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

Abstract

In vitro liver models are necessary tools for the development of new therapeutics. HepaRG cells are a commonly used cell line to produce hepatic progenitor cells and hepatocytes. This study demonstrates for the first time the suitability of 3% silk scaffolds to support HepaRG growth and differentiation. The modulus and pore size of 3% silk scaffolds were shown to be within the desired range for liver cell growth. The optimal seeding density for HepaRG cells on silk scaffolds was determined. The growth and maturation of scaffolded HepaRG cells was evaluated for 28 days, where the first 14 days of culture were a proliferation period and the last 14 days of culture were a differentiation period using dimethyl sulfoxide (DMSO) treatment. After the first 14 days of culture, the scaffolded HepaRG cells exhibited increased metabolic activity and albumin secretion compared to monolayer cultured controls and preserved these attributes through the duration of culture. Additionally, after the first 14 days of culture, the scaffolded HepaRG cells displayed a significantly reduced expression of genes associated with hepatocyte maturation. This difference in expression was no longer apparent after 28 days of culture, suggesting that the cells underwent rapid differentiation within the scaffold. The functionalization of silk scaffolds with extracellular matrix (ECM) components (type I collagen and/or an arginylglycylaspartic acid (RGD)-containing peptide) was investigated to determine the impact on HepaRG cell attachment and maturation. The inclusion of ECM components had no noticeable impact on cell attachment but did significantly influence CYP3A4 expression and albumin secretion. Finally, the matrix support provided by the 3% silk scaffolds could prime the HepaRG cells for steatosis liver model applications, as evidenced by lipid droplet accumulation and expression of steatosis-related genes after 24 h of exposure to oleic acid. Overall, our work demonstrates the utility of silk scaffolds in providing a modifiable platform for liver cell growth.

Hep3Gel: A Shape-Shifting Extracellular Matrix-Based, Three-Dimensional Liver Model Adaptable to Different Culture Systems

“…In the past years, numerous in vitro models of the liver have been proposed, not only for fundamental research but also to support pharmaceutical development with reliable high-throughput platforms. , In this context, two-dimensional models of the liver, intended as monolayers of hepatic cells grown on flat substrates such as Petri dishes or multiwell plates, were the first platforms exploited to evaluate the hepatotoxic response to the administration of active pharmaceutical ingredients in vitro. These types of models, which can rely both on primary cells and cell lines, thus providing robust and reproducible results, led the way for the production of more complex models. , Up to now, some recent models have been based on three-dimensional cultures, where the cells are cultured within matrices mimicking the hepatic extracellular environment .…”

Influence of lyophilization primary drying time and temperature on porous silk scaffold fabrication for biomedical applications

“…After 14 days of culture, the cells were passaged using 0.05% trypsin–EDTA. The HepaRG cell line was used for these experiments as it is a model liver cell line that has been previously used for liver TE applications 48–50 …”

“…The HepaRG cell line was used for these experiments as it is a model liver cell line that has been previously used for liver TE applications. [48][49][50]…”

Bioinspired silk fibroin materials: From silk building blocks extraction and reconstruction to advanced biomedical applications

“…demonstrated the suitability of SF lyophilized scaffolds to support the growth and differentiation of HepaRG cell, which is a commonly used cell line to produce hepatic progenitor cell and hepatocyte [ 156 ]. Results of the lipid droplet accumulation and expression of steatosis-related genes after exposure to oleic acid showed that the matrix support fabricated by 3% SF combined with HepaRG cells could be suitable for the construction of steatosis liver model [ 156 ]. By using a complex RSF scaffold fabricated by blending mulberry ( Bombyx mori ) SF with cell adhesion motif contained non-mulberry ( Antheraea assamensis ) SF, Janani et al.…”