Characterization of trophoblast cell isolations by a modified flow cytometry assay.
The purity of isolated trophoblast cells is an important checkpoint in in vitro studies on the human placenta. Maintaining viability of primary cells for a prolonged period, or achieving cell proliferation in primary cell cultures, is often a matter of concern. In this paper we present a method for characterizing the purity of isolated trophoblast cells based on the expression of cytoskeletal proteins, as well as for assessing their cell cycle status, by flow cytometry. We show that after a simple permeabilization and fixation step in 70 per cent methanol, staining for cytokeratin 7 and vimentin could discriminate between trophoblast cells and contaminating populations. The method was applicable to trophoblast cells both from villous and extravillous origin. By staining the proliferation-related antigen Ki-67 and DNA, information was gained about the cell cycle status and viability of freshly isolated and cultured villous trophoblast cells. This method may help to quickly and quantitatively characterize preparations of isolated trophoblast, as well as to search for culture conditions favouring long-term survival and proliferation.
Pötgens
A J
AJ
Department of Anatomy, University of Technology Aachen, Wendlingweg 2, D-52057 Aachen, Germany. apotgens@post.klinikum.rwth-aachen.de
Gaus
G
G
Frank
H G
HG
Kaufmann
P
P
D002453:Cell Cycle; D002455:Cell Division; D002463:Cell Membrane Permeability; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005260:Female; D005404:Fixatives; D005434:Flow Cytometry; D006801:Humans; D053552:Keratin-7; D007633:Keratins / Q000032:analysis; D019394:Ki-67 Antigen / Q000032:analysis; D000432:Methanol; D011247:Pregnancy; D014327:Trophoblasts / Q000166:cytology*; D014746:Vimentin / Q000032:analysis
D005404:Fixatives; C507497:KRT7 protein, human; D053552:Keratin-7; D019394:Ki-67 Antigen; D014746:Vimentin; D007633:Keratins; D004247:DNA; D000432:Methanol