Three-dimensional Co-culture of Primary Human Liver Cells in Bioreactors for In Vitro Drug Studies: Effects of the Initial Cell Quality on the Long-term Maintenance of Hepatocyte-specific Functions

Abstract

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (P450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and nonparenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.

Improvement of C3A cell metabolism for usage in bioartificial liver support systems

“…While albumin synthesis and gluconeogenesis capacity were slightly lower in preconditioned C3A cells than in porcine hepatocytes (respectively 3 and 2.6-fold), ureogenesis was similar. Interestingly, it has been shown that the value for albumin synthesis from primary human hepatocytes was approximately the same as what we obtained with our preconditioned C3A cells [33]. Moreover, in that same study, urea synthesis capacity was approximately 10 times lower than what we obtained with the preconditioned C3A cells.…”

Liver Grafts Preserved in Celsior Solution as Source of Hepatocytes for Drug Metabolism Studies: Comparison With Surgical Liver Biopsies

“…In the present study, the suitability of liver grafts perfused and maintained in CS for preparing metabolically competent hepatocyte cultures has been examined. Similar to previous reports for other solutions (Thomas et al, 1995;Zeilinger et al, 2002;Serralta et al, 2003), notable differences in basal P450 expression were found between human hepatocytes prepared from CS-preserved liver grafts and cultures prepared from other sources of liver tissue. Compared with hepatocytes obtained from SLRs, reduced levels of specific mRNAs and catalytic activities of the major P450 enzymes were observed in hepatocyte cultures from the CS group (Tables 2 and 3).…”

“…CYP1A2 and CYP3A4 activities in human hepatocytes from CS liver grafts exposed to model inducers. After 24 h of culture, human hepatocytes prepared from liver grafts cold stored in CS were exposed to 2 Thomas et al, 1995;Zeilinger et al, 2002;Serralta et al, 2003). A major difference in the procurement of both groups of liver samples is that livers from organ donors are perfused in situ with a cold preservation solution.…”

Lab on a chip-based hepatic sinusoidal system simulator for optimal primary hepatocyte culture

“…There is a body of evidence suggesting that flow applied to various culture systems can improve a variety of cellular functions, including mass transport (Hansen and Quake 2003), maintenance of enzymatic activities for long-term culture of hepatocytes (Schmitmeier et al 2006;Zeilinger et al 2002), and metabolism of xenobiotics (Kane et al 2006;Viravaidya et al 2004). In addition, many studies have reported that mRNA expression of genes involved in xenobiotic metabolism and transport, including CYP1A1, 1 A2, 2B6, 2C9, 3 A4, multidrug resistance protein 1, MRP2, and UDP-glucuronosyltransferase, was upregulated by medium flow (Baudoin et al 2014;Dash et al 2013;Vinci et al 2011).…”