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Published on: March 4, 2018
Development of a cryopreservation protocol for testicular interstitial cells with the account of temperature
T M Gurina1, A V Pakhomov, A L Kyryliuk
1Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, Ukraine.
Abstract:
A long course of anticancer therapy may lead to testicular steroidogenesis destruction. Cryopreservation of testicular interstitial cells (TIC) would be a strategy to protect hormonal and fertile potential of pre-pubertal boys treated with chemo - or radiotherapy. The aim of this research was to optimize protocols for freezing of TIC. Essential physical processes associated with the presence of dimethyl sulphoxide (Me(2)SO) in the cryoprotectant solution take place at the temperatures below -60°С. These processes are the eutectic crystallization at the stage of freezing and the recrystallization before the melting of the eutectic mixture at the stage of heating. Both of the processes affect the viability of the cells subjected to cryopreservation. Temperature intervals when these processes take place were determined by the method of thermoplastic deformation for 10% Me(2)SO selected for cryopreservation of TIC. Rat TIC were cryopreserved using five different protocols which varied in cooling rates within the chosen temperature intervals. Post-thaw cell viability and metabolic activity were evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining assays. Leydig cell recovery after cryopreservation was measured by 3-beta-hydroxysteroid dehydrogenase reaction. Based on the obtained results, the authors developed a cryopreservation protocol for TIC which makes it possible to achieve great cell viability due to using controlled cooling rates within the temperature intervals below -60°С.
Insights
Cryopreservation of testicular interstitial cells (TIC) can preserve fertility in young cancer patients. Optimizing freezing protocols, especially cooling rates below -60°C, is key to maintaining cell viability.
Area of Science:
- Reproductive Biology
- Cell Biology
- Cryobiology
Background:
- Anticancer therapies can impair testicular function and fertility.
- Cryopreservation of testicular interstitial cells (TIC) offers a strategy to preserve hormonal and reproductive potential in prepubertal boys undergoing treatment.
- Optimizing cryopreservation protocols for TIC is crucial for successful fertility preservation.
Purpose of the Study:
- To optimize cryopreservation protocols for testicular interstitial cells (TIC).
- To investigate the effects of cooling rates on TIC viability during cryopreservation.
- To identify optimal temperature intervals for cryopreservation processes involving dimethyl sulphoxide (Me(2)SO).
Main Methods:
- Thermoplastic deformation was used to determine critical temperature intervals for Me(2)SO-mediated cryopreservation processes.
- Rat TIC were cryopreserved using five protocols with varying cooling rates within identified temperature intervals.
- Cell viability, metabolic activity, and Leydig cell recovery were assessed using Trypan Blue, MTT assays, and 3-beta-hydroxysteroid dehydrogenase reaction, respectively.
Main Results:
- Critical temperature intervals for eutectic crystallization and recrystallization in 10% Me(2)SO solutions were identified.
- Controlled cooling rates within specific sub-zero temperature intervals significantly impacted TIC viability post-thaw.
- The developed protocol demonstrated high cell viability, metabolic activity, and Leydig cell recovery.
Conclusions:
- A novel cryopreservation protocol for TIC was established, enhancing cell viability.
- Controlled cooling rates below -60°C are essential for successful TIC cryopreservation.
- This optimized method holds promise for fertility preservation in young males undergoing gonadotoxic cancer therapies.
