Human Spermatogonial Stem Cell culture and effective cryopreservation – a step towards clinical application.
BAUS ePoster online library. Sandher R. 06/29/16; 131969; P7-15
Ms. Raveen Sandher
Ms. Raveen Sandher
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Abstract
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P7-15

900 boys are diagnosed with cancer each year but happily survival rates are high. One of the harms of chemo-radiation treatment is depletion of spermatogonial stem cells (SSCs) leading to subfertility in adulthood. Some centres are cryopreserving testicular biopsies prior to treatment with the hope of autotransplantation, requiring viable and stable SSCs. An alternative approach considered in our laboratory is to perform preliminary separation and ex-vivo culture of SSCs to then be cryopreserved for subsequent re-population of seminiferous tubules. The aim of this work was to develop rapid, reliable and reproducible method for human SSC culture and cryopreservation.

 

SSCs were isolated from testicular biopsies taken from azoospermic men undergoing sperm retrieval. Co-culture with testicular somatic cells facilitated SSCs clumping as chains and finally colonies. Immunofluorescence-labeling using antibodies against stem-cell markers GFRα1 and PLZF combined with confocal imaging, confirmed SSC signature. To date cultures have been maintained for up to 20 weeks. SSCs colonies were cryopreserved using vitrification. Post-thaw proliferation and cell death was assessed using Ki67 and TUNEL immunofluorescence-labelling; demonstrating an average post thaw viability rate of 90%.

 

Findings indicate our novel technique of firstly establishing a niche-like environment in-vitro is successful in maintaining long-term cultures of human SSCs. Subsequent cell-based cryostorage can then be used until the patient is ready for subsequent re-population of damaged seminiferous tubules.  This work is an important practical step forward in the male germline stem cell field, and undoubtedly will advance the future clinical application of SSCs restoring fertility to male childhood cancer survivors.

P7-15

900 boys are diagnosed with cancer each year but happily survival rates are high. One of the harms of chemo-radiation treatment is depletion of spermatogonial stem cells (SSCs) leading to subfertility in adulthood. Some centres are cryopreserving testicular biopsies prior to treatment with the hope of autotransplantation, requiring viable and stable SSCs. An alternative approach considered in our laboratory is to perform preliminary separation and ex-vivo culture of SSCs to then be cryopreserved for subsequent re-population of seminiferous tubules. The aim of this work was to develop rapid, reliable and reproducible method for human SSC culture and cryopreservation.

 

SSCs were isolated from testicular biopsies taken from azoospermic men undergoing sperm retrieval. Co-culture with testicular somatic cells facilitated SSCs clumping as chains and finally colonies. Immunofluorescence-labeling using antibodies against stem-cell markers GFRα1 and PLZF combined with confocal imaging, confirmed SSC signature. To date cultures have been maintained for up to 20 weeks. SSCs colonies were cryopreserved using vitrification. Post-thaw proliferation and cell death was assessed using Ki67 and TUNEL immunofluorescence-labelling; demonstrating an average post thaw viability rate of 90%.

 

Findings indicate our novel technique of firstly establishing a niche-like environment in-vitro is successful in maintaining long-term cultures of human SSCs. Subsequent cell-based cryostorage can then be used until the patient is ready for subsequent re-population of damaged seminiferous tubules.  This work is an important practical step forward in the male germline stem cell field, and undoubtedly will advance the future clinical application of SSCs restoring fertility to male childhood cancer survivors.

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