NDLI: Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

Content Provider	Springer Nature Link
Author	Caperna, Thomas J. Blomberg, Le Ann Garrett, Wesley M. Talbot, Neil C.
Copyright Year	2011
Abstract	A serum-free, feeder cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1-wk-old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 μM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several wk. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiocyte monolayers. Serum protein secretion in conditioned media was analyzed by Western blot and indicated that albumin, transferrin, and haptoglobin levels were maintained in hepatocytes while albumin and haptoglobin declined over time in cholangiocytes. Quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte differentiation and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.
Starting Page	218
Ending Page	233
Page Count	16
File Format	PDF
ISSN	10712690
Journal	In Vitro Cellular & Developmental Biology - Animal
Volume Number	47
Issue Number	3
e-ISSN	1543706X
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-02-07
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Cell culture Cholangiocyte Duct Hepatocyte P-450 γ-Glutamyl transpeptidase Serum-free Cell Biology Animal Genetics and Genomics Cell Culture Stem Cells Developmental Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Developmental Biology Medicine

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