Characterization of two subpopulations of the PICM-19 porcine liver stem cell line for use in cell-based extracorporeal liver assistance devices.
Two cell lines, PICM-19H and PICM-19B, were derived from the bipotent PICM-19 pig liver stem cell line and assessed for their potential application in artificial liver devices (ALD). The study included assessments of growth rate and cell density in culture, morphological features, serum protein production, gamma-glutamyltranspeptidase (GGT) activity and hepatocyte detoxification functions, i.e., inducible P450 activity, ammonia clearance, and urea production. The PICM-19H cell line was derived by temperature selection at 33-34 degrees C. After each passage, PICM-19H cells grew to a nearly confluent monolayer of cells of hepatocyte morphology, i.e., cuboidal cells with centrally located nuclei joined by biliary canaliculi. No differentiation and self-organization into multi-cellular bile ductules, as observed in the parental PICM-19 cell line, occurred within the PICM-19H cell monolayers. The PICM-19H cells contained numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies and occasional lipid vacuoles. The cells had a doubling time of 48-72 h and reached a final density of 1.5 x 10(5) cells/cm(2) at approximately10 d post-passage from a 1:6 split ratio. PICM-19H cells displayed inducible P450 activity, cleared ammonia, and produced urea in a glutamine-free medium. The PICM-19B cells were colony-cloned after spontaneous generation from the PICM-19 parental cell line. PICM-19B cells grew as a tightly knit dome-forming monolayer with no visible biliary canaliculi. Their doubling time was 48-72 h with a final cell density of 2.6 x 10(5) cells/cm(2). Ultrastructural analysis of the PICM-19B monolayers showed the roughly cuboidal cells displayed basal-apical polarization and were joined by tight junction-like complexes. Other ultrastructure features were similar to those of PICM-19H cells except that they possessed numerous cell bodies resembling mucus vacuoles. The PICM-19B cells had relatively high levels of GGT activity, but did retain some inducible P450 activity, and some ammonia clearance and urea synthesis ability. PICM-19B cells produced markedly less serum proteins than PICM-19H cells. These data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an ALD.
Talbot
Neil C
NC
US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Maryland, USA. neil.talbot@ars.usda.gov
Caperna
Thomas J
TJ
Willard
Ryan R
RR
Meekin
John H
JH
Garrett
Wesley M
WM
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line / Q000166:cytology* / Q000378:metabolism; D049109:Cell Proliferation; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D008099:Liver / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D019164:Liver, Artificial; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013552:Swine