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Effect of Equilibration Time and Temperature on Murine Spermatogonial Stem Cell Cryopreservation.
- Source :
-
Biopreservation and biobanking [Biopreserv Biobank] 2020 Jun; Vol. 18 (3), pp. 213-221. Date of Electronic Publication: 2020 Mar 27. - Publication Year :
- 2020
-
Abstract
- Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene ( Bcl6b , Erm , Dazl , and Sycp1 ) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.
- Subjects :
- Animals
Cell Culture Techniques
Cell Proliferation drug effects
Cell Survival drug effects
Dimethyl Sulfoxide pharmacology
Male
Mice
Spermatogonia drug effects
Spermatogonia metabolism
Stem Cells cytology
Stem Cells drug effects
Stem Cells metabolism
Temperature
Time Factors
Trehalose pharmacology
Biomarkers analysis
Cryopreservation methods
Cryoprotective Agents pharmacology
Semen Preservation methods
Spermatogonia cytology
Subjects
Details
- Language :
- English
- ISSN :
- 1947-5543
- Volume :
- 18
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Biopreservation and biobanking
- Publication Type :
- Academic Journal
- Accession number :
- 32216643
- Full Text :
- https://doi.org/10.1089/bio.2019.0116