BeWo B30 Cells Research Articles

Preparation, characterization, and transport of dexamethasone-loaded polymeric nanoparticles across a human placental in vitro model

The purpose of this study was to prepare dexamethasone-loaded polymeric nanoparticles and evaluate their potential for transport across human placenta. Statistical modeling and factorial design was applied to investigate the influence of process parameters on the following nanoparticle characteristics: particle size, polydispersity index, zeta potential, and drug encapsulation efficiency. Dexamethasone and nanoparticle transport was subsequently investigated using the BeWo b30 cell line, an in vitro model of human placental trophoblast cells, which represent the rate-limiting barrier for maternal-fetal transfer. Encapsulation efficiency and drug transport were determined using a validated high performance liquid chromatography method. Nanoparticle morphology and drug encapsulation were further characterized by cryo-transmission electron microscopy and X-ray diffraction, respectively. Nanoparticles prepared from poly(lactic-co-glycolic acid) were spherical, with particle sizes ranging from 140 to 298 nm, and encapsulation efficiency ranging from 52 to 89%. Nanoencapsulation enhanced the apparent permeability of dexamethasone from the maternal compartment to the fetal compartment more than 10-fold in this model. Particle size was shown to be inversely correlated with drug and nanoparticle permeability, as confirmed with fluorescently labeled nanoparticles. These results highlight the feasibility of designing nanoparticles capable of delivering medication to the fetus, in particular, potential dexamethasone therapy for the prenatal treatment of congenital adrenal hyperplasia.

Assessment of an in vitro transport model using BeWo b30 cells to predict placental transfer of compounds

The human ex vivo placental perfusion model has regularly been used to study the transplacental transport of compounds. However, this method is laborious and dependent on the presence of fresh human placenta, hampering its use for the assessment of large numbers of compounds. An in vitro model for the placental barrier using BeWo b30 cells may provide an alternative to the ex vivo system. The present study aims to assess whether such an in vitro model could be used to reliably predict placental transfer. To this end, BeWo b30 cells, derived from a human choriocarcinoma, were grown on transwell insert to form a cell layer, separating an apical maternal compartment from a basolateral fetal compartment. For a set of nine selected model compounds, including the reference compound antipyrine, the transport velocity from the apical to the basolateral compartment was determined. Relative transport rates obtained were compared with the transfer indices (a measure for the transport relative to antipyrine) of these compounds obtained in ex vivo placental perfusion studies as reported in the literature. The relative transport rates in the in vitro BeWo model were in good correlation (R² = 0.95) with the transfer indices reported for the ex vivo model. This indicates that the BeWo model could be a valuable in vitro model for prediction of placental transfer of compounds.

Fate of the teratogenic and carcinogenic ochratoxin A in human perfused placenta

Ochratoxin A (OTA) is one of the most frequent mycotoxins detected in human blood worldwide. Apart from its well known nephrotoxicity, OTA-induced teratogenicity and carcinogenicity proven in animals are potential effects also in humans. Pregnant women have been exposed to this food contaminant via dietary exposure in a continuous and widespread manner. Although the transplacental transfer of OTA has been demonstrated in laboratory animals and the presence of OTA in human fetal samples has been reported, little is known about the role of human placenta in OTA toxicokinetics. In this study, human perfused placenta was used to reveal the actual placental toxicokinetics of OTA using concentrations found in serum of pregnant women. Moreover, the effect of protein concentration and biological significance of placental transporters on the OTA transfer in human placenta were also determined. Our study is the first to pursue the transfer of OTA through perfused human placenta. The transfer of OTA through term human placenta was barely detectable in all perfusions. Inhibitors of neither ABCG2 nor ABCC2 increased the transport of OTA to fetal circulation in placental perfusion, and thus these transporters apparently do not have biological significance in inhibiting transplacental transfer of OTA. Human albumin has inhibited OTA transfer through a tight monolayer of BeWo b30 cells. Finding from this study clearly contradict the existing epidemiological studies reporting higher OTA levels in fetal than in maternal circulation in vivo.

Modeling placental transport: Correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion

The placental passage of three compounds with different physicochemical properties was recently investigated in ex vivo human placental perfusion experiments (caffeine, benzoic acid, and glyphosate) [Mose, T., Kjaerstad, M.B., Mathiesen, L., Nielsen, J.B., Edelfors, S., Knudsen, L.E., 2008. Placental passage of benzoic acid, caffeine, and glyphosate in an ex vivo human perfusion system. J. Toxicol. Environ. Health, Part A 71, 984–991]. In this work, the transport of these same three compounds, plus the reference compound antipyrine, was investigated using BeWo (b30) cell monolayers. Transport across the BeWo cells was observed in the rank order of caffeine > antipyrine > benzoic acid > glyphosate in terms of both the apparent permeability coefficient and the initial slope, defined as the linear rate of substance transferred to the fetal compartment as percent per time, a parameter used to compare the two experimental models. The results from the in vitro studies were in excellent agreement with the ex vivo results (caffeine ≈ antipyrine > benzoic acid > glyphosate). However the transfer rate was much slower in the BeWo cells compared to the perfusion system. The advantages and limitations of each model are discussed in order to assist in the preparation, prediction, and performance of future studies of maternal–fetal transfer.

IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): Evidence for MAP kinase-dependent and MAP kinase-independent mechanisms

Objective IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model. Design The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo b30 choriocarcinoma cells using the non-metabolizing amino acid analog, [ 3H]-α-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation. Results Maximal stimulating concentrations of IGF-I (100 ng/ml) stimulated AIB transport by 30–40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-I-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 μM), an inhibitor of MAP kinase activation, inhibited 20–30% of basal AIB transport but did not inhibit IGF-I-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms. Conclusions These data demonstrate that IGF-I stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo b30 cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs.

Type I insulin-like growth factor receptors in the BeWo choriocarcinoma cell (b30 clone) during cell differentiation

The expression of insulin-like growth factor (IGF) receptors in the differentiating human trophoblast was studied using the b30 clone of the BeWo choriocarcinoma cell line (BeWo b30) as a model system. This clonally derived cell line differentiates over 48–72 h, in culture, to form syncytiotrophoblasts when intracellular cAMP levels are elevated by exposure to 100 μ m forskolin (FSK). IGF receptors were studied at various times during the differentiation process by measuring the specific binding of [ 125I]-IGF-I and [ 125I]-IGF-II to attached cells. First, [ 125I]-IGF-I bound to a single class of binding sites in the untreated cells ( K D ≈ 1−2 × 10 −10 m) that exhibited binding specificity characteristic of the type I IGF receptor (IGF-I ≥ IGF-II ⪢ Insulin FSK treatment resulted in a two- to threefold increase in the number of these binding sites. Increased receptor expression was observed as early as 24 h after FSK treatment and remained elevated for at least 72 h. Next, [ 125I]-IGF-II bound to two classes of binding sites in the untreated cells, a high-affinity ( K D 2.5 x 10 −10 m), low-capacity site and a low-affinity ( K D ≈ 6 × 10 −9 m), high-capacity site. The B max and K D of the high-affinity site suggested that it represented the type I IGF receptor. Competition studies revealed that 15–20 per cent of total [ 125I]-IGF-II binding only was sensitive to IGF-I competition in the untreated cells. After FSK treatment, however, unlabelled IGF-I inhibited 60–70 per cent of specific [ 125I]-IGF-II binding. Scatchard analysis revealed a two- to fourfold increase in the number of both binding sites with no change in their respective binding affinities. Cross-linking analysis demonstrated that [ 125I]-IGF-II bound to two structurally distinct binding sites in the untreated BeWo b30 cell consistent with both the type I and II IGF receptors. After FSK treatment, however, there was an increase in the relative amount of [ 125I]-IGF-II associated with the higher affinity type I IGF receptor. The BeWo b30 cells expressed no insulin receptors at any stage of differentiation. These data demonstrate that the BeWob3o choriocarcinoma cell line expresses both type I and II IGF receptors. Induction of cell differentiation is associated with an increase in type I IGF receptors expressed at the cell surface. These receptors bind IGF-II with high-affinity, providing additional binding capacity for locally available IGF-II. These data are consistent with specific roles for the type I IGF receptor in regulating differentiated trophoblast cell function. Furthermore, the early rise in type I IGF receptor number suggests they may play a regulatory role in the differentiation process itself.