CN106032526A - An animal cell culture medium, a preparing method thereof and applications of the culture medium - Google Patents
An animal cell culture medium, a preparing method thereof and applications of the culture medium Download PDFInfo
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Abstract
An animal cell culture medium, a preparing method thereof and applications of the culture medium are provided. The culture medium comprises 150-230 parts by weight of amino acids or salts thereof, 180-280 parts by weight of carbohydrates, 160-260 parts by weight of inorganic salts, 1-2 parts by weight of vitamins and 0.002-0.003 part by weight of trace elements. The trace elements comprise manganese chloride tetrahydrate, sodium metavanadate, selenious acid, germanium dioxide, potassium bromide, chromium chloride hexahydrate, ammonium metavanadate, rubidium chloride, cadmium chloride, cobalt chloride hexahydrate, barium acetate, zirconyl chloride octahydrate, sodium fluoride, ammonium molybdate tetrahydrate, copper chloride dihydrate, lithium chloride and aluminium chloride hexahydrate. The culture medium is simple and definite in components, convenient to prepare and use, stable in quality, and low in batch differences, and can be particularly used for culturing a plurality of animal cells. Cell growth states are good and stable.
Description
Technical field
The present invention relates to a kind of cell culture medium, particularly relate to a kind of Zooblast culture medium and preparation thereof
Methods and applications.
Background technology
Cell culture medium is to cultivate to supply cytotrophy and the basic thing promoting germiparity to breed in cell
Matter, is also to cultivate cell growth and the living environment of breeding.The composition of cell culture medium generally includes amino
Acid, vitamin, carbohydrate, inorganic ions etc.；Wherein, aminoacid is the basic of constitutive protein matter
Unit, vitamin is the bioactive substance maintaining cell growth, plays regulation and control in cellular metabolism
Effect, carbohydrate is the main energy sources of cell growth, and inorganic ions is that cell composition institute is necessary
Element and participate in cellular metabolism.
Cell culture medium can be divided into containing blood serum medium, serum-free medium, protein-free culture and change
Learn defined medium four type.Serum is one of most important component in cell culture medium, and it is as ammonia
Base acid, protein, vitamin, carbohydrate, lipid, hormone, somatomedin, mineral and micro-
The source of secondary element, plays the important effect being even difficult to and substituting to the growth and breeding of cell.But,
Serum composition is complicated, and differs greatly between each batch, not only to the separation of cultured products, purification,
Detection causes certain difficulty, has an effect on the stability that cell is cultivated；Additionally, serum origin is limited, cost
Height, limits it to a certain extent and uses in a large number.
Serum-free medium does not contains serum, and it is interpolation insulin in basal medium, transferrins,
The components such as bovine serum albumin are formed, although it can increase stablizing of cell cultivation to a certain extent
Property, and make cultured products be prone to purification and Downstream processing, but albumen contained in its composition is easily by machine
The impact of the factors such as tool and chemistry, therefore preservation and the application of culture medium are more inconvenient；Additionally, such training
Supporting base specific aim relatively strong, usual a kind of serum-free medium is suitable only for the cultivation of a certain class cell.
Protein-free medium does not contains animal proteinum, and some protein-free mediums add plant hydrolyzed thing at present
Substitute the effect of zoohormone, somatomedin.Such as during Authorization Notice No. is CN101173246B
State's patent discloses a kind of serum-free medium without albumen for cultivating mammalian cell, this culture medium
Comprise the soya hydrolysate more than 10% weight based on culture medium gross dry weight, this soya hydrolysate
Molecular weight≤1000 dalton.Although this culture medium can be used in cultivating mammalian cell, but still deposits
In the problem such as medium component structure is indefinite.
Chemically defined culture medium is not only without serum, animal proteinum and plant hydrolyzed thing etc., and cultivates
In base, the structure of each composition is all clear and definite, this culture medium steady quality, and beneficially cell cultivation is steady
Qualitative and the purification of cultured products and analysis.The Chinese patent of such as Publication No. CN102533634A
Disclosing a kind of Chinese hamster ovary celI serum-free without protein chemistry culture medium, it includes inorganic salt, aminoacid, dimension
Raw element, trace element and biological small peptide A and biological small peptide B.This culture medium specific aim is relatively strong, the suitableeest
Close the cultivation of CHO series of cell；Additionally, it needs to synthesize specific small peptide, not only prepare inconvenience, and
And cost is high, it is unfavorable for large-scale production and use.
Summary of the invention
The present invention provides a kind of Zooblast culture medium, and its composition is simple and clear and definite, steady quality.
The present invention also provides for the preparation method of above-mentioned Zooblast culture medium, and the method is simple to operate, be prone to
Controlling, and each composition is uniformly dispersed in the medium, between each batch cultivation base, difference is little.
The present invention also provides for the application of above-mentioned Zooblast culture medium, and it is above-mentioned that this application is presented as that one utilizes
Zooblast culture medium carries out the cultural method of zooblast, and the method can realize many animals cell
Cultivate, and the zooblast growth conditions cultivated is good.
The Zooblast culture medium of the present invention, including the aminoacid of 150～230 weight portions or its salt,
The carbohydrate of 180～280 weight portions, 160～260 inorganic salt, 1～2 weight portions of weight portion
Vitamin and the trace element of 0.002～0.003 weight portion, wherein, described trace element includes four hydrations
Manganous chloride, sodium metavanadate, Monohydrated selenium dioxide, germanium dioxide, potassium bromide, six hydrated chromium trichlorides, inclined vanadium
Acid ammonium, Rubinorm (Ifi)., Caddy (Cleary), cobalt chloride hexahydrate, Barium acetate, eight water zirconium oxychlorides, sodium fluoride,
Ammonium Molybdate Tetrahydrate, Copper dichloride dihydrate, lithium chloride and Aluminium chloride hexahydrate.
Specifically, the Zooblast culture medium of the present invention does not contains serum, is i.e. the animal of a kind of serum-free
Cell culture medium.More specifically, the Zooblast culture medium of the present invention does not contains albumen, plant hydrolyzed thing
Etc. the uncertain composition of structure, it it is i.e. a kind of chemically defined Zooblast culture medium.
In the Zooblast culture medium of the present invention, described trace element includes the composition of following weight portion:
In the Zooblast culture medium of the present invention, described aminoacid can select this area to be conventionally used for animal
The aminoacid of cell culture medium, particularly selects L-type aminoacid, and described amino acid salts can be hydrochlorate；
Further, described aminoacid or its salt can include the composition of following weight portion:
In the Zooblast culture medium of the present invention, described carbohydrate can select this area to be conventionally used for
The carbohydrate of Zooblast culture medium, such as monosaccharide, can be glucose, particularly D-further
Glucose.
In the Zooblast culture medium of the present invention, described inorganic salt can select this area to be conventionally used for animal
The inorganic salt of cell culture medium；Further, described inorganic salt can include the composition of following weight portion:
In the Zooblast culture medium of the present invention, described vitamin can select this area to be conventionally used for animal
The vitamin of cell culture medium；Further, described vitamin can include the composition of following weight portion:
Further, the Zooblast culture medium of the present invention can also include 0.008～0.012 weight portion
Glutathion, 0.00008～0.00012 hydrocortisone, 0.03～0.05 weight portion time yellow of weight portion
Purine, 0.0008～0.0012 weight portion linoleic acid and the insulin of 0.024～0.036 weight portion.
The purity of each composition employed in Zooblast culture medium of the present invention all >=98%, further
>=99%；Each composition all can be by common commercially available acquisition.Further, the zooblast of the present invention is not being trained
Supporting under the precondition that base adversely affects when cultivating zooblast, this Zooblast culture medium also may be used
To include other cellar culture based component, the consumption of these cellar culture based components can be this area
Conventional amount used.
The present invention also provides for a kind of Zooblast culture medium, including the first component, second component, the 3rd group
Divide and the 4th component；
Described first component includes the composition of following weight portion:
Described second component includes the composition of following weight portion:
Described 3rd component includes the composition of following weight portion:
Described 4th component includes the composition of following weight portion:
In a concrete scheme, this Zooblast culture medium can by the first component, second component, the 3rd
Component and the 4th component composition.
The present invention also provides for the preparation method of above-mentioned Zooblast culture medium, comprises the steps:
1) after each composition in the first component being mixed according to weight, it is dried, prepares first group
Point；
2) after each composition in second component being mixed according to weight, it is dried, prepares second group
Point；
3), after each composition in the 3rd component being made solution, it is mixed to form first according to weight and mixes
Close solution, after adding excipient in described first mixed solution, be dried, prepare the 3rd component；
4), after each composition in the 4th component being made solution, it is mixed to form second according to weight and mixes
Close solution, after adding excipient in described second mixed solution, be dried, prepare the 4th component；
5) after described first component, second component, the 3rd component and the 4th component being mixed according to weight portion
Ball milling, prepares described Zooblast 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..It is possible to further control excipient mass content in described 3rd component it is
85～95%, and to control excipient mass content in described 4th component be 85～95%.
The present invention is to step 1) and step 2) in be dried do not make considered critical, this area can be used normal
The drying means of rule, such as drying, lyophilization, spray drying etc..In concrete scheme of the present invention,
Step 1) and step 2) in dry be drying, wherein step 1) in described be dried at 70～90 DEG C
At a temperature of carry out, and drying time is 2～3 hours；Step 2) in described be dried 105～
Carry out at a temperature of 120 DEG C, and drying time is 3～5 hours；Step 3) and step 4) in institute
State to be dried and be vacuum lyophilization.
Step 3 in the present invention) in, can use according to the dissolution properties of each composition in the 3rd component
One in water, acid solution, aqueous slkali is dissolved, and each composition can individually dissolve, it is also possible to
Make the composition that dissolution properties is identical be dissolved in the one in water, acid solution, aqueous slkali according to weight portion simultaneously
In, in the first mixed solution being mixed to form, the weight portion of each composition is it suffices that each in the 3rd component
The weight requirement of composition, and in the 3rd component, each composition contains in the gross mass of the first mixed solution
Amount can be 5～15%.
Step 4 in the present invention) in, can use according to the dissolution properties of each composition in the 4th component
One in water, acid solution, aqueous slkali is dissolved, and at the second mixed solution being mixed to form
In, the weight portion of each composition it suffices that the weight requirement of each composition in the 4th component, and
In four components, each composition can be 5～15% at the gross mass content of the second 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, when preparing the 3rd component and four components, can be according to the weight portion system expanded
It is standby, so that each composition disperses more uniform in the medium；Further, in preparation the 3rd component and the 4th group
After Fen, can require mix with the first component and second component according to weight portion, and make final the most prepared
In Zooblast culture medium, the weight portion of each composition meets above-mentioned weight portion requirement.
Further, control described ball milling upon mixing and make the average particle of described Zooblast culture medium
Degree is more than 120 mesh；Now the time of ball milling can be 2～4 hours.
The present invention also provides for any of the above-described described Zooblast culture medium answering in animal cell culture
With.This Zooblast culture medium is when for animal cell culture, it is not necessary to add the composition such as serum, albumen
Zooblast well-grown can be made and stablize.
The present invention also provides for a kind of Zooblast cultivation method, by any of the above-described described animal cell culture
After culture fluid made by base, zooblast is cultivated, wherein, control zooblast in described culture fluid
The mass content of culture medium is 1～3%, such as 2%.Further, when zooblast is cultivated, dynamic
The inoculum density of thing cell can be conventional density, such as 1～10 × 106Individual cell/ml, the most permissible
It is 5 × 106Individual cell/ml.
Further, any of the above-described described Zooblast culture medium is made culture fluid, including:
After any of the above-described described Zooblast culture medium is dissolved in water, addition sodium bicarbonate, stirring and evenly mixing,
Prepare culture fluid；Wherein it is possible to controlling the mass content of sodium bicarbonate in described culture fluid is 2～3%.
The pH value of the culture fluid prepared is 6.8～7.4.
Further, the zooblast cultivated include but not limited to BHK-21 cell, Chinese hamster ovary celI,
VERO cell, mdck cell, MARC-145 cell etc., the most preferably BHK-21 cell and
Chinese hamster ovary celI.
The enforcement of the present invention, at least has the advantage that
1, the Zooblast culture medium of the present invention does not contains serum, animal proteinum, plant hydrolyzed thing etc., its
Composition is simple and clearly, and each composition all by the most commercially available and be readily available, thus beneficially can exist
Actual application in animal cell culture.
2, the preparation method of the Zooblast culture medium of the present invention is simple to operate, easily controllable, uses each
The mode that component carries out preparing respectively not only contributes to ensure that each composition is not destroyed, and is conducive to protecting
Demonstrate,proving each composition in the medium dispersed, between each batch cultivation base of preparation, difference is little, product matter
Amount is stable.
3, the suitability when application of the Zooblast culture medium of the present invention is strong, can be used for most animal
Cell is cultivated, 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；Additionally, this Zooblast culture medium application time not to target
Product separation purification and analysis detection impact, and application prospect is extensive.
Accompanying drawing explanation
Fig. 1 is the microscope of the BHK-21 cell of serum-free medium 1 cultivation of the embodiment of the present invention 4
Observed result.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, attached below in conjunction with the present invention
Figure and embodiment, be clearly and completely described the technical scheme in the embodiment of the present invention, it is clear that
Described embodiment is a part of embodiment of the present invention rather than whole embodiments.Based on the present invention
In embodiment, the institute that those of ordinary skill in the art are obtained under not making creative work premise
There are other embodiments, broadly fall 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；
Each inorganic salt: injection stage, purity >=99%, purchased from sigma；
Each vitamin: pharmaceutical grade, purity >=99%, purchased from sigma；
Each trace element: SILVER REAGENT, purity >=98%, purchased from traditional Chinese medicines group；
Each zooblast: from Nat'l Pharmaceutical & Biological Products Control Institute (examining institute in abbreviation).
Embodiment 1
The Zooblast culture medium of the present embodiment, by the first component, second component, the 3rd component and the 4th
Component forms；Wherein:
First component comprises the following components in parts by weight:
Second component comprises the following components in parts by weight:
3rd component comprises the following components in parts by weight:
4th component comprises the following components in parts by weight:
This Zooblast culture medium can be prepared via a method which to obtain:
1, the first component is prepared
The each composition in the first component is weighed respectively, by each composition mix homogeneously according to above-mentioned weight
After, it is placed in electric drying oven with forced convection and is dried, controlling baking temperature is 80 DEG C, and drying time is
2.5 hours, prepare the first component.
2, second component is prepared
The each composition in second component is weighed respectively, by each composition mix homogeneously according to above-mentioned weight
After, it is placed in electric drying oven with forced convection and is dried, controlling baking temperature is 110 DEG C, and drying time is
4 hours, prepare second component.
3, preparation the 3rd component
By the choline chloride in the 3rd component, inositol, D-VB5 calcium, sodium selenite, nicotiamide, to ammonia
Yl benzoic acid, pyridoxal hydrochloride and pyridoxine hydrochloride are dissolved in ultra-pure water, stirring and evenly mixing, prepare first molten
Liquid；Insulin in 3rd component is dissolved in the hydrochloric acid solution that concentration is 10%, stirring and evenly mixing, prepares
Second solution；Remaining composition in 3rd component is dissolved in the sodium hydroxide solution that concentration is 20%, stirs
Mix mixing, prepare the 3rd solution；
Respectively the first solution, the second solution and the 3rd solution are mixed to form according to above-mentioned weight
One mixed solution, adds mannitol (excipient) in the first mixed solution, wherein controls the 3rd component
In each composition gross mass content in the first mixed solution be 10%, mannitol is in the first mixed solution
Mass content be 90%, after stirring and evenly mixing, use freezer dryer carry out vacuum lyophilization, prepare
3rd component.
4, preparation the 4th component
Germanium dioxide in 4th component being dissolved in the sodium hydroxide that concentration is 15% and holds this, stirring is mixed
Even, prepare the 4th solution；Remaining composition in 4th component is dissolved in ultra-pure water, stirring and evenly mixing, system
Obtain the 5th solution；
Respectively by the 4th solution, that the 5th solution is mixed to form the second mixing according to above-mentioned weight is molten
Liquid, adds mannitol in the second mixed solution, wherein controls each composition in the 4th component and mixes second
Gross mass content in solution is 10%, and mannitol mass content in the second mixed solution is 90%,
After stirring and evenly mixing, use freezer dryer to carry out vacuum lyophilization, prepare the 4th component.
5, Zooblast culture medium is prepared
According to above-mentioned weight portion by the first component, second component, the 3rd component and the 4th component mix homogeneously
After, using ball mill ball milling about 3 hours, prepared particle mean size is the zooblast training of more than 120 mesh
Support base.
Embodiment 2
The Zooblast culture medium of the present embodiment, by the first component, second component, the 3rd component and the 4th
Component forms；Wherein:
First component comprises the following components in parts by weight:
Second component comprises the following components in parts by weight:
3rd component comprises the following components in parts by weight:
4th component comprises the following components in parts by weight:
The preparation method of this Zooblast culture medium, except preparation the first composition step controls baking temperature be
90 DEG C, drying time is 2 hours；Preparing control baking temperature in second component step is 105 DEG C, dry
The dry time is 5 hours；Prepare that to control each composition in the 3rd component in the 3rd composition step molten in the first mixing
Gross mass content in liquid is 15%, and mannitol mass content in the first mixed solution is 85%；System
Controlling each composition gross mass content in the second mixed solution in the 4th component in standby 4th composition step is
5%, outside mannitol mass content in the second mixed solution is 95%, remaining and embodiment 1 phase
With, the particle mean size of the Zooblast culture medium prepared is more than 120 mesh.
Embodiment 3
The Zooblast culture medium of the present embodiment, by the first component, second component, the 3rd component and the 4th
Component forms；Wherein:
First component comprises the following components in parts by weight:
Second component comprises the following components in parts by weight:
3rd component comprises the following components in parts by weight:
4th component comprises the following components in parts by weight:
The preparation method of this Zooblast culture medium, except preparation the first composition step controls baking temperature be
70 DEG C, drying time is 3 hours；Preparing control baking temperature in second component step is 120 DEG C, dry
The dry time is that outside 3 hours, remaining is same as in Example 1, Zooblast culture medium average prepared
Granularity is more than 120 mesh.
Embodiment 4
The method using embodiment 1 prepares the Zooblast culture medium of three batches of embodiments 1, is designated as training respectively
Support base 1～culture medium 3.
Respectively each for 2g culture medium is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 2g bicarbonate
Sodium, stirring and evenly mixing, prepare each culture fluid；The culture fluid that culture medium 1～culture medium 3 are made is remembered respectively
Make serum-free medium 1～serum-free medium 3.
Meanwhile, the DMEM/F12 cell culture medium of 2g is dissolved in 100ml ultra-pure water, stirring and evenly mixing
After, add 5g 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 BHK-21 cell suspension, inoculum density is 5 × 106Individual cell/ml, under 37 DEG C of temperature conditionss
Suspension culture is carried out with the rotating speeds of 120 revs/min.
After cultivating 48 hours, examine under a microscope cell, the BHK-21 that serum-free medium 1 is cultivated
The microscopy results of cell is as shown in Figure 1.Micrograph results shows: serum-free medium 1～depletion of blood
The equal form of BHK-21 cell that clear culture fluid 3 is cultivated is full, and stand density is high, and growth conditions is good.
Beckman cell counter is used to carry out cell counting and measure cell viability (cell viability is for total thin
Percentage ratio shared by living cells in born of the same parents), the results are shown in Table 1.
Embodiment 5
The method using embodiment 2 prepares the Zooblast culture medium of three batches of embodiments 2, is designated as training respectively
Support base 4～culture medium 6.
Respectively each for 1g culture medium is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 2g bicarbonate
Sodium, stirring and evenly mixing, prepare each culture fluid；The culture fluid that culture medium 4～culture medium 6 are made is remembered respectively
Make serum-free medium 4～serum-free medium 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 Chinese hamster ovary celI suspension, inoculum density is 5 × 106Individual cell/ml, under 37 DEG C of temperature conditionss with
The rotating speed of 120 revs/min carries out suspension culture.
After cultivating 48 hours, examining under a microscope cell, result shows: serum-free medium 4～nothing
The equal form of Chinese hamster ovary celI that serum free culture system liquid 6 is cultivated is full, and stand density is high, and growth conditions is good.This
Outward, cell counting and cell viability the results are 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, Zooblast culture medium prepared by the present invention can preferably cultivate BHK-21 cell and CHO
Cell, cell growth state is good, and cell proliferation multiple is high, and can reach conventional containing blood serum medium
Cultivation level.
2, each batch Zooblast culture medium prepared by present invention training when zooblast is cultivated
Supporting and have good stability, the cell number difference cultivated is less, and the Zooblast culture medium matter of the present invention is described
Amount is stable.
Embodiment 6
The method using embodiment 3 prepares the Zooblast culture medium of three batches of embodiments 3, is designated as training respectively
Support base 7～culture medium 9.
Respectively each for 3g culture medium is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 3g bicarbonate
Sodium, stirring and evenly mixing, prepare each culture fluid；The culture fluid that culture medium 7～culture medium 9 are made is remembered respectively
Make serum-free medium 7～serum-free medium 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 VERO cell suspension, inoculates MDCK to the Tissue Culture Flask equipped with serum-free medium 8 thin
Born of the same parents' suspension, inoculates MARC-145 cell suspension to the Tissue Culture Flask equipped with serum-free medium 9, respectively
The inoculum density of cell is 5 × 106Individual cell/ml, under 37 DEG C of temperature conditionss with 120 revs/min turn
Speed carries out suspension culture.
After cultivating 48 hours, examining under a microscope cell, result shows: each serum-free medium is cultivated
The equal form of each cell full, stand density is high, and growth conditions is good.Additionally, the cytometer of each cell
Number and cell viability the results are shown in Table 2.
The cell counting of each cell of table 2 and cell viability result
As shown in Table 2:
The Zooblast culture medium of the present invention can be used for cultivate many animals cell, such as VERO cell,
Mdck cell, MARC-145 cell etc., and each zooblast growth conditions cultivated is good, says
The Zooblast culture medium suitability of the bright present invention is strong, and application prospect is extensive.
Reference examples 1
With the animal cell culture being only made up of the first component, second component and the 3rd component in embodiment 1
Base is as control medium 1 (i.e. not containing the 4th component in embodiment 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 BHK-21 cell suspension to Tissue Culture Flask, inoculum density is 5 × 106Individual
Cell/ml, under 37 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.Cultivate 48 little
Cell counting and cell viability time after the results are shown in Table 3.
Reference examples 2
In addition to the 4th component does not contains sodium metavanadate, Monohydrated selenium dioxide, six hydrated chromium trichlorides and sodium fluoride,
Other composition is identical with the Zooblast culture medium of embodiment 1, using this 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 Chinese hamster ovary celI suspension to Tissue Culture Flask, inoculum density is 5 × 106Individual cell
/ ml, under 37 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.After cultivating 48 hours
Cell counting and cell viability the results are shown in Table 3.
Reference examples 3
Except the 4th component does not contains germanium dioxide, Rubinorm (Ifi)., cobalt chloride hexahydrate and eight water zirconium oxychlorides
Outside, other composition is identical with the Zooblast culture medium of embodiment 1, using this culture medium as comparison training
Support base 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 VERO cell suspension to Tissue Culture Flask, inoculum density is 5 × 106Individual carefully
Born of the same parents/ml, under 37 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.Cultivate 48 hours
After cell counting and cell viability the results are shown in Table 3.
Reference examples 4
In addition to the 4th component does not contains four hydration manganous chloride, Caddy (Cleary), Barium acetate and lithium chloride,
Other composition is identical with the Zooblast culture medium of embodiment 2, using this culture medium as control medium 4.
Respectively 1g 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 mdck cell suspension to Tissue Culture Flask, inoculum density is 5 × 106Individual carefully
Born of the same parents/ml, under 37 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.Cultivate 48 hours
After cell counting and cell viability the results are shown in Table 3.
Reference examples 5
Except the 4th component does not contains potassium bromide, ammonium metavanadate, Ammonium Molybdate Tetrahydrate, Copper dichloride dihydrate
Outside Aluminium chloride hexahydrate, other composition is identical with the Zooblast culture medium of embodiment 3, with this training
Support base as control medium 5.
Respectively 3g control medium 5 is dissolved in 100ml ultra-pure water, after stirring and evenly mixing, adds 3g carbon
Acid hydrogen sodium, stirring and evenly mixing, prepare comparison culture fluid 5.
Aseptically, with sterilizing pipettor, the comparison culture fluid 5 of 50ml is transferred to 250ml
In Tissue Culture Flask, inoculating MARC-145 cell suspension to Tissue Culture Flask, inoculum density is 5 × 106
Individual cell/ml, under 37 DEG C of temperature conditionss, the rotating speed with 120 revs/min carries out suspension culture.Cultivate 48
Cell counting and cell viability after hour 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 training of the 4th component in Zooblast culture medium of the present invention or several compositions in the 4th component
Support base density and vigor of cell when for cultivating each zooblast to be decreased obviously.Thus illustrate, above-mentioned
Each composition in 4th component is all indispensable in the Zooblast culture medium of the present invention, and it is to respectively
The cultivation planting zooblast is respectively provided with remarkable effect.
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 Zooblast culture medium, it is characterised in that include the aminoacid of 150～230 weight portions
Or its salt, 180～280 carbohydrate, 160～260 inorganic salt, 1～2 of weight portion of weight portion
The vitamin of weight portion and the trace element of 0.002～0.003 weight portion, wherein, described trace element bag
Include four hydration manganous chloride, sodium metavanadate, Monohydrated selenium dioxide, germanium dioxide, potassium bromide, six trichloride hydrate
Chromium, ammonium metavanadate, Rubinorm (Ifi)., Caddy (Cleary), cobalt chloride hexahydrate, Barium acetate, eight water zirconium oxychlorides,
Sodium fluoride, Ammonium Molybdate Tetrahydrate, Copper dichloride dihydrate, lithium chloride and Aluminium chloride hexahydrate.
Zooblast culture medium the most according to claim 1, it is characterised in that described trace element
Composition including following weight portion:
Zooblast culture medium the most according to claim 1 and 2, it is characterised in that also include
The glutathion of 0.008～0.012 weight portion, 0.00008～0.00012 weight portion hydrocortisone,
The hypoxanthine of 0.03～0.05 weight portion, 0.0008～0.0012 weight portion linoleic acid and 0.024～
The insulin of 0.036 weight portion.
4. a Zooblast culture medium, it is characterised in that include the first component, second component, the 3rd
Component and the 4th component；
Described first component includes the composition of following weight portion:
Described second component includes the composition of following weight portion:
Described 3rd component includes the composition of following weight portion:
Described 4th component includes the composition of following weight portion:
5. the preparation method of the Zooblast culture medium described in claim 4, it is characterised in that include as
Lower step:
1) after each composition in the first component being mixed according to weight, it is dried, prepares first group
Point；
2) after each composition in second component being mixed according to weight, it is dried, prepares second group
Point；
3), after each composition in the 3rd component being made solution, it is mixed to form first according to weight and mixes
Close solution, after adding excipient in described first mixed solution, be dried, prepare the 3rd component；
4), after each composition in the 4th component being made solution, it is mixed to form second according to weight and mixes
Close solution, after adding excipient in described second mixed solution, be dried, prepare the 4th component；
5) after described first component, second component, the 3rd component and the 4th component being mixed according to weight portion
Ball milling, prepares described Zooblast culture medium.
Preparation method the most according to claim 5, it is characterised in that control excipient described the
Mass content in three components is 85～95%, and controls excipient quality in described 4th component
Content is 85～95%.
Preparation method the most according to claim 5, it is characterised in that step 1) in described dry
Dry carry out at a temperature of 70～90 DEG C, and drying time is 2～3 hours；Step 2) in described
It is dried and carries out at a temperature of 105～120 DEG C, and drying time is 3～5 hours；Step 3) and step
Rapid 4) described being dried in is vacuum lyophilization.
Preparation method the most according to claim 5, it is characterised in that control described ball milling and make institute
The particle mean size stating Zooblast culture medium is more than 120 mesh.
9. a Zooblast cultivation method, it is characterised in that by arbitrary described in Claims 1-4
Zooblast culture medium make culture fluid after, zooblast is cultivated, wherein, controls described training
In nutrient solution, the mass content of Zooblast culture medium is 1～3%.
Zooblast cultivation method the most according to claim 9, it is characterised in that described animal
Cell is BHK-21 cell, Chinese hamster ovary celI, VERO cell, mdck cell or MARC-145
Cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110272864A (en) * | 2019-07-25 | 2019-09-24 | 北京鼎持生物技术有限公司 | A kind of Vero33 cell strain adapting to serum free suspension culture and its acclimation method and application |
| CN113817666A (en) * | 2021-10-13 | 2021-12-21 | 无锡多宁生物科技有限公司 | CD culture medium for full suspension culture of BHK-21 cells and shake flask culture process thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033458A (en) * | 2006-03-07 | 2007-09-12 | 上海旭太生物工程有限公司 | Serum-free animal cell culture medium and preparing method thereof |
| CN102115728A (en) * | 2009-12-31 | 2011-07-06 | 北京清大天一科技有限公司 | Serum-free animal cell culture medium dry powder, liquid culture medium and preparation method thereof |
| CN104073464A (en) * | 2014-07-08 | 2014-10-01 | 西藏天虹科技股份有限责任公司 | Serum-free CHO cell culture medium and preparation method thereof |
-
2015
- 2015-03-12 CN CN201510108840.4A patent/CN106032526A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033458A (en) * | 2006-03-07 | 2007-09-12 | 上海旭太生物工程有限公司 | Serum-free animal cell culture medium and preparing method thereof |
| CN102115728A (en) * | 2009-12-31 | 2011-07-06 | 北京清大天一科技有限公司 | Serum-free animal cell culture medium dry powder, liquid culture medium and preparation method thereof |
| CN104073464A (en) * | 2014-07-08 | 2014-10-01 | 西藏天虹科技股份有限责任公司 | Serum-free CHO cell culture medium and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 杨颜慈等: "无血清培养基的成分改进及应用现状", 《生物学杂志》 * |
| 陈锋: "无血清细胞培养基的主要补充因子及研究进展", 《无血清细胞培养基的主要补充因子及研究进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110272864A (en) * | 2019-07-25 | 2019-09-24 | 北京鼎持生物技术有限公司 | A kind of Vero33 cell strain adapting to serum free suspension culture and its acclimation method and application |
| CN113817666A (en) * | 2021-10-13 | 2021-12-21 | 无锡多宁生物科技有限公司 | CD culture medium for full suspension culture of BHK-21 cells and shake flask culture process thereof |
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