Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells
The purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria
2022-12-01
|
| Series: | Bulgarian Journal of Veterinary Medicine |
| Subjects: |
| _version_ | 1811253774849671168 |
|---|---|
| author | R. Asadpour S. Kalantari A. A. Shahbazfar R. Jafari-Jozani |
| author_facet | R. Asadpour S. Kalantari A. A. Shahbazfar R. Jafari-Jozani |
| author_sort | R. Asadpour |
| collection | DOAJ |
| description | The purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium containing 200 mM trehalose, 40 µg/mL Vit C, and a combination of both for 3 weeks. Cell viability, colony number and diameter and mRNA levels of Bax, and Bcl-2 genes were evaluated before and after cryopreservation with quantitative real-time PCR. The results showed that cells cryopreserved in freezing medium containing 200 mM of trehalose plus 40 µg/mL Vit C had considerably greater cell viability than the control group (P<0.0001). Up to the 3rd week of cell cul-ture, supplementation of freezing medium with 200 mM trehalose resulted in significantly lower colonies diameters than in the control group. No significant differences were observed during the 1st to 2nd weeks in colony diameter and number of colonies between cells cryopreserved in the freezing medium containing either Vit C or trehalose compared with the control groups. Following thawing, the mRNA level of Bax in the Vit C + trehalose group was drastically lower than in those treated with trehalose or Vit C only (P<0.0001). High expression of Bcl-2 in the 40 µg/mL Vit C group was re-corded in the thawed cells compared to the control group (P<0.0001). These findings indicate that the concomitant use of antioxidants and sugar in the freezing medium can improve the quality and viability of SSCs after freezing via different mechanisms. Further studies are needed to clarify apoptosis and cell biomarkers in SSCs during freezing and thawing. |
| first_indexed | 2024-04-12T16:57:11Z |
| format | Article |
| id | doaj.art-ffc19ec6c9f74fe09292c70f8157a9af |
| institution | Directory Open Access Journal |
| issn | 1311-1477 1313-3543 |
| language | English |
| last_indexed | 2024-04-12T16:57:11Z |
| publishDate | 2022-12-01 |
| publisher | Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria |
| record_format | Article |
| series | Bulgarian Journal of Veterinary Medicine |
| spelling | doaj.art-ffc19ec6c9f74fe09292c70f8157a9af2022-12-22T03:24:12ZengFaculty of Veterinary Medicine, Trakia University, Stara Zagora, BulgariaBulgarian Journal of Veterinary Medicine1311-14771313-35432022-12-0125459360210.15547/bjvm.2020-0068Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cellsR. Asadpour0S. Kalantari1A. A. Shahbazfar 2R. Jafari-Jozani3Faculty of Veterinary Medicine, University of Tabriz, Tabriz, IranFaculty of Veterinary Medicine, University of Tabriz, Tabriz, IranFaculty of Veterinary Medicine, University of Tabriz, Tabriz, IranFaculty of Veterinary Medicine, University of Tabriz, Tabriz, IranThe purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium containing 200 mM trehalose, 40 µg/mL Vit C, and a combination of both for 3 weeks. Cell viability, colony number and diameter and mRNA levels of Bax, and Bcl-2 genes were evaluated before and after cryopreservation with quantitative real-time PCR. The results showed that cells cryopreserved in freezing medium containing 200 mM of trehalose plus 40 µg/mL Vit C had considerably greater cell viability than the control group (P<0.0001). Up to the 3rd week of cell cul-ture, supplementation of freezing medium with 200 mM trehalose resulted in significantly lower colonies diameters than in the control group. No significant differences were observed during the 1st to 2nd weeks in colony diameter and number of colonies between cells cryopreserved in the freezing medium containing either Vit C or trehalose compared with the control groups. Following thawing, the mRNA level of Bax in the Vit C + trehalose group was drastically lower than in those treated with trehalose or Vit C only (P<0.0001). High expression of Bcl-2 in the 40 µg/mL Vit C group was re-corded in the thawed cells compared to the control group (P<0.0001). These findings indicate that the concomitant use of antioxidants and sugar in the freezing medium can improve the quality and viability of SSCs after freezing via different mechanisms. Further studies are needed to clarify apoptosis and cell biomarkers in SSCs during freezing and thawing.cryopreservationspermatogonial stem celltrehalosevitamin c |
| spellingShingle | R. Asadpour S. Kalantari A. A. Shahbazfar R. Jafari-Jozani Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells Bulgarian Journal of Veterinary Medicine cryopreservation spermatogonial stem cell trehalose vitamin c |
| title | Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells |
| title_full | Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells |
| title_fullStr | Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells |
| title_full_unstemmed | Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells |
| title_short | Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells |
| title_sort | co supplementation of freezing media with trehalose and vitamin c on cell viability and apoptotic gene expression in ovine spermatogonial stem cells |
| topic | cryopreservation spermatogonial stem cell trehalose vitamin c |
| work_keys_str_mv | AT rasadpour cosupplementationoffreezingmediawithtrehaloseandvitaminconcellviabilityandapoptoticgeneexpressioninovinespermatogonialstemcells AT skalantari cosupplementationoffreezingmediawithtrehaloseandvitaminconcellviabilityandapoptoticgeneexpressioninovinespermatogonialstemcells AT aashahbazfar cosupplementationoffreezingmediawithtrehaloseandvitaminconcellviabilityandapoptoticgeneexpressioninovinespermatogonialstemcells AT rjafarijozani cosupplementationoffreezingmediawithtrehaloseandvitaminconcellviabilityandapoptoticgeneexpressioninovinespermatogonialstemcells |