Characterization of two subpopulations of the PICM-19 porcine liver stem cell line for use in cell-based extracorporeal liver assistance devices

Authors: Talbot, Neil; Caperna, Thomas; Willard, Ryan; MEEKIN, JOHN - HEPALIFE; Garrett, Wesley

Submitted to: International Journal of Artificial Organs
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2009
Publication Date: 6/1/2010
Citation: Talbot, N.C., Caperna, T.J., Willard, R.R., Meekin, J.H., Garrett, W.M. 2010. Characterization of two subpopulations of the PICM-19 porcine liver stem cell line for use in cell-based extracorporeal liver assistance devices. International Journal of Artificial Organs. 33(6):348-361.

Interpretive Summary: Two new cell lines, PICM-19H and PICM-19B, were derived from the parental PICM-19 pig liver stem cell line. The new cell lines were tested for their potential use in bioartificial liver devices. The study included assessments of cell growth rate, their morphological features, and their liver detoxification functions. The PICM-19H cell line was derived by temperature selection at 33-34ºC, and they were not able to form liver bile ducts like the parental PICM-19 cells could. Instead, PICM-19H cells looked exclusively like hepatocytes, the main cell type that forms liver tissue. The PICM-19H cells grew slowly and reached a final density of 160,000 cells/cm2. PICM-19H cells displayed several key liver functions. The PICM-19B cell line was derived from cells that spontaneously appeared in the parental PICM-19 cell culture. The PICM-19B cells grew relatively slowly, too, and reached a final cell density of 240,000 cells/cm2. Morphologically, PICM-19B cells resembled PICM-19H cells except that they did not have the special cell-to-cell unions found between PICM-19H cells that are characteristic of liver cells. PICM-19B cells also contained numerous bodies resembling mucus vacuoles. The PICM-19B cells had high levels of an enzyme found in liver bile duct cells, but also retained some liver detoxification functions. The data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an artificial liver device.

Technical Abstract: 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. The study included assessments of growth rate and cell density in culture, morphological features, 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 33-34ºC. 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 monolayer even after six months of maintenance culture. 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 1.6 x 105 cells/cm2 at ~10 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. After each passage, the PICM-19B cells grew out as tightly knit dome-forming monolayers with no visible biliary canaliculi. Their doubling time was 48-72 h with a final cell density of 240 x 105 cells/cm2. 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 possessed relatively high levels of GGT activity, but did retain some inducible P450 activity, and some ammonia clearance and urea synthesis ability. These data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an artificial liver device.