Effect of amine content and chemistry on long-term, three-dimensional hepatocyte spheroid culture atop aminated elastin-like polypeptide coatings.

Culture conditions that induce hepatic spheroidal aggregates sustain liver cells with metabolism that mimics in vivo hepatocytes. Here we present an array of elastin-like polypeptide conjugate coating materials (Aminated-ELPs) that are biocompatible, have spheroid-forming capacity, can be coated atop traditional culture surfaces, and maintain structural integrity while ensuring adherence of spheroids over long culture period. The Aminated-ELPs were synthesized either by direct conjugation of ELP and various polyelectrolytes or by conjugating both ELP and various small electrolytes to the reactive polymer poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA). Spheroid morphology, cellular metabolic function, and liver-specific gene expression over the long-term, 20-day culture period were assessed through optical microscopy, measurement of total protein content and albumin and urea production, and quantitative real-time (qRT) PCR. We found that the amine content of the Aminated-ELP coatings dictated the initial hepatocyte attachment, but not the subsequent hepatocyte spheroid formation and their continued attachment. A lower amine content was generally found to sustain higher albumin production by the spheroids. Out of the 19 Aminated-ELP coatings tested, we found that the lysine-containing substrates comprising ELP-polylysine or ELP-PVDMA-butanediamine proved to consistently culture productive spheroidal hepatocytes. We suggest that the incorporation of lysine functional groups in Aminated-ELP rendered more biocompatible surfaces, increasing spheroid attachment and leading to increased liver-specific function. Taken together, the Aminated-ELP array presented here has the potential to create in vitro hepatocyte culture models that mimic in vivo liver functionality and thus, lead to better understanding of liver pathophysiology and superior screening methods for drug efficacy and toxicity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 377-388, 2017.