Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking

Abstract

Background: The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. Results: The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. Conclusions:The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function.

Evaluation of human primary intestinal monolayers for drug metabolizing capabilities

“…While the two scaffolds possess many distinctions, a major difference is that of the gradient cross-linked scaffold which possesses a stiffness similar to that of the in vivo intestine, whereas the conventional scaffold displays a stiffness several orders of magnitude greater than that of native intestine, a stiffness more characteristic of fibrotic tissue [38, 39]. Both culture systems support growth of cell monolayers covering the scaffold with cells forming an impermeable barrier to small molecules.…”

“…In this work, we investigate two previously described simple and customizable scaffolding systems on the impact of drug metabolism by primary, human, small intestinal epithelium with a comparison to in vivo human small intestine [38]. Cells cultured as a monolayer on a gradient cross-linked scaffold or a conventional scaffold were assessed for their ability to synthesize drug metabolizing proteins as well as to metabolize exogenously added commonly used drugs.…”

“…Cells cultured as a monolayer on a gradient cross-linked scaffold or a conventional scaffold were assessed for their ability to synthesize drug metabolizing proteins as well as to metabolize exogenously added commonly used drugs. The gradient cross-linked scaffold consisted of a soft collagen hydrogel (1.2 mm thick, stiffness of 230 ± 140 Pa) that closely resembles the in vivo intestinal properties (640 ± 340 Pa) [38, 39]. The conventional scaffold was comprised of a very thin collagen film (< 900 nm) overlying a stiff surface (net stiffness of 1.50 MPa ± 0.27 MPa) [38].…”

“…The gradient cross-linked scaffold consisted of a soft collagen hydrogel (1.2 mm thick, stiffness of 230 ± 140 Pa) that closely resembles the in vivo intestinal properties (640 ± 340 Pa) [38, 39]. The conventional scaffold was comprised of a very thin collagen film (< 900 nm) overlying a stiff surface (net stiffness of 1.50 MPa ± 0.27 MPa) [38]. In prior experiments, the monolayers in the two scaffolds demonstrated differing abilities to transport drugs, with the gradient cross-linked scaffold monolayer most closely resembling in vivo intestinal drug transport relative to that of the conventional scaffold monolayer [38].…”

“…The conventional scaffold was comprised of a very thin collagen film (< 900 nm) overlying a stiff surface (net stiffness of 1.50 MPa ± 0.27 MPa) [38]. In prior experiments, the monolayers in the two scaffolds demonstrated differing abilities to transport drugs, with the gradient cross-linked scaffold monolayer most closely resembling in vivo intestinal drug transport relative to that of the conventional scaffold monolayer [38]. Thus, it is possible that the scaffold monolayers may also demonstrate significantly differing capacities for drug metabolism making one more suitable for assays of human intestine-drug interactions.…”