Obtaining Undifferentiated Cells From Testis Biopsy

The purpose of this study is to isolate and culture stem cell from adult human testicular biopsies for future transplantation in fertility preservation.

Testicular biopsies were obtained from azoospermic patients as part of their diagnosis and work-up after obtaining a signed informed consent. A portion of each sample was mechanically dissected in PBS with 1% Pen/Strep. For enzymatic digestion samples were incubated with 0.5g/ml Collagenase type 1A for 20min and TripLE Select for 15min at 37ºC on a shaker and then filtered with 30µm and centrifuged at 1,000rpm. The pellets were seeded and incubated in embryonic stem cell media (hESC) with glial cell line-derived neurotrophic factor (GDNF 4ng/mL) for 5-7 days at 37ºC (1st culture). To identify and isolate SSCs they were marked with CD49f antibody and FACS sorted. Positive cells were cultured in gelatine plates containing hESC media with leukaemia inhibitory factor (LIF1000U/mL) (2nd culture).

Obtention of excess tissue from testicular biopsies and maintained in 4ºC until procedures. Digestion of the biopsies through controlled enzymes Replication of the cells Selection undifferentiated cells Expansion of the purification cells population Characterization of the cell population Assessment of pluripotent and germinal cell potential. Possible maturation of the cells.

•The spermatogonial cells isolated from testicular biopsies of azoospermic patients will be matured in vitro for thegeneration of sperm for infertility treatment. * Undifferentiated cells of the testis biopsy will be characterized as degrees of plasticity for proof that they are capable of regeneration spermatogenesis.