| Methods to build and analyze realistic in vitro models of in vivo cellular microenvironments are critical for understanding and engineering biological tissues. To date, studies of cell biology have been conducted primarily on 2D surfaces, often in the absence of extracellular matrix (ECM) molecules (for example using tissue culture polystyrene). The reconstitution of naturally-derived extracellular matrix materials as 3D gels provides platforms for studying cell biology in more native-like microenvironments and for engineering replacement tissues. However, 3D ECM in vitro culture models, even the most well established such as the type I collagen gel, are less well defined compared to traditional tissue culture materials such as 2D polystyrene, and thus pose numerous challenges for both engineering tissues and studying cell biology with consistent results. Further, 3D ECMs consisting of a limited number ECM components do not fully recapitulate the complex compositions and structures of native tissue microenvironments.;Numerous studies have investigated ways to modulate the structure of collagen-based ECMs in vitro, to control such properties as mesh size, fiber morphology, and fiber alignment, in order to recreate the nano- to microscale- structural properties of native tissues. Further, because tissues can be complex on the microscale, consisting of numerous different types of cells and ECM, methods are being developed to integrate multiple types of cell-seeded ECM together. In particular, microfabricated systems, which offer a high degree of control over many physical properties, are increasingly well-suited for engineering complex tissues or developing more realistic models for biological studies.;This thesis develops methods for engineering 3D collagen-based cellular microenvironments, by engineering the microstructure and stably interfacing multiple types of collagen-based materials, and by dynamically controlling matrix structural properties after fabrication. First, the use of collagen-based biomaterials for tissue engineering and biological studies is reviewed (Chapter 2). Following, a method to microfabricate and stably interface collagen-based ECMs is developed (Chapter 3). A method to reversibly switch ECM structure to permit or restrict cell migration is presented (Chapter 4). The tissue interfacing method developed in Chapter 2 is characterized (Chapter 5). Finally, the methods are applied towards the engineering of vascularized tissues (Chapter 6). The methods developed herein provide high resolution spatial and temporal control over collagen-based ECM structural properties, enabling precision control over cell behaviors such as migration, networking, and differentiation. These techniques should prove useful for engineering complex tissues or studying 3D cellular microenvironments in vitro. |
