Porcine spermatogonia stem cells (pSSCs) are the only type of somatic stem cell that can pass genetic information to the successive generations. Little is known about pSSCs vitality in vitro, and due to their increasing importance in stem cell research, here, we optimized a protocol to culture pSSCs and explored their potential fate in vitro. Utilizing a feeder-free culture system with a 2D peptide-coating and small chemical molecules (including CHIR99021, Repsox, vitamin C, folic acid, and CD lipid concentrate), we were able to prolong the culture time of pSSCs by at least three months compared with previous methods. Moreover, we found that pSSCs could proliferate and self-renew in the seminiferous tubules of infertile mice. However, they could not perform meiosis. Our study shows that this feeder-free culture system optimizes cell culture and may facilitate advanced research on SSC biology and genetic manipulation of germ cells.