Abstract

Tissue engineering is a dichotomy of scaffold-based and scaffold-free cultures. Scaffold-based cultures form highly organotypic structures but with low uniformity and throughput. Conversely, scaffold-free cultures create consistently sized and shaped cell aggregates with limited spheroid-like structure and function, thus restricting their use for accurate disease modeling. We hypothesized that combining aspects of each culturing format, we would produce highly organotypic structures of consistent size and shape for use in pulmonary modeling. First, to improve on culture consistency and output, we created a novel easily scalable, minimalistic design for a micropatterned hydrogel dish that increases reliability and efficiency in 3D cell culture. This dish design features three times more efficient media change relative to commercially available plates. Moreover, we discovered that formation of consistently sized and shaped cell aggregates depended on hydrogel stiffness. Second, we developed a biocompatible 3D printing resin using poly(ethylene glycol) diacrylate (PEGDA) monomer with avobenzone as the UV absorber instead of 2-nitrophenyl phenyl sulfide (NPS). The polymerized resin could be surface activated to promote cell adhesion. This resin could be used in high-resolution printing of miniature devices for microfluidic and nanofluidic cell culture and cell assays. Third, we show a unique improvement on current methods to produce organotypic aggregates via suspension culture. By using soluble non-gelling concentration of basement membrane (BM), we created an organotypic lung model from three stable cells representing epithelial, vascular, and fibroblast cell populations within 14 days of culture. We observed that soluble BM promotes emergence of lumina comparable to mammalian lung airspaces. Using hypoxia induction techniques, we provide evidence for formation of branching, perfusable vasculature in pulmonary aggregates supplemented with soluble BM. Aside from these structural traits, we observed increased proliferation, survival, and 3D growth of aggregates. These results were supported by proteomic studies. As proof of concept, we applied this method in modeling of lung fibrosis using bleomycin induction followed by testing one investigational antifibrotic drug. Our results demonstrate a novel 3D culture method that creates organotypic models from stable cell lines. We anticipate this technology to pioneer creation of novel suspension-based organoids fostering consistent, expedited 3D culture. In summary, these three technologies highlighted in this dissertation improved on the 3D culture status quo. We view these technologies to have the potential to expedite creation of patient-derived organoids for personalized drug screening using lung-on-a-chip assays.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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