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Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

ABSTRACT: To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs.In vitro human testicular tissues.Academic research unit.Adult testicular tissues (n=4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n=3).Testicular tissue versus single cell suspension cryopreservation.Cell viability, total cell recovery per milligram of tissue, as well as viable and SSEA-4+ cell recovery.Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs, whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation.Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient's age, type of samples cryopreserved, and end points of therapeutic applications.

SUBMITTER: Yango P

PROVIDER: S-EPMC4608674 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

Yango Pamela P Altman Eran E Smith James F JF Klatsky Peter C PC Tran Nam D ND

Fertility and sterility 20140918 5

<h4>Objective</h4>To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs.<h4>Design</h4>In vitro human testicular tissues.<h4>Setting</h4>Academic research unit.<h4>Patient(s)</h4>Adult testicular tissues (n=4) collected from subjects with normal sper ...[more]

PMID: 25241367

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Project description:ObjectiveApplications 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 methodsIn 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.ResultsHaematoxylin-eosin (H and 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.ConclusionSpermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.

Dataset Information

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

Publications

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

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