De-epithelialization of porcine tracheal allografts as an approach for tracheal tissue engineering

Abstract

Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.

Computational fluid dynamics for enhanced tracheal bioreactor design and long-segment graft recellularization

“…S2 C), suggested homogenous distribution of cells in the trachea at a rotational speed of 1 rpm. This is in contrast to cell distribution at a rotational speed of 5 rpm 9 – 11 . Encouraged by the ability of the simulation model to predict cell deposition patterns, both axially and circumferentially, at low rotation speeds, we carried out biological experiments (cell repopulation studies on de-epithelialized tracheal grafts) using simulation results as a guide.…”

“…De-epithelialization was performed as previously described 11 . Briefly, a 1% SDS (w/v) in deionized water (diH 2 0) was run through the inner luminal chamber of the rotating bioreactor for 3 h to remove the tracheal epithelial cells.…”

“…BEAS-2B cells (CRL-9609, ATCC, USA) were cultured as per previous studies in our lab 11 . Briefly, cells were cultured in DMEM media supplemented with 10% (v/v) FBS, and 1% (v/v) penicillin/streptomycin.…”

“…Cell seeding on de-epithelialized tracheal grafts was performed as previously described by our group 11 . Briefly, de-epithelialized tracheal grafts were decontaminated in HBSS containing 2% (w/v) BSA and a broad-spectrum antibiotic solution for 48 h. Grafts were then reattached to the bioreactor and a 1 mL suspension containing 1 × 10 6 cells BEAS-2Bs per cm 2 of graft BEAS-2Bs was injected into the tracheal lumen.…”

“…However, successful epithelialization of tracheal scaffolds remains a challenge. We have previously demonstrated the use of a double-perfusion bioreactor system for recellularization of acellular porcine tracheal grafts 10 and repopulation of partially decellularized (de-epithelialized) tracheal grafts 11 . Our objective for this study was to enhance our previously developed bioreactor system 10 , by improving upon design limitations such as a lack of parameter control and monitoring (temperature, flow rate, rotational speed, etc.…”