Abstract
Development of a cryopreservation protocol for Leydig cells
Chen,Guo-Rong; Ge,Renshan; Lin,Han; Dong,Lei; Sottas,Chantal M.; Hardy,Matthew P.
Human Reproduction 22(8): 2160-2168
Publication date: 2007
Background
In the present study, we describe a procedure to cryopreservethe postnatal members of the Leydig cell lineage, includingprogenitor (PLC), immature (ILC) and adult (ALC) Leydig cellsfrom, respectively 21-, 35- and 90-day-old rats.
Methods
The cells were resuspended in a culture medium supplementedwith 1% bovine serum albumin (Dulbecco's Modified Eagle's Medium[DMEM]/F12) to a final concentration of 2 x 10cells/ml andthe effects of varying concentrations of dimethylsulfoxide (DMSO)(5, 10, 15 or 20%) were assessed after freezing at -70°Cand then storing in liquid nitrogen. After 12 months of frozenstorage, these cells were thawed rapidly at 37°C and TrypanBlue exclusion staining and attachment to culture dishes wereassessed 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 frozenstorage, Leydig cell steroidogenic capacity in response to arange of LH doses, (0.01-100 ng/ml) was unchanged comparedwith freshly isolated control cells. Furthermore, the steady-statemRNA levels for Leydig cell specific transcripts were maintained.
Conclusions
This study demonstrates that purified rat Leydig cells at arange of developmental stages can be frozen and that the cryopreservedcells retain normal function.
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