Suspension culture of hepatocyte-derived reporter cells in presence of albumin to form stable three-dimensional spheroids.

Several studies in the past have formed 3-dimensional (3D) spheroids of primary hepatocytes in suspension culture. Unfortunately, primary hepatocytes in a suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel suspension culture system, by seeding H35 rat hepatoma cells, a hepatocyte-derived cell line, in a 24-well tissue culture polystyrene (TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long-chain polymer, bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on TCPS surfaces placed in suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well-defined spheroids with little spheroid settling or "flattening" of cell colonies in either static or suspension configurations. In BSA-coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24-well plate suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state.

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.

Synthesis and Characterization of an Array of Elastin-like Polypeptide-Polyelectrolyte Conjugates with Varying Chemistries and Amine Content for Biomedical Applications.

Many structural variants of elastin-like polypeptides (ELPs), the genetically engineered equivalents of part of human elastin, currently are being investigated for drug delivery and tissue engineering. Here, we report preparation of six different aminated ELP conjugates via two strategies. In the first, a direct linking strategy was used to couple hydrophobic ELP with either polyethyleneimine, polylysine, or polyarginine. In the second, conjugates were made by attaching ELP onto the reactive polymer, poly(2-vinyl-4,4-dimethyl azlactone), and then exhaustively reacting residual azlactone groups with either ethylenediamine, 1,4-butanediamine, or arginine. Molecular size and chemistry of the resulting six aminated-ELP conjugates were confirmed through gel electrophoresis, FTIR spectroscopy, and mass spectrometry. Dynamic light scattering analysis showed that the conjugates prepared using the "direct reaction scheme" formed small aggregates as well as retained their inverse volume-phase transition temperature behavior. The conjugates prepared using the "reactive polymer linker scheme" also retained this transition temperature behavior. o-Phthalaldehyde assay was used to measure the relative primary amine content of the ELP conjugates. Overall, we prepared an array of aminated-ELPs with independently varying amine content and chemistry (i.e., the same amine content for different materials and different amine contents for the same material). Synthesis of such amphiphilic ELP structures that otherwise cannot be prepared through genetic engineering has the potential to further extend the versatility of the ELPs for many biomedical applications.

Spheroid organization kinetics of H35 rat hepatoma model cell system on elastin-like polypeptide-polyethyleneimine copolymer substrates.

Though two-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the three-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide (ELP) chemically conjugated to positively charged polyethyleneimine (PEI). We have achieved a conjugation ratio of 30 mol %, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol % and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool.