HepaRG Maturation in Silk Fibroin Scaffolds: Toward Developing a 3D In Vitro Liver Model

In vitro liver modelsare necessary tools forthe development of new therapeutics. HepaRG cells are a commonly usedcell line to produce hepatic progenitor cells and hepatocytes. Thisstudy demonstrates for the first time the suitability of 3% silk scaffoldsto support HepaRG growth and differentiation. The modulus and poresize of 3% silk scaffolds were shown to be within the desired rangefor liver cell growth. The optimal seeding density for HepaRG cellson silk scaffolds was determined. The growth and maturation of scaffoldedHepaRG cells was evaluated for 28 days, where the first 14 days ofculture were a proliferation period and the last 14 days of culturewere a differentiation period using dimethyl sulfoxide (DMSO) treatment.After the first 14 days of culture, the scaffolded HepaRG cells exhibitedincreased metabolic activity and albumin secretion compared to monolayercultured controls and preserved these attributes through the durationof culture. Additionally, after the first 14 days of culture, thescaffolded HepaRG cells displayed a significantly reduced expressionof genes associated with hepatocyte maturation. This difference inexpression was no longer apparent after 28 days of culture, suggestingthat the cells underwent rapid differentiation within the scaffold.The functionalization of silk scaffolds with extracellular matrix(ECM) components (type I collagen and/or an arginylglycylasparticacid (RGD)-containing peptide) was investigated to determine the impacton HepaRG cell attachment and maturation. The inclusion of ECM componentshad no noticeable impact on cell attachment but did significantlyinfluence CYP3A4 expression and albumin secretion.Finally, the matrix support provided by the 3% silk scaffolds couldprime the HepaRG cells for steatosis liver model applications, asevidenced by lipid droplet accumulation and expression of steatosis-relatedgenes after 24 h of exposure to oleic acid. Overall, our work demonstratesthe utility of silk scaffolds in providing a modifiable platform forliver cell growth.