Cell seeding on de-epithelialized tracheal grafts was performed as previously described by our group11. 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 × 106 cells BEAS-2Bs per cm2 of graft BEAS-2Bs was injected into the tracheal lumen. Recellularization was done by bidirectional perfusion cell seeding which involves the use of the peristaltic flow pump to inject the cell suspension into the inner chamber through the stop-cocks connected to silicone tubing for this chamber. Peristaltic bidirectional flow was utilized for the first 2 h allowing for cell adherence following which unidirectional flow was utilized towards the reservoir containing media. Media in the inner circuit (tracheal lumen) was changed every 24 h and half of the media in the outer circuit was changed every 48 h.

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