CN116694575B - Method for suspension culture of Marc145 cells - Google Patents

Method for suspension culture of Marc145 cells Download PDF

Info

Publication number
CN116694575B
CN116694575B CN202310961691.0A CN202310961691A CN116694575B CN 116694575 B CN116694575 B CN 116694575B CN 202310961691 A CN202310961691 A CN 202310961691A CN 116694575 B CN116694575 B CN 116694575B
Authority
CN
China
Prior art keywords
cells
culture
marc145
cell
suspension
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202310961691.0A
Other languages
Chinese (zh)
Other versions
CN116694575A (en
Inventor
侯瑞娟
张国仁
杨云
蔡仕君
罗顺
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Aosikang Biopharmaceutical Co ltd
Original Assignee
Shanghai Aosikang Biopharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Aosikang Biopharmaceutical Co ltd filed Critical Shanghai Aosikang Biopharmaceutical Co ltd
Priority to CN202310961691.0A priority Critical patent/CN116694575B/en
Publication of CN116694575A publication Critical patent/CN116694575A/en
Application granted granted Critical
Publication of CN116694575B publication Critical patent/CN116694575B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2527/00—Culture process characterised by the use of mechanical forces, e.g. strain, vibration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The application provides a method for domesticating adherent Marc145 cells into suspension cells, which comprises the steps of obtaining a resistance cell strain for stably expressing Siat7e gene through hygromycin resistance gene pressurization screening, performing single cell domestication in a serum-free culture medium to realize proliferation of full suspension cells, and obtaining a full suspension Marc145 monoclonal cell line with high yield and sustainable stable passage growth through limiting dilution selection. The Marc145 suspension cells of the application can effectively improve the cell growth cell density by optimizing key components such as glutamine, glutamic acid, aspartic acid, asparagine and the like in a serum-free culture medium, and the suspension culture can realize the highest cell density reaching 6.0 multiplied by 10 6 cells/mL~8.0×10 6 The method lays a foundation for suspension culture of Marc145 cells and industrialized production of vaccine, and also provides a new way for industrialized mass production of porcine reproductive and respiratory syndrome inactivated vaccine.

Description

Method for suspension culture of Marc145 cells
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for suspension culture of Marc145 cells.
Background
Marc145 cells, namely monkey embryo kidney epithelial cells, are derived from monkey kidney cells, are clone strains of maternal cells (MA 104 cells), are subjected to anchorage-dependent growth, can be continuously subjected to subculture, have no tumorigenicity, are particularly sensitive to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and are widely used for producing porcine reproductive and respiratory syndrome vaccines at present.
Conventional Marc145 cell culture adopts a two-dimensional single cell layer adherence production method, however, in the industrial scale-up production process, the adherence performance is limited, the digestion process also increases the complexity, the production time and the cost of the process, the quality is unstable, and the batch difference exists, so that the Marc145 cell adherence growth is necessary to be acclimatized into suspension growth. Although the prior art CN102453700a describes the transfection of MDCK cells with an expression vector for the sial 7e gene, there is no description of the transfer of the sial 7e gene into Marc145 cells and the suspension culture used to acclimate Marc145 cells, and there is no improvement in the cell density of the culture.
While the prior art CN102453699a describes stable transfection of Marc145 cells or MA104 cells with expression vectors containing the siat7e gene, it does not describe how to specifically acclimate adherent Marc145 cells into suspension cells. Meanwhile, the prior art has the technical problems of low cell growth density, low titer of subsequent vaccine preparation and the like in the culture process. Therefore, the existing method for suspension culture of Marc145 cells has great limitation in terms of production cost, production efficiency and market demand, and especially cannot meet the requirement of expanded industrial production in terms of density.
Disclosure of Invention
Based on the above, the application provides a method for suspension culture of Marc145 cells, which aims to solve the technical problems of high production cost and low growth density of suspension cells in the process of suspension culture of Marc145 cells in industrial production.
The technical scheme of the application is that a method for suspension culture of Marc145 cells is provided, which comprises the following steps:
constructing an adherent type Marc145 cell strain for stably expressing the Siat7e gene, domesticating and culturing the adherent type Marc145 cell strain for stably transfecting the Siat7e gene, and domesticating the adherent type Marc145 cell strain into a suspension type Marc145 cell strain.
The acclimation culture comprises a suspension acclimation stage with a serum-containing culture medium and a suspension acclimation stage with a serum-free culture medium which are sequentially carried out.
In one embodiment, the serum-free medium is CDMarc145 medium.
In one embodiment, the acclimatization culture includes the steps of:
and S1, performing adherence culture on the adherence type Marc145 cell strain of the stably transfected siat7e gene in a complete culture medium containing a resistance screening drug.
And S2, inoculating the adherent cell strain subjected to passage S1 after digestion and centrifugation in a serum-free culture medium containing a resistance screening drug and 1.5-2.5 v/v% of newborn bovine serum for suspension culture.
And step S3, inoculating the cells cultured in the step S2 into a serum-free culture medium containing a resistance screening drug and 0.5-1 v/v% of newborn bovine serum for suspension culture.
And S4, inoculating the cells cultured in the step S3 into a serum-free culture medium containing a resistance screening medicament for suspension culture.
And S5, inoculating the cells cultured in the step S4 into a serum-free culture medium for suspension culture.
In one embodiment, the serum-free medium comprises one or more of glutamine, glutamic acid, aspartic acid, and asparagine.
In one embodiment, the glutamine content is 0.5 mM-1.5 mM; the glutamic acid content is 4 mM-6 mM; the content of the aspartic acid is 4 mM-6 mM; the content of the asparagine is 1.5 mM-2.5 mM.
In one embodiment, the cell seeding density in step S2 is 0.5X10 6 cells/mL~1.0×10 6 cells/mL; or/and, in step S3, the cell inoculation density is 0.8X10 6 cells/mL~1.2×10 6 cells/mL; or/and, in step S4, the cell inoculation density is 1.0X10 6 cells/mL~1.5×10 6 cells/mL; or/and, in step S5, the cell inoculation density is 1.4X10 6 cells/mL~1.7×10 6 cells/mL。
In one embodiment, the suspension culture is shaking culture.
Optionally, the shaking culture is shaking culture, and the rotating speed is 180 rpm/min-220 rpm/min.
In one embodiment, the continuous subculture is carried out for 5-10 times in the culture process of the steps S1-S4, and the subculture interval is 3-4 days.
In one embodiment, the resistance-screening drug comprises one or more of hygromycin-B phosphotransferase, aminoglycoside phosphotransferase, xanthine-guanine phosphoribosyl transferase, and puromycin.
In one embodiment, the method of stably transfecting the siat7e gene comprises one or more of electrotransfection, chemical transfection, microinjection, virus mediated method.
The application provides a method for domesticating adherent Marc145 cells into suspension cells, which comprises the steps of obtaining a resistance cell strain stably expressing Siat7e gene through resistance screening genes, performing suspension domestication by a serum-containing culture medium and performing domestication by a serum-free culture medium sequentially to realize proliferation of the whole suspension cells, and obtaining a high-yield and sustainable stable passage-growing whole suspension Marc145 monoclonal cell line through limiting dilution selection. The Marc145 suspension cells of the application can effectively improve the cell growth cell density by optimizing key components such as glutamine, glutamic acid, aspartic acid, asparagine and the like in a serum-free culture medium, and the monoclonal suspension cell culture can realize the highest cell density reaching 6.0x10 6 cells/mL~8.0×10 6 The Marc145 suspension cells of the application reach a viral titer of 10 after infection on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) at cells/mL 9 ~10 10 /mL TCID 50 Compared with the adherence process and the high-glutamine culture medium, the virus titer is improved by 10-20 times. The method lays a foundation for suspension culture of Marc145 cells and industrialized production of vaccines, and also provides a new way for industrialized mass production of porcine reproductive and respiratory syndrome inactivated vaccines.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.
FIG. 1 shows the fluorescence of the selected resistant clone eGFP after transfection of Marc145 cells according to the application;
FIG. 2 shows eGFP fluorescence after Marc145 cell acclimation and suspension in accordance with the present application;
FIG. 3 shows the state of Marc145 cells after complete acclimation according to the present application;
FIG. 4 shows a Marc145 cell acclimation and growth map of the present application.
Detailed Description
The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Terminology
As used in this disclosure, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.
In the present application, "suitable" in "suitable combination manner", "suitable manner", "any suitable manner", etc., are used to implement the technical scheme of the present application, solve the technical problem of the present application, and achieve the technical effects expected by the present application.
In the present application, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present application.
In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.
In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1-10, and where t is an integer selected from any one of the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
The technical scheme of the application is that a method for domesticating adherent Marc145 cells into suspension cells is provided, which comprises the following steps:
constructing an adherent type Marc145 cell strain for stably expressing the Siat7e gene, domesticating and culturing the adherent type Marc145 cell strain for stably transfecting the Siat7e gene, and domesticating the adherent type Marc145 cell strain into a suspension type Marc145 cell strain.
The acclimation culture comprises a suspension acclimation stage with a serum-containing culture medium and a suspension acclimation stage with a serum-free culture medium which are sequentially carried out.
In a specific example, the serum-free medium is CD Marc145 medium.
In a specific example, the transfer from a serum-containing medium to a serum-free medium specifically comprises the steps of:
and S1, performing adherence culture on the adherence type Marc145 cell strain of the stably transfected siat7e gene in a complete culture medium containing a resistance screening drug. Specifically, the Marc145 cell strain of the stably transfected siat7e gene is continuously subjected to subculture for 5 times in a10 v/v% serum DMEM medium containing 250-500 mug/mL hygromycin.
And S2, inoculating the adherent cell strain subjected to passage S1 after digestion and centrifugation in a serum-free culture medium containing a resistance screening drug and 1.5-2.5 v/v% of newborn bovine serum for suspension culture. The method comprises the following steps: and (3) after the cells subjected to passage S1 are digested and centrifuged, inoculating the cells into a serum-free culture medium containing 450-500 mu g/mL hygromycin, adding 1.5-2.5 v/v% of newborn bovine serum into a suspension culture container for shake culture, and continuously carrying out passage at intervals of 4-8 days for 5-10 times.
And step S3, inoculating the cells cultured in the step S2 into a serum-free culture medium containing a resistance screening drug and 0.5-1 v/v% of newborn bovine serum for suspension culture. The method comprises the following steps: inoculating the cells cultured in the step S2 into a serum-free culture medium containing 450-500 mu g/mL hygromycin, adding 0.5-1 v/v% of new born calf serum, and re-suspending the new born calf serum into a suspension culture container for shake culture, wherein the culture is carried out for 5-10 times at intervals of 3-4 days.
And S4, inoculating the cells cultured in the step S3 into a serum-free culture medium containing a resistance screening medicament for suspension culture. The method comprises the following steps: and (3) inoculating the cells cultured in the step (S3) into a serum-free culture medium containing 450-500 mu g/mL hygromycin for shake culture, and continuously carrying out passage at intervals of 3-4 days for 5-10 times. The method comprises the following steps: and (3) inoculating the cells cultured in the step (S4) into a serum-free culture medium for shake culture, and continuously carrying out subculture for 5-10 times at intervals of 2-3 days.
And S5, inoculating the cells cultured in the step S4 into a serum-free culture medium for suspension culture.
Further optionally, the serum-free medium further comprises one or more of glutamine, glutamic acid, aspartic acid, and asparagine.
In a specific example, the glutamine content is 0.5mm to 1.5mm; the glutamic acid content is 4 mM-6 mM; the content of the aspartic acid is 4 mM-6 mM; the content of the asparagine is 1.5 mM-2.5 mM.
Further alternatively, the inoculation density in step S2 is 0.5X10 6 cells/mL~1.0×10 6 cells/mL. For example, it may be 0.5X10 6 cells/mL、0.6×10 6 cells/mL、0.7×10 6 cells/mL、0.8×10 6 cells/mL、0.9×10 6 cells/mL、1.0×10 6 cells/mL。
In a specific example, the inoculation density in step S3 is 0.8X10 6 cells/mL~1.2×10 6 cells/mL. For example, it may be 0.8X10 6 cells/mL、0.9×10 6 cells/mL、1.0×10 6 cells/mL、1.1×10 6 cells/mL、1.2×10 6 cells/mL。
In a specific example, the inoculation density in step S4 is 1.0X10 6 cells/mL~1.5×10 6 cells/mL. For example, it may be 1.0X10 6 cells/mL、1.1×10 6 cells/mL、1.2×10 6 cells/mL、1.3×10 6 cells/mL、1.4×10 6 cells/mL、1.5×10 6 cells/mL。
In a specific example, the inoculation density in step S5 is 1.4X10 6 cells/mL~1.7×10 6 cells/mL. For example, it may be 1.4X10 6 cells/mL、1.5×10 6 cells/mL、1.6×10 6 cells/mL、1.7×10 6 cells/mL。
Optionally, the suspension culture is shaking culture, so that the cells are in a non-static state; for example, the culture may be shake culture, and the rotation speed of the shake culture is 180rpm/min to 220rpm/min.180rpm/min to 220rpm/min. For example, the rotation speed is 180rpm/min, 190rpm/min, 200rpm/min, 210rpm/min, 220rpm/min.
In one embodiment, the continuous subculture is carried out for 5-10 times in the culture process of the steps S1-S4, and the subculture interval is 3-4 days.
In one embodiment, the resistance-screening drug comprises one or more of hygromycin-B phosphotransferase, aminoglycoside phosphotransferase, xanthine-guanine phosphoribosyl transferase, and puromycin.
In one embodiment, the method of transfection comprises one or more of electrotransfection, chemical transfection, microinjection, virus mediated methods.
The Marc145 cytochemical transfection method comprises the following steps: marc145 adherent cells were digested with pancreatin at 1.0 to 2.0X10 5 cell/well density was inoculated into 24-well plates in the presence of DMEM containing 10% serum at 37deg.C and 5% CO 2 After Marc145 cells are plated for 24 hours, the grown monolayer confluence reaches 80% -90% and is used for transfection; respectively diluting DNA and liposome with a serum-free DMEM culture medium, wherein the amount of the DNA is 1-3 mug, and the ratio of the DNA to the liposome comprises 1:1, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 and 1:5, and incubating for 10-15 minutes to obtain a transfection complex; the transfection complex is added into a 24-hole plate cell for culturing, and after 4-6 hours of transfection, the cell is replaced by a DMEM recovery medium containing 10% of serum, wherein the culture condition is 37 ℃ and 5% of CO 2 。
The Marc145 cell electrotransformation method comprises the following steps: digesting Marc145 cells with pancreatin, centrifuging at low speed, and diluting the obtained cells with transfection medium DMEM to 6.0-8.0X10 6 Adding electric rotating cup after cell/0.4. 0.4 mL, setting electric rotating program, transferring cells to T75-T175 square bottle for culturing at 37deg.C under 5% CO 2 。
In a specific example, the method further comprises the steps of stable passaging and freezing of the domesticated Marc145 suspension cell strain, wherein the density reaches 4.0X10 when the domesticated cell passaging reaches 3 days 6 cells/mL~6.0×10 6 The cell/mL has the activity rate of more than 95%, and the suspension Marc145 cell strain is obtained and frozen after larger lumps are cut off with good dispersibility.
Alternatively, the cell density upon cryopreservation is 20.0X10 6 cells/mL~30.0×10 6 cells/mL. For example, 20X 10 is possible 6 cells/mL、21×10 6 cells/mL、22×10 6 cells/mL、23×10 6 cells/mL、24×10 6 cells/mL、25×10 6 cells/mL、26×10 6 cells/mL、27×10 6 cells/mL、28×10 6 cells/mL、29×10 6 cells/mL、30×10 6 cells/mL。
Further alternatively, the freezing and storing step is to use a program cooling box in a refrigerator at the temperature of minus 80 ℃ for 22-26 hours, and then transfer a freezing and storing tube into a liquid nitrogen tank for long-term storage. For example 22h, 23h, 24h, 25h or 26h.
Optionally, limiting dilution is also carried out to select monoclonal after the frozen Marc145 suspension cell strain is obtained, and the specific method is as follows: and (3) carrying out one round of limiting dilution with the cell density of 0.5-1.0, adding direct cell image evidence, sequentially transferring the selected monoclonal cells to a shake flask for culturing, and selecting stable proliferation and better-growth monoclonal cryopreservation.
Optionally, the specific steps are as follows:
(1) Marc145 cell gene engineering: transferring eukaryotic expression vector Siat7e gene plasmid into Marc145 adherent cells, and screening with culture medium containing resistance screening medicine to obtain drug-resistant cell strain. Specifically, eukaryotic expression vector Siat7e gene (ST 6GALNAC5 cDNA ORF Clone, human, C-GFPSpark 1) plasmid is transferred into Marc145 adherent cells, and hygromycin of 250-500 mug/mL is used for screening to obtain drug-resistant cell strains.
(2) Screening and establishing Marc145 cell strain stably expressing Siat7e gene: and continuously screening Marc145 cell clones with drug resistance in the culture medium containing the drug for resistance screening, and obtaining Marc145 cell strains stably expressing Siat7e genes after passage. Specifically, plasmid ST6GALNAC5 cDNA ORF Clone, human, C-GFPSpark1 is transfected into Marc145 cells, and 10% serum DMEM culture medium with the resistance gene hygromycin concentration of 250-500 mu g/mL is subjected to continuous pressurized screening to obtain Marc145 cell clones with hygromycin resistance; digesting positive clones by pancreatin, placing the positive clones in a T25-T75 cell culture square bottle, and continuing to proliferate under continuous dosing; the cells are continuously cultured for 5-10 generations under pressure, obvious green fluorescence of eGFP is observed under a fluorescence inversion microscope, and the cells are subjected to expansion culture after being digested by pancreatin and frozen by a conventional method.
(3) Domestication culture of Marc145 cells: the Marc145 cell strain stably transfected with siat7e gene is cultured by transferring the culture medium containing serum into a serum-free culture medium.
(4) The domesticated suspension cell strain is stably passaged and built into a warehouse, the growth state is stable, the high-density growth is realized, the dispersibility is good, and the domesticated suspension cell strain is completely suitable for the cells which grow in a suspension manner in a serum-free culture medium, and the number of the cells is 20.0x10 6 cells/mL~30.0×10 6 Freezing cell density of cells/mL, storing in a refrigerator at-80 ℃ for 24 hours by using a program cooling box, and transferring to a liquid nitrogen tank for long-term storage.
(5) Limiting dilution selection of the monoclonal. In a specific example, the resistance-screening drug is 250 μg/mL to 500 μg/mL hygromycin.
The specific screening process is as follows: digesting Marc145 adherent cells with pancreatin according to 1.0-2.0X10 5 The density of cells/hole is inoculated in a 24-hole culture plate, when the culture plate is plated for 24 hours, the culture medium is prepared by changing the liquid into a DMEM culture medium containing 10% of serum to dilute hygromycin with the final concentration of 50 mug/mL, 100 mug/mL, 250 mug/mL, 500 mug/mL, 750 mug/mL and 5 concentration gradient culture, and the minimum concentration capable of killing all cells within 7-14 days is 250 mug/mL to 500 mug/mL.
After 24-48 h of chemical transfection or electrotransfection of Marc145 cells, changing a 10% serum DMEM screening medium containing 250-500 mug/mL hygromycin, changing the screening medium every 3-4 days, observing under a fluorescence microscope, and after 2-3 weeks of continuous screening, growing the resistant cells, and allowing the non-resistant cells to die until fluorescent resistant cloned cells are formed, thus obtaining the drug-resistant cell strain.
Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
The serum-free medium CD Marc145 (DP 848) described in the present application was developed independently from Gansu Jianshun biotechnology Co. Marc145 adherent cells, source: ATCC number CRL-12231.
The specific formulation of CD Marc145 is shown in table 1 below:
TABLE 1-1
TABLE 1-2
The cell library is established after the cells are amplified and cultured in Gansu Jianshun biotechnology limited company, and the number of the cell library is JSB-0479-031.
The Marc145 cell strain which is cultivated and adapted to serum-free full suspension cultivation is named as Marc145.S strain.
Example 1
Marc145 adherent cells were digested with pancreatin according to 1.5X10 5 cell/well density was inoculated into 24-well plates in the presence of DMEM containing 10% serum at 37deg.C and 5% CO 2 After Marc145 cells are plated for 24 hours, the grown monolayer confluence reaches 80% -90% and is used for transfection.
Respectively diluting the DNA and the liposome with a serum-free DMEM culture medium, wherein the amount of the DNA is 2 mug, the ratio of the DNA to the liposome is 1:2.5, and incubating for 15 minutes to obtain a transfection complex; the transfection complex is added into a 24-hole plate cell for culturing, and after 4-6 hours of transfection, the cell is replaced by a DMEM culture medium containing 10% of serum, wherein the culture condition is 37 ℃ and 5% of CO 2 。
After Marc145 cells are transfected for 24-48 hours, a 10% serum DMEM screening medium containing 250-500 mug/mL hygromycin is changed, the screening medium is changed every 3-4 days, and after continuous screening for 2-3 weeks, the cells grow, no resistant cells die, until fluorescent resistant cloned cells are formed, and drug resistant cell positive clones are obtained.
And digesting positive clones by using pancreatin, placing the positive clones into a T25-T75 cell culture square bottle, continuously proliferating the positive clones under continuous dosing, and culturing cells under pressure by using 10% serum with the concentration of resistance gene hygromycin of 250-500 mug/mL.
The DMEM screening culture medium is continuously transmitted for 5-10 generations, and the eGFP fluorescence shown in the figure 1 is obvious when the DMEM screening culture medium is observed under a fluorescence inversion microscope.
Using pancreatin digestion to stably express Siat7e gene Marc145 cell strain, low-speed centrifuging, obtaining cell, using serum-free culture medium CD Marc145 (healthy cis-organism) with hygromycin concentration of 500 mug/mL, adding 2% new born calf serum to resuspend into suspension culture container TPP, gently blowing off cell to make cell be in single cell dispersion state, inoculating with density of 0.5X10 6 cells/mL~1.0×10 6 cells/mL, working volume of 1/2-1/3 of TPP, culture conditions of 37 ℃ and 5% CO 2 RH is more than or equal to 80%, the rotation speed of a shaking table is 200rpm/min, the culture is carried out for 4-8 days at intervals for continuous subculture for 5-10 times.
CD Marc145 (healthy organism) with hygromycin concentration of 500 ug/mL and 1% fresh bovine serum were added to perform TPP culture at a rotation speed of 200rpm/min, and the inoculation density was 0.8X10 6 cells/mL~1.2×10 6 The cells/mL are continuously subjected to subculture for 5-10 times, and the time between passages is 5-6 days.
CD Marc145 (healthy organisms) with hygromycin concentration of 500. Mu.g/mL) TPP culture was performed at a rotation speed of 200rpm/min, and the inoculation density was 1.0X10 6 cells/mL~1.5×10 6 And continuously subculturing the cells/mL for 5-10 times, wherein the time between passages is 3-4 days.
Serum-free medium CD Marc145 (Jiansheng organism) was subjected to TPP culture at a rotation speed of 200rpm/min at a seed density of 1.5X10 6 The cells/mL are continuously subjected to subculture for 5-10 times, and the time between passages is 3 days.
After the cells are acclimatized according to the steps, the fully suspended Marc145 cells are obtained and cultured for 72 hours to reach 6.0x10 6 About cells/ml, the activity rate is more than 95%, and the dispersion is good, and no large lump is cut, so that the continuous passage is stable. Therefore, the serum-free full suspension culture Marc145 cells obtained by the culture of the application have excellent performance.
Domesticating the above steps to obtain suspended Marc145 cells, placing in shake flask for expansion culture, and culturing for 72 hr until cell density reaches 5.0X10% 6 cells/mL~6.0×10 6 About 20.0X10 at cell/mL 6 cells/mL~30.0×10 6 Freezing cell density of cells/mL, storing in a refrigerator at-80 ℃ for 24 hours by using a program cooling box, and then transferring a freezing tube into a liquid nitrogen tank for long-term storage.
Example 2
Marc145 cells were digested with pancreatin and centrifuged at low speed, and the resulting cells were diluted to 6.0X10 with DMEM as a transfection medium 6 cells/0.4 mL~8.0×10 6 After cells/0.4. 0.4 mL, an electric rotating cup is added, and an electric rotating program is set: transferring cells to T75-T175 square bottles for culture after electric transfer under the conditions of 37 ℃ and 5% CO at the voltage of 300V and the voltage of 950 mu F of an exponential wave 2 。
After Marc145 cells are transfected for 24-48 hours, a 10% serum DMEM screening medium containing 250-500 mug/mL hygromycin is changed, the screening medium is changed every 3-4 days, and after continuous screening for 2-3 weeks, the cells grow, no resistant cells die, until fluorescent resistant cloned cells are formed, and drug resistant cell positive clones are obtained.
And (3) digesting positive clones by using pancreatin, placing the positive clones into a T25-T75 cell culture square bottle, continuously proliferating the positive clones under continuous dosing, continuously transferring 5-10 generations of the positive clones into a 10% serum DMEM screening culture medium with the concentration of resistance gene hygromycin of 250-500 mu g/mL, and observing the positive clones under a fluorescence inversion microscope to see the green fluorescence of eGFP which is obviously shown in figure 2.
Using pancreatin digestion to stably express Siat7e gene Marc145 cell strain, low-speed centrifuging, obtaining cell, using serum-free culture medium CD Marc145 (healthy cis-organism) with hygromycin concentration of 500 mug/mL, adding 2% new born calf serum to resuspend into suspension culture container TPP, gently blowing off cell to make cell be in single cell dispersion state, inoculating with density of 0.5X10 6 cells/mL~1.0×10 6 cells/mL, working volume of 1/2-1/3 of TPP, culture condition of 37 ℃ and 5% CO 2 RH is more than or equal to 80%, the rotation speed of a shaking table is 200rpm/min, the culture is carried out for 4-8 days at intervals for continuous subculture for 5-10 times.
Serum-free medium CD Marc145 (Jianshun) containing hygromycin at 500 μg/mL and 1% fresh bovine serum were added for TPP culture at 200rpm/min, inoculation density was 0.8X10 6 cells/mL~1.2×10 6 The cells/mL are continuously subjected to subculture for 5-10 times, and the time between passages is 5-6 days.
Serum-free medium CD Marc145 (Jianshun) containing hygromycin at 500 μg/mL was subjected to TPP culture at a rotation speed of 200rpm/min, and inoculated at a density of 1.0X10 6 cells/mL~1.5×10 6 And continuously subculturing the cells/mL for 5-10 times, wherein the time between passages is 3-4 days.
Serum-free medium CD Marc145 (Jiansheng organism) was subjected to TPP culture at a rotation speed of 200rpm/min at a seed density of 1.5X10 6 The cells/mL are continuously subjected to subculture for 5-10 times, and the time between passages is 3 days.
After the cells are acclimatized according to the steps, the fully suspended Marc145 cells are obtained and cultured for 72 hours to reach 6.0x10 6 About cells/ml, the activity rate is more than 95%, and the dispersion is good, and no large lump is cut, so that the continuous passage is stable. Therefore, the serum-free full suspension culture Marc145 cells obtained by the culture of the application have excellent performance.
Domesticating the above steps to obtain suspended Marc145 cells, and placing inShake flask expansion culture, after 72h of culture, as shown in FIG. 3, until cell density reaches 5.0X10 6 cells/mL~6.0×10 6 About 20.0X10 at cell/mL 6 cells/mL~30.0×10 6 Freezing cell density of cells/mL, storing in a refrigerator at-80 ℃ for 24 hours by using a program cooling box, and transferring a freezing tube into a liquid nitrogen tank for long-term storage, wherein the Marc145 cell domestication growth map is shown in FIG. 4.
Example 3
Suspension Marc145.S cells were grown for the first day, supernatant culture was collected by centrifugation, after centrifugation of the supernatant at 3000rpm for 5min, the supernatant was removed aseptically and filtered using a 0.22 μm syringe filter, labeled Conditioned Medium + cell name.
Continuous dilution is carried out by using suspended Marc145.S cells in the logarithmic growth phase, the cells are finally diluted to 5 cells/mL-10 cells/mL by using a limiting dilution plating cell culture medium Conditioned Medium +cloning medium+ACF for the last time, and the cells are added into a sterile sample adding tank and are mixed evenly in a light shaking way. 100 mu L of the cell mixture is sucked by an 8-channel row gun and added into a 96-well plate, the mixture is gently blown and evenly mixed while adding, the sample adding operation is repeated for 8 times, 8 96-well plates, namely 100 mu L/well (1 cell/well) are paved in total, and each plate is provided with a focusing observation hole.
Taking photos every day by using CSI (CloneSelect Imager cell growth analysis system) on days 0-3, taking photos every 7 days from the 7 th day, and supplementing 50-100 mu L of fresh culture medium on days 3, 7, 14 and 21 respectively; the cells are cultured by 96-pore plates, 24-pore plates and 12-pore plates, then the cells which are well grown are screened and transferred to 6-pore plates for culture, the cells are transferred to 24-pore plates and 24-pore plates to 12-pore plates in 96-pore plates, the cells are amplified according to the proportion of 3 times dilution, and the cells are transferred to 6-pore plates from 12-pore plates and amplified according to the proportion of 2 times dilution; after the cells are cultured in a 6-hole plate for 5-7 days, sampling is performed to determine the cell density and the cell activation rate, and the cells which are good in growth and quick in multiplication time are selected and transferred into a shake flask or a TPP container for passage screening and amplification.
Clone cell inoculation density at passage was 0.3X10 6 cells/mL~1.0×10 6 cells/mL, passaging every 3 days, maximum cell density 6.0×10 6 cells/mL~8.0×10 6 The cell/mL doubling time is 20-25 hours, and the cell density is improved by 1-2 times compared with that of the original cell.
Example 4
Subculturing with cloned Marc145.S suspension cells in serum-free medium, inoculation density was 0.8X10 6 cells/mL~1.2×10 6 The cells/mL, the culture vessel is a shake flask, the culture condition is 37 ℃ and 5% CO 2 、125rpm/min~140rpm/min。
The cell density was 6.0X10 when cultured for 72 hours 6 cells/mL~8.0×10 6 cells/mL, key components glutamine, glutamic acid, aspartic acid and asparagine in the formulation were optimized in shake flasks by single factor and DOE experimental design.
Experimental results show that the glutamine is reduced to 1mM, the glutamic acid is increased to 5mM, the aspartic acid is increased to 5mM, and the cell density can be increased to 10.0 cells/mL-12.0X10 when the asparagine is 2mM 6 cells/mL。
Example 5
Subculturing with cloned Marc145.s suspension cells in serum-free medium containing 1mM glutamine, 5mM glutamic acid, 5mM aspartic acid and 2mM asparagine, inoculation density was 0.8X10 6 cells/mL~1.2×10 6 The cells/mL, the culture vessel is a shake flask, the culture condition is 37 ℃ and 5% CO 2 、125rpm/min~140rpm/min。
Clone Marc145.s suspension cells which are continuously passaged for 48-72 hours are taken out, and cell liquid is taken to detect the living cell density (multiplied by 10) 6 cells/mL) and cell viability (%), the cell density was diluted to 2.0X10 according to the second order process 6 cells/mL~5.0×10 6 cells/mL。
Directly accessing Porcine Reproductive and Respiratory Syndrome (PRRSV) seed viruses according to MOI infection complex number of 0.005-0.05, wherein the Porcine Reproductive and Respiratory Syndrome (PRRSV) vaccine has a virus content of more than or equal to 10 per milliliter 6.0 TCID 50 Culturing for 48-96 hours, and harvesting virus liquid.
The virus liquid prepared according to the method is subjected to virus titer detection, and the virus detection method comprises the following steps:
respectively carrying out serial dilution of 10 times on each sample to be detected,from 10 -1 Diluting to 10 -9 Different dilutions were inoculated into 96-well cell culture plates of Marc145 cells grown as monolayers, each dilution was inoculated in 6 wells, 100 μl per well, and 6 wells were additionally provided with only the inoculation of the dilution as a negative control, 100 μl per well. After 1 hour of adsorption, 100. Mu.L of the maintenance solution was added to each well, and the mixture was placed at 37℃with 5% CO 2 Culturing in incubator for 6 days, observing and recording cytopathic condition of each well every day, and calculating TCID according to Reed-Muench method 50 As a result, the virus titer was
10 9 ~10 10 /mL TCID 50 Compared with the adherence process and the high-glutamine medium, the virus titer is improved by 10-20 times, and the specific results are shown in table 2.
TABLE 2
The above examples merely illustrate a few embodiments of the present application, which are convenient for a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims (8)

1. A method of suspension culturing a Marc145 cell line, the method comprising the steps of:
constructing an adherent Marc145 cell strain which stably expresses the Siat7e gene, and,
domesticating and culturing the adherent Marc145 cell strain stably expressing the siat7e gene, and domesticating the adherent Marc145 cell strain into a suspension Marc145 cell strain;
wherein, the domestication culture comprises the following steps:
step S1, the adherent Marc145 cell strain which stably expresses the siat7e gene is subjected to adherent culture in a complete culture medium containing a resistance screening drug;
s2, inoculating the adherent cell strain subjected to passage S1 after digestion and centrifugation in a serum-free culture medium containing a resistance screening drug and 1.5-2.5 v/v% of newborn bovine serum for suspension culture;
step S3, inoculating the cells cultured in the step S2 into a serum-free culture medium containing a resistance screening drug and 0.5-1 v/v% of newborn bovine serum for suspension culture;
step S4, inoculating the cells cultured in the step S3 into a serum-free culture medium containing a resistance screening drug for suspension culture;
and S5, inoculating the cells cultured in the step S4 into a serum-free culture medium for suspension culture.
2. The method of claim 1, wherein the serum-free medium comprises glutamine, glutamic acid, aspartic acid, and asparagine.
3. The method for suspension culture of Marc145 cell line according to claim 2, wherein the glutamine content is 0.5 mM-1.5 mM; the glutamic acid content is 4 mM-6 mM; the content of the aspartic acid is 4 mM-6 mM; the content of the asparagine is 1.5 mM-2.5 mM.
4. The method for suspension culture of Marc145 cell line as set forth in claim 1, wherein,
the cell seeding density in step S2 was 0.5X10 6 cells/mL~1.0×10 6 cells/mL; or-And, a step of, in the first embodiment,
the cell seeding density in step S3 was 0.8X10 6 cells/mL~1.2×10 6 cells/mL; or/and the combination of the two,
the cell seeding density in step S4 was 1.0X10 6 cells/mL~1.5×10 6 cells/mL; or/and the combination of the two,
the cell seeding density in step S5 was 1.4X10 6 cells/mL~1.7×10 6 cells/mL。
5. The method of claim 1, wherein the suspension culture is shaking culture;
the shaking culture is shaking culture, and the rotating speed is 180 rpm/min-220 rpm/min.
6. The method for suspension culture of Marc145 cell lines according to claim 1, wherein the culture process of steps S1 to S4 is continuously conducted for 5 to 10 times at a 3 to 4 day interval.
7. The method of claim 1, wherein the resistance selection drug comprises one or more of hygromycin-B phosphotransferase, aminoglycoside phosphotransferase, xanthine-guanine phosphoribosyl transferase, and puromycin.
8. The method of claim 1, wherein the method of stably expressing the siat7e gene comprises one or more of electrotransfection, chemical transfection, microinjection, and virus mediated method.
CN202310961691.0A 2023-08-02 2023-08-02 Method for suspension culture of Marc145 cells Active CN116694575B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310961691.0A CN116694575B (en) 2023-08-02 2023-08-02 Method for suspension culture of Marc145 cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310961691.0A CN116694575B (en) 2023-08-02 2023-08-02 Method for suspension culture of Marc145 cells

Publications (2)

Publication Number Publication Date
CN116694575A CN116694575A (en) 2023-09-05
CN116694575B true CN116694575B (en) 2023-12-01

Family

ID=87837763

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310961691.0A Active CN116694575B (en) 2023-08-02 2023-08-02 Method for suspension culture of Marc145 cells

Country Status (1)

Country Link
CN (1) CN116694575B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114591885B (en) * 2022-03-05 2024-02-23 河南普华基因科技有限公司 LLC-PK1Sa cell domestication method suitable for serum-free suspension culture and application

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102453699A (en) * 2010-10-18 2012-05-16 北京清大天一科技有限公司 Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells
CN102453700A (en) * 2010-10-18 2012-05-16 北京清大天一科技有限公司 Method for suspension culture of MDCK cells and for production of virus vaccines by using MDCK cells
CN105950544A (en) * 2016-05-17 2016-09-21 西北民族大学 Domestication method of full suspension culture type Marc-145 cell line
CN108865967A (en) * 2017-05-11 2018-11-23 华威特（江苏）生物制药有限公司 A kind of quick domestication attached cell is the method for full suspension cell line
WO2019119521A1 (en) * 2017-12-18 2019-06-27 中山大学 Marc-145 cell line against porcine reproductive and respiratory syndrome and preparation method and use thereof
CN110373379A (en) * 2019-07-25 2019-10-25 北京鼎持生物技术有限公司 A kind of method that the full suspension cell of Marc-145 directly cultivates PRRS virus vaccine
CN110564765A (en) * 2019-09-19 2019-12-13 中国农业科学院兰州兽医研究所 Homologous recombination vector and recombinant cell for expressing eGFP, and preparation method and application thereof
CN111394315A (en) * 2020-03-13 2020-07-10 成都天邦生物制品有限公司 Suspended Marc145 cell line expressing porcine CD163 and CD169 molecules
CN113684175A (en) * 2021-09-14 2021-11-23 山东恒业生物技术有限公司 Method for domesticating suspension cells by adherent cells
CN114591885A (en) * 2022-03-05 2022-06-07 河南普华基因科技有限公司 LLC-PK1Sa cell domestication method adapted to serum-free suspension culture and application

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102453699A (en) * 2010-10-18 2012-05-16 北京清大天一科技有限公司 Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells
CN102453700A (en) * 2010-10-18 2012-05-16 北京清大天一科技有限公司 Method for suspension culture of MDCK cells and for production of virus vaccines by using MDCK cells
CN105950544A (en) * 2016-05-17 2016-09-21 西北民族大学 Domestication method of full suspension culture type Marc-145 cell line
CN108865967A (en) * 2017-05-11 2018-11-23 华威特（江苏）生物制药有限公司 A kind of quick domestication attached cell is the method for full suspension cell line
WO2019119521A1 (en) * 2017-12-18 2019-06-27 中山大学 Marc-145 cell line against porcine reproductive and respiratory syndrome and preparation method and use thereof
CN110373379A (en) * 2019-07-25 2019-10-25 北京鼎持生物技术有限公司 A kind of method that the full suspension cell of Marc-145 directly cultivates PRRS virus vaccine
CN110564765A (en) * 2019-09-19 2019-12-13 中国农业科学院兰州兽医研究所 Homologous recombination vector and recombinant cell for expressing eGFP, and preparation method and application thereof
CN111394315A (en) * 2020-03-13 2020-07-10 成都天邦生物制品有限公司 Suspended Marc145 cell line expressing porcine CD163 and CD169 molecules
CN113684175A (en) * 2021-09-14 2021-11-23 山东恒业生物技术有限公司 Method for domesticating suspension cells by adherent cells
CN114591885A (en) * 2022-03-05 2022-06-07 河南普华基因科技有限公司 LLC-PK1Sa cell domestication method adapted to serum-free suspension culture and application

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Marc-145 细胞无血清培养驯化、代谢分析及 PRRSV 增殖能力比较;冯磊等;《江苏农业科学》;第40卷(第7期);182-185 *
Marc-145细胞无血清悬浮培养及猪蓝耳病毒高效扩增用工程细胞株的开发;程成;《中国优秀硕士学位论文全文数据库_基础科学辑》(第2期);A006-1593 *
Suspended cell lines for inactivated virus vaccine production;Jiayou Zhang等;《EXPERT REVIEW OF VACCINES 》;第22卷(第1期);468-480 *
猪FcγRⅢ和γ链共转染Marc-145细胞系的建立;邬成业;史平玲;乔松林;王爱萍;郝慧芳;杜晓明;王林林;张改平;;华北农学报(01);6-10 *

Also Published As

Publication number Publication date
CN116694575A (en) 2023-09-05

Similar Documents

Publication Publication Date Title
CN105950544B (en) Domestication method of full suspension culture type Marc-145 cell line
US11629338B2 (en) Method for acclimating and suspending Vero and second order production process for virus
CN116694575B (en) Method for suspension culture of Marc145 cells
US11696949B2 (en) Coronavirus-targeting universal DC cell vaccine, and preparation method and use thereof
CN110713971B (en) Serum-free suspension culture type 293T cell, preparation method thereof and virus packaging method
WO2022267856A1 (en) Wayne293 lvpro cells adapted to serum-free medium environment and use thereof
CN111826341A (en) Method for in vitro culture of HEK293 cells for transient expression of proteins
US20190345441A1 (en) Preparation and application of immortalized alpha-1,3-galactosyltransferase gene knockout pig hepatocyte cell line
CN104894054B (en) A kind of suspension adapted strains of monkey embryo renal epithelial cell Marc 145 and its application in culture reproductive and respiratory syndrome virus, the blue ear viral vaccine of production
CN114774351A (en) Paralichthys olivaceus egg primary stem cell culture solution, in-vitro culture method of Paralichthys olivaceus egg primary stem cells and application of culture solution
Tang et al. Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy
CN102453699A (en) Method for suspension culture of sensitive cells and method for producing blue ear disease vaccine by using sensitive cells
CN106834209A (en) The BHK21 cell clones of high density suspension culture
WO2024051022A1 (en) Hek293 monoclonal cell population culturing method
CN106916780A (en) The BHK21 cell clones of high density suspension culture
CN110699326A (en) Immortalized human hepatic stellate cell line and preparation method thereof
CN116218763A (en) Full suspension cell culture method for porcine epidemic diarrhea virus strain
CN106754753B (en) Virus culture method
CN113980888B (en) Pure suspension culture cell and application and preparation method thereof
CN111876383B (en) Quasi-organ lung cancer PDXO model, EGFR (epidermal growth factor receptor) engineering modification and application of PDXO model in tumor drug pharmacodynamic research
CN106939318A (en) A kind of single cell clone separation method
WO2007040242A1 (en) Isolated human cell, method for obtaining the same and their use
CN102453697A (en) Vero cell by suspension culture and method for producing viral vaccine by using Vero cell thereof
CN102453698A (en) Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells
CN107488633B (en) Insect cell strain for efficiently proliferating baculovirus and construction method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant