3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro

3d stem cell niche engineering via two-photon laser polymerization

A strategy to modulate the behavior of stem cells in culture is to mimic structural aspects of the native cellâ\u80\u93extracellular matrix (ECM) interaction. An important example of such artificial microenvironments for stem cell culture is the so-called â\u80\u9csynthetic niche.â\u80\u9d Synthetic niches can be defined as polymeric culture systems mimicking at least one aspect of the interactions between stem cells and the extracellular surroundings, including biochemical factors (e.g., the delivery of soluble factors) and/or biophysical factors (e.g., the microarchitecture of the ECM). Most of the currently available approaches for scaffold fabrication, based on self-assembly methods, do not allow for a submicrometer control of the geometrical structure of the substrate, which might play a crucial role in stem cell fate determination. A novel technology that overcomes these limitations is laser two-photon polymerization (2PP). Femtosecond laser 2PP is a mask-less direct laser writing technique that allows manufacturing three dimensional arbitrary microarchitectures using photosensitive materials. Here, we report on the development of an innovative culture substrate, called the â\u80\u9cnichoid,â\u80\u9d microfabricated in a hybrid organicâ\u80\u93inorganic photoresist called SZ2080, to study mesenchymal stem cell mechanobiology