Microvessel structure formation in a 3D perfused co-culture of rat hepatocytes and liver endothelial cells

Abstract

Many liver physiological and pathophysiological behaviors are not adequately captured by current in vitro hepatocyte culture methods. A 3D perfused microreactor previously demonstrated superior hepatic functional maintenance than conventional 2D cultures, and was hypothesized to provide an environment favorable to endothelial cell maintenance and morphogenesis. This dissertation focuses on characterizing the 3D perfused co-culture of primary hepatocyte fraction with primary rat liver endothelial isolate. Scanning electron microscopy revealed significantly higher numbers of pore-like structures on the co-culture tissue surface resembling liver sinusoids compared to cultures containing only the hepatocytes fraction (mono-culture). EGFP-labeled endothelial cells proliferated moderately and organized into microvessel-like structures as observed by in situ multi-photon microscopy. By mixing female endothelial cells with male hepatocytes, the female cell population increased from initially -7% on day 1 to -12% on day 13, as determined by quantitative PCR on genomic DNA. The maintenance and morphogenesis of endothelial cells were not observed in parallel 2D collagen gel sandwich cultures. Immunohistochemistry further confirmed the presence of sinusoidal endothelia within the 3D co-culture tissue, as well as other non-parenchymal cells in both 3D mono-culture and co-culture.

(cont.) Global transcriptional profiling confirmed the loss of endothelia in 2D culture as the comparison between mono-culture and co-culture showed substantial differential expression levels only in the 3D format. The majority of the genes expressed substantially higher in 3D co-culture than mono-culture was found to be endothelia-specific. A group of key liver metabolism genes, however, do not show significant expression differences between the 3D cultures. This study concludes that the 3D perfused microreactor maintains non-parenchymal cells better than the 2D format, and the retention of non-parenchymal cells in the primary hepatocyte fraction likely contributes to the maintenance of key hepatic function gene expression. Additional endothelial cells organize into microvessel-like structures in this environment, but exert little influence on the gene expression of most key liver transcription factors and metabolism enzymes. Therefore 3D cultures may eliminate the need of co-cultures for applications focusing on metabolic behaviors of hepatocytes, and 3D endothelial-hepatocyte co-cultures may prove useful in studies where proper endothelium structure is required, such as cancer metastasis.