In Vitro Models for Studying Trophoblast Transcellular Transport

Bode, Claudia J.; Ji, Hong; Rytting, Erik; Silverstein, Peter S.; Young, Amber; Audus, Kenneth L.

doi:10.1385/1-59259-989-3:225

Cited by 64 publications

(81 citation statements)

References 34 publications

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“…Trophoblast cultures can be prepared from term placentas; however, the primary trophoblast monolayers are difficult to use for transport studies. Instead of forming a tight-junctioned monolayer appropriate for transport studies, these nonproliferative, multinucleated cells form aggregates with large intercellular spaces when grown on semi-permeable membranes (30). This drawback was overcome by Hemmings et al (31) who have prepared confluent cell layers of syncytiotrophoblasts on semi-permeable supports which function as an effective barrier to low and high molecular weight molecules (31).…”

Section: Discussionmentioning

confidence: 99%

The Role of Megalin in the Transport of Gentamicin Across BeWo Cells, an In Vitro Model of the Human Placenta

Akour

Kennedy

Gerk

2015

AAPS J

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Abstract. Aminoglycosides (AG) are known to readily cross the placenta, although the mechanisms responsible for placental transport have not been characterized. Megalin is expressed in human placenta, and it is reasonable to speculate, given its role in renal AG uptake, that it is similarly involved in placental transport. However, the role of megalin in placental AG uptake has not been established. An in vitro model to study megalin-mediated placental transport has also not been previously described. The objectives of this study, therefore, were to evaluate the human choriocarcinoma (BeWo) cell line as a model to study megalin-mediated placental transport and to assess the uptake kinetics of gentamicin, an AG antibiotic, using this in vitro model. BeWo cells were grown on Transwell® plates, and megalin expression and functional activity were assessed. Uptake of 3 H-gentamicin was also evaluated in the presence and absence of megalin inhibitors. Expression of megalin protein and mRNA in BeWo cells were confirmed via immunoblot and qPCR analysis. Uptake of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) (a megalin substrate) was time-, concentration-, and temperature-dependent consistent with a transporter-mediated process. FITC-BSA uptake was also significantly reduced in the presence of unlabeled gentamicin (a megalin substrate) and sodium maleate (to induce megalin shedding) suggesting that megalin is functionally active in BeWo cells. Gentamicin uptake exhibited time and temperature dependence, saturability and Michaelis-Menten kinetics, all of which suggest a transporter-mediated process. Gentamicin uptake was also significantly reduced in the presence of the megalin inhibitors RAP and EDTA suggesting that megalin is likely involved in gentamicin uptake.

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Section: Discussionmentioning

confidence: 99%

The Role of Megalin in the Transport of Gentamicin Across BeWo Cells, an In Vitro Model of the Human Placenta

Akour

Kennedy

Gerk

2015

AAPS J

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“…Cells were revived from an aliquot stored in liquid nitrogen (passage number 29) and grown in a 95% humidified incubator with 5% CO 2 at 37°C. At confluence (80-90%), cells were seeded at a density of 224,000 cells/ml in polycarbonate Transwell ® inserts (pore size 3 μm, growth area 1.12 cm 2 , apical volume 0.5 ml, basolateral volume 1.5 ml) coated with human placental collagen (Type IV, Sigma) in preparation for transport studies as described previously [25]. Cell culture media was changed daily.…”

Section: Cell Culturementioning

confidence: 99%

Transport of digoxin-loaded Polymeric Nanoparticles Across BeWo cells, an In Vitro Model of Human Placental Trophoblast

et al. 2015

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Background: Fetal arrhythmias can lead to fetal congestive heart failure and hydrops fetalis. Digoxin (the first-line treatment) has low transplacental permeability and high risk of maternal side effects. Biodegradable digoxin-loaded PEGylated poly(lactic-co-glycolic acid) nanoparticles may increase digoxin transport across BeWo b30 cell monolayers (an in vitro model of trophoblast in human placenta) by reducing the drug's interaction with P-gp. Results/methodology: The nanoparticles showed high encapsulation efficiency and sustained release over 48 h. Transport studies revealed significantly increased permeability across BeWo cell layers of digoxin-loaded nanoparticles when compared with free digoxin. P-gp inhibition also increased the permeability of digoxin, but not digoxin-loaded nanoparticles. Conclusion: This represents a novel treatment strategy for fetal cardiovascular disease which may improve maternal and fetal outcomes.

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“…Protocols regarding the revival, maintenance, passage, and growth of BeWo cells for uptake studies were followed as provided in the literature [14]. Briefly, BeWo cells between passage numbers 31 and 49 were seeded in 12-or 24-well plates at a density of 12,500 cells/cm 2 in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine, and 1% non-essential amino acids.…”

Section: Cell Culture and Uptake Experimentsmentioning

confidence: 99%

Contributions of phosphorylation to regulation of OCTN2 uptake of carnitine are minimal in BeWo cells

Rytting

Audus

2008

Biochemical Pharmacology

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Physiological functions of organic cation transporters (OCTs) in the placenta include transporting essential nutrients from the maternal to fetal circulations. OCTN2 transports carnitine with high affinity, and the transport of several drugs has also been shown to be mediated by this transporter. In this work, the role of phosphorylation and dephosphorylation mechanisms in regulating OCTN2 was investigated by observing the effects of various activators and inhibitors of kinases and phosphatases on the uptake of carnitine in BeWo cells, a human choriocarcinoma trophoblast cell line frequently used as an in vitro model of the rate-limiting barrier for maternal-fetal exchange. Preincubation with genistein resulted in significant increases in both alkaline phosphatase (ALP) activity and carnitine uptake. Levamisole, an ALP inhibitor, caused a more substantial decrease in carnitine uptake than expected from its corresponding decrease in ALP activity. It was determined that levamisole competitively inhibits carnitine uptake, with a K i value of 1.01 ± 0.05 mM, and this effect has a greater role in decreasing carnitine uptake than any indirect effects of ALP inhibition upon OCTN2 function. Progesterone also competitively inhibited carnitine uptake (K i = 48.6 ± 5.0 μM), but had no effect on ALP activity in BeWo cells.

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In Vitro Models for Studying Trophoblast Transcellular Transport

Cited by 64 publications

References 34 publications

The Role of Megalin in the Transport of Gentamicin Across BeWo Cells, an In Vitro Model of the Human Placenta

The Role of Megalin in the Transport of Gentamicin Across BeWo Cells, an In Vitro Model of the Human Placenta

Transport of digoxin-loaded Polymeric Nanoparticles Across BeWo cells, an In Vitro Model of Human Placental Trophoblast

Contributions of phosphorylation to regulation of OCTN2 uptake of carnitine are minimal in BeWo cells

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