A Novel, Efficient Method to Isolate Chicken Primordial Germ Cells from Embryonic Blood Using Cell Culture Inserts.

Primordial germ cells (PGCs) play a crucial role in preserving poultry genetic resources and conducting transgenic research. A system for the rapid isolation of PGCs from single chicken embryonic blood was established in this paper. We found that PGCs can migrate to the lower layer of chicken embryonic fibroblasts (CEFs) through pores smaller than their diameter, while blood cells cannot, when co-cultured with CEFs of passages two to three. Based on the characteristics of PGCs, we developed a new PGC isolation method (cell culture insert/CEF adhesion method) that utilizes a 3 μm cell culture insert and CEFs of passages two to three. Using this method, approximately 700 PGCs can be isolated from the blood of a single chicken embryo at Hamburger and Hamilton (H&H) stage 17 of development. The separation rate achieved was 87.5%, with a separation purity of 95%. The separation rate of this method was 41.4% higher than the common Percoll density gradient centrifugation method and 33.6% higher than lysis with ACK buffer. PGCs isolated from embryonic blood could proliferate 37-fold within 2 weeks when cultured in a feeder-free culture system. They also continued to express the