Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception

Testicular cell suspensions were prepared from obstructive and non-obstructive azoospermic men and were cultured in vitro for 96 h as (i) mixed cell populations and (ii) isolated homogeneous populations of primary spermatocytes, round spermatids and elongating spermatids. The cells lost their viability gradually during the first 24 h period. By 72 h almost 90% of the cells were non-viable. Isolated pure fractions showed better viability at each time interval (P < 0.0005). Throughout the culture period primary spermatocytes, elongating spermatids and other non-spermatogenic cells showed no change in their morphology, but almost 22% of round spermatids showed growth of flagella. Most of the round spermatids developed their flagella during the first 4-8 h period of culture. Isolated pure round spermatids showed better flagellar growth compared with mixed cell suspensions (P < 0.0005). The spermatogenic cells were successfully cryopreserved. However, when mixed spermatogenic cell suspensions were cryopreserved, more cells lost their viability compared with when isolated pure fractions were cryopreserved (P < 0.0005).