3D Chitosan-Based Microenvironments to Regulate Cellular Fate For Tissue Engineering and Drug Screening Applications

Abstract

Conventional two-dimensional (2D) cell culture systems fail to recapitulate the spatial organization, mechanical cues, and biochemical complexity of native tissues, limiting their predictive value in disease modeling, drug discovery, and regenerative medicine. Although three-dimensional (3D) culture platforms improve physiological relevance, widely used matrices such as Matrigel remain xenogeneic, compositionally undefined, mechanically unstable, and poorly suited for scalable manufacturing or clinical translation. These limitations underscore the need for tunable, xeno-free biomaterial systems capable of not only supporting cells structurally but actively regulating cellular plasticity and tissue morphogenesis.This dissertation advances a microenvironment engineering strategy based on tunable 3D chitosan-based porous scaffolds designed to function as active regulators of cell fate. Cellular plasticity—the ability of cells to alter phenotype, transcriptional programs, or lineage identity in response to environmental cues—plays a central role in cancer progression, stem cell reprogramming, and tissue development. The central hypothesis of this work is that precisely engineered 3D scaffold architectures can modulate mechanical, spatial, and biochemical signals to control cellular state transitions while maintaining translational feasibility. To establish this framework, freeze-drying–based fabrication strategies were systematically investigated to define how processing parameters—including polymer concentration, solution depth, mold geometry, freezing temperature, freezing direction, and cooling rate—govern pore size, anisotropy, and mechanical properties. By linking thermal gradients and ice crystal dynamics to scaffold microstructure, an integrated design framework was developed to enable predictable customization of 3D microenvironments tailored to specific biological applications. Building on this architectural foundation, three major applications were pursued. First, tunable chitosan–hyaluronic acid porous scaffolds were developed as high-throughput platforms for glioblastoma modeling and drug screening. Controlled modulation of pore size and structural organization revealed that scaffold architecture regulates tumor cell morphology, gene expression, phenotypic heterogeneity, and therapeutic response, demonstrating that 3D microenvironmental cues directly influence cancer cell plasticity. Second, a virus-free nanoparticle–scaffold system was engineered by integrating polymeric gene delivery nanoparticles with a 3D chitosan microenvironment. This platform significantly enhanced human induced pluripotent stem cell (hiPSC) reprogramming efficiency and enabled a continuous, selection-free workflow. Transcriptomic analyses revealed that 3D scaffold culture reshapes transcriptional programs by suppressing inflammatory and extracellular matrix–associated pathways while promoting chromatin remodeling and pluripotency networks, thereby facilitating controlled cell-state transitions and improving reprogramming stability. Third, a xeno-free chitosan–alginate scaffold was developed to support hiPSC-derived epithelial–mesenchymal recombination for dental organoid formation and early tooth regeneration. The scaffold enabled coordinated epithelial–mesenchymal interactions, progressive odontogenic differentiation, and mineralized matrix deposition in vitro. Following orthotopic implantation, cell-laden scaffolds supported early tooth-like tissue organization and localized mineral formation, demonstrating the potential of engineered 3D scaffolds to guide hard tissue morphogenesis. Collectively, this work establishes design principles for engineering 3D porous scaffolds as active regulators of cellular plasticity across disease modeling, cell reprogramming, and regenerative tissue formation. By integrating scaffold architecture control, gene delivery technologies, and organoid-based regeneration within a unified microenvironment engineering framework, this dissertation provides a translationally relevant strategy for next-generation biomaterial platforms in biomedical engineering.