Discipline: Biological Sciences
Subcategory: Physiology and Health
Patresia Payton - Tuskegee University
Co-Author(s): Jennifer Luyando, Temesgen Samuel, and Gemechu Wirtu, Tuskegee University, Tuskegee, AL
The current clinical applications of stem cells involves overnight shipment of tissue from a sick animal to a laboratory for extraction of cells, which are then shipped back to the clinic. This method of shipment likely compromises the viability of the tissue and the cells, thus compromising the efficacy of the stem cell therapy. The overall goal of the study was to establish optimal transportation conditions for testes, which will yield the most viable, functional spermatogonial stem cells (SSCs). Testes were obtained from the Alabama Animal Alliance. They were exposed to five shipping conditions to evaluate effects of duration (day 0, day 1), controlled temperature (cooled/refrigerated, room temperature), and uncontrolled ambient temperature during overnight shipment. Tissues in each treatment were frozen and processed post-thaw to test the effects of cryopreservation. Processing for extraction involved two-step collagenase/collagenase-trypsin digestion of tissue slices. Initial viability and stemness potential (adipogenic, osteogenic, CD73 and CD90 staining) were evaluated. Overall, better growth was observed in day 0 samples. No growth was seen in the day 1 room temperature or shipped samples. Established cell lines were frozen-thawed and subjected to induced differentiation or cell surface marker analysis. Three cells lines from both fresh (unfrozen) and frozen-thawed tissue showed positive staining for CD73 and CD90, with more cells from frozen thawed tissue expressing CD90 (61% vs 20%) when compared with cell lines originating from tissues processed fresh (without freezing). In the induced differentiation assay, we did not observe adipogenic differentiation in two groups of cells from an animal; however osteogenic differentiation was observed in a cell line from frozen-thawed tissue but not in those from fresh tissue. In conclusion, we have demonstrated canine testicular cells are very sensitive to high temperature with poor viability and growth after overnight, room temperature storage or courier shipment; hence, cold storage or shipment is recommended whenever immediate processing is not possible. Additionally, cells from frozen-thawed testes appear to be more enriched in SSCs suggesting the latter may be more cryotolerant than other testicular cells.
Funder Acknowledgement(s): The IBS program directors, NSF Award Number 1263207, RCMI Core Grant #G12MD007585-23, Jason White, Alabama Animal Alliance Inc., VetiCell/Alphacord.
Faculty Advisor: Gemechu Wirtu,