title: Development of a cryopreservation protocol for Leydig cells.
abstract: 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.
authors.affiliation: Department of Pathology of the 1st Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000, People's Republic of China.
authors.forename: Guo-Rong
authors.initials: GR
authors.lastname: Chen
authors.forename: Ren-Shan
authors.initials: RS
authors.lastname: Ge
authors.forename: Han
authors.initials: H
authors.lastname: Lin
authors.forename: Lei
authors.initials: L
authors.lastname: Dong
authors.forename: Chantal M
authors.initials: CM
authors.lastname: Sottas
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Hardy
mesh_terms: 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
chemical_list: D015113:Androstane-3,17-diol; D013739:Testosterone; D007986:Luteinizing Hormone; D000738:Androsterone; D004121:Dimethyl Sulfoxide