Cryopreservation of canine ovarian and testicular fibroblasts.
To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.
Yu
Il-Jeoung
IJ
College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University, Korea. iyu@chonbuk.ac.kr
Leibo
S P
SP
Songsasen
Nucharin
N
Dresser
Betsy L
BL
Kim
In-Shik
IS
D000818:Animals; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004285:Dogs; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008297:Male; D010053:Ovary / Q000166:cytology*; D013737:Testis / Q000166:cytology*
D003451:Cryoprotective Agents; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide