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

Abstract

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.

Keywords: Automation; Controlled rate freezer; Decellularization; Horse; Regenerative medicine; Tendon; Tissue engineering.

Fig. 1

Temperature profiles of Auto-Protocol 1 (group 1) (a) and Auto-Protocol 2 (group 2) (b). Representative graphics for group 1 (a) (Auto-Protocol 1; cooling/heating rate of 50 °C/min) and for group 2 (b) (Auto-Protocol 2; cooling/heating rate of 20 °C/min). Blue curves represent actual values and brown curves show target values of the temperature. Both graphics are prepared on the basis of printed temperature records of the biological controlled rate freezer (PLANER® Kryo 360–1.7) by the use of Adobe® Illustrator® CS6 software

Fig. 2

Visible nuclei count (a) and DNA content (b) of decellularized tendon samples (n = 10). Mean values of residual nuclei count (a) and residual DNA (b) in % relative to the controls (n = 10). The vertical error bars indicate the confidence interval of 95%. There were no significant differences in the number of residual nuclei and in the amount of DNA content among tendon samples of both automated protocols (group 1 and 2) and the manually performed protocol (group 3) for decellularization

Fig. 3

Histological assessment of decellularization effectiveness. Representative images of hematoxylin and eosin stained equine superficial digital flexor tendon samples of group 1 (Auto-Protocol 1) (a), group 2 (Auto-Protocol 2) (b), group 3 (Manual Protocol) (c), showing an apparent reduction of visible nuclei compared with tendon samples of the internal controls (no decellularization) (d). Decellularized tendon samples of all groups reveal regularly aligned collagen fibrils and interfibrillar tissue gaps instead of resident cells

Fig. 4

Histological assessment of cytocompatibility. Representative images of hematoxylin and eosin stained equine superficial digital flexor tendon samples after decellularization by automated (a) and manual (b) freeze-thaw cycles, re-seeding with equine adipose tissue-derived mesenchymal stromal cells and 3 days of culture. A successful re-seeding procedure of the tendon surface is indicated by the dense cell layer adhering to the sample surface, with a lower number of cells penetrating deeper tissue structures

Fig. 5

Fluorescence microscopic assessment of cytocompatibility. Representative panels of LIVE/DEAD® staining of equine superficial digital flexor tendon specimens decellularized by automated (a) and manual (b) freeze thaw cycles. Decellularized scaffolds were re-seeded with equine adipose tissue derived mesenchymal stromal cells and a fluorescence microscopic evaluation was performed after 3 days of culture. Vital cells are indicated by green fluorescence (display of intracellular esterase activity), cells with defect cellular membranes show a red fluorescence signal of their nucleus