CN107227291A - A kind of culture medium of culture hepatocyte and preparation method thereof - Google Patents

A kind of culture medium of culture hepatocyte and preparation method thereof Download PDF

Info

Publication number
CN107227291A
CN107227291A CN201710470100.4A CN201710470100A CN107227291A CN 107227291 A CN107227291 A CN 107227291A CN 201710470100 A CN201710470100 A CN 201710470100A CN 107227291 A CN107227291 A CN 107227291A
Authority
CN
China
Prior art keywords
culture
vitamin
culture medium
hepatocyte
medium
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.)
Pending
Application number
CN201710470100.4A
Other languages
Chinese (zh)
Inventor
翟岱青
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.)
Qingdao Biochemical Equipment Co Ltd Of Gold Allusion Quotation
Original Assignee
Qingdao Biochemical Equipment Co Ltd Of Gold Allusion Quotation
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 Qingdao Biochemical Equipment Co Ltd Of Gold Allusion Quotation filed Critical Qingdao Biochemical Equipment Co Ltd Of Gold Allusion Quotation
Priority to CN201710470100.4A priority Critical patent/CN107227291A/en
Publication of CN107227291A publication Critical patent/CN107227291A/en
Pending legal-status Critical Current

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
    • C12N5/067—Hepatocytes
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • 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
    • 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
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/71—Oxidoreductases (EC 1.)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

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)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (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 present invention relates to cell culture medium technical field, more particularly to a kind of culture medium of culture hepatocyte and preparation method thereof, including basal medium and addition the additive in the basal medium, the composition of the additive with final concentration, including：2~10mg/L of cholesterol, the μ g/L of lipoic acid 30~100, the 0.5mg/L of chondroitin sulfate 0.2, 13 16 μM of antibiotic, the 1.3mg/L of insulin 0.9, the 17mg/L of catalase 10, the μ g/L of ATRA 2~7, the μ g/L of FTN 14 22, 180~420mg/L of lipid type bovine serum albumin, the μ g/L of 2 mercaptoethanol 10~60, the μ g/L of rapamycin 3~12, the μ g/L of folic acid 3~9, 0.2~1.6mg/L of progesterone, 5~23 μ g/L of trace element, the μ g/L of Porcine HGF 20 35, in the culture medium of the present invention, stem cell growth is good, cellular morphology, density is suitable with the culture medium containing serum, overcome the defect of traditional serum-containing media；The culture medium collocation of the present invention is reasonable, is acted synergistically between each composition, can provide the sufficient nutrition needed for growth and proliferation of cell and good environment, improve the Cell viability of liver cell.

Description

A kind of culture medium of culture hepatocyte and preparation method thereof
Technical field
The present invention relates to culture medium and its preparation side of cell culture medium technical field, more particularly to a kind of culture hepatocyte Method.
Background technology
Verify that the test of compound toxicological experiment is so far the most efficiently and feasible using animal cell culture technology Method.As biotechnology is in the toxicologically safe continuous expansion for evaluating application field and market demand of compound, improve Animal cell culture and toxicology test, extension are held time to obtain reliable results, as optimizing animal cell cultivation process With the core of toxicological experiment.
It is well known that the economy and efficiency of animal cell culture and the effect of toxicology test play a decisive role be Culture medium, the optimization of culture medium is the core link for setting up efficient animal cell culture and toxicology test process.Therefore, root According to nutritional need of the cell during growth, metabolism and toxicology test expression, nutrients in designing animal cell culture medium Composition, content and proportioning be work important content.
Domestic and international many commercialized culture mediums-as be made up of basal medium and serum or substitute, significantly affect thin Intracellular growth, metabolism and toxicological test phenomenon express these culture mediums-as only can ensure that the stable passage of cell, can not be preferable The expression of cell growth and toxicological test effect is supported on ground, thus hinders the popularization and application of cell toxicology experiment.China Patent application CN102311938A discloses a kind of serum free medium for hepatocyte cultures, its comprising basal medium and AddO-on therapy, wherein, the addO-on therapy includes：The μ g/mL of insulin 0.1~10, the μ g/mL of transferrins 0.5~10, selenous acid The μ g/L of sodium 5~10,1~100ng/mL of EGF, 1~100ng/mL of HGF, FTN 0.1~ 1 μ g/mL, 0.1~10nmol/mL of dexamethasone and the μ g/mL of hyperglycemic factor 0.05~5.Chinese patent application CN105087465A discloses a kind of hepatocyte serum-free medium, and it includes following components：Basal medium 500mL；Silk gum Albumen 0.05~0.5%；0.1~1000nmol/mL of dexamethasone；5~20ng/mL of HGF；Epidermal growth factor 10~50ng/mL of son；Mycillin 100U/mL, these are used for the serum free medium of hepatocyte cultures, still, there is price Costliness, be unfavorable for daily use, be widely popularized and liver cell industrialization culture the problems such as.
Commercially available business serum free medium in terms of blood serum substituting more has good performance, but in sertoli cell culture Still there are many defects with terms of high efficient expression toxicological effects.Therefore, it is many when carrying out animal cell culture and toxicological experiment Number serum free medium is difficult to fully, evenly to the nutriment needed for cell offer.Culture medium utilization rate is very low, causes pole Big waste.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of culture medium of culture hepatocyte, the present invention The culture medium of the culture hepatocyte of offer does not contain hyclone, without any animal origin composition, can provide cell growth increasing Sufficient nutrition and good environment needed for growing.
To solve the above problems, the invention provides a kind of culture medium of culture hepatocyte, including basal medium and add Be added in the additive in the basal medium, the composition of the additive with final concentration, including：2~10mg/L of cholesterol, Lipoic acid 30~100 μ g/L, chondroitin sulfate 0.2-0.5mg/L, 13-16 μM of antibiotic, insulin 0.9-1.3mg/L, peroxide Change hydrogen enzyme 10-17mg/L, ATRA 2~7 μ g/L, FTN 14-22 μ g/L, lipid type bovine serum albumin 180 ~420mg/L, the μ g/L of 2 mercapto ethanol 10~60, the μ g/L of rapamycin 3~12, the μ g/L of folic acid 3~9, progesterone 0.2~ 1.6mg/L, trace element 5~23 μ g/L, Porcine HGF 20-35 μ g/L.
It is preferred that, the composition of the additive with final concentration, including：5~8mg/L of cholesterol, the μ g/ of lipoic acid 42~82 L, chondroitin sulfate 0.2-0.5mg/L, 13-16 μM of antibiotic, insulin 0.9-1.3mg/L, catalase 10-17mg/L, The μ g/L of ATRA 3~6, the μ g/L of FTN 15~18, lipid type bovine serum albumin 240~360mg/L, 2- sulfydryl The μ g/L of ethanol 25~42, the μ g/L of rapamycin 5~8, folic acid 4~7 μ g/L, 0.7~1.3mg/L of progesterone, trace element 5~23 μ g/L, Porcine HGF 20-35 μ g/L.
The culture medium for the culture hepatocyte that the present invention is provided, including basal medium and addition are in the basal medium Additive, basal medium can provide the existence of liver cell and minimum physiological activity.Cholesterol is used as a kind of lipid, ginseng With forming cell membrane, lipoic acid acts synergistically with the present invention other raw materials used, it is possible to increase the Cell viability of liver cell, separately Outside, lipoic acid also acts as oxidation resistant effect, and catalase can remove free radical, protect liver cell from superoxide radical Infringement etc., insulin can be by acting on the insulin receptor of surface of hepatocytes, intake and utilization of the enhancing liver cell to the energy, While promoting the synthesis of the RNA in liver cell, protein and aliphatic acid, suppress Apoptosis, so as to strengthen the vigor of liver cell With function, FTN can promote sticking for liver cell, and its adherent growth, and chondroitin sulfate can increase the courier of cell The biosynthesis of ribonucleic acid and DNA and the effect with promotion cell metabolism, coordinating with other raw materials makes With, can reach preferably improve liver cell proliferation times and Cell viability effect.
The present invention does not have special requirement to the species of basal medium, can often know for those skilled in the art, example Such as, the basal medium is F12 or the culture mediums of RMPI 1640.
Porcine HGF can promote the propagation of liver cell, and adjust the function of liver cell.It is preferred that, the cell Growth factor is made up of EGF and HGF, and the Porcine HGF is by EGF and liver The weight ratio of Porcine HGF is 1:(1.2~3.6).
It is preferred that, also containing trace element in culture medium of the invention, the trace element for minor metallic element and/or Vitamin, the minor metallic element is in iron, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium, tin, silicon, strontium, boron, rubidium At least one.
It is preferred that, the vitamin be vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, At least one of vitamin B5, vitamin B6, vitamin B12, orotic acid, pangamic acid, p-aminobenzoic acid.
It is preferred that, also contain in the additive：Glu, adenine, guanine, uracil, thymidine, born of the same parents At least one of pyrimidine, ribose, deoxyribose.
A kind of preparation method of the culture medium of culture hepatocyte, comprises the following steps：
(1) cholesterol, lipoic acid, chondroitin sulfate, antibiotic, insulin, hydrogen peroxide are added into basal medium Enzyme, ATRA, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, Trace element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 6.7-7.3, temperature be 35~38 DEG C, gas concentration lwevel be 5~60mL, humidity be 60~85% under conditions of cultivate 2 weeks；
(3) product of step (2) is filtered with micron membrane filter, it is degerming after, produce.
It is preferred that, the micron membranes are the filter membrane that aperture is 0.1~0.3 micron.
Compared with prior art, the present invention has following technique effect：
The culture medium specific chemical components of the culture hepatocyte of the present invention, in the culture medium of the present invention, stem cell growth Well, cellular morphology, density are suitable with the culture medium containing serum, and cell function and vigor are substantially better than containing serum free culture system Base, overcomes the defect of traditional serum-containing media；The culture medium collocation of culture hepatocyte of the present invention is reasonable, is cooperateed between each composition Effect, can provide the sufficient nutrition needed for growth and proliferation of cell and good environment, promote the propagation of liver cell, improve liver cell Cell viability.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, following examples, to present invention progress It is further described.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit The present invention.
Embodiment 1
The invention provides a kind of culture medium of culture hepatocyte, including basal medium F12 culture mediums and addition are in institute State the additive in basal medium, the composition of the additive with final concentration, including：Cholesterol 6mg/L, the μ of lipoic acid 60 G/L, chondroitin sulfate 0.3g/L, 15 μM of antibiotic, insulin 1.2mg/L, catalase 14mg/L, the μ of ATRA 4 G/L, the μ g/L of FTN 16, lipid type bovine serum albumin 300mg/L, the μ g/L of 2 mercapto ethanol 30, the μ g/L of rapamycin 6, The μ g/L of folic acid 5, progesterone 1.0mg/L, micro- 15 μ g/L, the μ g/L of Porcine HGF 30；The Porcine HGF by EGF and HGF composition, the Porcine HGF is by EGF and hepatocyte growth factor The weight ratio of son is 1:2.
The trace element is iron, copper, zinc, cobalt, manganese；
Also contain in the additive：Glu, adenine；
Present invention also offers the preparation method of the culture medium of the culture hepatocyte, comprise the following steps：
(1) cholesterol, lipoic acid, chondroitin sulfate, antibiotic, insulin, hydrogen peroxide are added into basal medium Enzyme, ATRA, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, Trace element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 7.0, temperature be 36 DEG C, gas concentration lwevel be 40mL, it is wet Spend to cultivate 2 weeks under conditions of 70%；
(3) be 0.2 micron of membrane filtration by the product aperture of step (2), it is degerming after, produce.
Embodiment 2
The invention provides a kind of culture medium of culture hepatocyte, including the culture mediums of basal medium RMPI 1640 and add Be added in the additive in the basal medium, the composition of the additive with final concentration, including：Cholesterol 5mg/L, sulphur is pungent Acid 42 μ g/L, chondroitin sulfate 0.2mg/L, 13 μM of antibiotic, insulin 0.9mg/L, catalase 10mg/L, total trans dimension The μ g/L of formic acid 3, the μ g/L of FTN 15, lipid type bovine serum albumin 240mg/L, the μ g/L of 2 mercapto ethanol 25, rapamycin 5 μ g/L, the μ g/L of folic acid 4, progesterone 0.7mg/L, micro- 5 μ g/L, the μ g/L of Porcine HGF 20；The cell growth factor Son is made up of EGF and HGF, and the Porcine HGF is given birth to by EGF and liver cell The weight ratio of the long factor is 1:1.2.
The trace elements iron, copper, vitamin A, vitamin D；
Also contain in the additive：Glu, adenine, guanine, uracil；
Present invention also offers the preparation method of the culture medium of the culture hepatocyte, comprise the following steps：
(1) cholesterol, lipoic acid, chondroitin sulfate, antibiotic, insulin, hydrogen peroxide are added into basal medium Enzyme, ATRA, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, Trace element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 6.7, temperature be 35 DEG C, gas concentration lwevel be 5mL, humidity To cultivate 2 weeks under conditions of 60%；
(3) be 0.1 micron of membrane filtration by the product aperture of step (2), it is degerming after, produce.
Embodiment 3
The invention provides a kind of culture medium of culture hepatocyte, including basal medium F12 culture mediums and addition are in institute State the additive in basal medium, the composition of the additive with final concentration, including：Cholesterol 8mg/L, the μ of lipoic acid 82 G/L, chondroitin sulfate 0.5mg/L, 16 μM of antibiotic, insulin 1.3mg/L, catalase 17mg/L, ATRA 6 μ g/L, the μ g/L of FTN 18, lipid type bovine serum albumin 360mg/L, the μ g/L of 2 mercapto ethanol 42, the μ g/L of rapamycin 8, The μ g/L of folic acid 7, progesterone 1.3mg/L, micro- 23 μ g/L, the μ g/L of Porcine HGF 35；The Porcine HGF by EGF and HGF composition, the Porcine HGF is by EGF and hepatocyte growth factor The weight ratio of son is 1:3.6.
The trace element is vitamin, and the vitamin is vitamin A, vitamin D, vitamin E, vitamin K, dimension life Plain B1, vitamin B2；
Also contain in the additive：Glu, adenine；
Present invention also offers the preparation method of the culture medium of the culture hepatocyte, comprise the following steps：
(1) cholesterol, lipoic acid, chondroitin sulfate, antibiotic, insulin, hydrogen peroxide are added into basal medium Enzyme, ATRA, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, Trace element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 7.3, temperature be 38 DEG C, gas concentration lwevel be 60mL, it is wet Spend to cultivate 2 weeks under conditions of 85%；
(3) be 0.3 micron of membrane filtration by the product aperture of step (2), it is degerming after, produce.
Embodiment 4
The invention provides a kind of culture medium of culture hepatocyte, including basal medium F12 culture mediums and addition are in institute State the additive in basal medium, the composition of the additive with final concentration, including：Cholesterol 2mg/L, the μ of lipoic acid 30 G/L, chondroitin sulfate 0.2mg/L, 13 μM of antibiotic, insulin 0.9mg/L, catalase 10mg/L, ATRA 2 μ g/L, the μ g/L of FTN 14, lipid type bovine serum albumin 180mg/L, the μ g/L of 2 mercapto ethanol 10, the μ g/L of rapamycin 3, The μ g/L of folic acid 3, progesterone 0.2mg/L, micro- 5 μ g/L, the μ g/L of Porcine HGF 20；The Porcine HGF is by table Skin growth factor and HGF composition, the Porcine HGF is by EGF and HGF Weight ratio be 1:1.2.
The trace element is chromium, selenium, iodine, nickel；
Also contain in the additive：Glu, adenine, guanine, uracil；
Present invention also offers the preparation method of the culture medium of the culture hepatocyte, comprise the following steps：
(1) cholesterol, lipoic acid, chondroitin sulfate, antibiotic, insulin, hydrogen peroxide are added into basal medium Enzyme, ATRA, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, Trace element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 6.7, temperature be 35 DEG C, gas concentration lwevel be 5mL, humidity To cultivate 2 weeks under conditions of 60%；
(3) be 0.1 micron of membrane filtration by the product aperture of step (2), it is degerming after, produce.
Embodiment 5
The invention provides a kind of culture medium of culture hepatocyte, including the culture mediums of basal medium RMPI 1640 and add Be added in the additive in the basal medium, the composition of the additive with final concentration, including：Cholesterol 10mg/L, sulphur Octanoic acid 100 μ g/L, chondroitin sulfate 0.5mg/L, 16 μM of antibiotic, insulin 1.3mg/L, catalase 17mg/L are all-trans The μ g/L of formula vitamin A acid 7, the μ g/L of FTN 22, lipid type bovine serum albumin 420mg/L, the μ g/L of 2 mercapto ethanol 60, thunder handkerchief The μ g/L of mycin 12, the μ g/L of folic acid 9, progesterone 1.6mg/L, micro- 23 μ g/L, the μ g/L of Porcine HGF 35；The cell Growth factor is made up of EGF and HGF, and the Porcine HGF is by EGF and liver The weight ratio of Porcine HGF is 1:3.6.
The trace element is iron, copper, zinc, cobalt, manganese, vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B5, vitamin B6；
Also contain in the additive：Glu, adenine, guanine；
Present invention also offers the preparation method of the culture medium of the culture hepatocyte, comprise the following steps：
(1) cholesterol, lipoic acid, chondroitin sulfate, antibiotic, insulin, hydrogen peroxide are added into basal medium Enzyme, ATRA, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, Trace element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 7.3, temperature be 38 DEG C, gas concentration lwevel be 60mL, it is wet Spend to cultivate 2 weeks under conditions of 85%；
(3) be 0.3 micron of membrane filtration by the product aperture of step (2), it is degerming after, produce.
Experimental method:
(1) by the rat hepatocytes of fresh separated, with the serum free medium of embodiment 1~5 and comparative example 1 and contain respectively Cell is resuspended William ' the s-E culture mediums of 10% (v/v) hyclone (FBS), and adjustment cell concentration to 1 × 106Cells/ml, then adds 20ml cell suspensions and is shaken to 40ml in culture glass dish, is placed on shake shaking table, speed is 10cpm, is sampled, analyzing rat hepatocyte function, experimental result such as table 1 for 24 hours, 48 hours and 72 hours in culture.
Table 1：Primary rat cell is into stem cell sphere ratio (cell concentration 5 × 106Cells/ml) balling ratio % (diameters> 60 microns)
24h 48h 72h
Embodiment 1 72±3.6 78±4.3 89.2±1.2
Embodiment 2 68±1.3 75±3.8 85.6±0.6
Embodiment 3 61±5.0 69±1.6 86.5±0.5
Embodiment 4 60.5±4.2 68±0.8 75.3±0.9
Embodiment 5 59.8±4.0 69±1.2 75.5±0.2
Containing serum 52.3±4.9 69.1±0.6 65.2±0.6
Serum free medium of the present invention can support liver cell high density suspension culture, and density is 5 × 106cells/ml× 107When between cells/ml, balling ratio is suitable with serum-containing media culture effect higher than 60%.
To sum up, the serum free medium specific chemical components that provide of the present invention and with low cost, without animal derived components, The culture of liver cell high density suspension can be supported to grow, hence it is evident that better than traditional serum-containing media and existing literature report without blood Clear culture medium, should with good market available for the treatment of cell transplantation, organizational project liver and biological artificial liver support system Use prospect.
Foregoing description is only the description to section Example of the present invention, not to any restriction of the scope of the invention, one's own profession The those of ordinary skill of industry can make improvement according to the present invention or change to above-described embodiment, but belong to present invention protection model Enclose.

Claims (9)

1. a kind of culture medium of culture hepatocyte, it is characterised in that including basal medium and addition in the basal medium In additive, the composition of the additive with final concentration, including：2~10mg/L of cholesterol, the μ g/L of lipoic acid 30~100, Chondroitin sulfate 0.2-0.5mg/L, 13-16 μM of antibiotic, insulin 0.9-1.3mg/L, catalase 10-17mg/L, entirely Retinotic acid 2~7 μ g/L, FTN 14-22 μ g/L, lipid type bovine serum albumin 180~420mg/L, 2- sulfydryl second The μ g/L of alcohol 10~60, the μ g/L of rapamycin 3~12, folic acid 3~9 μ g/L, 0.2~1.6mg/L of progesterone, 5~23 μ of trace element G/L, Porcine HGF 20-35 μ g/L.
2. the culture medium of culture hepatocyte according to claim 1, it is characterised in that the composition of the additive is with dense eventually Degree meter, including：5~8mg/L of cholesterol, lipoic acid 42~82 μ g/L, chondroitin sulfate 0.2-0.5mg/L, antibiotic 13-16 μ M, insulin 0.9-1.3mg/L, catalase 10-17mg/L, the μ g/L of ATRA 3~6, FTN 15~18 μ g/L, lipid type 240~360mg/L of bovine serum albumin, the μ g/L of 2 mercapto ethanol 25~42, the μ g/L of rapamycin 5~8, folic acid 4 ~7 μ g/L, 0.7~1.3mg/L of progesterone, trace element 5~23 μ g/L, Porcine HGF 20-35 μ g/L.
3. the culture medium of culture hepatocyte according to claim 1 or 2, it is characterised in that the basal medium is F12 Or the culture mediums of RMPI 1640.
4. the culture medium of culture hepatocyte according to claim 1 or 2, it is characterised in that the Porcine HGF by EGF and HGF composition, the Porcine HGF is by EGF and hepatocyte growth factor The weight ratio of son is 1:(1.2~3.6).
5. the culture medium of culture hepatocyte according to claim 1 or 2, it is characterised in that the trace element is micro Metallic element and/or vitamin, the minor metallic element be iron, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium, tin, At least one of silicon, strontium, boron, rubidium.
6. the culture medium of culture hepatocyte according to claim 5, it is characterised in that the vitamin is vitamin A, dimension Raw element D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, vitamin At least one of B13, pangamic acid, p-aminobenzoic acid.
7. the culture medium of culture hepatocyte according to claim 1 or 2, it is characterised in that also contain in the additive： At least one of Glu, adenine, guanine, uracil, thymidine, cytimidine, ribose, deoxyribose.
8. the preparation method of the culture medium of the culture hepatocyte as described in claim 1-7 any one, it is characterised in that including Following steps：
(1) cholesterol is added into basal medium, lipoic acid, chondroitin sulfate, antibiotic, insulin, catalase, entirely Retinotic acid, FTN, lipid type bovine serum albumin, 2 mercapto ethanol, rapamycin, folic acid, progesterone, micro member Element, Porcine HGF is well mixed, obtains mixed system 1；
(2) mixed system of step (1) is adjusted into pH to 6.7-7.3, temperature be 35~38 DEG C, gas concentration lwevel be 5~ 60mL, humidity be 60~85% under conditions of cultivate 2 weeks；
(3) product of step (2) is filtered with micron membrane filter, it is degerming after, produce.
9. the preparation method of the culture medium of culture hepatocyte according to claim 8, it is characterised in that the micron membranes are Aperture is 0.1~0.3 micron of filter membrane.
CN201710470100.4A 2017-06-20 2017-06-20 A kind of culture medium of culture hepatocyte and preparation method thereof Pending CN107227291A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710470100.4A CN107227291A (en) 2017-06-20 2017-06-20 A kind of culture medium of culture hepatocyte and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710470100.4A CN107227291A (en) 2017-06-20 2017-06-20 A kind of culture medium of culture hepatocyte and preparation method thereof

Publications (1)

Publication Number Publication Date
CN107227291A true CN107227291A (en) 2017-10-03

Family

ID=59935954

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710470100.4A Pending CN107227291A (en) 2017-06-20 2017-06-20 A kind of culture medium of culture hepatocyte and preparation method thereof

Country Status (1)

Country Link
CN (1) CN107227291A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257319A (en) * 2019-07-26 2019-09-20 苏州瑞徕生物科技有限公司 A kind of cells on microcarriers maintains the culture solution and method of culture

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063625A (en) * 1993-02-12 2000-05-16 Board Of Trustees Of Leland S, Stanford, Jr. University Regulated transcription of targeted genes and other biological events
CN105087465A (en) * 2015-08-26 2015-11-25 南方医科大学珠江医院 Hepatocyte serum-free culture medium
CN106434532A (en) * 2016-12-22 2017-02-22 叶宗耀 Culture medium for culturing hepatocytes and preparation method thereof
CN106754643A (en) * 2016-12-24 2017-05-31 严志海 A kind of serum free hepatocyte medium and preparation method thereof
CN106801030A (en) * 2016-12-13 2017-06-06 华东理工大学 It is suitable to the alternative serum composition of hepatic lineage in vitro culture and its application method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063625A (en) * 1993-02-12 2000-05-16 Board Of Trustees Of Leland S, Stanford, Jr. University Regulated transcription of targeted genes and other biological events
CN105087465A (en) * 2015-08-26 2015-11-25 南方医科大学珠江医院 Hepatocyte serum-free culture medium
CN106801030A (en) * 2016-12-13 2017-06-06 华东理工大学 It is suitable to the alternative serum composition of hepatic lineage in vitro culture and its application method
CN106434532A (en) * 2016-12-22 2017-02-22 叶宗耀 Culture medium for culturing hepatocytes and preparation method thereof
CN106754643A (en) * 2016-12-24 2017-05-31 严志海 A kind of serum free hepatocyte medium and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
李金存等: "雷帕霉素对小鼠原代肝细胞脂滴形态和脂滴表面蛋白表达的影响", 《现代生物医学进展》 *
李青旺主编: "《动物细胞工程与实践》", 30 June 2005 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257319A (en) * 2019-07-26 2019-09-20 苏州瑞徕生物科技有限公司 A kind of cells on microcarriers maintains the culture solution and method of culture

Similar Documents

Publication Publication Date Title
JP6177260B2 (en) Method for producing a polypeptide or virus of interest in continuous cell culture
CN104278009B (en) Serum free medium and application thereof and a kind of cultural method of CSFV
CN101724600B (en) Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density
CN105087465B (en) Hepatocyte serum-free medium
CN103911339A (en) Serum-free fibroblast cell culture medium and preparation method thereof
CN102827804A (en) Culture medium applicable to suspension and magnification cultivation of Vero cell microcarriers and method for suspension magnification cultivation of Vero cell microcarriers
CN102311938A (en) Serum-free medium for culturing hepatic cells
WO2013113196A1 (en) Culture medium for primary culture of hippocampus neurons of a neonatal rat and preparation method and use thereof
CN106434532A (en) Culture medium for culturing hepatocytes and preparation method thereof
CN107227291A (en) A kind of culture medium of culture hepatocyte and preparation method thereof
CN102405279B (en) Improve the method for single cell clone
CN109576308B (en) Method for improving detoxification function of human stem cell-derived liver-like cells and application thereof
CN112391335A (en) Monoclonal cell culture medium, application and method for culturing monoclonal cells
CN107043742A (en) A kind of serum free medium of culture hepatocyte and preparation method thereof
CN104593335B (en) The low cells of serum free culture system Marc 145 production pig breeding and the method for respiratory syndrome CH 1R strain virus
CN111662882A (en) Method for proliferating avian influenza virus by MDCK cell line
CN110373384A (en) A kind of cultural method of serum-free fat stem cell culture medium and fat stem cell
CN105087479A (en) Stem cell serum-free culture medium and stem cell culture method
CN112779220B (en) Culture medium for neural stem cell expansion
CN102653729B (en) Culture medium used for Chinese hamster ovary cells
CN114525239A (en) Serum-free cell culture medium and preparation method thereof
CN111304148B (en) Application of thiamine in promoting differentiation of human pluripotent stem cells to definitive endoderm cells
CN104212768A (en) Protein-free culture medium for culturing microencapsulated recombinant Chinese hamster ovary (CHO) cells and preparation method thereof
CN107012115A (en) Culture medium of sertoli cell high density suspension culture and preparation method thereof
CN113583940A (en) Liver oval cell immortalized culture medium and preparation 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
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20171003