Relationship between chemistry, surface properties, and hepatocyte spheroid culture performance of charged elastin-like polypeptide coatings.

摘要

Emerging biomedical applications poised to benefit from improved in vitro hepatic culture include: 1) in vitro spheroid culture models that accurately reflect in vivo liver functionality to help researchers screen drugs and study hepatic diseases; 2) development of a large-scale robust, differentiated hepatic culture system for use as a "liver bioreactor" to periodically mediate toxicity in the blood of liver patients (liver "dialysis"); and 3) in vitro growth of effective autologous 3D differentiated hepatic tissue constructs for therapeutic implantation within end stage liver disease patients.;To directly advance these technologies we addressed improvement of the in vitro hepatic culture surface itself. We engineered surfaces inducing long-term differentiated hepatic function that can be easily adjusted to achieve a range of desired culture morphologies.;The default morphology assumed in current liver cell culture models typically features a hepatocyte monolayer immersed in static or flowing media. In vitro conditions that encourage hepatocytes to aggregate and assume a spheroidal morphology, however, render highly differentiated liver cells with metabolism more closely reflecting that of in vivo hepatocytes. Furthermore, spheroidal cells have been shown to remain viable over relatively long culture periods (> 3 weeks) compared to cells of a monolayer. Unfortunately, currently available spheroid-inducing surfaces offer limited cytocompatibility and allow for easy surface detachment of anchorage-dependent spheroidal hepatocytes, rendering cultures of limited surface cell density and hepatic functionality. We sought to engineer surfaces to mitigate these issues, building on recent success developing, thoroughly characterizing, and testing a spheroid-inducing coating material based on elastin-like-polypeptides conjugated with polyethyleneimine (PEI).;To this end, ELP was expressed by culturing E. coli bacteria genetically modified to translate the ELP polypeptide sequence [VPGVG] 40. ELP was then chemically modified with primary amines conjugates. In the "Direct Reaction Scheme", ELP molecules were directly conjugated to polyethylenimine, polyarginine, or polylysine, while in the "PVDMA Reaction Scheme", ELP was conjugated to a poly(2-vinyl-4,4-dimethyl aziactone) (PVDMA) linker molecule and subsequently reacted to N-Boc-1,4-butanediamine, N-Boc ethylenediamine, or L-arginine molecules to render Charged-ELP conjugates. Chemical composition and molecular weights of synthesized ChargedELPs were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), Fourier transform infrared (FTIR) spectroscopy, and matrix-assisted laser desorption/ionization time of flight (MALDI TOF) spectroscopy. O-phthalaldehyde (OPA) fluorescence measures of the products proved an effective method to assess and control the relative primary amine "spheroid-inducing content" for all six materials. Selected OPA-based mixture ratios of pure ELP and Charged-ELP called "charge grades" were coated on tissue culture polystyrene (TCPS) surfaces for analysis of surface properties. Contact angle goniometry was used to develop Zisman plots that revealed no statistically significant relationship between the coating material's primary amine content and its surface free energy. Atomic force micrographs of dry coating surfaces revealed intricate patterns that formed as coatings deposited, though no definite trend in patterning was noted with change in conjugate type or primary amine content of material. Coating surfaces were shown to rearrange following immersion in culture media under culture conditions and ultimately reverted to a flat, featureless surface within a week under culture conditions. X-ray photoelectron spectroscopy (XPS) showed spatial variation in surface chemistry atop all coating surfaces. We next cultured primary rat hepatocytes atop 2 control surfaces and 22 total test coatings comprised of six Charged-ELP material groups and four main charge grades. Cell viability, differentiation, and quantifiable liver-specific metabolism over a 20 day culture period were assessed through optical microscopy and through measurement of total protein, rat albumin, and urea. By day 14 protein-normalized rat albumin production was maximum at the lowest charge grade atop each material except for ELP-polylysine coatings. Total albumin production on day 20 was higher atop coatings charged with lysine-like terminal groups (664 +/- 50 ng albumin/culture well/day atop ELP-polylysine grade 3.4 and 580 +/- 43 ng albumin/culture well/day atop ELP-PVDMA-butanediamine grade 1.1) than those charged with synthetic imine or using one of two reaction schemes to render six different spheroid-inducing "Charged-ELP" arginine-like terminal groups (411 +/- 111 ng albumin/culture well/day atop ELP-PEI grade 1.1, 304 +/- 65 ng albumin/culture well/day atop ELP-PVDMA-ethylenediamine grade 1.7, and less than 100 ng albumin/culture well/day atop ELP-polyarginine or ELP-PVDMA-arginine). In summary, we have developed an array of coating materials that are highly biocompatible (support long term culture), have tailorable spheroid-forming capacity to accommodate desired cell morphologies, are able to be quickly coated atop traditional culture surfaces, and maintains structural integrity while ensuring tight adherence of spheroids over long culture periods.