A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies

Abstract

Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as achieve tighter control of experimental variables than can be achieved with animals. Thus development of a primary in vitro colonic epithelium cultured as a high-resistance with transport protein expression and functional behavior similar to that of native colonic would be of enormous value for pharmaceutical research. A collagen scaffold in which the degree of collagen cross-linking was present as a gradient was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein) and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability (Papp) of < 5×10−7 cm s−1 while, propranolol demonstrated a Papp of 9.69×10−6 cm s-1. Rh123 was transported in a luminal direction (Papp, efflux/Papp, influx =7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo.

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

“…While these systems demonstrate gene expression at the mRNA level of known transporters, these non-natural polymer culture surfaces can have a profound impact of transporter activity since the underlying stiff surfaces do not replicate most features of the lamina propria [21, 22]. Thick collagen scaffolds mimicking critical features of the lamina propria, such as its stiffness, have been shown to support both human and mouse primary intestinal monolayers for short-term assay or long-term culture [23–25]. A recently described scaffolding utilizes a gradient of chemical cross-linking across a thick collagen hydrogel so that the luminal collagen surface in contact with cells mimics the stiffness of in vivo intestine while the more cross-linked or rigid basal surface prevents gross hydrogel deformation by the cells [25].…”

“…Thick collagen scaffolds mimicking critical features of the lamina propria, such as its stiffness, have been shown to support both human and mouse primary intestinal monolayers for short-term assay or long-term culture [23–25]. A recently described scaffolding utilizes a gradient of chemical cross-linking across a thick collagen hydrogel so that the luminal collagen surface in contact with cells mimics the stiffness of in vivo intestine while the more cross-linked or rigid basal surface prevents gross hydrogel deformation by the cells [25]. However, the cells on this newer physiologic scaffold have not been characterized with regard to their transporter protein expression and function.…”

“…Despite a growing interest, limited studies of the interplay of stiffness and stem cell self-renewal, cellular differentiation and cell function have been performed in intestinal epithelium. Organoid culture systems have shown optimal intestinal cell growth in stiff matrices and optimal differentiation in soft matrices, yet growth on a planar substrate has shown opposite behavior [23, 25, 32]. The effects of collagen/ECM stiffness on transporters such as BCRP and MRP2 has been studied in cancer cell models [14, 33, 34], but there is little reported on how matrix stiffness affects protein expression in primary intestinal cell systems.…”

Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models

“…Beyond chemical gradients, stem cell fate decisions have been partially ascribed to the mechanical properties (e.g., stiffness, porosity) of the extracellular matrix, 23 presenting opportunities for further studies of mechanotransduction, potentially with parallel gradients of stiffness. 63 Given that a standardized geometry (96-well) is already built into the design of this device, with further scaling and adoption of rapid, 3D microscopy techniques (e.g., spinning disc confocal, light sheet), a high content platform can be envisioned that is both user-friendly and human patient-relevant. then transferred to a 150 °C hot plate.…”

Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn’s Disease

“…Epithelial permeability was evaluated in 2D-cultured human primary IEC monolayers as we reported previously. 47 Primary IECs cultured on 48-well plates were passaged to 12-well cell culture inserts (353180; BD Falcon, San Jose, CA) coated with collagen scaffolds with a gradient of cross-linking 48 at a ratio of 1:1. Cells were expanded in EM for 4 days and stimulated with 10 ng/mL BMP9 alone or in combination with 100 ng/mL ALK1-Fc chimera protein in EM or cultured in differentiation medium (DM) as a positive control of colonocyte differentiation 44 and TEER increase for an additional 2 days.…”