Development of a cryopreservation protocol for Leydig cells

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Article (peer-reviewed)

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Background: In the present study, we describe a procedure to cryopreserve the postnatal members of the Leydig cell lineage, including progenitor (PLC), immature (ILC) and adult (ALC) Leydig cells from, respectively 21-, 35- and 90-day-old rats. Methods: The cells were resuspended in a culture medium supplemented with 1% bovine serum albumin (Dulbecco's Modified Eagle's Medium [DMEM]/ F12) to a final concentration of 2 × 106cells/ml and the effects of varying concentrations of dimethylsulfoxide (DMSO) (5, 10, 15 or 20%) were assessed after freezing at -70°C and then storing in liquid nitrogen. After 12 months of frozen storage, these cells were thawed rapidly at 37°C and Trypan Blue exclusion staining and attachment to culture dishes were assessed as measures of viability. Results: The trypan blue exclusion and attachment rates for Leydig cellstages were around 85% in the presence of 15% DMSO. After frozen storage, Leydig cell steroidogenic capacity in response to a range of LH doses, (0.01-100 ng/ml) was unchanged compared with freshly isolated control cells. Furthermore, the steady-state mRNA levels for Leydig cell specific transcripts were maintained. Conclusions: This study demonstrates that purified rat Leydig cells at a range of developmental stages can be frozen and that the cryopreserved cells retain normal function.