Three-Dimensional Culture of Mouse Spermatogonial Stem
Cells Using A Decellularised Testicular Scaffold
Applications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells.
Materials and Methods
In this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson’s trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC.
Haematoxylin-eosin (H&E) staining showed that the cells were successfully removed by SDS and Triton
X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This confirmed that our
decellularisation protocol was efficient. Masson’s trichrome and alcian blue staining respectively showed that
glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of
fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation
of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous
tubule. Analyses of gene expression using real-time PCR indicated that expression of the
Spermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.