A Novel Method for Extracting Testicular Tissue Ex-tracellular Matrix

Objective: The development of the decellularized extracellular matrix (ECM) scaffolds is able to open a new perspective to-wards regenerative medicine approaches. The use of transplant-ed testicular (TT) scaffolds with support of testicular cells may be an option for maintaining fertility in patients. Therefore, the purpose of this study was to prepare a decellularized testicular tissue as a suitable biomaterial for spermatogonial cell culture. Materials and Methods: Fragments of testicular tissues were decellularized using three protocols: NaCl buffer, NaCl buffer-Triton, and sodium dodecyl sulfate (SDS)-Triton (ST). In all groups, urea buffer was used for solubilization of testis ECM. After centrifuge, the supernatant was removed and dialyzed. Finally, the contents of the dialysis tubes were centrifuged to remove polymerized proteins, and the supernatant (viscous matrix) was collected. The removal of the cells from tissues was confirmed by DAPI and Hematoxylin and eosin (HandE) staining, and DNA content assay. Alcian blue, Orcein and Mas-son’s trichrome (MT) staining were used to confirm that ECM remained intact.Results: According to staining results, all protocols were able to completely decellularized testis tissue. Furthermore, no re-sidual nuclei were observed by DAPI and HandE staining. In NaCl buffer and NaCl-Triton groups, elastic, collagen fibers, and glycosaminoglycans were kept in decellularized testis tis-sue. But the structure of ECM was damaged in the ST group. In the NaCl group, DNA levels were significantly higher than other groups (P<۰.۰۵).Conclusion: As a result, our study showed a valid and highperformance protocol for testicular testis decellularization. The use of decellularized TT can be useful for testicular tissue engi-neering purposes.