The goal of this project is to investigate the effects of growth factors (GF) during in vitro liver injury. Micropatterned surfaces were created by mixing GF such as HGF, BMP7 and TGF-β with collagen I in solution and printing this ECM/GF mixture onto silane modified glass slides. Primary rat hepatocytes were seeded on top of the GF/ECM arrays forming clusters corresponding in size to the underlying protein spots (300 to 500 mm). Cell arrays were then injured in culture by exposure to 100 mM ethanol for 48h. Hepatocytes residing on GF spots were found to have less apoptosis then cells cultured on collagen-only spots. Least apoptosis (0.3 % as estimated by TUNEL assay) was observed on HGF/BMP7/collagen spots whereas most apoptosis (17.3%) was seen on collagen-only arrays. Interestingly, the extent of alcohol-induced apoptosis in hepatocytes varied based on the concentration of printed GF. In addition to preventing apoptosis, printed GFs contributed to maintenance of epithelial phenotype during alcohol injury as evidenced by higher levels of E-cadherin expression in HGF-protected hepatocytes.
Importantly, GF microarrays could be used to investigate heterotypic interactions in the context of liver injury. To highlight this, stellate cells -nonparenchymal liver cells involved in fibrosis - were added to hepatocytes residing on arrays of either HGF/collagen or collagen-only spots. Exposure of these cocultures to ethanol followed by RT-PCR analysis revealed that stellate cells residing alongside HGF-protected hepatocytes were significantly less activated (less fibrotic) compared to controls. Overall, our results demonstrate that micropatterned surfaces and GF microarray format can be used to screen anti-fibrotic and anti-apoptotic effects of growth factors as well as to investigate how signals delivered to a specific cell type modulate heterotypic cellular interactions.