Collaborative Research: High-throughput microliver platform for drug toxicity screening

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In vitro models of the human liver play a critical role in assessing the toxicity of drugs and industrial chemicals prior to human exposure. Such models are also useful for developing therapeutics against global diseases that affect the liver, such as hepatitis B/C viral infections, fatty liver disease, malaria, type 2 diabetes, and cancer. While considerable progress has been made over the last few years in utilizing engineering tools to fabricate higher functioning and longer-lasting human liver models, some key gaps and limitations still need to be addressed including increasing the throughput capability of the system, improving reproducibility in the production of the model, and including cancer cell lines in certain models. This research project is developing and optimizing a high-throughput "micro-liver" platform comprised of a biologically compatible gel, human liver cells, and additional biological molecules needed to support the functions of the cells. The investigators are using this platform to test the hypothesis that the microenvironment of this platform will imitate liver functions at levels that are significantly closer to actual physiological liver function. The investigators are also using clinically-relevant compounds to test the utility of these "micro-livers" for drug metabolism and toxicity screening. The educational efforts associated with this project using the findings and novel devices of this project to engage high school teachers and students in research experiences. Introducing cutting-edge research concepts earlier in high school is expected to better prepare students for a rigorous engineering curriculum at the college level. This research project is focused on creating the first high-throughput, three-dimensional (3D) microliver platform with a tunable extracellular matrix (ECM) microenvironment containing primary human hepatocytes (PHHs) and a complex mixture of liver stromal cells. The data generated will yield a fundamental understanding of the interactions of different types of human liver cells in a 3D context and the effects of drugs on such interactions. These findings will provide design criteria for the biomanufacturing of larger-scale 3D liver constructs for tissue engineering and regenerative medicine. The microgel platform and systematic exploration of cell-cell and cell-ECM interactions in the liver can also be broadly applicable to other tissue types being developed for integration into organs-on-a-chip systems. The project has two objectives: 1) develop and test a microfluidic platform for the high-throughput fabrication of 3D human liver microgels for investigating cell-cell and cell-ECM interactions and 2) investigate the effects of drugs on the morphology/functions of 3D human liver microgels. Objective 1 builds on a recently developed microfluidic flow-focusing device for generating microgels containing liver-inspired ECM, PHHs, and liver stromal cell types. The device will be used to test the hypothesis that a 3D microenvironment, which contains the complex liver-inspired ECM coupled with key liver stromal cell types, will enhance and stabilize for several months PHH functions at levels that are significantly closer to physiological outcomes than possible with existing systems. Use of a microfluidic system overcomes the problems associated with 3D culture methods using bulk collagen gels that are too labor-, time-, and cost-intensive to use in high throughput screening and, due to large size, have significant diffusion limitations for nutrients and signaling molecules. The system enables systemic investigation of the interactions of PHHs with stromal cells within a 3D ECM microenvironment, thus providing design principles for the construction of a human liver model that more accurately recapitulates human liver functions and drug responses.