Freeze-thaw sheep pericardium decellularization without detergents: A pilot study

Abstract

Decellularization in tissue engineering preserves the extracellular matrix for the development of bioequivalent tissues. Detergent-based decellularization protocols can adversely affect the extracellular matrix (ECM) by destroying structural proteins. Furthermore, some of them are associated with residual cytotoxicity. The freeze-thaw method is not toxic, but the influence of its parameters on the results of the treatment has not been studied. This study explores the influence of a wide range of parameters of freeze-thaw protocols on the efficacy of decellularization and structural and mechanical properties of the ECM. The samples of sheep pericardium were decellularized in liquid nitrogen and phosphate-buffered saline for 5–50 cycles and subjected to histological and immunofluorescence analysis and tensile testing. A local decellularization was prominent for protocols with 20 and 30 cycles with 2-minute freezing and 2 or 5-minute thawing. Increasing the freeze-thaw duration to 5 or 10 min did not improve the results, and all the protocols with a low number of cycles did not result in effective decellularization. The ECM was well preserved and its mechanical properties were not significantly altered. One of the protocols resulted in sufficient decellularization which demonstrates the ability to successfully decellularize tissue and highlights the further need to investigate the influencing factors.