Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.

RATIONALE: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

A comparison of tracheal scaffold strategies for pediatric transplantation in a rabbit model.

OBJECTIVES/HYPOTHESIS: Despite surgical advances, childhood tracheal stenosis is associated with high morbidity and mortality. Various tracheal scaffold strategies have been developed as the basis for bioengineered substitutes, but there is no consensus on which may be superior in vivo. We hypothesized that there would be no difference in morbidity and mortality between three competing scaffold strategies in rabbits. STUDY DESIGN: Pilot preclinical study. METHODS: Tracheal scaffolds were prepared by three methods that have been applied clinically and reported: preserved cadaveric ("Herberhold") allografts, detergent-enzymatically decellularized allografts, and synthetic scaffolds (nanocomposite polymer [polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU)]). Scaffolds were implanted into cervical trachea of New Zealand White rabbits (n = 4 per group) without cell seeding. Control animals (n = 4) received autotransplanted tracheal segments using the same technique. Animals underwent bronchoscopic monitoring of the grafts for 30 days. Macroscopic evaluation of tissue integration, graft stenosis, and collapsibility and histological examinations were performed on explants at termination. RESULTS: All surgical controls survived to termination without airway compromise. Mild to moderate anastomotic stenosis from granulation tissue was detected, but there was evidence suggestive of vascular reconnection with minimal fibrous encapsulation. In contrast, three of the four animals in the Herberhold and POSS-PCU groups, and all animals receiving decellularized allografts, required early termination due to respiratory distress. Herberhold grafts showed intense inflammatory reactions, anastomotic stenoses, and mucus plugging. Synthetic graft integration and vascularization were poor, whereas decellularized grafts demonstrated malacia and collapse but had features suggestive of vascular connection or revascularization. CONCLUSIONS: There are mirror-image benefits and drawbacks to nonrecellularized, decellularized, and synthetic grafts, such that none emerged as the preferred option. Results from prevascularized and/or cell-seeded grafts (as applied clinically) may elucidate clearer advantages of one scaffold type over another. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E449-E457, 2017.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.

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.

Tracheal Replacement Therapy with a Stem Cell-Seeded Graft: Lessons from Compassionate Use Application of a GMP-Compliant Tissue-Engineered Medicine.

Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.