Use of three-dimensional spheroids of hepatocyte-derived reporter cells to study the effects of intracellular fat accumulation and subsequent cytokine exposure.

Non-alcoholic fatty liver disease (NAFLD) is a family of liver diseases associated with obesity. Initial stage of NAFLD is characterized by a fatty liver, referred to as steatosis, which progresses in some individuals to non-alcoholic steatohepatitis (NASH) and liver failure. In order to study and treat the many liver diseases such as NAFLD, an improved in vitro cellular model is needed. Several studies in the past have attempted to elucidate these mechanisms using primary hepatocytes or relevant hepatoma cell lines in two-dimensional (2D) monolayer in vitro cultures. These 2D planar culture systems, unfortunately, do not represent the complex architecture of hepatic tissue in vivo. Therefore, we have engineered an elastin-like polypeptide (ELP)-polyethyleneimine (PEI) copolymer and shown that ELP-PEI coated surfaces influenced H35 rat hepatoma cell morphology to create 3D spheroids. Our reporter cell model recapitulates many cellular features of the human disease, including fatty acid uptake, intracellular triglyceride accumulation, decreased proliferation, decreased liver-specific function, and increased reactive oxygen species accumulation. Finally, to demonstrate the utility of the reporter cells for studying transcriptional regulation, we compared the transcriptional dynamics of nuclear factor κB (NFκB) in response to its classical inducer (tumor necrosis factor-α, TNF-α) under lean and fatty conditions in both 2D and 3D culture configurations. We found that, in 3D spheroids, linoleic acid treatment activated NFκB at earlier time points during the development of steatosis, but suppressed the TNF-mediated NFκB activation at later time points. These studies therefore provide a good starting point to evaluate such relationships observed during NAFLD in a 3D in vitro cell culture.

Production of hyaline-like cartilage by bone marrow mesenchymal stem cells in a self-assembly model.

A scaffoldless or self-assembly approach to cartilage tissue engineering has been used to produce hyaline cartilage from bone marrow-derived mesenchymal stem cells (bMSCs), but the mechanical properties of such engineered cartilage and the effects the transforming growth factor (TGF) isoform have not been fully explored. This study employs a cell culture insert model to produce tissue-engineered cartilage using bMSCs. Neonatal pig bMSCs were isolated by plastic adherence and expanded in monolayer before being seeded into porous transwell inserts and cultured for 4 or 8 weeks in defined chondrogenic media containing either TGF-beta1 or TGF-beta3. Following biomechanical evaluation in confined compression, colorimetric dimethyl methylene blue and Sircol dye-binding assays were used to analyze glycosaminoglycan (GAG) and collagen contents, respectively. Histological sections were stained with toluidine blue for proteoglycans and with picrosirius red to reveal collagen orientation, and immunostained for detection of collagen types I and II. Neocartilage increased in thickness, collagen, and GAG content between 4 and 8 weeks. Proteoglycan concentration increased with depth from the top surface. The tissue contained much more collagen type II than type I, and there was a consistent pattern of collagen alignment. TGF-beta1-treated and TGF-beta3-treated constructs were similar at 4 weeks, but 8-week TGF-beta1 constructs had a higher aggregate modulus and GAG content compared to TGF-beta3. These results demonstrate that bMSCs can generate functional hyaline-like cartilage through a self-assembling process.