RESUMEN
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.
Asunto(s)
Coriocarcinoma/genética , Coriocarcinoma/patología , Espectroscopía Dieléctrica , Perfilación de la Expresión Génica , Modelos Biológicos , Placenta/citología , Placenta/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Femenino , Humanos , EmbarazoRESUMEN
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.
Asunto(s)
Matriz Extracelular/metabolismo , Mucosa Intestinal/metabolismo , Membrana Basal/metabolismo , Células CACO-2 , Espectroscopía Dieléctrica , Impedancia Eléctrica , Humanos , MicroARNs/genética , Mucina 3/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Neoplasias Colorrectales/genética , Transportadores de Sodio Acoplados a la Vitamina C/genética , Transportador 8 de Zinc/genética , Células CACO-2 , Técnicas de Cultivo de Célula , Membrana Celular/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Impedancia Eléctrica , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación Neoplásica de la Expresión Génica , HumanosRESUMEN
The mistletoe lectin viscumin (MLI) is a ribosome-inactivating protein from Viscum album widely used in cancer therapy. Its antitumor properties are due to its immunomodulating action, previously demonstrated in experiments involving intravenous, subcutaneous, and oral administration of viscumin. To investigate whether viscumin has a cytotoxic effect on the intestinal epithelium, its safety was assessed using (i) impedance spectroscopy to measure the integrity of the colorectal adenocarcinoma Caco-2 cell monolayer after exposure to viscumin and (ii) a novel technique of determining the portion of viscumin-inactivated ribosomes. It was shown that inactivation of at least 20% of the ribosomes within 6 h did not lead to disruption of the Caco-2 cell monolayer or alter the physicochemical parameters of enterocyte membranes.