Primary Human Hepatocytes Maintain Long-term Functions in Porous Silk Scaffolds Containing Extracellular Matrix Proteins

The shortage of donor organs for transplantation has prompted the development of alternative implantable human liver tissues; however, the need for a clinically viable liver tissue that can be fabricated using physiologically-relevant primary human hepatocytes (PHHs) is unmet. Purified silk proteins provide desirable features for generating implantable tissues, such as sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation; however, the utility of such scaffolds to generate human liver tissues using PHHs remains unclear. Here, we show that the incorporation of type I collagen during the fabrication and/or autoclaving of silk scaffolds was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 +/- 25 {micro}m) promoted higher PHH functions than large pores (235 +/- 84 {micro}m). Further incorporation of growth-arrested 3T3-J2 fibroblasts into scaffolds enhanced PHH functions up to 5-fold for 5 months in culture, an unprecedented longevity, and functions were better retained than 2D configurations. Lastly, encapsulating PHHs within Matrigel while housed in the silk/collagen scaffold led to higher functions than Matrigel or silk/collagen alone. In conclusion, porous silk scaffolds are useful for generating long-term PHH +/- fibroblast tissues which may ultimately find applications in regenerative medicine and drug development.