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

Development of novel strategies for the isolation of piglet testis cells with a high proportion... 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 × 10 6 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Reproduction Fertility and Development CSIRO Publishing

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

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References (46)

Publisher
CSIRO Publishing
Copyright
CSIRO
ISSN
1031-3613
eISSN
1448-5990
DOI
10.1071/RD09316
pmid
20797343
Publisher site
See Article on Publisher Site

Abstract

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 × 10 6 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.

Journal

Reproduction Fertility and DevelopmentCSIRO Publishing

Published: Aug 20, 2010

Keywords: enzymatic cell isolation, pig, spermatogonial stem cell.

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