Automated freeze-thaw cycles for decellularization of tendon tissue: a pilot study

Background: Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. Methods: In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficial digital flexor tendons. Additional tendon specimens underwent manually performed freeze-thaw cycles based on an established procedure. Tendon decellularization was completed by using non-ionic detergent treatment (Triton X-100). Effectiveness of decellularization was assessed by residual nuclei count and calculation of DNA content. Cytocompatibility was evaluated by culturing allogeneic adipose tissue-derived mesenchymal stromal cells on the tendon scaffolds. Results: There were no significant differences in decellularization effectiveness between samples decellularized by the automated freeze-thaw procedure and samples that underwent manual freeze-thaw cycles. Further, we inferred no significant differences in the effectiveness of decellularization between two different cooling and heating rates applied in the automated freeze-thaw process. Both the automated protocols and the manually performed protocol resulted in roughly 2% residual nuclei and 13% residual DNA content. Successful cell culture was achieved with samples decellularized by automated freeze-thawing as well as with tendon samples decellularized by manually performed freeze-thaw cycles. Conclusions: Automated freeze-thaw cycles performed by using a liquid nitrogen-based controlled rate freezer were as effective as previously described manual freeze-thaw procedures for decellularization of equine superficial digital flexor tendons. The automation of this key procedure in decellularization of large tendon samples is an important step towards the processing of large sample quantities under standardized conditions. Furthermore, with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine, the automation of key procedural steps is highly required to develop manufacturing processes under GMP conditions