Robust bioengineered 3D functional human intestinal epithelium

Abstract

Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

Figure 1. Human intestines on 3D porous silk scaffolds.

(a,b) Schematics of the fabrication process for building silk-based porous scaffolds for 3D human intestine engineering. Silk scaffolds with hollow channels were prepared using a sequential six step process involving silk regeneration, cylindrical PDMS mold casting, insertion of Teflon-coated wires or Nylon screw across the cylinder, application of the silk solution, lyophilization and β-sheet induction. Upon completion of this process, the Teflon-coated wires or Nylon screw was removed, leaving a 3D, porous scaffold with a 2 mm diameter of non-patterned or screw-patterned (patterned, ridge like features with a height of 400 μm) hollow channels spanning the length of the scaffold and a bulk space that contained interconnected pores surrounding the channel. The scaffolds were then reproducibly trimmed along the axis of the hollow channels into 5 mm diameter × 8 mm long (mm) cylinders (b, Scale bars, 4 mm). (c) Schematics show the 3D non-patterned and patterned intestine system layout. (d,e) F-actin stain of 3D scaffolds without patterns (upper panel) and with patterns (lower panel) demonstrates how intestinal epithelial cells and myofiblasts localize in the 3D silk scaffolds. Scale bar, 1 mm. (f–k) 3D confocal images of ZO-1 immunostaining, SEM, and ALP staining on the epithelial cells seeded in the scaffold lumens demonstrated fully polarized epithelial cells. Scale bars, 100 μm (f,g), 1 μm (h,i), 200 μm (j,k). (l,m) 3D confocal view of immunostaining of SM22α on cells seeded in the bulk space (week 8) showed H-InMyoFibs keep their phenotype on the scaffolds. Scale bar, 50 μm.

Figure 2. 3D silk scaffolds increase the thickness of the mucus layer of the intestinal epithelial cells.

(a–c) Confocal microscopy images of immunostaining of MUC-2, ZO-1, and DAPI of Caco-2/HT29-MTX cultured on 2D transwells (a), and non-patterned (b) and patterned (c) 3D silk scaffolds 21 days post cell seeding. MUC2 is visualized as red, ZO-1 as green, and DAPI as blue. Scale bar = 200 μm. (d,e) Light microscopy of toluidine blue stained frozen sections across the transwell inserts (d), non-patterned (e) and patterned (f) 3D silk scaffolds. Outer mucus layers on 3D scaffolds are pointed by the black arrowheads, while inner mucus layers are pointed by yellow arrowheads. Scale bar = 100 μm. (g) Measurement of mucus thickness yielded by the epithelium grown on 2D and 3D systems. n = 6 in each group, ***p < 0.001.

Figure 3. 3D bioengineered human intestinal tissues mimic in vivo luminal oxygen levels.

(a) Confluent intestinal epithelial monolayers cultured on transwell inserts under the aerobic condition. (b) Concentration profile of oxygen at different depths down the lumen of 3D tissues (with plain luminal surfaces) demonstrated ready access to microaerobic and nanaerobic oxygen concentrations. (c) The increased epithelial area in 3D tissues achieved by patterning the luminal surfaces further extended the low end of the oxygen profile to below the anaerobic threshold. All measurements were performed on day 21 post-confluence. (d) Red fluorescence of the yopE reporter (arrowheads) on transwell inserts, in the absence of frdA-driven green fluorescence (GFP or miniSOG), verified that bacteria experience aerobic conditions. (e) Yellow fluorescence (arrowheads) (mixed color of red anti-Yersinia and green GFP or miniSOG) in non-patterned lumens indicates bacteria experience microaerobic conditions. (f) Red Yersinia, detected by antisera, only expressed miniSOG (arrowheads), revealing bacteria experience strictly anaerobic conditions in patterned lumens. Scale bar = 50 μm.

Figure 4. 3D architecture and multicellular co-culture system significantly enhanced the overall differentiation level of the epithelial cells and supported longer-term static cultures of intestinal epithelium.

(a,b) Quantification of alkaline phosphatase activity (a) and mucus production (b) of intestinal epithelial cells cultured on 2D transwell and 3D silk scaffolds. (c–f) Gene expression levels of cell junction-related genes and intestinal epithelial biomarkers, including ZO-1 (c), E-cadherin (d), Villin (e), and SI (f), were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Data is presented as mean ± SEM, n = 5 in each group, p < 0.001.