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The introduction of trypsinisation in the 1950's was a paradigm shift which helped instigate cell culture. We demonstrate here that human hepatocyte cell line HepG2–C3A needs at least 18 days after trypsinisation to re-establish key ultrastructural and physiological traits. After trypsinisation, cells start to recover these traits at similar rates in both monolayer (2D) or spheroid (3D) growth environments. While this development is restarted by trypsinisation of 2D cultures (typically after 5 days), recovery continues in 3D cultures up until 15–18 days when changes in growth rate, adenylate kinase, ATP, urea and cholesterol all suggest that spheroids undergo some type of physiological transition. Several other cell lines (e.g. Caco-2, HT 29, MDCK, MCF-10A and HepG2 used to model the small and large intestine, kidney, breast acini and liver respectively) are reported in the literature to exhibit very similar changes, on a similar timescale to those reported here. These changes may thus represent a ubiquitous recovery process after trypsinisation rather than differentiation. This would partially explain the common observation that cells grown in 3D exhibit physiological capabilities that are closer to those seen in the intact tissue or organ.

toxicology research

After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver