College of Animal Science and Technology, Northwest A&F University, 22 Xi-nong Road, Yangling, Shaanxi, 712100, People's Republic of China.
J Assist Reprod Genet. 2013 Jul;30(7):945-52. doi: 10.1007/s10815-013-0031-0. Epub 2013 Jun 19.
To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells.
Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors.
The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10 ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population.
In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.
研究血清和生长因子对猪雄性生殖干细胞（MGSCs）体外增殖的影响，并建立这些干细胞的培养体系。
通过机械分离和胶原酶-胰蛋白酶消化获得新生仔猪的新鲜睾丸细胞。差速贴壁后，将非贴壁细胞培养在添加不同浓度血清（0、1%、2%、5%、10%）的培养基中。原代培养 10 天后，用添加不同浓度生长因子（碱性成纤维细胞生长因子和表皮生长因子分别为 1、5、10ng/ml）的培养基维持细胞培养。在每种处理中确定具有不同大小的 MGSC 衍生集落的数量，以评估血清浓度和生长因子的影响。
在存在 1%而不是 10%胎牛血清（FBS）的情况下，MGSC 衍生集落的数量显著增加。1、5ng/ml 的碱性成纤维细胞生长因子（bFGF）和 5、10ng/ml 的表皮生长因子（EGF）显著促进集落形成。免疫细胞化学、逆转录-聚合酶链反应（RT-PCR）和异种移植实验表明，培养细胞群体中存在功能干细胞。
在 1%FBS 存在和生长因子补充的情况下，可以维持猪 MGSCs 的体外增殖达 1 个月。
