3D micro-addressable tissue models to understand spatiotemporal heterogeneity in transcriptional regulation Advanced In vitro cell culture platforms have the potential to reveal the complex transcriptional and epigenetic regulation of cellular stress response and adaptation dynamics, which is challenging or impossible to study in vivo due to the inherent complexity of animal models, inability to experimentally manipulate the vast majority of tissue parameters, and a lack of high spatiotemporal resolution measurement techniques. Because of advances in tissue engineering and biomaterials, these platforms furthermore provide an ever-closer approximation of the physiological tissue microenvironment, reducing the need for in vivo experiments at earlier stages of research. Such studies will have important applications in our understanding of antibiotic resistance, personalized cancer medicine, and the development of effective and safe stem cell therapies. However, illustration of the complex molecular mechanisms behind the phenotype changes has been highly limited, and enabling tools to study the dynamics of such processes at high spatiotemporal resolution will provide new windows into previously inaccessible biology. While micro fabrication strategies have enabled well-defined heterogeneous model tissues, broad-spectrum genetic or epigenetic analysis of cells residing within micro scale tissue niches has not been possible. Our broad hypothesis is that spatiotemporal heterogeneity at micron scales impacts cellular stress response via transcriptional mechanisms, and that expanding the capabilities of physiologically relevant in vitro platforms will provide a powerful, broad spectrum, high-resolution tool to understand these dynamics. We will work towards our goals by pursuing two synergistic paths: 1) build on our prior microfluidic vascular tissue models to develop a brain tumor tissue mimic exhibiting the key chemo-mechano-cellular features regulating drug distribution, metabolism and chemoresistance development in vivo, and 2) extend our ability to analyze transcription level regulation via chromatin immunoprecipitation (ChIP), which we have already demonstrated on as few as 50 cells, to measure the role played by NF-kB in the interplay between spatiotemporal oxygen variations and cytotoxic stress response. The capabilities developed in this project will greatly enhance the utility of 3D cell culture models, and will provide access to the transcriptional machinery underlying stress response in a broad range of contexts.
We will greatly enhance the interrogation capabilities possible with physiologically relevant experimental tissue platforms, which are currently limited to optical measurements or analysis of secreted soluble factors due to incompatibility of platforms containing a small number of cells with traditional cell-based molecular analyses. This project will leverage in vitro micro fabricated 3D tissue mimics, in combination with a novel microfluidic chromatin immunoprecipitation (ChIP) technique, to understand transcriptional dynamics involving small numbers of cells evolving within heterogeneous tissues. These tools will be broadly useful for studies of cell signaling and stress response in well-defined, yet complex physiological contexts.
