Three-Dimensional Culture of Mouse Spermatogonial Stem Cells Using A Decellularised Testicular Scaffold
Objective: 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 spe...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Royan Institute (ACECR), Tehran
2019-08-01
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| Series: | Cell Journal |
| Subjects: | |
| Online Access: | https://celljournal.org/journal/article/fulltext/three-dimensional-culture-of-mouse-spermatogonial-stem-cells-using-a-decellularized-testicular-scaffold.pdf |
| Summary: | Objective: 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.
Results: 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 Plzf gene was unchanged
over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture,
suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial
stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules.
Conclusion: Spermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the
decellularised testicular scaffolds |
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| ISSN: | 2228-5806 2228-5814 |