Rebecca L. Carrier
Shashi Krishna Murthy, Jeffrey W. Ruberti, Ali Khademhosseini
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Engineering. Department of Chemical Engineering.
chemical engineering, biomimesis, extracellular matrix, intestinal cell, topography
Intestines - Cytology, Biomimetics
Biological Phenomena, Cell Phenomena, and Immunity | Molecular, cellular, and tissue engineering
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 μm in diameter and 120 μm 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 μm 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.
Wang, Lin, "Biomimesis of intestinal crypt micro-topography and extracellular matrix chemistry for intestinal cell culture" (2011). Chemical Engineering Dissertations. Paper 9. http://hdl.handle.net/2047/d20001041
Click button above to open, or right-click to save.