Generation of proliferating human hepatocytes using upcyte^®technology: characterisation and applications in induction and cytotoxicity assays

Abstract

1. We have developed a novel technique which causes primary human hepatocytes to proliferate by transducing them with genes that upregulate their proliferation. 2. Upcyte(®) hepatocytes did not form colonies in soft agar and are not immortalised anchorage-independent cells. Confluent cultures expressed liver-specific proteins, produced urea and stored glycogen. 3. CYP activities were low but similar to that in 5-day cultures of primary human hepatocytes. CYP1A2 and CYP3A4 were inducible; moreover, upcyte(®) hepatocytes predicted the in vivo induction potencies of known CYP3A4 inducers using the "relative induction score" prediction model. Placing cells into 3D culture increased their basal CYP2B6 and CYP3A4 basal activities and induction responses. 4. Phase 2 activities (UGTs, SULTs and GSTs) were comparable to activities in freshly isolated hepatocytes. 5. Upcyte(®) hepatocytes were markedly more sensitive to the hepatotoxin, α-amanitin, than HepG2 cells, indicating functional OATP1B3 uptake. The cytotoxicity of aflatoxin B(1), was decreased in upcyte(®) hepatocytes by co-incubation with the CYP3A4 inhibitor, ketoconazole. Upcyte(®) hepatocytes also differentiated between ten hepatotoxic and eight non-hepatotoxic compounds. 6. In conclusion, upcyte(®) hepatocyte cultures have a differentiated phenotype and exhibit functional phase 1 and 2 activities. These data support the use of upcyte(®) hepatocytes for CYP induction and cytotoxicity screening.

Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds

“…However, limited availability and large variability in DME expression and activity remain shortcomings of PHH, in particular for routine screening applications in drug discovery. In contrast, the recently introduced upcyte human hepatocytes exhibit an extended proliferative aptitude (Burkard et al, 2012).…”

“…Cell yields of donor 10-03 were significantly reduced (2.4 Â 10 7 cells), essentially due to an increased collagen formation (communicated by the vendor), thus impeding subcultivation and passaging. UHH from all tested donors shared a common morphologic phenotype, as described by Burkard et al (2012), clearly distinguishable from plated PHH (Supplemental Fig. 1).…”

“…al, 2015). In contrast, expression of adult hepatic markers (albumin and cytokeratin 8 and 18) and mature hepatic functions, such as storage of glycogen; urea production; basal phase II and P450 activity; responsiveness to prototypical inducers of CYP1A2, CYP3A4, and CYP2B6; as well as functional polarization were reported for differentiated UHH (Burkard et al, 2012;Levy et al, 2015).…”

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

“…We have been using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-derived cell types as an alternative to stem cells. This has already been demonstrated for human hepatocytes to be a very powerful procedure [11][12][13][14].…”

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

“…Other approaches to coax hepatocytes into the third dimension make use of either solid scaffold materials such as polystyrene scaffolds (Bokhari et al, 2007;Burkard et al, 2012;Schutte et al, 2011b) or de-cellularized tissues (linke et al, 2007).…”