Impact of Post-Thaw Enrichment of Primary Human Hepatocytes on Steatosis, Inflammation, and Fibrosis in the TruVivo® System

<b>Background</b>: Liver diseases are a global health concern. Many in vitro liver models utilize cryopreserved primary human hepatocytes (PHHs), which commonly undergo post-thaw processing through colloidal silica gradients to remove debris and enrich for a viable PHH population. Post-thaw processing effects on healthy PHHs are partially understood, but the consequences of applying disease-origin PHHs to post-thaw density gradient separation have not been described. <b>Methods</b>: Using the TruVivo^® system, diseased, type 2 diabetes mellitus (T2DM), and fibrotic PHHs were cultured for 14 days after initially being subjected to either low-density (permissive) or high-density (selective) gradients using Percoll-based thawing medium. <b>Results</b>: Changes in functionality, including albumin and urea secretion and CYP3A4 activity, were measured in diseased, T2DM, and fibrotic PHHs enriched in low Percoll compared to PHHs enriched in high Percoll. Lipogenesis increased in the PHHs enriched in low Percoll. Higher expression of CK18 and TGF-β, two fibrotic markers, and changes in expression of the macrophage markers CD68 and CD163 were also measured. <b>Conclusions</b>: The use of Percoll for the enrichment of PHHs post-thaw results in differences in attachment and functionality, along with changes in diseased phenotypes, in the TruVivo^® system.