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Hum. Reprod. Advance Access published online on June 27, 2007
Human Reproduction, doi:10.1093/humrep/dem169
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Development of a cryopreservation protocol for Leydig cells

Guo-Rong Chen1,{dagger}, Ren-Shan Ge2,{dagger}, Han Lin3, Lei Dong1, Chantal M. Sottas2 and Matthew P. Hardy2,4

1 Department of Pathology of the 1st Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000, People's Republic of China 2 Population Council, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA 3 Department of Anesthesiology of the 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000, People's Republic of China

4 Correspondence address. Tel: +1-212-327-8754; Fax: +1-212-327-7678; E-mail: m-hardy{at}popcbr.rockefeller.edu

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 106 cells/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 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.

Key words: Leydig cells/cryopreservation/hypogonadism/testosterone/LH response

{dagger} Authors contributed equally

Submitted on February 9, 2007; resubmitted on May 12, 2007; accepted on May 16, 2007.


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