INTELLECTUAL MERIT: This proposal describes the development of liver-mimetic cellular architectures using self-assembled polymeric scaffolds. There is currently no generally applicable methodology to layer liver cells in vitro. Although two dimensional (2D) cell cultures and co-cultures are used extensively as model systems, they do not recapitulate key spatial, geometric, and physiological characteristics of cellular architectures found in vivo. The specific features of this proposal are the design of detachable polyelectrolyte multilayer (PEM) films, the ability to incorporate cell-adhesive ligands such as galactose, tuning the physical properties of the scaffold, and the design of nanoporous detachable polyelectrolyte multilayer films. The specific objectives of the proposal are: (1) Design detachable chitosan/hyaluronan PEM films and demonstrate the feasibility of using them to assemble highly functional 3D liver-mimetic cellular architectures. (2) Tune PEM scaffold film properties to control cell growth and function. (3) Design and assemble nanoporous PEM scaffolds with tunable porosity for efficient transport of nutrients and metabolites. Such liver-mimetic tissues can serve as an accurate model for applications in bioreactor devices, understanding disease pathogenesis, toxicity evaluations, and in the testing of pharmaceuticals and drugs. The detachable PEM-scaffold can be modified, both chemically, and physically to incorporate a diverse range of cell types.
BROADER IMPACTS: Hepatocyte tissue culture has challenged the regenerative medicine field for some time. This proposal holds promise for facilitating a range of investigations of liver pathogenesis, toxicity, and, ultimately, liver-like bioreactor devices. The PI and Co-PI are both active participants in outreach and educational initiatives on the Virginia Tech campus. The PI will continue to participate in the VT STARS program, a 3-year residential program focusing on low-income, under-represented youth from South West Virginia, and she has just completed a term as Chair of the AIChE Women's Initiative Committee. The proposal also details her outreach to female middle and high school students designed to acquaint them with career opportunities in science and engineering. The Co-PI is a long time participant in the VT Summer Undergraduate Research Program supported by NSF REU and IGERT awards. Continued commitment to these and other educational initiatives can be anticipated.
The liver is responsible for several important physiological functions such as metabolism, protein production and transformation of xenobiotics. Although the liver has a great capacity to regenerate inside the body, once the cells are cultured in a laboratory environment, they rapidly lose their characteristics. Therefore, there is a critical need to develop in vitro models of the liver that closely mimic its structure to understand mechanisms through which the liver functions. In this project, we developed a polymeric membrane that resembles an interfacial region found in the liver. Development of this membrane was critical to our ability to assemble in vitro liver tissues and to conduct subsequent investigations into liver function. We incorporated three of the four major liver cell types to assemble an engineered hepatic tissue. We conducted investigations into the material, physical and biocompatibility properties of the polymeric membrane. Subsequent to culturing the liver tissue, we would separate each cell type to determine the extent to which cells divided in these cultures. We monitored the detoxification capacity of the assembled liver tissue, and protein production. We were able to show that in this engineered tissue, ratios of cells were very close to what is found inside the body. We disseminated our results to the scientific community via peer-reviewed journal publications, presentations at national conferences, seminars at universities and in publicly available graduate theses.
