Effects of storage and passage of bovine luteal endothelial cells on endothelin-1 and prostaglandin F2alpha production.
To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) alpha-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2alpha in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFalpha (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2alpha by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2alpha by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFalpha increased (P<0.05) ET-1 and PGF2alpha production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.
Acosta
Tomas J
TJ
Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Japan.
Yoshioka
Shin
S
Komiyama
Junichi
J
Lee
Seung-Hyung
SH
Grazul-Bilska
Anna T
AT
Skarzynski
Dariusz J
DJ
Okuda
Kiyoshi
K
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000166:cytology*; D015237:Dinoprost / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism*; D005260:Female; D005615:Freezing; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D019332:Endothelin-1; D014409:Tumor Necrosis Factor-alpha; D015237:Dinoprost