Submitted to: Cytotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 4, 2013
Publication Date: January 13, 2014
Citation: Talbot, N.C., Caperna, T.J. 2014. A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion. Cytotechnology. 66(1):1-7. Interpretive Summary: The ARS-PICM-19 pig liver stem cell line could be useful for in vitro, i.e., “in the petri dish,” pharmacological and toxicological assays of liver function and liver damage, respectively. The cell line could also be useful as the biological component, i.e., live cell component, of an artificial liver device for the treatment of people suffering from acute liver failure. For agricultural purposes, the cell line is an in vitro liver model that could be useful for testing man-made genetic changes to the liver function of pigs, and it could also be useful for acting as the “nucleus donor” in the somatic cell cloning technique – the method whereby precise genetic changes to pigs might be made. Finally, the PICM-19 cell line could be useful as a reagent for “cellular augmentation”, where PICM-19 cells, genetically engineered to produce growth stimulating substances, would be injected into newborn pigs in order to enhance their growth. All of the above potential applications would be greatly enhanced or enabled by devising a cell culture method for the PICM-19 cells that does not require the use of so-called “feeder-cells”. Previously, a “feeder-cell-free” method for the continuous culture of the PICM-19 cell line was devised, but the feeder-free PICM-19 cells stopped dividing after approximately one year. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-cell free PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over two years without any sign of its continuous replication ever stopping. The PICM-FF cells closely resemble the finite feeder-free PICM-19 cultures previously reported, and, as before, they maintained their vital liver-specific functions. The PICM-19FF cells therefore appear to be immortal in their growth potential, and this should enhance their experimental usefulness in general and may also improve their application to toxicological/pharmacological assays and artificial liver devices.
Technical Abstract: A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a one year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to approximately 90% confluency after each passage at 2 week intervals and the cells maintained a relatively high cell density after 2 years and over 45 passages of culture (average of 2.6 x 106 cells/T25 flask or 1x 105 cells/cm2). Morphologically the PICM-FF cells closely resembled finite feeder-independent PICM-19 cultures previously reported, and as before no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. The PICM-19FF subpopulation was continuously cultured for over two years (48th passages at final freeze-down) with no decline in growth potential. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line, and this should enhance their experimental usefulness in general and may also improve their application to toxicological/pharmacological assays and artificial liver devices.