title: An updated method for the isolation and culture of primary calf hepatocytes.
abstract: Primary hepatocytes are commonly used during in vitro studies, but care must be taken with isolation and culture of the cells to ensure their viability. In this study, hepatocytes were isolated from the liver (caudate process) of a newborn calf by the collagenase perfusion and digestion method. The trypan blue exclusion method was used to determine total cell number and the survival rate of hepatocytes, while hepatocyte function was assessed by measuring lactate dehydrogenase, albumin and urea in culture medium supernatants at 24, 48, 72, 96, 120, 144 and 168 h. Results showed that the number of viable cells/g of liver (wet weight) averaged 1.12×10(7) cells/g, with an average hepatocyte viability of 85.7% (range 83-92%). After 48 h of culture, the hepatocytes solidly adhered to the well culture plate and were spread in an epithelioid shape, with clear cell boundaries between the cells and biliary ductule-like structures formed which persisted for up to 10 days. Hepatocyte function was optimal at 72 h after isolation and culture. This simple and economical procedure for the isolation and culture of viable cells may be useful for in vitro bovine hepatocyte studies.
authors.affiliation: College of Veterinary Medicine, Northeast Agriculture University, Harbin 150030, China.
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Zhang
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.forename: Guo-Wen
authors.initials: GW
authors.lastname: Liu
authors.forename: Tao
authors.initials: T
authors.lastname: Kong
authors.forename: Yan-Fei
authors.initials: YF
authors.lastname: Li
authors.forename: Hong-Bin
authors.initials: HB
authors.lastname: Wang
authors.forename: Cai
authors.initials: C
authors.lastname: Zhang
authors.forename: Zhe
authors.initials: Z
authors.lastname: Wang
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Zhang
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000662:veterinary*; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D000418:Albumins; D014508:Urea; D007770:L-Lactate Dehydrogenase