Development of a cryopreservation protocol for Leydig cells.
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 x 10(6)cells/ml and the effects of varying concentrations of dimethylsulfoxide (DMSO) (5, 10, 15 or 20%) were assessed after freezing at -70 degrees C and then storing in liquid nitrogen. After 12 months of frozen storage, these cells were thawed rapidly at 37 degrees 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 cell stages 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.
Chen
Guo-Rong
GR
Department of Pathology of the 1st Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000, People's Republic of China.
Ge
Ren-Shan
RS
Lin
Han
H
Dong
Lei
L
Sottas
Chantal M
CM
Hardy
Matthew P
MP
D015113:Androstane-3,17-diol / Q000096:biosynthesis; D000738:Androsterone / Q000096:biosynthesis; D000818:Animals; D049109:Cell Proliferation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D007985:Leydig Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000494:pharmacology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013739:Testosterone / Q000096:biosynthesis
D015113:Androstane-3,17-diol; D013739:Testosterone; D007986:Luteinizing Hormone; D000738:Androsterone; D004121:Dimethyl Sulfoxide