG C of temperature conditionss
Suspension culture is carried out with the rotating speeds of 120 revs/min.
After cultivating 2 weeks, Beckman cell counter is used to carry out cell counting and measure cell viability (carefully
Born of the same parents' vigor is the percentage ratio in total cell shared by living cells), the results are shown in Table 1.
Embodiment 5
Respectively culture medium 4 to culture medium 6 prepared by 1g embodiment 2 is dissolved in 100ml ultra-pure water, stirs
After mixing mixing, add 2g sodium bicarbonate, stirring and evenly mixing, prepare serum-free medium 4～serum-free culture
Liquid 6.
Meanwhile, the MEM cell culture medium of 1g is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds
Enter 10g serum, prepare containing serum free culture system liquid 2.
Aseptically, with sterilizing pipettor respectively by serum-free medium 4～the serum-free of 50ml
Culture fluid 6 and transfer to, in 250ml Tissue Culture Flask, divide to each Tissue Culture Flask containing serum free culture system liquid 2
Not Jie Zhong sf21 cell suspension, inoculum density is 0.5 × 106Individual cell/ml, 27 ± 2 DEG C of temperature conditionss
Under carry out suspension culture with the rotating speed of 120 revs/min.
After cultivating 2 weeks, Beckman cell counter is used to carry out cell counting and measure cell viability, knot
Fruit is shown in Table 1.
Cell counting after the cultivation of table 1 each culture fluid and cell viability result
As shown in Table 1:
1, the serum-free insect cell culture medium that prepared by the present invention can preferably cultivate sf9 cell and sf21
Cell, cell growth state is good, and cell proliferation multiple is high, and after cultivation, cell number and cell viability are the highest
In the routine cultivation level containing blood serum medium.
2, the lipid composition steady quality that prepared by the present invention, is unlikely to deteriorate, and adds fat after storing 12 months
When the serum-free insect cell culture medium of matter component and just preparation, the cultivation to insect cell is on close level, and says
The long-time stability of this lipid composition bright and serum-free insect cell culture medium are good.
Embodiment 6
Respectively culture medium 7 to culture medium 9 prepared by 3g embodiment 3 is dissolved in 100ml ultra-pure water, stirs
After mixing mixing, add 3g sodium bicarbonate, stirring and evenly mixing, prepare serum-free medium 7～serum-free culture
Liquid 9.
Aseptically, with sterilizing pipettor respectively by serum-free medium 7～the serum-free of 50ml
Culture fluid 9 is transferred in 250ml Tissue Culture Flask, to the Tissue Culture Flask equipped with serum-free medium 7
Inoculation sf9 cell suspension, inoculates sf21 cell suspension to the Tissue Culture Flask equipped with serum-free medium 8,
High Five cell suspension, the inoculation of each cell is inoculated to the Tissue Culture Flask equipped with serum-free medium 9
Density is 0.5 × 106Individual cell/ml, enters with the rotating speed of 120 revs/min under 27 ± 2 DEG C of temperature conditionss
Row suspension culture.
After cultivating 2 weeks, Beckman cell counter is used to carry out cell counting and measure cell viability, knot
Fruit is shown in Table 2.
The cell counting of each cell of table 2 and cell viability result
As shown in Table 2:
It is thin that the serum-free insect cell culture medium of the present invention can be used for cultivating various insects cell, such as sf9
Born of the same parents, sf2 cell, High Five cell etc., and each insect cell growth cultivated is in good condition, says
The serum-free insect cell culture medium suitability of the bright present invention is strong, and application prospect is extensive.
Reference examples 1
In addition to not containing the lipid composition of embodiment 1 serum-free insect cell culture medium, other and embodiment
The serum-free insect cell culture medium of 1 is identical, cultivates using this serum-free insect cell culture medium as comparison
Base 1.
2g control medium 1 is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 2g bicarbonate
Sodium, stirring and evenly mixing, prepare comparison culture fluid 1.
Aseptically, with sterilizing pipettor, the comparison culture fluid 1 of 50ml is transferred to 250ml
In Tissue Culture Flask, inoculating sf9 cell suspension to Tissue Culture Flask, inoculum density is 0.5 × 106Individual cell
/ ml, under 27 ± 2 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.After cultivating 2 weeks
Cell counting and cell viability the results are shown in Table 3.
Reference examples 2
In addition to lipid composition does not contains cholesterol, arachidonic acid, oleic acid and vitamin D, other
Composition is identical with the serum-free insect cell culture medium of embodiment 1, with this serum-free insect cell culture medium
As control medium 2.
2g control medium 2 is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 2g bicarbonate
Sodium, stirring and evenly mixing, prepare comparison culture fluid 2.
Aseptically, with sterilizing pipettor, the comparison culture fluid 2 of 50ml is transferred to 250ml
In Tissue Culture Flask, inoculating sf9 cell suspension to Tissue Culture Flask, inoculum density is 0.5 × 106Individual cell
/ ml, under 27 ± 2 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.After cultivating 2 weeks
Cell counting and cell viability the results are shown in Table 3.
Reference examples 3
In addition to not containing linolenic acid, Palmic acid, stearic acid and vitamin E in lipid composition, other becomes
Divide identical with the serum-free insect cell culture medium of embodiment 1, make with this serum-free insect cell culture medium
For control medium 3.
2g control medium 3 is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 2g bicarbonate
Sodium, stirring and evenly mixing, prepare comparison culture fluid 3.
Aseptically, with sterilizing pipettor, the comparison culture fluid 3 of 50ml is transferred to 250ml
In Tissue Culture Flask, inoculating sf21 cell suspension to Tissue Culture Flask, inoculum density is 0.5 × 106Individual carefully
Born of the same parents/ml, under 27 ± 2 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.Cultivate 2 weeks
After cell counting and cell viability the results are shown in Table 3.
Reference examples 4
In addition to lipid composition does not contains linoleic acid, myristic acid, palmitoleic acid and vitamin A,
Other composition is identical with the serum-free insect cell culture medium of embodiment 1, using this culture medium as comparison training
Support base 4.
Respectively 2g control medium 4 is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 2g carbon
Acid hydrogen sodium, stirring and evenly mixing, prepare comparison culture fluid 4.
Aseptically, with sterilizing pipettor, the comparison culture fluid 4 of 50ml is transferred to 250ml
In Tissue Culture Flask, inoculating High Five cell suspension to Tissue Culture Flask, inoculum density is 0.5 × 106
Individual cell/ml, under 27 ± 2 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.Cultivate
Cell counting and cell viability after 2 weeks the results are shown in Table 3.
Table 3 respectively compares the cell counts after culture fluid is cultivated
As shown in Table 3:
Lack the lipid composition in serum-free insect cell culture medium of the present invention or several one-tenth in lipid composition
The culture medium divided density of cell when for cultivating each insect cell is decreased obviously.Thus illustrate, above-mentioned
Each composition in lipid composition is all indispensable in the serum-free insect cell culture medium of the present invention,
It is respectively provided with remarkable effect to the cultivation of various insect cells.
Last it is noted that various embodiments above is only in order to illustrate technical scheme, rather than right
It limits；Although the present invention being described in detail with reference to foregoing embodiments, this area common
Skilled artisans appreciate that the technical scheme described in foregoing embodiments still can be repaiied by it
Change, or the most some or all of technical characteristic is carried out equivalent；And these are revised or replace
Change, do not make the essence of appropriate technical solution depart from the scope of various embodiments of the present invention technical scheme.
Claims (10)
1. a serum-free insect cell culture medium, it is characterised in that include basic components, protein component,
Lipid composition and auxiliary agent, wherein said basic components includes the inorganic salt of 9～14 weight portions, 3.3～5 weights
The amount aminoacid of part or its salt, 0.17～0.26 water soluble vitamins, 0.0016～0.0024 weight of weight portion
The hormone of amount part and the trace element of 0.0024～0.0036 weight portion, described protein component includes 1.6～2.4
The animal proteinum hydrolysate of weight portion, 2.4～3.6 weight portion vegetable protein hydrolyzate and 2.4～3.6 weights
Amount part yeast extract, described lipid composition include the fatty acid of 0.14～0.22 weight portion, 0.028～
The fatsoluble vitamin of 0.042 weight portion and the cholesterol of 0.24～0.36 weight portion, described auxiliary agent includes
The defoamer of 7.2～10.8 weight portions and the surfactant of 2.6～4 weight portions.
Serum-free insect cell culture medium the most according to claim 1, it is characterised in that described fat
Fat acid includes the composition of following weight portion:
Serum-free insect cell culture medium the most according to claim 1, it is characterised in that described fat
Soluble vitamin includes the composition of following weight portion:
Vitamin A 0.008～0.012 weight portion
Vitamin D 0.004～0.006 weight portion
Vitamin E 0.016～0.024 weight portion.
Serum-free insect cell culture medium the most according to claim 1, it is characterised in that described dynamic
One or more in tryptone, meat peptone and bone peptone of thing protolysate；Described vegetable protein hydrolyzate
Selected from soybean protein hydrolyate, wheat protein hydrolysate, Potato protein concentrate hydrolysate and Semen Pisi sativi protein hydrolysate
In one or more.
5., according to the arbitrary described serum-free insect cell culture medium of Claims 1-4, its feature exists
The composition of following weight portion is included in, described inorganic salt:
6., according to the arbitrary described serum-free insect cell culture medium of Claims 1-4, its feature exists
In, described aminoacid or its salt include the composition of following weight portion:
7., according to the arbitrary described serum-free insect cell culture medium of Claims 1-4, its feature exists
The composition of following weight portion is included in, described water soluble vitamins:
8., according to the arbitrary described serum-free insect cell culture medium of Claims 1-4, its feature exists
The composition of following weight portion is included in, described trace element:
9. the preparation method of the arbitrary described serum-free insect cell culture medium of claim 1 to 8, its feature
It is, comprises the steps:
After each composition in described lipid composition being mixed according to weight, add organic solvent, stir
Mix to being completely dissolved, prepare the first solution；
Excipient is soluble in water, prepare the second solution；
Under stirring, described first solution is slowly added in described second solution, forms mixing molten
Liquid, to described mixed solution vacuum lyophilization, prepares described lipid composition；
According to weight by other in described lipid composition and described serum-free insect cell culture medium
Composition mixes, and prepares described serum-free insect cell culture medium.
10. an Insect cellculture method, it is characterised in that by institute arbitrary in claim 1 to 8
After the serum-free insect cell culture medium stated makes culture fluid, insect cell is cultivated, wherein, control
Making the mass content of serum-free insect cell culture medium in described culture fluid is 1～3%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510172756.9A CN106148268A (en) | 2015-04-13 | 2015-04-13 | A kind of serum-free insect cell culture medium and its preparation method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367379A (en) * | 2016-12-07 | 2017-02-01 | 天康生物股份有限公司 | Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof |
| CN106676053A (en) * | 2016-12-31 | 2017-05-17 | 山东金周生物科技有限公司 | Low-serum cell culture medium with universal adaptability and preparation method thereof |
| WO2021030125A1 (en) * | 2019-08-09 | 2021-02-18 | Voyager Therapeutics, Inc. | Cell culture medium for use in producing gene therapy products in bioreactors |
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| CN1498267A (en) * | 2000-06-23 | 2004-05-19 | 巴斯福股份公司 | 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 |
| CN103146648A (en) * | 2013-03-14 | 2013-06-12 | 北京京蒙高科干细胞技术有限公司 | Animal source-free and serum-free culture medium of lymphocyte |
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| CN1498267A (en) * | 2000-06-23 | 2004-05-19 | 巴斯福股份公司 | 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 |
| CN103146648A (en) * | 2013-03-14 | 2013-06-12 | 北京京蒙高科干细胞技术有限公司 | Animal source-free and serum-free culture medium of lymphocyte |
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| 曹翠平等: "昆虫细胞无血清培养", 《细胞生物学杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367379A (en) * | 2016-12-07 | 2017-02-01 | 天康生物股份有限公司 | Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof |
| CN106367379B (en) * | 2016-12-07 | 2019-09-10 | 天康生物股份有限公司 | A kind of insect cell maintaining liquid and its preparation method improving swine fever virus E2 recombinant protein expression quantity |
| CN106676053A (en) * | 2016-12-31 | 2017-05-17 | 山东金周生物科技有限公司 | Low-serum cell culture medium with universal adaptability and preparation method thereof |
| CN106676053B (en) * | 2016-12-31 | 2020-08-25 | 山东甲骨文生物科技有限公司 | Low-serum cell culture medium with wide adaptability and preparation method thereof |
| WO2021030125A1 (en) * | 2019-08-09 | 2021-02-18 | Voyager Therapeutics, Inc. | Cell culture medium for use in producing gene therapy products in bioreactors |
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