Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells

Abstract

Mouse embryonic stem (mES) cells were induced to form intact monolayers in cell culture inserts, using combinations of extracellular matrix (ECM) components and growth factors (GFs). Progressive formation of intact monolayers was monitored using transepithelial electrical resistance (TEER) and passage of paracellular permeability (PP) markers. The mES cells were initially inoculated on inactivated mouse embryonic fibroblasts (MEFs) plus leukemia inhibitory factor (LIF). At 75% confluence, cells were passaged in the absence of MEF and LIF to stimulate formation of rounded multicellular aggregates (MA). After 4 days, cultures containing MA were transferred to culture inserts coated with ECM components only, and grown in the presence of selected individual GFs. An additional 10 to 14 days revealed confluent monolayers with TEER values of 500-700 ohmscm 2 (Ω-cm 2 ). Monolayers grown on inserts coated with ECM components, such as fibronectin or collagen-IV, in the presence of epidermal growth factor or keratinocyte growth factor in the medium, yielded the highest TEER measurements when compared to cultures grown without GFs or ECM. Acute cytotoxicity (AC) studies with confluent monolayers of mES cells in 96-well plates indicated that there is a high correlation (R 2 =0.91) between cell viability and TEER for 24-h exposure time. Also, decrease in TEER is inversely proportional with increase in PP of markers. In comparison to standardized Registry of Cytotoxicity (RC) data and TEER measurements, MTT IC 50 values for mES cells are lower. Thus, at equivalent concentrations for the same chemicals, cell viability decreases before the integrity of the monolayer is compromised. This system represents a novel approach for the manipulation of mES cells toward specific intact monolayers, as an in vitro model for biological monolayer formation, and most importantly, for applications to cytotoxicity testing.

Toxicity of antimony, copper, cobalt, manganese, titanium and zinc oxide nanoparticles for the alveolar and intestinal epithelial barrier cells in vitro

“…TEER measurements are used not only for epithelial barrier research but also for indications of the destruction of the extracellular matrix; for example, of neoplastic formations (Calabro et al 2008;Buchert et al 2012). To assess the effect of NPs on epithelial membrane integrity, we used the transepithelial resistance measurement of intact Caco2 and A549 epithelial cell monolayer.…”

Water-soluble organic solubilizers for in vitro drug delivery studies with respiratory epithelial cells: Selection based on various toxicity indicators

“…Based on TEER, sodium fluorescein permeation, and actin staining studies, DMSO, Cremophor EL, and PEG 400 (≤ 10%), as well as DMA and MPA (≤ 0.5%) appeared suitable for in vitro studies. Although TEER and paracelluar marker permeation are generally accepted indicators of tight junction permeability, these methods do not detect non-tight junction related epithelial membrane perturbation and as such are not good indicators of cell viability (Calabro et al, 2008). Therefore, more accurate indicators of cytotoxicity such as MTT and LDH were recommended as additional toxicity screening tools during in vitro studies (Sambruy et al, 2001).…”

Effect of metals on β-actin and total protein synthesis in cultured human intestinal epithelial cells

“…Cultures were confluent at 7-days, but maximum resistance values (between 750–1000 Ω-cm 2 ) were achieved in intact monolayers after 15-days. Trans-membrane specific electrical resistance (TMER) was measured as previously described (Calabro et al ., 2008) using the Millicell-ERS® resistance system (Millipore) before and after 24-, 48- and 96-hr incubation with test chemicals. Blanks (inserts without cells containing media and chemical) were used to determine baseline values, and are expressed as percent TMER of untreated control groups.…”