Submitted to: In Vitro Cellular and Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2010
Publication Date: 7/7/2010
Citation: Talbot, N.C., Blomberg, L., Garrett, W.M., Caperna, T.J. 2010. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line. In Vitro Cellular and Developmental Biology. 46(9):746-757. 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”. The present report describes a simple “feeder-cell-free” method for the continuous culture of the PICM-19 cell line. That is, the PICM-19 cells were grown for almost one year without feeder-cells. Most importantly, it was demonstrated that the “feeder-independent” PICM-19 cells maintained their vital liver-specific functions over this period.
Technical Abstract: The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology, and function were lost if the cells were cultured without STO feeder cells. A method for the feeder-independent continuous culture of PICM-19 cells (FI-PICM-19) is presented. PICM-19 cells were maintained and grown without feeder cells on collage I-coated tissue culture plastic for 26 passages (P26) with initial split ratios of 1:3 that diminished to split ratios of less than 1:2 after P16. Once plated, the FI-PICM-19 cells were overlaid with a 1:12 to 1:50 dilution of Matrigel or related extracellular matrix product. Growth of the cells was stimulated by daily refeedings with STO feeder cell conditioned medium. The FI-PICM-19 cells grew to approximately 50% confluency prior to each passage at 2 week intervals. Growth curve analysis showed their average cell number doubling time to be ~96 h. Morphologically, the feeder-independent cells closely resembled PICM-19 cells grown on feeder cells. However, no spontaneous multicellular ductules formed in the monolayers of FI-PICM-19 cells. Ultrastructureal subcellular features of the FI-PICM-19 cells were similar to PICM-19 cells cultured on feeder cells and biliary canalicui were observed at cell-to-cell junctions. The FI-PICM-19 cells produced a spectrum of serum proteins and expressed many liver/hepatocyte-specific genes. Importantly, cytochrome P450 (EROD), ammonia clearance, and urea production activities were maintained in the feeder-independent cells. This simple method for the propagation of the PICM-19 cell line without feeder cells should simplify the generation and selection of functional mutants within the population and enhanced their potential use for toxicological/pharmacological screening assays and for their use in an artificial liver device.