Trophoblast Cultures

Advances in cell culture techniques have allowed the use of trophoblast cell cultures to evaluate the various transporter and metabolic systems of the placenta. Many studies have used primary cultures of undifferentiated cytotro-phoblasts isolated from placentas, whereas others have used trophoblast cell lines as a model. Trophoblast cell lines can be generated from normal tissues or malignant tissues and also from embryonal carcinomas exhibiting trophoblast differentiation [48], 

Since transport across the syncytiotrophoblast layer is the rate-limiting step in the absorption of substances from the maternal circulation to the fetal, these cells can provide information on uptake processes which are subject to intracellular regulatory mechanisms or affected by intracellular metabolism. However, it is very difficult to isolate this layer because of its syncytial nature. As a result, the undifferentiated precursor cytotrophoblast cells have been isolated and cultured. These cells do not proliferate in culture, but aggregate and spontaneously differentiate into syncytiotrophoblasts [49], 

Morphologically, this is demonstrated by the development of a multinuclear syncytium with apical microvilli [49], while functional differentiation is associated with induction and synthesis of hormones, such as human chorionic gonadotropin (hCG) and human placental lactogen (hPL) [50, 51]. Cultures of primary cytotrophoblasts have been used to study functional expression of P-gp, amino acid uptake, and hormonal stimulation of amino acid uptake and 

Either Transwell inserts or side-by-side diffusion chambers can be used for transport studies. Bode et al. have provided an excellent review on this subject [60], Briefly, cells are incubated for 30-60 min with a buffer solution. To initiate the transport study, a transport buffer containing the drug under investigation is added to either the apical or the basal chamber depending on the transport direction of interest. At predetermined time points, the respective receiver chamber is sampled and the withdrawn volume is replaced with the same volume of fresh buffer. The permeability coefficient (Papp) is calculated and the ratio of /apP in the basolateral-to-apical direction versus that in the apical-to-basolateral direction gives the efflux ratio. These sort of transport experiments are well suited to determine if drugs/xenobiotics are substrates of the placental efflux proteins. 

The choriocarcinoma cell line JAr bears close resemblance to early trophoblast, as their secretion pattern of hCG and steroids is similar, and the cells can differentiate into syncytiotrophoblasts in vitro [71], In a study comparing efflux transporter expression and activity in trophoblast cell lines and choriocarcinoma cells, the expression profiles indicated that while BeWo are more 

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D.G. Hemmings, B. Lowen, R. Sherburne, G. Sawicki, and LJ. Guilbert. Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles. Placenta. 22 70-79 (2001). 

Keywords Placenta Trophoblast Multidrug resistance Transport Cell culture... 

Xu et al. [76] have showed that human embryonic stem cells by treatment with bone morphogenetic protein-4 can be driven to differentiate into tro-phoblasts which have the ability to syncytialize and form confluent mono-layers. The differentiated cells express a number of trophoblast markers and secrete placental hormones and thus may provide an alternative placental model. Under the culture conditions used, however, the cells propagated poorly. 

H.E. Meyer Zu Schwabedissen, M. Grube, B. Heydrich, K. Linnemann, C. Fusch, H.K. Kroemer, and G. Jedlitschky. Expression, localization, and function of MRP5 (ABCC5), a transporter for cyclic nucleotides, in human placenta and cultured human trophoblasts Effects of gestational age and cellular differentiation. Am J Pathol. 166 39 18 (2005). 

A. King, L. Thomas, and P. Bischof. Cell culture models of trophoblast II Trophoblast cell lines—a workshop report. Placenta. 21 Suppl A S113-S119 (2000). 

P.I. Karl, K.L. Alpy, and S.E. Fisher. Amino acid transport by the cultured human placental trophoblast Effect of insulin on AIB transport. Am J Physiol. 262 C834—C839 (1992). 

Evaluate the function of human and macaque granulosa, trophoblast and endometrial cells, and macaque embryos in response to 2,3,7,8-TCDD while being cultured in vitro, and the cellular mechanisms by which primate reproductive cells sustain toxic damage... 

Twenty years ago, a high-affinity-binding site for uPA was demonstrated on the surface of peripheral blood monocytes and cultured cells of the human histiocytic lymphoma cell line, U937 [46]. The expression of uPAR on the cell surface of many cell types has since then been demonstrated, including a variety of neoplastic cell lines as well as nonneoplastic cells such as neutrophils, macrophages, keratinocytes, placental trophoblasts, endothelial, and smooth muscle cells [7, 33, 47-51]. The human uPAR gene has been mapped to chromosome 19ql.3 [52]. 

Recent advances in cell and tissue culture techniques provide the potential for evaluation of drug transport or metabolism processes at the placenta. Techniques are available for culturing trophoblasts of both animal and human origin.106 However, our focus here is primarily on human systems. Primary explant and isolated cell cultures of human cytotrophoblasts have been well described 106-109 however, these systems do not form confluent monolayer systems adequate for transcellular transport studies.105... 

Fig. 4 (A) In vitro modeling of colon cancer (B) production of cord blood cells (C) astrocyte culture for cell-based therapy of Parkinson s disease and (D) placenta model using trophoblast cells for transport.

Ma, T. Fiber-Based Bioreactor Systems In Mammalian Cell Culture and Tissue Engineering Human Trophoblast Cells Dissertation The Ohio State University Columbus, OH, 1999. 

Johnston, J.O., C.L. Wright, and B.W. Metcalf (1984). Time-dependent inhibition of aromatase in trophoblastic tumor cells in tissue culture. J. Steroid Biochem. 20, 1221-1226. 

Lehman, L.D., Poisner, A.M., 1984. Induction of metallothionein synthesis in cultured human trophoblasts by cadmium and zinc. J. Toxicol. Environ. Health 14, 419-432. 

Cultured trophoblasts Grow in monolayer, express corticotrophin-relea.sing factor Placental hormone secretion Petraglia et al. (1989)... 

Primary trophoblast cells (and ahso most cell lines of tro-phoblast origin) do not grew into confluent monolayers in culture (Cariappa et ai, 2003). This inhibits studies on polarized transport of nutrients and other compounds. They are difficult to culture because of contamination by other cell types and poor viability (Choy et ai, 2000). Villous and extravillous cytotrophoblasts differ in their function and characteristics. As studied by immunofluorescence, Ockleford and coworkers (2004) have shown a much higher cytokeratin expression in extravillous cytotrophoblasts compared to villous cytotrophoblasts,... 

Sooranna SR, Patel S, Das I. The effect of garlic on cell growth and cell division in cultured trophoblast and endothelial cell lines. Biochem Soc Trans 1997 25 456S. 

Atay, S., Gercel-Taylorb, C., Kesimerc, M., Taylor, D.D., 2011. Morphologic and proteo-mic characterization of exosomes released by cultured extravillous trophoblast cells. Exp. Cell Res. Available from http //dx.doi.Org/10.1016/j.yexcr.2011.01.014. 

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of a blastocyst stage embryo, which consists of a layer of trophoblast cells lining the ICM and blastocoel or blastocyst cavity. The ICM and trophoblast cells give rise to the embryo proper and extra-embryonic tissue, respectively. Thirthy years ago the in vitro culture of mouse ES (mES) cells was first described (Evans and Kaufman, 1981 Martin, 1981) and later in 1998 also human ES (hES) cells were derived (Thomson et al. 1998). ES cells are characterized by the unique properties of unlimited self-renewal without senescence and pluripotency. The latter infers that ES cells give rise to all cell types of the body. These specific properties led to the great scientific interest in ES cell either for their potential medical applications or as models to address more fundamental questions in development. 

Avila E, Halhali A, Larrea F, Barrera D (2014) Regulation of CYP27B1 and CYP24A1 gene expression by recombinant pro-inflammatory cytokines in cultured human trophoblasts. J Steroid 2624. Biochem Mol Biol 144 106-109. doi 10.1016/j. jsbmb.2013.12.007... 

By culturing specific cells from cloned embryos, scientists can make embryonic stem cell (ESC) cultures. During mammalian development, two distinct cell populations form after the first few days of embryonic development. The trophoblast, or the flattened, outer layer of cells, will eventually form the placenta and its associated structures. The inner cell mass (IGM) is the round, inner clump of cells that develop to form the embryo proper and a few structures associated with the placenta. If IGM cells are isolated and cultured on feeder cells, a layer of nondividing skin cells that secrete a cocktail of growth-promoting chemicals, the IGM cells will grow and spread over the surface of the culture dish. Such a culture is an embryonic stem cell culture, and these cells are pluripotent, which means that they can differentiate into any cell type in the adult body. 

Ma et al. [1997] have discussed an in vitro human placenta model for drug testing. This was a two-compartment perfusion system using human trophoblast cells attached to a chemically modified polyethylene terephthalate fibrous matrix as a cell culture scaffold. This system is a CCA in the same sense as the two-compartment system used to estimate response to dioxin. 

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