Isolation and characterization of a bovine blastocyst-derived trophoblastic cell line, BT-1: development of a culture system in the absence of feeder cell.
We established a trophoblastic cell line, bovine trophoblast-1 (BT-1), derived from in vitro matured and fertilized blastocyst. While several trophoblastic cell lines have been previously reported using feeder cell, BT-1 could be cultured in the absence of feeder cell. BT-1 was cultured for more than 18 months (over 75 passage) in the absence of feeder cells, using bovine endometrial fibroblast-conditioned medium (fibroblast-conditioned medium). We found that the cell growth was accelerated in fibroblast-conditioned medium. In bromodeoxyuridine incorporation analysis, BT-1 cells growth rate in fibroblast-conditioned medium was about two-fold higher than that in conventional medium. Furthermore, fibroblast-conditioned medium accelerated attachment of BT-1 cells to culture dishes following plating. BT-1 showed epithelial morphology and expressed cytokeratin. During continuous culture, cells accumulated fluid under the cell sheet and form dome-like structure that eventually transformed into free floating vesicles. Reverse transcription polymerase chain reaction analysis and immunoblot analysis demonstrated that BT-1 cells expressed interferon-tau as well as placental lactogen (PL). Immunofluorescence analysis demonstrated that a small number of cells were PL-positive, and these cells were binucleate. The BT-1 trophoblastic cell line could serve as a powerful model system for the study of trophoblast cell lineage and proliferation.
Shimada
A
A
Laboratory of Reproductive Biology and Technology, Department of Developmental Biology, National Institute of Agrobiological Sciences, Ibaraki, Japan.
Nakano
H
H
Takahashi
T
T
Imai
K
K
Hashizume
K
K
D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D001973:Bromodeoxyuridine / Q000378:metabolism; D002417:Cattle; D018929:Cell Culture Techniques; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D017077:Culture Media, Conditioned; D005347:Fibroblasts / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D015151:Immunoblotting; D007370:Interferon Type I / Q000235:genetics; D010928:Placental Lactogen / Q000032:analysis / Q000235:genetics / Q000276:immunology; D011257:Pregnancy Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014327:Trophoblasts / Q000166:cytology* / Q000502:physiology
D017077:Culture Media, Conditioned; D007370:Interferon Type I; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau; D010928:Placental Lactogen; D001973:Bromodeoxyuridine