Crypt-Villus Scaffold Architecture for Bioengineering Functional Human Intestinal Epithelium

Abstract

Crypt-villus architecture in the small intestine is crucial for the structural integrity of the intestinal epithelium and maintenance of gut homeostasis. We utilized three-dimensional (3D) printing and inverse molding techniques to form threedimensional (3D) spongy scaffold systems that resemble the intestinal crypt-villus microarchitecture. The scaffolds consist of silk fibroin protein with curved lumens with rows of protruding villi with invaginating crypts to generate the architecture. Intestinal cell (Caco-2, HT29-MTX) attachment and growth, as well as long-term culture support were demonstrated with cell polarization and tissue barrier properties compared to two-dimensional (2D) Transwell culture controls. Further, physiologically relevant oxygen gradients were generated in the 3D system. The various advantages of this system may be ascribed to the more physiologically relevant 3D environment, offering a system for the exploration of disease pathogenesis, host−microbiome interactions, and therapeutic discovery.

A vascularized crypt-patterned colon model for high-throughput drug screening and disease modelling

“…Microvilli were present on both apical and basal surfaces of the crypt cross section, indicating mature Caco-2 tissues were spread throughout the length of the crypt-axis, as seen in other works. 24,37 Interestingly, we also observed fibrin gel compaction at the cell-matrix interface around the patterned crypts as opposed to the more porous matrix in the bulk gel. This is likely due to physical compression of the gel in the stamping process.…”

“…Patterned crypt structures have been shown to drive colon tissue maturation in vitro. 8,22,24,25 However, existing methods relying on PDMS molds or stereolithography printing are not scalable to 384-wellplate formats. 24,25 Our aim was to use a 3D printed stamp (TopoStamp) to rapidly imprint an array of hydrogel scaffolds inside microwells (Fig.…”

“…A similar study by Rudolph et al , used inverse molding to create a scaffold with both crypts and villi. 24 The study also used Caco-2 cells to form patterned intestinal epithelium and demonstrated increased expression of enterocyte specific sucrase isomaltase marker in the patterned scaffold. Taken together, the recent works highlighted the ability for colon organoid and Caco-2 cells to recognize colon-specific patterning in scaffolds.…”

“…Limitations for use in high-throughput compound screens could be that custom chip platforms are difficult to scale up for supporting a high number of replicates. 22,24 Wang et al, utilizing transwell inserts performed a drug screen, but was done in 6-and 12-wellplates requiring multiple plates. 18,25 Integrating crypt-patterning within a 384-wellplate format could emulate colon epithelium for use in high-throughput compound screening.…”

“…Fibroblasts not only enable vascularization, but directly support cultured colon epithelium by secreting R-spondin, Wnt, and epidermal growth factor (EGF) to crypt bases. 24,32 These stromal cells also provide support through secretion of ECM scaffolding to influence tissue remodelling. The benefit conferred by fibroblast integration into colon models is absent in membrane-based cultures utilizing only endothelial lined channels.…”