title: Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.
abstract: Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK; Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, UK.
authors.forename: Colin R
authors.initials: CR
authors.lastname: Butler
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Hynds
authors.affiliation: Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Crowley
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Kate H C
authors.initials: KH
authors.lastname: Gowers
authors.affiliation: Department of Haematology, Royal Free Hospital and University College London, London, UK.
authors.forename: Leanne
authors.initials: L
authors.lastname: Partington
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Hamilton
authors.affiliation: Department of Haematology, Royal Free Hospital and University College London, London, UK.
authors.forename: Carla
authors.initials: C
authors.lastname: Carvalho
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
authors.forename: Manuela
authors.initials: M
authors.lastname: Platé
authors.affiliation: Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, UK.
authors.forename: Edward R
authors.initials: ER
authors.lastname: Samuel
authors.affiliation: Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, UK; Department of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Burns
authors.affiliation: Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, UK.
authors.forename: Luca
authors.initials: L
authors.lastname: Urbani
authors.affiliation: UCL Ear Institute, The Royal National Throat Nose and Ear Hospital, London, UK.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Birchall
authors.affiliation: Department of Haematology, Royal Free Hospital and University College London, London, UK.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Lowdell
authors.affiliation: Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, UK. Electronic address: p.decoppi@ucl.ac.uk.
authors.forename: Paolo
authors.initials: P
authors.lastname: De Coppi
authors.affiliation: Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK. Electronic address: s.janes@ucl.ac.uk.
authors.forename: Sam M
authors.initials: SM
authors.lastname: Janes
mesh_terms: D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods; D002474:Cell-Free System / Q000737:chemistry*; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005109:Extracellular Matrix / Q000737:chemistry* / Q000648:ultrastructure*; D006801:Humans; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods; D054457:Tissue Scaffolds; D014132:Trachea / Q000166:cytology* / Q000254:growth & development* / Q000648:ultrastructure; D014618:Vacuum
keywords: Epithelial cells; Tissue engineering; Tissue scaffolds; Trachea; Transplantation