Proposal for a non-adhesive single-cell culture technology for primary hepatocytes

Primary hepatocytes (PHs) are indispensable for studying liver function, drug screening, and regenerative medicine. However, freshly isolated PHs only survive for a few hours in non-adherent suspension culture. This study proposes treatment with PEG-GRGDS, a polymer-peptide conjugate comprising polyethylene glycol (PEG) and the pentapeptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS), to sustain the viability of dispersed single PHs under non-adherent conditions. As a proof of concept, PHs treated with the PEG-GRGDS molecule were cultured in a microarray with single-cell-sized microwells. After 24 h of culture, enhanced cell survival was confirmed via esterase activity alongside activity for Cytochrome P450 1A1 (CYP1A1). Some liver-specific functionalities, including albumin secretion, were observed in the treated PHs. Additionally, it was observed that the length of the PEG-chain in the conjugates influenced the maintenance of single-cell dispersion and the levels of polymerized actin in the cells. These findings suggest that treatment with a polymer-peptide like PEG-GRGDS might provide a promising platform for the short-term culture of non-adherent single PHs.