Assessing the compatibility of primary human hepatocyte culture within porous silk sponges

Donor organ shortages have prompted the development of alternative implantable human liver tissues for patients suffering from end-stage liver failure. Purified silk proteins provide desirable features for generating implantable tissues, including sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation. While different cell types were previously cultured for weeks within silk-based scaffolds, it remains unclear whether such scaffolds can be used to culture primary human hepatocytes (PHH) isolated from livers. Therefore, here we assessed the compatibility of PHH culture within porous silk scaffolds that enable diffusion of oxygen/nutrients through the pores. We found that incorporation of type I collagen during the fabrication and/or autoclaving of porous silk scaffolds, as opposed to simple adsorption of collagen onto pre-fabricated silk scaffolds, was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 ± 25 μm) promoted larger PHH spheroids and consequently higher PHH functions than large pores (235 ± 84 μm) for at least 1 month in culture. Further incorporation of supportive fibroblasts into scaffolds enhanced PHH functions up to 5-fold relative to scaffolds with PHHs alone and 2D co-cultures on plastic. Lastly, encapsulating PHHs within protein hydrogels while housed in the silk scaffold led to higher functions than protein hydrogel-only or silk-only controls. In conclusion, porous silk scaffolds containing extracellular matrix proteins can be used for the culture of PHHs ± supportive non-parenchymal cells, which can be further built on in the future to create optimized silk-based liver tissue surrogates for cell-based therapy.