Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Abstract

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two-dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell-specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three-dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen-coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver-cell-specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver-specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real-time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well-preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF-4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re-established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO-1-positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes.

Physiologically Low Oxygen Enhances Biomolecule Production and Stemness of Mesenchymal Stem Cell Spheroids

“…For example, primary hepatocytes and chondrocytes will lose their normal phenotype in monolayer culture, but this loss can be reversed simply by growing these cells into 3D spheroids. 9,10 Aggregation of cells such as myoblasts has also been shown to promote cell viability and proliferation. 11 Human mesenchymal stem cells (hMSCs) are multipotent cells with many useful attributes such as their proliferative capacity in vitro and immunomodulatory abilities in vivo.…”

Recent advances and future applications of microfluidic live-cell microarrays

“…These studies show that 3D culture techniques based on aggregates, spheroids and tissue scaffolds or hydrogels help cells to retain their natural functionality. For instance, it was demonstrated that hepatocytes can exclusively maintain their physiologically relevant phenotype within a three-dimensional context (Bierwolf et al, 2011).…”

Cell Detachment for Engineering Three-Dimensional Tissues

“…Compared with conventional monolayer culture, spheroid cultures are considered to provide microenvironments more closely related to in vivo conditions [16,17]. For example, primary hepatocytes and articular chondrocytes maintained their organ-specific functions when they were cultured as spheroids but not when grown in monolayer cultures [18]. Various spheroid fabrication techniques that employ nonadherent surfaces [19], spinner flasks [20], hanging drop methods [21], micropatterning of cell-adhesive/non-adhesive surfaces [22,23], and micromolding [24,25] have been reported.…”