OBJECTIVEThe culture of human spermatogonial stem cells (SSC) has not been studied in detail yet. Here we tried to explore the optimized culture method of human SSC by using several different co-culture systems.

METHODSThe alpha6 +Thy-1 +c-kit- cells acquired by the immunomagnetic beads sorting technique were cultured in different co-culture systems. Their morphological, biological characteristics and survival rates were intensively observed by microscopic or immunocytochemical assay. The long-term survival rate of human SSC during culture period was evaluated by germ cell transplantation technique.

RESULTSThe alpha6 +Thy-1 +c-kit- cells could stably survive in the DMEM and DMEM/F12 mediums with fetal bovine serum (FBS) on feeder layer. The survival rates within 1 week were more than 90%. The long-time culture showed the cells were gradually attached on the surface of Sertoli cells by the manner of scattered single cell or accumulated masses. Part of the SSC became more tightly attachment with Sertoli cells or mounted among the Sertoli cells. They could survive or even proliferate for more than 3 months in vitro. Germ cells transplantation study showed that some alpha6 +Thy-1 +c-kit- cells labeled by PKH26 could resided on the basal membrane of seminiferous tubule of nude mice, appearing as single or coupled cells 2 months later after transplantation. The function evaluation of the cultured cells by counting the fluorescent cells in the seminiferous tubule showed 54.9% and 9.2% of SSC in the alpha6 +Thy-1 +c-kit- cells were still remained after cultured for 2 and 4 weeks, respectively.

CONCLUSIONHuman SSC could maintain survival in vitro for more than 3 months, but it was still need to seek for a more optimized and successful culture system for its efficient expansion and proliferation. Thus it will open up a wide prospect for the understanding of the biology of human SSC and the treatment of male sterility.