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 three-dimensional (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.

Keywords: 3D printing; crypts; intestine tissue; oxygen profile; silk; tissue engineering; villi.

Figure 1.

3D half-scaffold system with crypts and villi for intestinal tissue engineering was fabricated using 3D printing techniques and silk fibroin. (A) Schematics of the fabrication process for generating silk-based porous half-scaffolds with crypts and villi for 3D human intestine engineering. Silk-based scaffolds are constructed by (1) designing a 3D model using 3D CAD software, (2 and 3) 3D printing resin molds, (4) casting PDMS reverse molds by placing the resin molds into liquid PDMS, (5) curing and obtaining the PDMS molds with heat exposure, (6) pipetting 7% aqueous silk solution into PDMS molds, (7) vacuuming and centrifuging silk filled molds then removing excess silk to leave a thin silk film on PDMS mold, (8) drying silk film then water annealing to achieve a smooth surface prior to filling with 6% silk solution, and (9) freezing, lyophilizing, and inducing β-sheets via autoclave. (B) Transverse cross section of half-scaffold constructs showing dimensions of crypt/villi height and diameter at both the base and the tip. (C) Longitudinal cross section of half-scaffold constructs showing distances between villi and crypts (scale bar: 1 mm). (D) Macroscopic top view of PDMS mold for casting silk scaffolds. (E) Macroscopic top view of silk scaffolds with crypts and villi (scale bar: 1 mm).

Figure 2.

Caco2 and HT-29 cell lines formed confluent and functional monolayer in 2D Transwells and 3D silk half-scaffolds. (A–C) Illustration of cell culture with Caco2 and HT-29 cell lines and primary myofibroblasts (A), cell seeding strategies for Transwells (B), and 3D half-scaffolds with crypts and villi (C). (D) Top-view photos of silk half-scaffolds seeded with Caco2/HT29 and myofibroblasts prior to fixing and staining (scale bars: 4 mm). (E) Representative confocal z-stack of DAPI staining (blue) on luminal surfaces of the scaffolds showing crypts and villi well covered with a cell monolayer, outlined by white dashed lines (scale bar: 200 μm).

Figure 3.

SEM images on the 3D scaffolds seeded with cells (week 3 post cell seeding) revealed a full coverage of epithelial cells on the scaffold luminal surface and the apical surface of the epithelial cells were covered with dense microvilli. (A) SEM images of the luminal surface of the seeded 3D scaffold with crypt/villus pattern (scale bar: 500 nm). (B–D) SEM images with higher magnifications of the villi in the seeded 3D scaffold lumen. (B) Scale bar: 100 μm, (C) scale bar: 20 μm, (D) scale bar: 2 μm. (E–G) SEM images with higher magnifications of the crypt in the seeded scaffold lumen. (E) Scale bar: 200 μm, (F) scale bar: 20 μm, (G) scale bar: 10 μm.

Figure 4.

Intestinal epithelial cells (Caco2 and HT29-MTX) formed a confluent and functional monolayer in 3D silk half-scaffolds. Schematic of Caco2/HT29-MTX monolayer and primary myofibroblasts. (A–F) Representative confocal z-stack of the immunostained luminal surface of the scaffold (scale bar: 200 μm). Confocal immunofluorescence images of the epithelia for ZO-1 (A, B), Muc-2 (C, D), and SI (E, F). Scale bars: 20 μm. (A, C, E) 3D reconstruction of z-stack of fluorescence microscope images of the scaffolds. (B, D, F) Confocal maximum projection fluorescence microscope images of the scaffolds with higher magnification on the luminal surface.

Figure 5.

3D architecture and multicellular co-culture system significantly improved the overall differentiation and function of epithelial cells. (A–F) Gene expression level of intestinal epithelial biomarkers, including SI (A), Villin (B), and Muc-2 (C), and cell–cell junction-related genes, including ZO-1 (D), Occludin-1 (E), and Claudin-4 (F), evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Data are presented as mean ± SEM (n = 3, ***p < 0.001, *p < 0.05).

Figure 6.

3D bioengineered intestinal tissues generated low luminal oxygen levels. (A) Scaffolds cultured in an upward position generated microaerobic conditions in the lumen (pO₂ between 5 and 7%), stable for up to 48 h. (B) Scaffolds cultured in a downward position generated anaerobic conditions (pO₂ ~1 to 2%) stable for up to 48 h.