[Impedance Spectroscopy and Transcriptome Analysis of Choriocarcinoma BeWo b30 as a Model of Human Placenta].

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.

Comparison of Profiles of Extracellular MicroRNA Secreted by Caco-2 Cells from the Apical Side of the Membrane under Static and Microcirculation Conditions.

Extracellular microRNA are one of the indicators of the functional state of cells. Culturing of Caco-2 cells under the conditions of microcirculation in a Homunculus microfluidic device allows better simulating natural environment of the body in comparison with static culturing. Impedance spectroscopy (BioClinicum Research Center) was used for non-invasive estimation of the monolayer quality and changes in the cell apical membrane due to the formation of microvilli. Under static conditions, Caco-2 cells release more microRNA from the apical membrane than under microcirculation conditions, while secretion of miR-320a, miR-24-3p, and miR-221-3p microRNA under static conditions can indicate stress of the cells and activation of inflammatory response. Under microcirculation conditions, the expression of laminin-α1 (LAMA1) was lower than under static conditions, which indicates deeper differentiation of cells.

Application of Impedance Spectroscopy for the Control of the Integrity of In Vitro Models of Barrier Tissues.

We compared available methods for monitoring the integrity of in vitro models of barrier tissues and studied the possibility of using impedance spectroscopy to solve this problem. It was demonstrated (theoretically and experimentally) that TEER measurements are not sufficiently sensitive to detect small defects in the cell barrier that significantly affect its permeability. For obtaining reliable results, it is necessary to set a sufficiently high threshold TEER, which leads to the loss of many intact samples. At the same time, impedance spectroscopy has all advantages of the classical method of measuring TEER (it is rapid and non-invasive method), while its application in combination with the methods of machine learning allows reliable detection of defects in the cell barrier.

Oxyquinoline-Dependent Changes in Claudin-Encoding Genes Contribute to Impairment of the Barrier Function of the Trophoblast Monolayer.

Natural response to hypoxia critically depends on rapid stabilization of hypoxia-inducible factor (HIF). Under normoxic conditions, HIF-prolyl hydroxylases mark α-subunits of HIF for degradation, while hypoxia results in stabilization of HIF-α. Oxyquinoline derivatives suppress activity of HIF-prolyl hydroxylases leading to HIF activation in the cell. Here we show that 24-h incubation of BeWo b30 choriocarcinoma cells (a model of trophoblast in the placental barrier) with oxyquinoline derivative leads to a decrease in transepithelial electrical resistance (TEER) of the cell monolayer, while the permeability of the monolayer for FITC-dextran (70 kDa) remains unchanged. These findings suggest that the overall barrier function is preserved, while the structure of intercellular tight junctions can undergo minor changes. Using Affymetrix Human Transcriptome Array 2.0, we showed that the treatment with oxyquinoline derivative was followed by a decrease in the expression of claudins 6 and 7 (CLDN6, CLDN7), occludin (OCLN), contact adhesion molecule 3 (JAM3), and angiomotinlike protein 1 (AMOTL1).

Non-Invasive Evaluation of Extracellular Matrix Formation in the Intestinal Epithelium.

Differentiation of colorectal cancer Caco-2 cells was assessed using Affymetrix Human Gene 1.0 ST arrays and by the main electrical parameters measured by bioimpedance spectroscopy. Transepithelial electrical resistance (TEER) was maximum on day 7, then decreased by day 11, and remained stable. The baseline resistance was maximum on day 4, minimum on day 7, but then gradually increased over 2 weeks, which can be explained by the formation of the basement membrane components or the apical mucous layer. Caco-2 cells express components of laminin-111 and laminin-511. A synchronous increase in the expression of mucin 3 mRNA (MUC3A/MUC3B) and mucin 17 mRNA (MUC17) and reduced expression of miR-21 and miR-622 microRNA genes were observed. Possible use of the described approach for studying the formation of extracellular matrix is discussed.

[Expression of SLC30A10 and SLC23A3 Transporter mRNAs in Caco-2 Cells Correlates with an Increase in the Area of the Apical Membrane].

Drug bioavailability studies commonly employ in vitro barrier tissue models consisting of epithelial and endothelial cells. These experiments require that the cell barrier quality be assessed regularly, which is usually performed using various labeled substrates and/or evaluation of transepithelial (transendothelial) electrical resistance (TEER). This technique provides information on the integrity of the monolayer, but not on differentiation-induced changes in the cell morphology. The present work shows that impedance spectroscopy can be applied to monitor both the integrity of the monolayer and the morphological changes of Caco-2 cells. The growth kinetics of the apical membrane was determined by calculating the electrical capacitance of the cell monolayer. In the course of differentiation, the most pronounced changes in the expression levels were observed for the mRNAs that encode SLC30A10 and SLC23A3 transporters. Their increase correlated with an increase in the apical membrane area, indicating that SLC30A10 and SLC23A3 mRNA levels assessed by qRT-PCR may be employed as cell differentiation biomarkers in Caco-2 models.

Effect of Circulation Parameters on Functional Status of HepaRG Spheroids Cultured in Microbioreactor.

We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.

A Model of Cadmium Uptake and Transport in Caco-2 Cells.

We created a physiologically substantiated kinetic model of cadmium transport and toxicity in intestinal cell model (Caco-2 cells). Transcriptome profiling of Caco-2 cells revealed high content of transporter DMT1 and ZIP14 and intensive expression of some calcium channels of the CACN family. The mathematical model describing three types of transporters, as well as intracellular cadmium binding with metallothionein and excretion through the basolateral membrane allowed us to construct cadmium uptake and transport curves that approximated the previously obtained experimental data. Using the proposed model, we determined toxic intracellular cadmium concentration leading to cell death and impairing the integrity of cell monolayer and described cadmium transport in this case.

Mathematical and Experimental Model of Oxygen Diffusion for HepaRG Cell Spheroids.

3D cell cultures are extensively used to study in vitro toxic effect of xenobiotics. When using multicellular spheroids, the question about their optimal size should be solved: small spheroids are difficult to manipulate, while large size of spheroids impairs the transport of nutrients and oxygen into the center. Mathematical models describing the distribution of substances in multicellular spheroids numerical procedure for solving differential equation system, which complicates their use in laboratory practice. We proposed and experimentally evaluated a new mathematical model describing oxygen distribution in HepaRG cell spheroids. Markers of functional activity were studied in spheroids of different size. The maximum size of spheroids that can be maintained in culture for 9 days without necrosis was determined.

Transport and Toxicity of Silver Nanoparticles in HepaRG Cell Spheroids.

We studied the effects of silver nanoparticles (10 nm) on HepaRG cell spheroids simulating liver tissue. The mathematical model was proposed that describes nanoparticle diffusion in a spheroid consisting of 5000 cells depending on the external nanoparticle concentration. It was demonstrated that cells in the 3D model were less sensitive to the toxic effects of nanoparticles in comparison with 2D cultures. Impaired integrity of the cell membrane did not deteriorate cell viability (according to MTT test).

Localization and Expression of Nucleoside Transporters ENT1 and ENT2 in Polar Cells of Intestinal Epithelium.

Сасо-2 cell line is widely used in in vitro studies of the intestinal wall permeability for drugs, but transport activity of these cells has not been thoroughly studied. We analyzed localization and expression of nucleoside transporters of the ENT family transferring a number of anticancer and antiviral drugs. Immunocytochemical staining revealed apical localization of ENT1 and integral expression of ENT2 in Caco-2 cells. Based on these data and on the value of hypoxanthine uptake by these cells, we created a mathematical model allowing quantification of transporter expression on the apical and basolateral membranes of Caco-2 cells. The discrepancy between gene and protein expression of the transporter complicating prediction of patient's sensitivity to the drug upon individual therapy selection is discussed.