Isolation, Culture and Characterization Stem Cells From Testis Biopsy

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).