HepaRG Maturation in Silk Fibroin Scaffolds: Toward Developing a 3D In Vitro Liver Model
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.
Graphical presentation of culture timeline and culturing conditions for HepaRG cells. (A) Culture timeline for HepaRG cells. (B) Conditions utilized for HepaRG cell culturing.
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, their work demonstrates the utility of silk scaffolds in providing a modifiable platform for liver cell growth.
Silk scaffold characterization. (A) SEM images of 5% silk scaffold used for initial studies and 3% silk scaffold used for maturation studies. (B) Median Feret diameter of 3% and 5% silk scaffolds. Data are presented as median ± SD of four independent scaffolds. An asterisk indicates a significantly greater Feret diameter in 3% silk scaffolds compared to 5% silk as determined by the Mann–Whitney test (* p < 0.05). (C) Young’s modulus of 3% and 5% silk scaffolds. Data are presented as mean ± SD of three independent experiments. The Young’s modulus of the 5% scaffolds was significantly greater than Young’s modulus of the 3% scaffolds as determined by a Student’s t test (** p < 0.01).
The liver is the largest internal organ, making up 1/40th of the total body mass and receiving 25% of the cardiac output.It has over 500 functions ascribed to it including xenobiotic metabolism, lipid, and protein synthesis, glycogen storage, bile production, and exogenous (e.g., drugs) and endogenous (e.g., bilirubin, the byproduct of red blood cell breakdown) detoxification. However, a broad range of conditions including hepatitis, drug-induced liver injury, nonalcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma can severely compromise liver function. Worldwide, over 600 million people are affected by liver disease, resulting in the death of over 1 million people each year.
Determination of optimal HepaRG cell seeding number. (A) Confocal images (40×) of silk scaffolds seeded with 10 000–60 000 HepaRG cells. Blue = nuclei/silk. Green = cytoskeleton. Red = lipids/silk. (B) Normalized fluorescence of resazurin after incubation with HepaRG cells on Day 14. All values were normalized to average fluorescence for 10 000 TCP HepaRG cells and are presented as mean ± SD from four independent samples. No significant difference was observed between scaffolded and TCP HepaRG cells.
In vitro models serve as an essential research tool for furthering their understanding of the mechanisms behind disease progression as well as improving the development of new therapies. Primary human hepatocytes (PHHs) are commonly used for in vitro liver models. However, in 2D cultures, PHHs rapidly dedifferentiate and lose liver functions, such as cytochrome P450 activity and transporter functions. Numerous methods have been developed to improve and extend PHH culture including sandwich cultures, coculture models containing other liver cell types, spheroid systems, and liver-on-a-chip technologies.Induced pluripotent stem cell (iPSC)-derived hepatocytes have been studied as an alternative, but developing mature hepatic phenotypes from iPSCs remains a major challenge. As an alternative, immortalized cell lines, such as HepG2, Huh7, and HepaRG, are widely used to conduct in vitro studies. HepaRG cells have demonstrated stable liver-like functions, including the expression of cytochrome p450 enzymes at levels comparable to cultured PHH, and are commonly used in liver metabolism and toxicity models.
Response of undifferentiated HepaRG cells to scaffolded and TCP culturing conditions. (A) Normalized fluorescence of resazurin after incubation with HepaRG cells on Days 7 and 14. All values were normalized to average fluorescence for Day 7 TCP HepaRG cells and are presented as mean ± SD from three independent experiments. No significant difference was seen between scaffolded HepaRG cells and TCP HepaRG cells by t test. (B) Representative DNA content from Days 1, 7, and 14. Data are presented as mean ± SD from six independent samples. DNA content from TCP HepaRG cells was significantly greater than DNA content from scaffolded HepaRG cells as determined by a Student’s t test (** p < 0.01, *** p < 0.001). (C) Gene expression normalized to Day 14 TCP HepaRG cells. Scaffolded HepaRG cells expressed significantly less ALB, CYP1A1, and CYP3A4 than TCP HepaRG cells as determined by a Student’s t test (* p < 0.05, ** p < 0.01, **** p < 0.0001). (D) Average albumin secretion over cell culture period. Data are presented as mean ± SD from three independent experiments. Albumin secreted from scaffolded HepaRG cells was significantly greater than from TCP HepaRG cells as determined by a Student’s t test (* p < 0.05).
HepaRG cells grown in a monolayer (2D) are commonly used in disease models to enable high-throughput assays. However, it is widely accepted that 2D models are insufficient to accurately recapitulate pathophysiological responses seen in vivo.HepaRG cells have been demonstrated to display improved hepatocyte functions in 3D culture conditions, through improved recapitulation of the liver microenvironment. However, some studies show stark differences in the functional ability between HepaRG cells obtained through current culture methods as compared to mature liver cells.Typically, a 3D culture of HepaRG cells relies on the formation of spheroids within a confined culture space. However, these methods are limited in size and do not allow control over the biochemical and biomechanical environment around the cells. The inclusion of a biomaterial within the culture system could be used to address these issues. Prior 3D culturing of HepaRG cells has focused on nanofibrillar cellulose, hyaluronan-gelatin, and PuraMatrix.When grown on these hydrogel-based biomaterials, HepaRG cells form spheroids that initially display higher levels of hepatocyte maturity (e.g., increased CYP3A4 expression) but cannot maintain that expression by the differentiated cells.
Differentiation of scaffolded and TCP HepaRG cells. (A) Normalized metabolic activity of HepaRG cells on Days 21 and 28. All values were normalized to average fluorescence for Day 7 TCP HepaRG cells and are presented as mean ± SD from three independent experiments. Metabolic activity of scaffolded HepaRG cells was significantly greater than the metabolic activity of TCP HepaRG cells on Day 21 as determined by a Student’s t test (* p < 0.05). (B) DNA content from Days 21 and 28. Data are presented as mean ± SD from three independent experiments. DNA content from TCP HepaRG cell control was significantly greater than DNA content from scaffolded HepaRG cells on Day 28 as determined by a Student’s t test (* p < 0.05). (C) Confocal microscopy image of HepaRG cells on silk scaffold to demonstrate cell phenotype. Blue = nuclei/silk. Green = cytoskeleton. Red = lipids/silk. Arrows denote lipid droplets. (D) Gene expression data normalized to Day 14 TCP HepaRG cell values. No significant difference in Day 28 scaffolded and TCP HepaRG cell gene expression as determined by a Student’s t test. (E) Average albumin secretion over the cell culture duration. Data are presented as mean ± SD from three independent experiments. Albumin secreted from scaffolded HepaRG cells was significantly greater than from TCP HepaRG cell control on Day 28 as determined by a Student’s t test (* p < 0.05). (F) Gene expression data normalized to Day 14 TCP or scaffolded HepaRG values. Data are presented as mean ± SD from three independent experiments. Scaffolded HepaRG cells expressed significantly more PCK2 than TCP HepaRG cells by a Student’s t test (* p < 0.05).
The use of a porous biomaterial scaffold that provides structural support for 3D culturing could be advantageous for the differentiation of HepaRG cells as well as the maintenance of a differentiated phenotype. Silk fibroin (silk) has been used for the development of numerous 3D in vitro tissue and disease models including cornea, kidney, gastrointestinal tissues, and various cancer types.The lyophilization of silk solutions produces porous scaffolds with mechanics that can be tuned to suit liver modeling applications. In addition to its ability to provide mechanical support for the culture environment, silk scaffolds can be functionalized through the incorporation of molecules into the bulk solution or after scaffold formation to provide biochemical signaling to the cells. Silk materials, such as films, electrospun fibers, and porous scaffolds, have been evaluated for liver modeling and favorably support PHHs and iPSC-derived hepatocyte-like cells.However, the use of silk scaffolds has not yet been applied to the HepaRG cell line.
Potential for steatosis model. (A) Day 14 confocal microscopy image (63×) of scaffolded HepaRG cells and Day 28 fluorescent image of TCP HepaRG cells. Blue = nuclei/silk; Green = cytoskeleton; Red = lipids/silk. Arrows denote lipid droplets. (B) Representative gene expression of scaffolded HepaRG cells normalized to Day 14 TCP HepaRG cells. Data are presented as the mean (bar) from two independent samples (circle = TCP; triangle = silk). (C) Scaffolded HepaRG cells confocal microscopy image (63×) after 24 h of oleic acid induction. Blue = nuclei/silk; Green = cytoskeleton; Red = lipids/silk. (D) Representative gene expression of TCP HepaRG cells and scaffolded HepaRG cells after exposure to 150 or 300 μM oleic acid for 24 h. Samples normalized to untreated TCP or scaffolded HepaRG cells. Data are presented as the mean (bar) from two independent samples (dots).
In the present study, they utilize lyophilized silk scaffolds to provide a 3D culture environment for the HepaRG cell line as progress toward developing a facile method to model liver tissue and disease in vitro. After determining the optimal seeding density and culture conditions, HepaRG cell maturation was examined over a 28 day culture period. Changes in the metabolic activity, DNA content, hepatic-associated gene expression, and albumin secretion were evaluated every 7 days during the culture period. They compared this scaffolded culture system to two current HepaRG cell culture methods: tissue culture plastic (2D) and spheroids. In addition, they investigated the impact of the bioactive molecule functionalization of the silk scaffolds on HepaRG cell behavior. The effect of incorporating type I collagen within the bulk scaffold and functionalizing scaffold surfaces with an RGD peptide was evaluated.
Finally, they evaluate lipid droplet accumulation as a potential indicator of utility as a steatosis model. To their knowledge, this work represents the first use of silk scaffolds to investigate HepaRG cell growth and differentiation.
HepaRG Maturation in Silk Fibroin Scaffolds: Toward Developing a 3D In Vitro Liver Model Alycia Abbott and Jeannine M. Coburn ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.0c01584