SBIR-STTR Award

Formation of three-dimensional, tissue-like structures in vitro has not previously been possible. We have developed a novel gelatin covered perfluorocarbon substrate which supports growth of more than 15 layers of Hela cells compared to monolayers formed in conventional conditions. The aim of this project is to use this novel oxygenation system for culture of liver parenchymal cells. Work in Phase I will determine whether improved oxygenation of the cultures will enable liver cells to grow in multilayers with normal function, as determined by albumin in synthesis and pentabarbital induction of cytochrome P450. In Phase II, hepatocytes will be cocultured with non-parenchymal cells, known to supply hepatocyte growth factors and extracellular matrix materials, to grow functional histotypic structures in vitro. These structures would provide improved systems for studying factors that contribute to cellular injury in liver diseases. such as alcoholism. Tissue fragments from in vitro could also be used as extra-corporeal transplants to assist patients with compromised liver function.Proposed commercial application:This technology makes it possible to grow viable cells in tissue-like multi-layers. This is the first step toward the development of complex tissues in vitro and may ultimately make it possible to grow replacement organs. Immediate applications for this technology are to co-culture hepatocytes with other liver cells. This will provide superior systems for studying alcohol-induced liver damage and other liver diseases in vitro.National Institute on Alcohol Abuse and Alcoholism (NIAAA)

The long term goal of this project is to develop an oxygenation system which can provide hepatocytes with the oxygen they need to maintain long term proliferation in vitro. Adequate oxygenation of these cells in culture is prevented by the slow rate oxygen diffuses from air through the overlaying liquid medium. The system being developed overcomes this limitation. Cells are grown on a perfluorocarbon conjugated gelatin substratum over a layer of perfluorodecalin (PFD). This provides oxygen directly to the cells at the interface. Hela cells on this substratum grew to form a tissue like structure of more than 19 layers. These areas of work are being undertaken to adapt this method for hepatocyte growth: 1) development of a system for re-oxygenation PFD, 2) development of suitable perfluoro-conjugates for collagens, and 3) development of an enriched culture media containing factors needed to sustain proliferation. This new oxygenation system for hepatocyte culture will provide greatly improved systems for testing drugs and studying the effects of alcohol on the liver. Co-culture of hepatocytes with other liver cells will provide heterotypic tissue like structures which could be used as extra-corporeal implants in patients with compromised-liver function.Proposed Commercial Applications:There are three commercial products being developed in this project. The first involves a liver cell culture media which outperforms commercially available media. The second involves new conjugated perfluorocarbons which will be used for protein attachment. The last and most important includes the first two products along with a cell culture chamber which will be used to grow liver cells in 3D. These cells can be used to study alcohol and drug metabolism in normal hepatocytes.