A New Method for Isolating Viable Gonadal Germ Cells from 7-day-old Chick Embryos

A novel and simple method for isolating viable gonadal germ cells (GGCs) was developed in the domestic chicken, Gallus domesticus. Developing gonads were isolated from 7-day-old chick embryos and cultured for up to 24 hours at 37.8°C in phosphate buffered saline without Ca2+ and Mg2+ (PBS[−]). A discharge of GGCs from the gonad was observed within 30 minutes after introducing the embryonic gonad into PBS[−], and the number of discharged GGCs increased until 12 hours of incubation. The purity of the GGCs (number of discharged GGCs/total number of discharged cells) was approximately 50% for the initial 1.5 hours of incubation and decreased thereafter. The number of discharged GGCs from the left gonad was significantly higher than that from the right gonads in females. Fifty discharged GGCs in PBS[−] were injected into the blood stream of 2-day-old chick embryos after staining with PHK26 fluorescent dye. GGCs exhibiting a fluorescent signal were detected after incubating recipient embryos for 5 days.These results indicate that high-purity, viable GGCs can be collected by simply introducing isolated developing gonads into PBS[−].