Abstract
Human primary cytotrophoblast cell culture is a very useful model to study the endocrine and immunological functions of syncytiotrophoblasts, as well as the ion exchange between the mother and her fetus, like calcium. In this chapter, we expose the procedure to (1) isolate and purify the cytotrophoblast cells from human term placenta and (2) study syncytiotrophoblast calcium uptake. First, the methodology is based on the enzymatic dissociation of villous placental tissue, followed by Percoll gradient separation. Purity is assessed by flow cytometry using staining against cytokeratin-7, protein specific for trophoblast cells. Cell proliferation is evaluated by a Thiazolyl Blue Tetrazolium Bromide (MTT) assay, hormonal secretion is measured by enzyme-linked immunosorbent assay (ELISA), and fusion is estimated by immunofluorescence using staining against desmosomal proteins. Second, we describe the calcium uptake experiment using the cytotrophoblast cells in culture.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
J. D. Boyd (1970) The human placenta. Cambridge University Press, Cambridge, MA.
A. Malassine and L. Cronier (2002) Hormones and human trophoblast differentiation: a review. Endocrine 19, 3–11.
F. Teasdale and G. Jean-Jacques (1985) Morphometric evaluation of the microvillous surface enlargement factor in the human placenta from mid-gestation to term. Placenta 6, 375–81.
R. A. Olli, H. J. Rajaniemi, R. Rydbeck and K. Metsikko (1993) Polarity and fusion of JAR choriocarcinoma cells as assessed by enveloped viral glycoproteins. Exp Cell Res 206, 276–82.
A. Vargas, J. Moreau, F. Le Bellego, J. Lafond and B. Barbeau (2008) Induction of trophoblast cell fusion by a protein tyrosine phosphatase inhibitor. Placenta 29, 170–74.
H. J. Kliman, J. E. Nestler, E. Sermasi, J. M. Sanger and J. F. Strauss, III (1986) Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118, 1567–82.
G. C. Douglas and B. F. King (1989) Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J Immunol Methods 119, 259–68.
L. J. Guilbert, B. Winkler-Lowen, R. Sherburne, N. S. Rote, H. Li and D. W. Morrish (2002) Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments. Placenta 23, 175–83.
A. J. Potgens, H. Kataoka, S. Ferstl, H. G. Frank and P. Kaufmann (2003) A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity. Placenta 24, 412–23.
R. Moreau, G. Daoud, R. Bernatchez, L. Simoneau, A. Masse and J. Lafond (2002) Calcium uptake and calcium transporter expression by trophoblast cells from human term placenta. Biochim Biophys Acta 1564, 325–32.
F. M. Campbell, P. G. Bush, J. H. Veerkamp and A. K. Dutta-Roy (1998) Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta. Placenta 19, 409–15.
T. Haigh, C. Chen, C. J. Jones and J. D. Aplin (1999) Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. Placenta 20, 615–25.
Y. Kato and G. D. Braunstein (1989) Discordant secretion of placental protein hormones in differentiating trophoblasts in vitro. J Clin Endocrinol Metab 68, 814–20.
T. Nagamatsu, T. Fujii, T. Ishikawa, T. Kanai, H. Hyodo, T. Yamashita, Y. Osuga, M. Momoeda, S. Kozuma and Y. Taketani (2004) A primary cell culture system for human cytotrophoblasts of proximal cytotrophoblast cell columns enabling in vitro acquisition of the extra-villous phenotype. Placenta 25, 153–65.
J. Yui, M. Garcia-Lloret, A. J. Brown, R. C. Berdan, D. W. Morrish, T. G. Wegmann and L. J. Guilbert (1994) Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells. Placenta 15, 231–46.
D. W. Morrish, J. Dakour, H. Li, J. Xiao, R. Miller, R. Sherburne, R. C. Berdan and L. J. Guilbert (1997) In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium. Placenta 18, 577–85.
G. Daoud, M. Amyot, E. Rassart, A. Masse, L. Simoneau and J. Lafond (2005) ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta. J Physiol 566, 409–23.
T. M. Mayhew (2008) Taking tissue samples from the placenta: an illustration of principles and strategies. Placenta 29, 1–14.
P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76–85.
Acknowledgments
F. Le Bellego holds a fellowship from Canadian Institute of Health Research (CIHR) funded Strategic Training Initiative in Research in Reproductive Health Sciences (STIRRHS).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Le Bellego, F., Vaillancourt, C., Lafond, J. (2009). Isolation and Culture of Term Human Cytotrophoblast Cells and In Vitro Methods for Studying Human Cytotrophoblast Cells’ Calcium Uptake. In: Vaillancourt, C., Lafond, J. (eds) Human Embryogenesis. Methods in Molecular Biology, vol 550. Humana Press. https://doi.org/10.1007/978-1-60327-009-0_4
Download citation
DOI: https://doi.org/10.1007/978-1-60327-009-0_4
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-008-3
Online ISBN: 978-1-60327-009-0
eBook Packages: Springer Protocols