An intestinal model with a finger-like villus structure fabricated using a bioprinting process and collagen/SIS-based cell-laden bioink

Abstract

The surface of the small intestine has a finger-like microscale villus structure, which provides a large surface area to realize efficient digestion and absorption. However, the fabrication of a villus structure using a cell-laden bioink containing a decellularized small intestine submucosa, SIS, which can induce significant cellular activities, has not been attempted owing to the limited mechanical stiffness, which sustains the complex projective finger-like 3D structure. In this work, we developed a human intestinal villi model with an innovative bioprinting process using a collagen/SIS cell-laden bioink. Methods: A Caco-2-laden microscale villus structure (geometry of the villus: height = 831.1 ± 36.2 μm and diameter = 190.9 ± 3.9 μm) using a bioink consisting of collagen type-I and SIS was generated using a vertically moving 3D bioprinting process. By manipulating various compositions of dECM and a crosslinking agent in the bioink and the processing factors (printing speed, printing time, and pneumatic pressure), the villus structure was achieved. Results: The epithelial cell-laden collagen/SIS villi showed significant cell proliferation (1.2-fold) and demonstrated meaningful results for the various cellular activities, such as the expression of tight-junction proteins (ZO-1 and E-cadherin), ALP and ANPEP activities, MUC17 expression, and the permeability coefficient and the glucose uptake ability, compared with the pure 3D collagen villus structure. Conclusion: In vitro cellular activities demonstrated that the proposed cell-laden collagen/dECM villus structure generates a more meaningful epithelium layer mimicking the intestinal structure, compared with the pure cell-laden collagen villus structure having a similar villus geometry. Based on the results, we believe that this dECM-based 3D villus model will be helpful in obtaining a more realistic physiological small-intestine model.

Figure 1

Optical and scanning electron microscopy (SEM) images of the decellularized small intestinal submucosa (SIS), (A) sheet and (B) powder. (C) DNA content, (D) collagen, (E) α-elastin, and (F) glycosaminoglycan (GAG) contents of native and decellularized SIS (n = 5, ^*P < 0.5). (G) Immunofluorescence images (DAPI/collagen-type-I and DAPI/elastin) before and after decellularization.

Figure 2

Storage modulus (G') and complex viscosity (η^*) of the bioinks containing various concentrations of SIS (0, 10, 20, 30, and 40 mg/mL), (A) with and (B) without tannic acid (TA; 2 wt%). (C) G' of the bioinks at a frequency of 1 Hz. (D) Yield stress (σ_y) of the bioinks. (E) Optical images showing the relative viscosity of the bioinks after mixing for 150 min.

Figure 3

(A) Schematic of a vertically moved 3D printing process for fabricating Caco-2-laden 3D intestinal villi using collagen/SIS bioink. (B) Optical and live (green)/dead (red) images for various concentrations of the decellularized SIS (0, 10, 20, 30, and 40 mg/mL). (C) Diameter of the three different regions (regions a, b, and c) of the villus structures measured using the optical images, and (D) initial cell-viability calculated using the live/dead images for various SIS concentrations. (E) Optical images showing the nozzle and the villus structure after printing for 10 s and 120 s, and live (green)/dead (red) images for various weight fractions of TA (0, 1, 2, 3, and 4 wt%). (F) Height of the printed villus structure after various time points using the collagen/SIS bioink with various weight fractions of TA. (G) Initial cell-viability calculated using the live/dead images for various weight fractions of TA.

Figure 4

(A) Schematics showing various printed structure types ((i) non-continuous, (ii) stable, and (iii) coiled) using collagen (4 wt%)/SIS (10 mg/mL) bioink crosslinked with 2 wt% TA under different processing conditions. (B) SEM images showing the printed villus structures using various printing speeds on z-axis (2.5, 5, 7.5, and 10 mm/s) under a fixed condition (nozzle inner diameter (ID): 250 μm, extrusion time: 0.5 s, and pneumatic pressure: 275 kPa). (C, D) Processing diagrams demonstrating the fabricated 3D geometries vs. the printing speed. (E) SEM images of the printed villus structures for various pneumatic pressures (225, 275, 325, 375, and 425 kPa) under a fixed condition (nozzle inner diam. (ID): 250 μm, extrusion time: 0.5 s and printing speed: 0.5 mm/s). (F) Process diagram demonstrating the applied pneumatic pressure. (G) The Fabricated 3D geometries vs. the amount of bioink extrudate and SEM images of the printed villus structures for various amount of bioink extrudate (0.018, 0.024, and 0.034 μL).

Figure 5

(A) Optical and SEM images and (B) cell viability of the two fabricated villus structures using the bioinks.

Figure 6

(A) DNA content (1, 7, and 14 days of culture) and growth rate (14 days of culture) for the Caco-2-laden 3D intestinal villi with collagen (CLIV-C) and collagen/SIS (CLIV-CS). (B) DAPI (blue)/phalloidin (red) images (14 and 21 days of culture) and cell coverage area (%) of cytoskeleton calculated using the phalloidin images at 14 days for the 3D models. (C) SEM images showing the microvilli on the surface of the scaffolds. The immunofluorescence images show the formation of (D) basement membrane (laminin, green), (E) tight junction (ZO-1, red), and (F) adherent junction (E-cadherin, green) for the Caco-2 cells cultured in the 3D intestinal models at 21 days of culture. (G) Relative ZO-1 and E-cadherin expression area for the 3D models calculated using the immunofluorescence images at 21 days of culture.

Figure 7

(A) Surface, tip, and cross-sectional DAPI (blue))/MUC17 (green) images of the Caco-2 cells in the CLIV-C and CLIV-CS after 21 and 28 days of culture. (B) MUC17 area fraction of CLIV-C and CLIV-CS calculated using the MUC17 images after 21 days of culture. Enzymatic results, (C) ALP activities (7, 14, and 21 days of culture) and ALP staining optical images, and (D) ANPEP activities (7, 14, and 21 days of culture) in CLIV-C and CLIV-CS.

Figure 8

(A) Permeability coefficient (20 and 30 days of cell culture) and (B) glucose uptake ability (30 days of culture) of the scaffolds, CLIV-C, and CLIV-CS.

Figure 9

(A) Schematic showing a 3D printing process supplemented with core/shell nozzle for fabricating 3D intestinal model with epithelium and capillaries using collagen/SIS bioinks. (B) Cell tracker image showing the fabricated 3D model consisting of epithelium region with Caco-2 cells (red) and capillary region with HUVECs (green) at 1 day of culture. (C) DAPI (blue)/MUC17 (green)/CD31 (purple) image showing the matured epithelium monolayer and capillaries at 28 days of culture.