| In small intestine, a monolayer of enterocytes rests on extracellular matrix (ECM) mainly comprised of collagen (Col), laminin (Ln) and fibronectin (Fn), with convoluted topography consisting of finger-like projections (villi) with deep invaginations (crypts) between the villi. The small intestinal epithelial cell phenotype varies with position relative to crypt-villus structure. It is hypothesized that a biomimetic cell culture substrate with intestinal ECM-like topography and chemical makeup may induce in vitro intestinal cell culture expressing a phenotype more similar to native enterocytes.;The goal of this thesis work is to develop a topologically, chemically, and physiologically biomimetic intestinal cell culture system; as well as elucidate the effect of physical and chemical biomaterial substrate properties on intestinal cell phenotype.;A poly(dimethylsiloxane) (PDMS) based cell culture substrate patterned with a crypt-like micro-well array (50, 100, and 500 mum in diameter and 120 mum in depth) was first fabricated. Intestinal epithelial Caco-2 cells were cultivated on Fn coated patterned PDMS, the influence of crypt-like topography on Caco-2 cell phenotype was studied. Cells grown on micro-well patterned surfaces had prominent cellular protrusions, defined progression of coverage over small diameter wells, the bypassing of sharp well corners, higher metabolic activity, and lower level of differentiation.;Secondly, a chemically and physically more biomimetic, and also permeable, type I collagen cell culture substrate was created. Type I collagen was patterned with micro-well array with 70, 500 mum diameter and further coated with ECM proteins (i.e. Fn and Ln). Crypt-like topography inhibited Caco-2 differentiation during early culture. Caco-2 cultured on Fn-coated collagen started to spread earlier and formed longer protrusions than on Ln-coated collagen. Pre-coating of Ln enhanced cell differentiation and maintained differentiated phenotype in long-term culture. Compared to substrate topography, coating with ECM protein had more prominent and longer term effect on cell behavior.;The broad significance of this work lies in understanding the synergic influence of topographic cues and pre-incubation of ECM proteins on intestinal cell function, and enabling a better design of biomimetic materials for better intestinal tissue engineering scaffolds or substrates for in vitro intestinal cell models for drug pharmacokinetics testing. |
