High resolution stereolithography fabrication of perfusable scaffolds to enable long-term meso-scale hepatic culture for disease modeling.

Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases.

Engineering Helical Modular Polypeptide-Based Hydrogels as Synthetic Extracellular Matrices for Cell Culture.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.