Impact of Temperature and Time Interval Prior to Immature Testicular-Tissue Organotypic Culture on Cellular Niche

Abstract

Cryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research.

Short-Term Hypothermic Holding of Mouse Immature Testicular Tissue Does Not Alter the Expression of DNA Methyltransferases and Global DNA Methylation Level, Post-Organotypic Culture

“…The holding phase interval was scheduled as 6 and 24 h, corresponding to short-range and long-range shipment. Post holding, these testes were processed for organotypic culture as described previously ( 12 ).…”

“…In this regard, studies have determined the optimal conditions for ITT such as tissue size, storage temperatures, and storage periods in various models including human tissue ( 7 – 11 ). Recently, our group has demonstrated that ITT manipulation at 4°C had a minimal negative impact on the organotypically cultured germ cell population when compared to room temperature and 37°C ( 12 ). However, we believe that it is important to address the epigenetic integrity of germ cells from cultured ITT as epigenetic aberrations may negatively affect the subsequent developmental process.…”

Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue

“…The other fragments were cultured in a modified testicular explant culture system established by Sato et al (2011) at 35 • C and 5% CO 2 [24,28,32]. For the gel stands, 0.07 g agarose (#A9639, Sigma-Aldrich, St. Louis, MO, USA) was heated in 10 mL cell culture grade deionized water using a water bath.…”