Developing New Approach Methodologies to Increase the Physiological Relevance and Utility of Scaffold-Based 3D Cell Culture Techniques.

Since their first introduction in the early 1900s, cell cultures have become a vital tool for experimental model systems, pathological studies, gene manipulation, biotechnology, and cellular differentiation studies. Historically, two-dimensional (2D) cultures have served as the go to method for culturing cells in vitro. However, there has been a growing notion that cells in 2D cultures demonstrate in vivo irrelevant environments and cellular responses. This led to the introduction of three-dimensional (3D) cell cultures, that demonstrate environments and cellular responses that better recapitulate cells in vivo. In chapter 2, we evaluate the compatibility of five indirect viability assays (calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay) originally designed for 2D cultures with 3D scaffold-based paper cultures. We present a guide for selecting indirect viability assays for 3D cell cultures based on the analytical figures of merit, hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. In chapter 3, we introduce a new form of scaffold-based 3D cell culture that improves the compatibility of scaffold-based cell cultures with complex experimental workflows, termed the supported gel slab (SGS). We characterize and validate the SGS culture system and demonstrate the compatibility with microscopy, predicting dose-response relationships, and the intra- and inter-experimental reproducibility and repeatability across multiple days, cell passages, and scientists. In chapter 4, we demonstrate the capabilities of the SGS system with methods that have made scaffold-based 3D cell culture, specifically paper-based cultures, powerful tools for probing cellular responses. Specifically, we demonstrate the compatibility with generating tumor-like structures, single- and multi-scaffold invasion assays, the enzyme stability in primary hepatocytes, and screening compounds. In chapter 5, we introduce a macrofluidic culture device that incorporates physiologically relevant nutrient and drug delivery into scaffold-based 3D cell culture methods termed the continuous flow delivery system (CFDS). We characterize and validate the flow profile of the medium through the flow cells along with the experimental capabilities and modularity afforded by the CFDS. Additionally, we demonstrating the ability to incorporate blood vessel- and tumor-like models with more in vivo relevant delivery.