Characterization of Two Subpopulations of the PICM-19 Porcine Liver Stem Cell Line for use in Cell-Based Extracorporeal Liver Assistance Devices

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 (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.

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

“…Studies using a substrate of living, but non-dividing "feeder cells" such as rat liver epithelial cell lines (Guguen-Guillouzo et al 1983), 3T3 cells (Joplin et al 1989;Kuri-Harcuch and Mendoza-Figueroa 1989), and human diploid fibroblasts (Michalopoulos et al 1979) have also demonstrated long-term in vitro maintenance of hepatocyte morphology and function. Our laboratory has previously demonstrated that feeder cells prepared from the STO mouse embryonic fibroblasts cell line were capable of maintaining replicating cultures of pig embryonic stem cellderived, and fetus-derived, porcine hepatocytes and cholangiocytes, the latter spontaneously self-organizing into multicellular ductal structures that displayed in vivo-like responses (Talbot et al 1994a(Talbot et al , b, 1996(Talbot et al , 2002(Talbot et al , 2010aWillard et al 2009). Thus, mitotically inactivated STO feeder cells may be a suitable substrate for the primary culture of cells from the livers of young or young-adult pigs.…”

Generation of induced pluripotent stem cells from domestic goats

“…For clonal, spontaneous in vitro differentiation, the giPSC cell lines were passaged onto STO feeder cells prepared as previously described (Talbot et al, ), except that the STO feeder cells were seeded at 2.5 × 10 4 cells/cm 2 in T12.5 tissue‐culture flasks (BD Biosciences, San Jose, CA). The giPSC cells were plated in 10% DMEM at clonal density (i.e., 100 cells per T12.5 flask).…”

“…Two‐dimensional electrophoretic analysis of the serum‐free, conditioned medium from giPSC B5‐derived yolk‐sac endoderm cells was performed as previously described (Talbot et al, ). Briefly, a T12.5 flask of the cells was washed four times with serum‐free medium (DMEM) and re‐fed with 2 ml of serum‐free medium.…”

“…After 72 hr of culture, the conditioned medium was collected, subjected to low speed centrifugation to pellet cell debris, and the supernatant was frozen at −80°C. A portion of conditioned medium (0.5 ml) was concentrated approximately 10‐fold using a Vivaspin 500, 3,000 MWCO apparatus (Sartorius Stedim Biotech, Bohemia, NY), and the constituent proteins were then separated by two‐dimensional‐gel electrophoresis using an IPGphor isoelectric focusing unit (GE Healthcare Life Sciences), as previously described (Talbot et al, ). The resulting protein spots were identified by mass spectrometry.…”

The Influence of Membrane Molecular Weight Cutoff on a Novel Bioartificial Liver

“…Although considerable expertise has been gained in the use of xenogenic hepatocyte-driven BAL (9)(10)(11)(12), serious concerns exist about their use. One of the main recognized disadvantages is immunological rejection.…”