Characterization and Functionalization of Lyophilized Silk Scaffolds for Use in 3D In Vitro Disease Modeling

Abbott, Alycia

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Characterization and Functionalization of Lyophilized Silk Scaffolds for Use in 3D In Vitro Disease Modeling

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Steatosis, or fatty liver disease, affects up to 35% of the general population and currently has no therapeutic treatments. In vitro models serve as an essential research tool for furthering our understanding of the mechanisms behind disease progression as well as improving our development of new therapies. Biomaterial-based 3D in vitro culture models present an attractive intermediate between traditional 2D (monolayer) cell culture environments and in vivo animal models due to the control allowed over the culture microenvironment. Additionally, manipulation of biomaterial properties results in tunable biophysical and biochemical cues capable of guiding cell behavior and allows study of biological processes, such as disease progression. In this work, lyophilized (freeze-dried) silk fibroin scaffolds were characterized and functionalized to aid in the development of 3D in vitro disease models. The ability of lyophilized 3% (w/v) silk scaffolds to support liver cell growth was evaluated and compared to spheroid culture as well as 2D culture. The 3% scaffolded liver cells demonstrated significantly improved liver cell phenotypes as determined through metabolic activity, cell proliferation, lipid accumulation, and expression of hepatic genes. Additionally, the scaffolded liver cells demonstrated responsiveness to known steatosis inducers and potential inhibitors, indicating usefulness for disease modeling. Manipulation of the biochemical environment was then explored and pre- and post-processing functionalization methods for the silk scaffolds were utilized. Direct incorporation of type I collagen was completed during pre-processing while a facile, electrostatic-based functionalization process utilizing an avidin-biotin system was developed for post-processing functionalization. The impact of these functionalization methods on liver cell attachment to and maturation on silk scaffolds was evaluated. Scaffold functionalization did not significantly increase liver cell attachment. However, scaffold functionalization significantly influenced liver cell phenotype. Manipulation of the biophysical environment was then explored through modification of the lyophilization process. We developed lyophilization protocols to investigate the influence of primary drying time and temperature on resultant scaffold properties. We determined that changes to primary drying significantly impact scaffold properties including Young’s modulus, pore Feret diameter, and degradation rate. Silk secondary structure was not significantly influenced by changes to primary drying during lyophilization. We then demonstrated the versatility and utility of the developed lyophilization protocols by creating scaffolds suitable for use in an in vitro kidney cancer model (ccRCC). We demonstrated 6% silk scaffolds mechanically match the ccRCC microenvironment and provide an improved culture environment for ccRCC. This improvement in ccRCC phenotype was shown to be dependent on the Young’s modulus of the silk scaffold. In summary, utilizing lyophilized silk scaffolds, we developed in vitro models for both liver and kidney disease. Additionally, we developed techniques to fabricate and functionalize silk scaffolds that will enable silk scaffolds to be tailored for specific disease applications in the future.

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