Imaging-guided bioreactor for de-epithelialization and long-term cultivation of ex vivo rat trachea

Abstract

Recent synergistic advances in organ-on-chip and tissue engineering technologies offer opportunities to create in vitro-grown tissue or organ constructs that can faithfully recapitulate their in vivo counterparts. Such in vitro tissue or organ constructs can be utilized in multiple applications, including rapid drug screening, high-fidelity disease modeling, and precision medicine. Here, we report an imaging-guided bioreactor that allows in situ monitoring of the lumen of ex vivo airway tissues during controlled in vitro tissue manipulation and cultivation of isolated rat trachea. Using this platform, we demonstrated partial removal of the rat tracheal epithelium (i.e., de-epithelialization) without disrupting the underlying subepithelial cells and extracellular matrix. Through different tissue evaluation assays, such as immunofluorescent staining, DNA/protein quantification, and electron beam microscopy, we showed that the epithelium of the tracheal lumen can be effectively removed with negligible disruption in the underlying tissue layers, such as cartilage and blood vessel. Notably, using a custom-built micro-optical imaging device integrated with the bioreactor, the trachea lumen was visualized at the cellular level, and removal of the endogenous epithelium and distribution of locally delivered exogenous cells were demonstrated in situ. Moreover, the de-epithelialized trachea supported on the bioreactor allowed attachment and growth of exogenous cells seeded topically on its denuded tissue surface. Collectively, the results suggest that our imaging-enabled rat trachea bioreactor and localized cell replacement method can facilitate creation of bioengineered in vitro airway tissue that can be used in different biomedical applications.

Fig. 1.. Overview of imaging-enabled bioreactor and de-epithelialization of ex vivo rat trachea:

(A) Schematic of the imaging-integrated rat trachea bioreactor platform. Cam: camera. CM: culture medium. IP: imaging probe. Vent: ventilation. (B) Three-dimensional (3D) representation of the trachea bioreactor. (C) Photograph of the bioreactor system. Cam: camera. (D) Schematic showing the procedure of de-epithelialization of rat trachea. EP: epithelium. BM: basement membrane. ECM: extracellular matrix. PBS: phosphate-buffered saline.

Fig. 2.. In situ visualization of the trachea lumen using custom-built micro-optical imaging device:

(A) (i-iii) GRIN lens (diameter: 500 μm) used for both bright-field and fluorescence imaging of the rat tracheal lumen. (iv) 1951 USAF test target imaged using the imaging device. (B) (i) Photograph and (ii) schematic showing the imaging probe being used for visual inspection of an in vitro-cultured rat trachea. Cam: camera. TL: tube lens. F: optical filter. DM: dichroic mirror. OL: objective lens. IP: imaging probe. (C) (i) Bright-field and (ii) fluorescence images of the interior of the rat trachea before CFSE-labelling of the epithelium. (D) Fluorescence images of (i) native and (ii) de-epithelialized (De-epi) rat trachea lumen that was labelled with CFSE.

Fig. 3.. Histologic analysis of native and de-epithelialized rat tracheas:

(A) H&E, (B) pentachrome, and (C) trichrome staining images of native and de-epithelialized rat tracheas with 2% and 4% SDS. The images of de-epithelialized tracheas show removal of the epithelium and preservation of ECM architecture. *Pentachrome: purple (cell cytoplasm), blue (cell nuclei), green (proteoglycans), yellow (collagen fibers). *Trichrome: pink (cell cytoplasm), dark blue (cell nuclei), blue (collagen). Arrowhead: tracheal lumen.

Fig. 4.. Evaluation of ECM components via immunohistochemistry and DNA/GAG quantification:

Immunostaining images of (A) epithelial cell adhesion molecule (EpCAM), (B) laminin, and (C) cluster differentiation 31 (CD31) showing removal of the tracheal epithelium and preservation of ECM components within the rat trachea treated with 2% and 4% SDS. Arrowhead: tracheal lumen. (D) DNA and (E) GAG quantifications (n = 5 each) decrease in DNA and sulfated GAG amounts. Error bars represent means ± SD of experimental values. **p < 0.01. ***p < 0.001.

Fig. 5.. Investigation of the luminal surface of de-epithelialized tracheas via SEM imaging:

SEM images obtained at (A) 30×, (B) 2000×, and (C) 4000× of magnifications showing the luminal surface of the ex vivo rat tracheas. (D) Porosity of basement membrane (BM) of de-epithelialized tracheas treated with 2% and 4% SDS. (E) Size distribution of pores generated in the BM following the SDS treatment.

Fig. 6.. Live and dead analysis of native and de-epithelialized trachea (4% SDS):

(A) fluorescent images of live and dead cartilage cells obtained via conventional fluorescence microscopy showing the viability of chondrocytes after de-epithelialization and two weeks of in vitro cultivation. (B) Quantification of the viable chondrocyte revealed that there was no significant difference between native and de-epithelialized tracheas (p = 0.18).

Fig. 7.. Evaluation of cytotoxicity of the rat tracheas de-epithelialized via 4% SDS detergent:

(A) Characterization of the GRIN lens imaging probe using MSCs (red) labeled with quantum dots and suspended in culture medium. (B) (i-iii) CFSE-labelled MSCs (green) were deposited onto the denuded rat trachea lumen using the GRIN lens imaging device. (C) Schematic and (D) photograph showing the experimental setup used for long-term in vitro culture (i.e., over one week) of MSC-seeded de-epithelialized rat tracheas. Arrows show flow directions. (E) (i-iii) Fluorescence images of CFSE-labelled MSCs (green) being cultured on the tissue surface over the course of 7 days. The fluorescence images were obtained using a conventional microscope. (iv) Density of the cells measured at different time points. Error bars represent means ± SD of experimental values. **p < 0.001. ***p < 0.0001