Cultivation of primary hepatocytes induces an inflammation-cholestasis response that resembles features of in vivo inflammation during damage and regeneration

Although is well known that cultivation of primary hepatocytes results in a dramatic loss of function and dedifferentiation with features of epithelial to mesenchymal transition, it is generally assumed that most of these alterations are largely repressed on 3D extracellular matrix. To gain a comprehensive insight on the alterations occurring in culture, we performed gene array analysis of primary mouse hepatocytes in the 3 most common cultivation systems: 2D monolayer cultures confluent (MC) and sub-confluent (MS), and 3D collagen sandwich (S), for a 7 day period, using freshly isolated hepatocytes (FH) as reference. Although S cultivation repressed typical dedifferentiation features induced in 2D cultures (mesenchymal morphology, loss of bile canaliculi) at the transcriptome level all cultures strongly deviated from FH (MS: 4287, MC: 4002; S: 3296. 2-fold cutoff). The majority of deregulated genes were triggered already at day 1 in culture in all culture systems. Genes typically induced in inflammatory responses were among the strongest up-regulated (e.g. Lipocalin, SAA3, Mt2, CEBP/d). In addition, down regulated genes were enriched in liver and bile salt metabolism clusters which included Bsep, Mrp2, Cyp7a1 and Cyp8b1, reflecting an inflammatory-cholestatic state. The extent of inflammatory features, assessed by comparing their transcriptional profile to that of an in vivo model of sterile inflammation (CCl4 i.p) at different times after injury, showed a strong correlation between all culture systems with early time points of in vivo inflammation, where monolayer confluent presented the strongest correlation (r=0,62 v/s 24h after CCl4), which was persistent throughout the 7 days in cultivation. Cultivation in 3D sandwich system only partially repressed the inflammatory response (r=0,524 for S v/s 24h after CCl4,). In conclusion, cultivation of primary hepatocytes results in activation of a program that resembles in vivo inflammation and cholestasis.

Primary hepatocytes - gene arrays - inflammation - signal transduction