Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications

Abstract

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd.

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

“…NZW rabbit donor tracheae (n = 4) were harvested and underwent decellularization using a detergent‐enzyme protocol . Following washing in sterile saline, tracheae were incubated in 0.2% Triton X‐100 (Sigma Aldrich, UK) and 0.2% sodium deoxycholate (FLUKA; Sigma Aldrich, Buchs, Switzerland) in PBS, at 37°C for 24 hours.…”

“…NZW rabbit donor tracheae (n 5 4) were harvested and underwent decellularization using a detergent-enzyme protocol. 15,16 Following washing in sterile saline, tracheae were incubated in 0.2% Triton X-100 (Sigma Aldrich, UK) and 0.2% sodium deoxycholate (FLUKA; Sigma Aldrich, Buchs, Switzerland) in PBS, at 378C for 24 hours. After a 48-hour wash in sterile water at 48C (wash water changed three times), tracheae were incubated with 2,000 Kunitz units/L DNAse (Sigma Aldrich, UK) and 0.1 g/L RNAse (Roche, Basel, Switzerland) at 378C for 24 hours to solubilize nuclear contents and degrade DNA.…”

In vivo implantation of a tissue engineered stem cell seeded hemi‐laryngeal replacement maintains airway, phonation, and swallowing in pigs

“…We used our previously developed protocol for de‐cellularisation, which has been successfully applied to human and porcine trachea (Lange et al, ; Lange, Shah, Birchall, Sibbons, & Ansari, ) and porcine larynx (Ansari et al, ). Briefly, the thawed tissue (hemilarynges) was incubated in 50 nmol/L Latrunculin B (Sigma‐Aldrich, UK) in high‐glucose Dulbecco's modified Eagle's medium (Gibco, Paisley, Scotland) for 2 hr at 37°C.…”

Characterization of a biologically derived rabbit tracheal scaffold

“…To address special prerequisites for the clinical setting a shorter decellularization protocol suitable for adult tracheal replacements based on porcine and human tissue was developed. 11 Congenital tracheal diseases as described above would need a scaffold which is appropriately sized matched for the pediatric patient and ideally can match the growth of the child. Engineered tissue theoretically could solve this problem contrary to simple allografts; as investigated in several studies.…”