Optimization of Isolation and Primary Culture of Hepatocytes in Crucian Carp (Carassius auratus)

doi:10.7668/hbnxb.2011.S2.044

Abstract

Abstract: In order to establish a stable primary culture model of hepatocytes in Carassius auratus,3 different isolation methods(tissue culture,mechanical speration and pancreatin digestion) and culture conditions of primary hepatocytes culture were compared in the present study.The survival rate was tested with trypan blue exclusion and the viability was assessed with WST-1;the morphological changes of cells in long-term culture were also observed.The results showed that in tissue culture,hepatocytes migrated from liver and proliferated after 4-5 days,compare with the other methods,it took much longer time for the hepatocytes to move out and it was difficult to collect the cells for further culture.The number and viability of the hepatocytes obtained by mechanical separation were obviously lower than by pancreatin digestion.The liver tissue digested by 0.1% trypsin for 30 min yielded 1.47×108 cell/g(liver weight) and the viability was 91.43%.Both culture medium and concentration of serum influenced the attachment and proliferation of the hepatocytes.The cell cultivated in M199 basal medium supplemented with 10% fetal calf serum at 28℃,4% CO2 for 8-9 days can sub-cultured

Key words: Carassius auratus, Hepatocytes, Trypsin, Primary culture

CLC Number:

S917

JIA Rui, CAO Li-ping, DING Wei-dong, DU Jin-liang, XU Pao, YIN Guo-jun. Optimization of Isolation and Primary Culture of Hepatocytes in Crucian Carp (Carassius auratus)[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(S2): 206-212. doi: 10.7668/hbnxb.2011.S2.044.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2011.S2.044

http://www.hbnxb.net/EN/Y2011/V26/IS2/206

References

[1] 张训蒲,朱伟义.普通动物学[M].北京:中国农业出版社,2000.
[2] Probst I,Schwartz P,Jungermann K.Induction in primary culture of 'gluconeogenic'and 'glycolytichepatocytes resembling periportal and perivenous cells[J].European Journal of Biochemistry,1982,126 (2):271-278.
[3] Tanaka K,Sato M,Tomita Y,et al.Biochemical studies on liver functions in primary cultured hepatocytes of adult rats[J].Journal of Biochemistry,1978,84 (4):937-938.
[4] Gerson R J,Shaikh Z A.Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures[J].Biochemical Journal,1982,208 (2):465-469.
[5] Pesonen M,Andersson T B.Fish primary hepatocyte culture; An important model for xenobiotic metabolism and toxicity studies[J].Aquatic Toxicology,1997,37 (2-3):253-267.
[6] 薛庆善.体外培养的原理与技术[M].北京:科学出版社,2001.
[7] Baksi S M,Frazier J M.Isolated fish hepatocytes-model systems for toxicology research.[J].Aquatic Toxicology,1990,16(4):229-256.
[8] 左文功.淡水鱼类细胞培养方法[J].淡水渔业,1991,2:37-40.
[9] 于淼,管华诗,郭华荣,等.鱼类细胞培养及其应用[J].Marine Sciencdes,2003,27(3):4-8.
[10] Birnbaum M J,Schultz J,Fain J N.Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes[J].American Journal of Physiology-Legacy Content,1976,231(1):191-195.
[11] Moon T W,Walsh P J,Mommsen T P.Fish hepatocytes:a model metabolic system[J].Canadian Journal of Fisheries and Aquatic Sciences,1985,42 (11):1772-1782.
[12] Klaunig J E.Establishment of fish hepatocyte cultures for use in in vitro carcinogenicity studies[J].National Cancer Institute Monograph,1984,65:163-168.
[13] Klaunig J E,Ruch R J,Goldblatt P J.Trout hepatocyte culture:isolation and primary culture[J].In Vitro Cellular & Developmental Biology-Plant,1985,21 (4):221-228.
[14] Braunbeck T,Segner H.Isolation and cultivation of teleost hepatocytes[J].The Hepatocyte Review,2000:49-72.
[15] Mommsen T P,Moon T W,Walsh P J.Hepatocytes:isolation,maintenance and utilization[J].Biochemistry and Molecular Biology of Fishes,1994,3:355-373.
[16] Segner H.Isolation and primary culture of teleost hepatocytes[J].Comparative Biochemistry and Physiology-Part A:Molecular & Integrative Physiology,1998,120 (1):71-81.
[17] 孟祥林,丁庆秋.鱼类肝胆综合症的发病原因及防治方法[J].水利渔业,2004,24 (003):65-65.
[18] R Bergh C M I,Lipsky M M.Toxicity of chloroform and carbon tetrachloride in primary cultures of rainbow trout hepatocytes[J].Aquatic Toxicology,1997,37 (2-3):169-182.
[19] Fent K.Fish cell lines as versatile tools in ecotoxicology:assessment of cytotoxicity,cytochrome p4501a induction potential and estrogenic activity of chemicals and environmental samples[J].Toxicology in Vitro,2001,15 (4-5):477-488.
[20] Hayashi S,Ooshiro Z.Primary culture of the eel hepatocytes in the serum-free medium[J].Bulletin of the Japanese Society of Scientific Fisheries,1986,52 (9):1641-1651.
[21] Ferraris M,Radice S,Catalani P,et al.Early oxidative damage in primary cultured trout hepatocytes:a time course study[J].Aquatic Toxicology,2002,59 (3-4):283-296.
[22] 喻文娟,杨先乐,唐俊,等,大口黑鲈肝细胞原代培养方法的建立[J].上海水产大学学报,2006,15(004):430-435.
[23] Khan E A,Dasmahapatra A K,Ghosh R.Evaluation of edta and fish skin extract in primary culture of fish liver cells[J].Methods in Cell Science,1997,19 (3):153-159.
[24] Berry M N,Edwards A M,Barritt G J,et al.Isolated hepatocytes:preparation,properties and applications[J].Laboratory Techniques in Biochemistry and Molecular Biology,1991,21 (1):433-439.
[25] Walton M J,Cowey C B.Gluconeogenesis by isolated hepatocytes from rainbow trout salmo gairdneri[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1979,62 (1):75-79.
[26] Hayashi S,Ooshiro Z.Primary culture of the freshly isolated liver cells of the eel[J].Bull Japan Soc Sci Fish,1985,51:765-771.
[27] Jung J O,Mitema E S,Gutzeit H O.Establishment and comparative analyses of different culture conditions of primary hepatocytes from nile tilapia (Oreochromis niloticus) as a model to study stress induction in vitro[J].In Vitro Cellular & Developmental Biology-Animal,2005,41(1):1-6.
[28] Yanhong F,Chenghua H,Guofang L,et al.Optimization of the isolation and cultivation of cyprinus carpio primary hepatocytes[J].Cytotechnology,2008,58 (2):85-92.
[29] Ferraris M,Radice S,Catalani P,et al.Early oxidative damage in primary cultured trout hepatocytes:a time course study[J].Aquatic Toxicology,2002,59 (3-4):283-296.
[30] Faisal M,Rutan B J,Sami-Demmerle S.Development of continuous liver cell cultures from the marine teleost,spot (leiostomus xanthurus,pisces:sciaenidae)[J].Aquaculture,1995,132 (1-2):59-72.
[31] 梁岳,马广智,方展强.剑尾鱼肝细胞原代培养[J].中国比较医学杂志,2006,16(3):185-187.
[32] Braunbeck T,Segner H.Isolation and cultivation of teleost hepatocytes[J].The Hepatocyte Review,2000,49-72.
[33] 谭凤霞.三株鱼类细胞系的建立和十二株鱼类细胞系对重金属毒性的敏感性研究[D].武汉:华中农业大学,2008.
[34] Ye H Q,Chen S L,Sha Z X,et al.Development and characterization of cell lines from heart,liver,spleen and head kidney of sea perch lateolabrax japonicus[J].Journal of Fish Biology,2006,69:115-126.
[35] 任国诚.几种重要海水养殖鱼类细胞系的建立,鉴定及应用[D].青岛:中国海洋大学,2007.
[36] 叶湘辉,刘汉勤,俞小牧.中华鲟组织培养的初步研究[J].水生生物学报,1999,23(6):566-571.
[37] Salvo L M,Malucelli M I C,Richartz R R T B,et al.Primary culture of hepatic cells from metynnis roosevelti (pisces,teleostei,characidae)[J].Brazilian Journal of Veterinary Research and Animal Science,2000,37 (5):10-16.
[38] Blair J B,Miller M R,Pack D,et al.Isolated trout liver cells:establishing short-term primary cultures exhibiting cell-to-cell interactions[J].In Vitro Cellular & Developmental Biology-Plant,1990,26 (3):237-249.
[39] Klaunig J E,Ruch R J,Goldblatt P J.Trout hepatocyte culture:isolation and primary culture[J].In Vitro Cellular & Developmental Biology-Plant,1985,21 (4):221-228.
[40] Duan C,Hanzawa N,Takeuchi Y,et al.Use of primary cultures of salmon hepatocytes for the study of hormonal regulation of insulin-like growth factor i expression in vitro[J].Zoological Science,1993,10 (3):473-480.
[41] Koban M.Can cultured teleost hepatocytes show temperature acclimation?[J].American Journal of PhysiologyRegulatory,Integrative and Comparative Physiology,1986,250(2):R211.
[42] Flouriot G,Vaillant C,Salbert G,et al.Monolayer and aggregate cultures of rainbow trout hepatocytes:longterm and stable liver-specific expression in aggregates[J].Journal of Cell Science,1993,105 (2):407-413.
[43] Braumbeck T,Storch V.Senescence of hepatocytes isolated from rainbow trout (oncorhynchus mkiss) in primary culture:an ultrastructural study[J].Protoplasma,1992,170(3-4):138-159.
[44] Haschemeyer A E V,Mathews R W.Temperature dependency of protein synthesis in isolated hepatocytes of antarctic fish[J].Physiological Zoology,1983,56 (1):78-87.
[45] Tocher D R,Sargen J R,Frerichs G N.The fatty acid compositions of established fish cell lines after long-term culture in mammalian sera[J].Fish Physiology and Biochemistry,1988,5 (4):219-227.
[46] 张莉萍,康格非.原代肝细胞培养的研究现状[J].国外医学:临床生物化学与检验学分册,2004,25(3):193-196.
[47] Pesonen M,Andersson T.Characterization and induction of xenobiotic metabolizing enzyme activities in a primary culture of rainbow trout hepatocytes[J].Xenobiotica,1991,21 (4):461-471.
[48] Pawlowski S,Islinger M,V(o)lkl A,et al.Temperature-dependent vitellogenin-mrna expression in primary cultures of rainbow trout (oncorhynchus mykiss) hepatocytes at 14 and 18℃[J].Toxicology in Vitro,2000,14(6):531-540.

Please choose a citation manager

Content to export