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Development of novel strategies for the isolation of piglet testis cells with a high proportion of gonocytes

生殖细胞 胶原酶 生物 男科 消化(炼金术) 间质细胞 干细胞 细胞培养 胰蛋白酶 细胞 生殖细胞 生物化学 细胞生物学 分子生物学 化学 激素 遗传学 医学 色谱法 促黄体激素 基因
作者
Yanfei Yang,Mehran Yarahmadi,Ali Honaramooz
出处
期刊:Reproduction, Fertility and Development [CSIRO Publishing]
卷期号:22 (7): 1057-1057 被引量:32
标识
DOI:10.1071/rd09316
摘要

Gonocytes have germline stem cell potential and are present in the neonatal testis, comprising 5–10% of freshly isolated testis cells. Maximising the number and proportion of gonocytes among freshly isolated testis cells will greatly facilitate their subsequent purification and in vitro study and manipulation. Seven experiments were conducted to evaluate the effects of multiple factors on the efficiency of testis cell isolation from neonatal pigs. We found that the use of a lysis buffer led to elimination of erythrocytes without adversely affecting testis cell isolation. Approximately ninefold as many live cells could be harvested by enzymatic digestion of testis tissues compared with mechanical methods. Digestion with collagenase–hyaluronidase–DNase followed by trypsin resulted in the highest recovery of live cells. However, the proportion of gonocytes (∼7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. Pretreatment of the tissue with cold enzymes increased the recovery of live testis cells. New strategies of combining a gentle enzymatic digestion with two rounds of vortexing resulted in the isolation of testis cells with very high gonocyte proportion. The efficiency of these novel methods could be further optimised to collect testis cells with a gonocyte proportion of approximately 40%. This novel three-step testis cell isolation strategy can be completed within 1 h and can harvest approximately 17 × 106 live gonocytes per g testis tissue. Therefore, in addition to elucidating the effects of several factors on testis cell isolation, we developed a novel strategy for the isolation of testis cells that yielded approximately 40% gonocytes in the freshly isolated cells (i.e. four- to eight-fold higher than the proportions obtained using current strategies). This strategy has instant applications in the purification of gonocytes.
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