CN102453698A - Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells - Google Patents
Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells Download PDFInfo
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Abstract
The invention provides a method for suspension culture of subculture cells, which comprises the following steps that: (1) expression vectors containing gene sequences, capable of weakening the cell adhesion property, are used for stably transfecting pig testis cells or pig kidney cells; (2) a stable transfection cell clone is screened and separated; (3) the gene expression in the stable transfection cell clone is detected; and (4) the stable transfection cell clone is subjected to suspension culture. The method further relates to a method for producing hog cholera vaccine by using the pig testicle cells or the pig kidney cells obtained through the suspension culture.
Description
Technical field
The present invention relates to the medicine bioengineering field, be specifically related to the method for suspension culture passage cell, said passage cell comprises pig testis cell or pig kidney cell; Further relate to the method for utilizing this cells produce swine Fever Vaccine.
Background technology
The cultural method of cell in vitro comprises adherent culture and suspension culture, and wherein suspension culture can be divided into microcarrier suspension culture and full suspension culture again.Compare with adherent culture, suspension culture has following advantage: (1) can enlarge turnout continuously; (2) nutritive substance that helps in the cell culture medium fully contacts with gas, and is easy to control culture condition (temperature, pH, oxygen partial pressure and CO
2Deng); (3) culture condition is stable, is tending towards homogeneous, is convenient to carry out quantitative examination; (4) be easy in airtight continuously system, carry out, reduced the chance of operation steps and pollution; (5) cultured continuously for a long time both can be saved manpower, made cell can continue to maintain logarithmic phase again; (6) suspension cultured cells still keeps original susceptibility and biological characteristics to virus.And compare with microcarrier suspension culture, full suspension culture has following advantage: need not expensive microcarrier, thereby effectively reduce production costs; Can save microcarrier cultivate in additional step like the digestion harvested cell etc., thereby simplify production process, shorten the PT, enhance productivity, and be easy to carry out enlarged culturing.
Existing research has found to influence some genes of cell adhesion.For example human siat7e gene is considered in control cell attaching degree, play certain function; The siat7e expression of gene that increases can reduce cell adhesion (Jaluria P; Betenbaugh M; Konstantopoulos K, Frank B, Shiloach J (2007) Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells.Metab Eng 9:241-251.).
Swine fever (HC) is that (it is popular extensively for Hog cholera virus, a kind of hyperinfection property disease of the pig that HCV) causes, and sickness rate, mortality ratio height have caused serious economy loss to pig industry by CSFV.The effective means of existing prevention and control swine fever is the inoculation swine Fever Vaccine
Be used for CSFV cultured cells matrix at present and mainly contain bull testis primary cell (BT cell), pig kidney cell (PK cell or IBRS-2 cell), pig testis cell (ST cell) etc.Because China cows are sick than ubiquity BVD/MBV, with the seedling of bull testis cell primary cell, be prone to cause cell external source virus pollution, and it is not high to produce malicious titre, differences between batches are big, bring difficulty to the raising of vaccine output, effectiveness.And the bull testis cell is primary cell, the limited service life of cell, and each seedling need regain cell.And pig kidney cell, pig testis cell are continuous cell line, draw materials to be easy to easily amplify and carry out mass-producing and cultivate, and the trend that replaces bull testis cells produce swine Fever Vaccine is gradually arranged.Non-patent literature 1 shows that PK, ST, MPK cell are responsive to CSFV, and multiple swine Fever Vaccine strain adapts on pig kidney cell or pig testis cell and obtains vaccine strain.
Non-patent literature 1: CSFV and vaccine research progress thereof, Chinese virusology, the 201st～207 page of the 11st the 3rd phase of volume, in September, 1996.
But; Have now and be rolling bottle adherent culture or microcarrier suspension culture as cytostromatic swine Fever Vaccine production technique with pig testis cell or pig kidney cell; Also fail to realize the full suspension culture of cell, all have big limitation in production cost, production efficiency and aspect meeting the need of market.
Summary of the invention
The objective of the invention is to solve the problem that exists in the method for cultured swine testicular cell in the prior art or pig kidney cell, the method for a kind of extensive suspension culture pig testis cell or pig kidney cell is provided.
The method of suspension culture pig testis cell of the present invention or pig kidney cell comprises the steps:
(1) utilization comprises the expression vector stable transfection pig testis cell or the pig kidney cell that can weaken cell adhesion performance gene order;
(2) screening and separating stable transfectional cell clone;
(3) genetic expression among the detection stable transfected cells clone;
(4) stable transfected cells clone's suspension culture.
Further aim of the present invention provides a kind of suspension culture pig testis cell or pig kidney cell to produce the method for vaccine.
Description of drawings
Fig. 1 representes the RT-PCR detection figure of stable transfection pig testis cell clone among the embodiment 1.Wherein, GAPDH and 18s RNA are internal control gene, contrast to be the pig testis cell without transfection.A, B and C are respectively the stable transfected cells clone who is separated to.RT-PCR result shows among A, B and three stable transfected cells clones of C the siat7e expression of gene is arranged all.
Fig. 2 representes the cell growth curve of stable transfection pig testis cell clone among the embodiment 1.The result shows that the growth characteristics of stable transfection pig testis cell clone are normal.
Embodiment
Through pig testis cell or pig kidney cell genome are carried out genetic modification, obtain to be suitable for the pig testis cell or the pig kidney cell of suspension culture, ins conjunction with cell cultures technology, realize through extensive suspension culture pig testis cell or pig kidney cell with the production vaccine.
The method of suspension culture pig testis cell of the present invention or pig kidney cell comprises the steps:
(1) utilization comprises the expression vector stable transfection pig testis cell or the pig kidney cell that can weaken cell adhesion performance gene order;
(2) screening and separating stable transfectional cell clone;
(3) genetic expression among the detection stable transfected cells clone;
(4) stable transfected cells clone's suspension culture.
In described step (1), described gene order is the siat7e gene; Carrier can be expression vector conventional in the present technique field, for example plasmid.Described expression vector both can prepare according to ordinary skill in the art means voluntarily, can also buy acquisition.
DNA is imported eukaryotic mode have two kinds: transient transfection and stable transfection.Recombinant DNA imports infectious strong clone to obtain the temporary transient but high-caliber expression of goal gene in transient transfection.The DNA of transfection needn't be integrated into host chromosome, and when a large amount of samples need be analyzed at short notice, especially harvested cell in 1 to 4 day after transfection when the lysate of gained is used for the testing goal expression of gene, can adopt the mode of transient transfection.The temporary transient expression of DNA is transient expression in the transient transfection.
Stable or persistent transfection is used to set up clone's clone, and the goal gene of transfection is incorporated into and and guides the synthetic of an amount of target protein in the chromosomal DNA in this clone.In general (by the cell type decision), the efficient that forms the efficiency ratio transient transfection of stable transfected cells is hanged down 1 to 2 one magnitude.Utilize selectable genetic marker to help in the background of non-transfected cell, isolating rare stable transfection body.The expression of integrator gene is stably express in the stable transfection.
Gene is imported eukaryotic method has many kinds, comprises the biochemical method transfection, physical method transfection and virus-mediated conversion etc.Particularly comprise DEAE (diethylaminoethyl-)-VISOSE mediated method, calcium phosphate mediated method, liposome mediated-method; Polycation-DMSO infection protocol, biomone mediated method, electroporation transfection method; Microinjection, virus-mediated method etc. preferably adopt liposome mediated-method.
Described step (1) further comprises the steps:
(a) with the human siat7e expression vector of total length transfection Escherichia coli DH5 α competent cell, plasmid purification; The method of plasmid purification can be the conventional technique means in this area, can take the method for test kit or non-test kit;
(b) switching 1 * 10
5-5 * 10
5Pig testis cell or pig kidney cell in Tissue Culture Plate, incubated cell 20-24h, about 40-90% degree of converging; Said pig testis cell or pig kidney cell can be that for example commodity are called ATCC, the commercial goods of Cat.No.CCL-34;
(c) DNA of getting 1-10 μ g is diluted in the 250 μ L transfection media (like the Opti-MEM substratum) mixing; 2-50 μ L liposome is diluted in the 250 μ L transfection media, and mixing leaves standstill 5-15min gently;
(d) solution with above-mentioned preparation mixes, and room temperature leaves standstill 10-20min; Each hole in the Tissue Culture Plate described in the step (b) is changed into the 0.5-2mL transfection media of serum-free antibiotic-free; Mixture in the above-mentioned steps (c) is changed in the cell cultures plate hole of the described incubated cell of step (b), mixing is rocked in front and back gently;
(e) 37 ℃ of cultivations behind the 1-24h, change the substratum in each hole into perfect medium in 37 ℃ of cultivations;
(f) behind the 24h, cell is reached in the Tissue Culture Flask from Tissue Culture Plate.
In said step (2), because picked-up, integration and expression foreign DNA are small probability events, usually according to new phenotypic screen stable transfection body.Generally speaking, this phenotype is provided by the gene of the coding antibiotics resistance of common transfection.Express the cell of the genetic marker on the dna molecular and often also express the genetic marker that another dna molecular carries.Therefore, the cell of stably express selected marker (like antibiotics resistance) also other dna sequence dnas on the expression vector dna probably.This physically disjunct gene is integrated into the phenomenon of expressing in the same transformant and is called cotransformation.Existing selection markers commonly used comprises glucosaminide phosphotransferase (G418 or Xin Meisu are had resistance), Totomycin-B phosphotransferase (Totomycin-B is had resistance), xanthine-guanine phosphoribosyl transferase (mycophenolic acid, AMT are had resistance) and tetracycline-N-acetyltransferase (tetracycline is had resistance).The present invention preferably adopts G418 pressurization screening.
Described step (2) further comprises the steps:
Behind the cell transfecting 24-48h, the perfect medium in the Tissue Culture Flask is replaced by selective medium, changed a subculture in later every 2-4 days, continue to cultivate 14-21 days, observe whether forming the clone, the independent cell clone separates, increases.The mark of said screening can be a selection markers well-known to those skilled in the art, includes but not limited to the G418 selection markers, and described selective medium is preferably the G418 selective medium.Utilize selective medium to promote the resistant cell growth, the non-resistant cell is promptly dead.
Described step (3) further comprises the steps:
Behind the clone to be formed; Picking the clone identify; Said authentication method is mainly the detection to rna level that is cloned in the insertion gene that obtains and protein level expression, and wherein the detection method of rna level mainly contains: RT-PCR (reverse transcription polymerase chain amplified reaction), quantitative fluorescent PCR (fluorescent quantitative poly chain type amplified reaction), Northern hybridization etc.The detection method of protein level mainly contains: Western hybridization, ELLSA (EUSA), immunofluorescence detect, immunohistochemistry technology is detected etc.
In said step (4), screening is obtained and tames to suspension culture through detecting cell clone that the human siat7e stable gene of proof expresses in Tissue Culture Flask (square vase or shake bottle).To tame suspended culture cell get a part frozen, another part in Tissue Culture Flask, continues the cultivation., vigor normal when cell growthhabit in Tissue Culture Flask is seeded to greater than 90% the time and shakes in the bottle.Amplification cultivation cell in shaking bottle and bio-reactor; Be seeded in the production-scale bio-reactor by suitable cell inoculation density at last; Parameters such as the temperature of adjusting bio-reactor, medium pH value, oxyty make cell grow in the optimum environment and carry out cell cultures.
The condition of the suspension culture of described step (4) is:
Container: the suspension culture container is preferably and shakes bottle (shake bottle), blender jar (Spinner Bottle), bio-reactor etc.;
Cell inoculation density: 1 * 10
5-1 * 10
6, preferred 2 * 10
5-6 * 10
5
Cell cultures temperature: 33-38 ℃, preferred 36-37 ℃;
PH value: 6.0-7.5, preferred 6.8-7.2;
Oxyty: 20%-100%, preferred 30%-80%.
The cell cultures mode can be batch cultivation, feeding culture and perfusion culture.
Said bio-reactor includes but not limited to stirring type bioreactor, airlift bioreactor, fixed bed and fluidized bed bio reactor drum, hollow fiber reactor, membrane bioreactor, disposable bioreactor etc.
In various embodiments of the present invention, the cell culture medium of being selected for use is not crucial, and all can be used in the pig testis cell or the used cell culture medium of pig kidney cell suspension culture can use in principle, are preferably serum-free cell culture medium.A lot of substratum all have been commercially produced products, can obtain through commercial sources.
The invention still further relates to suspension culture pig testis cell or pig kidney cell to produce the method for swine Fever Vaccine.
The pig testis cell or the pig kidney cell of extensive suspension culture stable transfection, the cell density propagation of treating pig testis cell or pig kidney cell is to 1 * 10
6When cell/mL is above, cell culture fluid is replaced by keeps nutrient solution, with viral suspension inoculation pig testis cell or pig kidney cell.Its condition is following:
(cell density is at least about 1 * 10 with the pig testis cell of virus infection propagation or pig kidney cell
6)
Virus infection plural number (m.o.i): 0.0001-10, preferred 0.001-5, more preferably 0.01-1;
Virus culture temperature: 33-38 ℃, preferred 36-37 ℃;
PH:6.5-8.0, preferred 7.2-7.8;
Dissolved oxygen: 20%-100%, preferred 30%-80%.
Can adopt batch cultivation, feeding culture and perfusion culture mode culturing cell propagative viruses, and can cultivate venom by disposable or continuous in good time harvested cell.Preferred feeding culture or perfusion culture.Promptly at virus infection pig testis cell or pig kidney cell after for some time, behind 6-18hr, can adopt stream to add or dabbling cultured continuously mode interpolation virus in the bio-reactor is kept liquid, constantly gather in the crops viral supernatant liquid.The viral liquid of results places below-15 ℃ and preserves.Can adopt the whole bag of tricks further to prepare vaccine, for example gather in the crops viral liquid and be positioned in 2-8 ℃, and be kept at-20 ℃, freeze thawing 1 time prepares vaccine after filtration, deactivation, the emulsification.
In this article, term " virus infection plural number MOI (multiplicity of infection) " is meant that each cell attendes the quantity of institute's infective virus.The infectious virus quantity of MOI=/cell total amount.
In various embodiments of the present invention, the virus strain of being selected for use can be obtained by production of vaccine producer or research institution for producing or develop the virus strain commonly used of said vaccine.
The present invention realized suspension culture pig testis cell or pig kidney cell and utilized this cells produce swine Fever Vaccine, compares with existing adherent culture cell or microcarrier suspension culture production technique to have following beneficial effect:
(1) the full suspension culture of realization pig testis cell and pig kidney cell need not to purchase microcarrier, has greatly reduced the swine Fever Vaccine production cost;
(2) can save in the microcarrier suspension culture additional step like the digestion harvested cell etc., thereby simplify production process, shorten the PT, enhance productivity;
(3) in bio-reactor microcarrier culture process, the microcarrier amplification technique has big technical difficulty, and has increased the complicacy of production technique.And realize pig testis cell and pig kidney cell suspension culture, then the amplification culture technology of cell is comparatively simple, is easy to realize and greatly simplified production technique.
(4) production technique is improved and can be satisfied the ever-increasing market requirement of swine Fever Vaccine.
Embodiment
Below further bright specifically to the present invention's work through embodiment and Comparative Examples, but not as limitation of the present invention.
Embodiment 1 utilizes genetic modification suspension culture ST cell
1) utilization contains siat7e expression carrier stable transfection ST cell;
(1) with the human siat7e expression vector of total length (Cat.No.EX-V1581-M03, Genecopoeia) transfection Escherichia coli DH5 α competent cell.Utilize no intracellular toxin plasmid extraction test kit (QIAGEN) to extract DNA.
(2) first days: switching 5 * 10
5ST cell (China Veterinery Drug Inspection Office provides) is hatched about 90% degree of converging of 24h cell in 6 orifice plates.
(3) second days, the DNA of getting 10 μ g was diluted in the 250 μ L Opti-MEM substratum mixing; 50 μ L liposomes are diluted in the 250 μ L Opti-MEM substratum, and mixing leaves standstill 15min gently.
(4) solution with above-mentioned preparation mixes, and room temperature leaves standstill 20min; Each hole in 6 orifice plates described in the step (2) is changed into the 2mL Opti-MEM substratum of serum-free antibiotic-free; 500 μ L change in 6 orifice bores with the mixture in the above-mentioned steps (3), and mixing is rocked in front and back gently.
Change the substratum in each hole into perfect medium DMEM (containing 10% calf serum) behind (5) 37 ℃ of cultivation 24h, 37 ℃ in CO
2Incubator is cultivated.
(6) behind the 24h, it is reached in the Tissue Culture Flask from 6 orifice plates.
2) G418 pressurization screening is to obtain stable expression cell strain;
Behind the 48h, the perfect medium in the Tissue Culture Flask is replaced by the G418 selective medium, changed a not good liquor in later per 4 days, continue to cultivate 14 days, observe every day, sees whether form the clone.After resistance clone occurs, obtain 5 strain monoclonal cell strains according to the unicellular separation and Culture of unicellular isolation cultivation method (levy in Hubei Province, and tissue culture and molecular cell learn a skill, the .100 of Beijing Publishing House～107, January calendar year 2001), respectively enlarged culturing.
3) stable transfection of checking gene;
Utilize RNA to extract test kit (Qiagen company) and extract the total RNA that obtains monoclonal cell and contrast ST cell.Following according to siat7e gene order design primer:
Forward primer: 5 '-ttactcgccacaagatgctg-3 '
Reverse primer: 5 '-gcaccatgccataaacattg-3 '
Utilize superscrpt reverse transcription-polysaccharase formula reaction kit (invitrogen company) to carry out reverse transcription-polysaccharase formula reaction check siat7e expression of gene in the monoclonal cell that obtains.Proof institute obtains that the siat7e gene all has expression (as shown in Figure 1) in the monoclonal cell.
Screening is obtained and through detecting cell clone that the human siat7e stable gene of proof expresses (square vase or shake bottle) domestication suspension culture in Tissue Culture Flask.
Draw cell growth curve: but after the cell stable suspersion is cultivated, get the growth conditions good cell, process cell suspension; With 4 * 10
5Cell/mL places 37 ℃ of incubators to cultivate in shaking bottle cell inoculation, and take out cell and count every day, computation of mean values.With the incubation time is transverse axis, and cell density is the longitudinal axis (logarithm), draws cell growth curve (as shown in Figure 2).
Embodiment 2-4
Except changing the experiment condition, carry out 1 identical operations with embodiment according to table 1.
Table 1
Embodiment 5 utilizes the method for the ST cells produce swine Fever Vaccine of large scale culturing genetic modification suspension culture
To pass through suspension culture checking cell (ST cell, embodiment 1), to get a part frozen, and another part continues cultivation in Tissue Culture Flask., vigor normal when cell growthhabit in Tissue Culture Flask is seeded to greater than 90% the time and shakes in the bottle.Amplification cultivation cell in shaking bottle is by 3 * 10
5Cell/mL cell density is seeded in the 650L bio-reactor (the clear big day Science and Technology Ltd. in Beijing), and wherein cell culture condition is following:
Working volume: 300L
Cell cultures temperature: 37 ℃
pH：7.0
Dissolved oxygen: 40%;
Cell culture fluid: improvement MEM cell culture medium MD611 (the clear big day Science and Technology Ltd. in Beijing)+5% serum
Cultivated 4 days, when cell density is 3 * 10
6During cell/mL, cell culture fluid being replaced by keeping nutrient solution, is 0.1 amount inoculation CSFV by virus infection plural number MOI: Chinese rabbitization attenuated vaccine strain (CSFV C strain).
Virus culture condition: temperature: 37 ℃; PH:7.4; Dissolved oxygen: 40%.
Cultivate and gather in the crops obtained vaccine after 3 days, detect virus titer 1gTCID
50/ mL is 8.9.
Embodiment 6～9, comparative example 1
Except changing the experiment condition according to table 2, carry out 5 identical operations, and inoculate different vaccines with embodiment, the titre result of vaccine is shown in table 2 in the lump.Wherein comparative example 1 is the contrast ST cell without transfection, carries out the bio-reactor microcarrier suspension culture.
Table 2
Can find out through the foregoing description and comparative example; The virus titer that embodiment and comparative example obtain is close; Thereby explain,, be applied to the large scale culturing of vaccine through the pig testis cell or the pig kidney cell that are suitable for suspension culture that genetic modification obtains; When saving production cost, enhancing productivity and simplifying production stage, kept susceptibility to virus.The present invention has very high using value aspect suspension culture pig testis cell or the pig kidney cell scale operation vaccine.
Claims (11)
1. the method for suspension culture pig testis cell or pig kidney cell, it comprises the steps:
(1) utilization comprises the expression vector stable transfection pig testis cell or the pig kidney cell that can weaken cell adhesion performance gene order;
(2) screening and separating stable transfectional cell clone;
(3) genetic expression among the detection stable transfected cells clone;
(4) stable transfected cells clone's suspension culture.
2. utilize in the method according to claim 1, wherein said step (1) and contain siat7e expression carrier stable transfection pig testis cell or pig kidney cell.
3. method according to claim 1, the method for said transfection comprise diethylaminoethyl--VISOSE mediated method, calcium phosphate mediated method; Liposome mediated-method, polycation-DMSO infection protocol, biomone mediated method; The electroporation transfection method, microinjection, virus-mediated method.
4. method according to claim 3, the method for said transfection are liposome mediated-method.
5. method according to claim 1; Wherein, after described step (2) comprised the steps: cell transfecting 24-48h, the perfect medium in the Tissue Culture Flask was replaced by selective medium; Changed a subculture in later every 2-4 days; Continue to cultivate 14-21 days, observe whether forming cell clone, the independent cell clone separates, increases.
6. method according to claim 1; Wherein, Described step (3) comprises the steps: to utilize rna level detection method or protein level detection method to detect the genetic expression among the stable transfected cells clone; Wherein, described rna level detection method comprises reverse transcription polymerase chain amplified reaction, fluorescent quantitative poly chain type amplified reaction, Northern hybridization; Described protein level detection method comprises that Western hybridization, EUSA, immunofluorescence detect, immunohistochemistry technology is detected.
7. method according to claim 1, wherein, the condition of the suspension culture of described step (4) is:
Container: suspension culture container;
Cell inoculation density: 1 * 10
5-1 * 10
6
Cell cultures temperature: 33-38 ℃;
PH value: 6.0-7.5;
Dissolved oxygen: 20%-100%.
8. method according to claim 7, wherein, described suspension culture container is for shaking bottle, bio-reactor; Said cell inoculation density is 2 * 10
5-6 * 10
5Said cell cultures temperature is 36-37 ℃; Said pH is 6.8-7.2; Said dissolved oxygen is 30%-80%.
9. method that adopts any described method obtains among the claim 1-8 pig testis cell or pig kidney cell to produce swine Fever Vaccine.
10. method according to claim 9, wherein, the condition of producing vaccine is:
With virus infection pig testis cell or pig kidney cell;
Virus infection plural number: 0.001-5;
Virus culture temperature: 33-38 ℃;
PH value: 6.5-8.0;
Dissolved oxygen: 20%-100%.
11. method according to claim 10, wherein, the culture density of said pig testis cell or pig kidney cell is at least 1 * 10
6Said virus infection plural number is 0.01-1; Said virus culture temperature is 36-37 ℃; Said pH is 7.2-7.8; Said dissolved oxygen is 30%-80%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105112352A (en) * | 2015-07-13 | 2015-12-02 | 郑州爱科生物科技有限公司 | ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method |
| CN106085946A (en) * | 2016-06-13 | 2016-11-09 | 金宇保灵生物药品有限公司 | Can be with the Pig testicular cell strain ST S of suspension culture and preparation method thereof and application |
| CN108795841A (en) * | 2017-05-03 | 2018-11-13 | 上海奥浦迈生物科技有限公司 | A kind of ST culture mediums and its application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112352A (en) * | 2015-07-13 | 2015-12-02 | 郑州爱科生物科技有限公司 | ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method |
| CN105112352B (en) * | 2015-07-13 | 2018-07-24 | 郑州爱科生物科技有限公司 | One plant of ST cell for adapting to the full culture that suspends and its application and the method for cultivating vaccine virus |
| CN106085946A (en) * | 2016-06-13 | 2016-11-09 | 金宇保灵生物药品有限公司 | Can be with the Pig testicular cell strain ST S of suspension culture and preparation method thereof and application |
| CN106085946B (en) * | 2016-06-13 | 2019-11-22 | 金宇保灵生物药品有限公司 | Can suspend culture Pig testicular cell strain ST-S and its preparation method and application |
| CN108795841A (en) * | 2017-05-03 | 2018-11-13 | 上海奥浦迈生物科技有限公司 | A kind of ST culture mediums and its application |
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Application publication date: 20120516 |