Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.
Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea.
Ki-Jung
KJ
Kim
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea.
Yong-An
YA
Lee
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea.
Bang-Jin
BJ
Kim
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea.
Yong-Hee
YH
Kim
Obstetrics, Gynecology & Reproductive Biology, Michigan State University, USA.
Byung-Gak
BG
Kim
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea.
Hyun-Gu
HG
Kang
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea.
Sang-Eun
SE
Jung
National Institute of Animal Science, RDA, Cheonan 331-801, Republic of Korea.
Sun-Ho
SH
Choi
Department of Science, Spokane Community College, 1810 N Greene St., Spokane, WA 99217-5399, USA.
Jonathan A
JA
Schmidt
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do 456-756, Republic of Korea. Electronic address: byryu@cau.ac.kr.
Buom-Yong
BY
Ryu
D053687:Adult Stem Cells / Q000502:physiology* / Q000637:transplantation*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods* / Q000662:veterinary; D003451:Cryoprotective Agents / Q000494:pharmacology*; D059247:Fertility Preservation / Q000379:methods / Q000662:veterinary; D005615:Freezing / Q000009:adverse effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011092:Polyethylene Glycols / Q000494:pharmacology; D013093:Spermatogonia / Q000166:cytology* / Q000187:drug effects; D013395:Sucrose / Q000494:pharmacology; D014183:Transplantation, Heterologous; D014199:Trehalose / Q000494:pharmacology
D003451:Cryoprotective Agents; D011092:Polyethylene Glycols; D013395:Sucrose; D014199:Trehalose
Bovine; Cryopreservation; Slow freezing; Spermatogonial stem cell; Trehalose