Homogeneous Distribution of Exogenous Cells onto De-epithelialized Rat Trachea via Instillation of Cell-Loaded Hydrogel

Abstract

Injured or diseased airway epithelium due to repeated environmental insults or genetic mutations can lead to a functional decline of the lung and incurable lung diseases. Bioengineered airway tissue constructs can facilitate in vitro investigation of human lung diseases and accelerate the development of effective therapeutics. Here, we report robust tissue manipulation modalities that allow: (i) selective removal of the endogenous epithelium of in vitro cultured airway tissues and (ii) spatially uniform distribution and prolonged cultivation of exogenous cells that are implanted topically onto the denuded airway lumen. Results obtained highlight that our approach to airway tissue manipulation can facilitate controlled removal of the airway epithelium and subsequent homogeneous distribution of newly implanted cells. This study can contribute to the creation of innovative tissue engineering methodologies that can facilitate the treatment of lung diseases, such as cystic fibrosis, primary ciliary dyskinesia, and chronic obstructive pulmonary disease.

Keywords: bioreactor; cell replacement; hydrogel; imaging; lung disease; tissue-on-a-chip.

Figure 1.

Overview of the epithelium removal and cell implantation. Schematics illustrating (A) epithelium clearance and (B) collagen-assisted cell seeding. (C) Photograph and (D) schematic showing the rat trachea bioreactor.

Figure 2.

Microscopic analysis of (A) native and (B) de-epithelialized (de-epith) rat tracheas via H&E staining, trichrome staining (cell cytoplasm: pink; nuclei: dark brown; and collagen: blue), SEM imaging, EpCAM (green), DAPI staining (blue), and live/dead staining (green/red). Arrowhead indicates the surface of the tracheal lumen. (C) DNA and (D) sGAG quantification of the rat tracheas before and after de-epithelialization. *p < 0.05.

Figure 3.

Topical deposition of fluorescent microparticles in a gelatin-based tubular structure. (A) Laser light sheet microscope and GRIN lens imaging system constructed for imaging the interior of the tube. (B, C) Microscopic images of the particles (green) deposited in the tube via PBS and different concentrations of collagens. (D) Particle seeding density measured in upper and lower half of the tube after 30 min of cell seeding. ***p < 0.001. ns: not significant.

Figure 4.

Topical deposition of exogenous cells onto de-epithelialized rat tracheal lumen. (A) Bright-field image of the trachea interior. (B) Fluorescent images of the denuded trachea lumen seeded with MSCs (red) via PBS or collagen. (C) Fluorescent images and (D) cell seeding density obtained in the upper and lower half of the tracheal lumen. ***p < 0.001. ns: not significant.

Figure 5.

Proliferation of seeded MSCs on the de-epithelialized rat tracheal lumen. Fluorescence images of the tracheas seeded with CFSE-labeled MSCs (green) obtained following (A) 1 day and (B) 4 days of in vitro cultivation. Cell densities measured in the upper and lower half of the trachea lumens following (C) 1 day and (D) 4 days of in vitro cultivation. (E) Circularity of the seeded cells measured over time. CM: culture medium. C: collagen. *p < 0.05. **p < 0.01. ***p < 0.001. ns: not significant.