Refined Cumulative Risk Assessment of Pb, Cd, and as in TCM Decoction Based on Bioavailability through In Vitro Digestion/MDCK Cells.

In this study, the oral bioavailability of Pb, Cd, and As in three types of traditional Chinese medicines (TCMs) and TCM decoctions were investigated through in vitro PBET digestion/MDKC cell model. Furthermore, a novel cumulative risk assessment model associated with co-exposure of heavy metal(loid)s in TCM and TCM decoction based on bioavailability was developed using hazard index (HI) for rapid screening and target organ toxicity dose modification of the HI (TTD) method for precise assessment. The results revealed that the bioavailability of Pb, Cd, and As in three types of TCM and TCM decoction was 5.32-72.49% and 4.98-51.97%, respectively. After rapid screening of the co-exposure health risks of heavy metal(loid)s by the HI method, cumulative risk assessment results acquired by TTD method based on total metal contents in TCMs indicated that potential health risks associated with the co-exposure of Pb, Cd, and As in Pheretima aspergillum (E. Perrier) and Oldenlandia diffusa (Willd.) Roxb were of concern. However, considering both the factors of decoction and bioavailability, TTD-adjusted HI outcomes for TCMs in this study were <1, indicating acceptable health risks. Collectively, our innovation on cumulative risk assessment of TCM and TCM decoction provides a novel strategy with the main purpose of improving population health.

An avian-origin internal backbone effectively increases the H5 subtype avian influenza vaccine candidate yield in both chicken embryonated eggs and MDCK cells.

Inactivated vaccines play an important role in preventing and controlling the epidemic caused by the H5 subtype avian influenza virus. The vaccine strains are updated in response to alterations in surface protein antigens, while an avian-derived vaccine internal backbone with a high replicative capacity in chicken embryonated eggs and MDCK cells is essential for vaccine development. In this study, we constructed recombinant viruses using the clade 2.3.4.4d A/chicken/Jiangsu/GY5/2017(H5N6, CkG) strain as the surface protein donor and the clade 2.3.4.4b A/duck/Jiangsu/84512/2017(H5N6, Dk8) strain with high replicative ability as an internal donor. After optimization, the integration of the M gene from the CkG into the internal genes from Dk8 (8GM) was selected as the high-yield vaccine internal backbone, as the combination improved the hemagglutinin1/nucleoprotein (HA1/NP) ratio in recombinant viruses. The r8GMΔG with attenuated hemagglutinin and neuraminidase from the CkG exhibited high-growth capacity in both chicken embryos and MDCK cell cultures. The inactivated r8GMΔG vaccine candidate also induced a higher hemagglutination inhibition antibody titer and microneutralization titer than the vaccine strain using PR8 as the internal backbone. Further, the inactivated r8GMΔG vaccine candidate provided complete protection against wild-type strain challenge. Therefore, our study provides a high-yield, easy-to-cultivate candidate donor as an internal gene backbone for vaccine development.

Evaluating the MDCK cell permeability of greenly synthesize bimetallic Ag/Zn Nanoparticles using leaf extract of Vallaris solanacea as a potential antipesticide-resistant agent.

Bimetallic nanoparticles, particularly Ag/Zn bimetallic nanoparticles, have gained increasing attention due to their unique properties, making them suitable for a variety of applications such as catalysis, water treatment, and environmental remediation. This study aimed to elucidate the use of bimetallic nanoparticles of Ag/Zn as an alternative to resistant pesticides for pest control. Furthermore, this research demonstrates that BNPs can target specific pollutants and degrade them through various mechanisms. BNP docking with the Nilaparvata lugens cytochrome P450 (CYP6ER1) protein exhibited the lowest binding energy of -7.5 kcal/mol. The cell permeability analysis of BNP in plant cells reveals that the BNP has 0 % permeability towards any cell at -10 kcal/mol energy, which is the lowest free energy translocation pathway. The harmful leftover residues of the pesticides have a higher chance of degradability in case of interaction with BNP validated by chemical-chemical interaction analysis. Additionally, MDCK permeability coefficient of small molecules based on the regression model was calculated for BNP which authenticated the efficiency of BNP. Moreover, Swiss ADMET simulated absorption using a boiled egg model with no blood-brain barrier and gastrointestinal crossing for the expected BNP molecule has been observed. Significantly, the findings indicate that employing bimetallic nanoparticles like Ag/Zn is a crucial strategy for bioremediation because they proficiently decompose pesticides while posing no risk to humans. Our results will facilitate the design of novel BNPs materials for environmental remediation and pest control ensuring human health safety that are predicated on bimetallic nanoparticles.

Qualification of In Vitro Dissolution Absorption System 2 (IDAS2) with Caco-2 and MDCK Cell Monolayers: Dose Sensitivity Study Using BCS Class I and III Drugs.

This study aimed to validate the In vitro Dissolution Absorption System 2 (IDAS2) containing a biological barrier of Caco-2 or Madin-Darby canine kidney (MDCK) cell monolayer through dose sensitivity studies. Metoprolol and propranolol were selected as Biopharmaceutics Classification System (BCS) Class I model drugs, and atenolol as a Class III model drug. The IDAS2 is comprised of a dissolution vessel (500 mL) and two permeation chambers (2 × 8.0 mL) mounted with Caco-2 or MDCK cell monolayer. One or two immediate-release tablet(s) of the model drug were added to the dissolution vessel, and the time profiles of dissolution and permeation were observed. Greater than 85% of metoprolol and propranolol (tested at two dosing concentrations) were dissolved by 15 min, and all drugs were fully dissolved by 30 min. All three drugs were more permeable across Caco-2 cells than MDCK cells with a linear increase in permeation across both cells at both dose concentrations. Thus, the dose sensitivity of the IDAS2 was demonstrated using both cell barriers. These results indicate a successful qualification of IDAS2 for the development/optimization of oral formulations and that MDCK cells can be utilized as a surrogate for Caco-2 cells.

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal.

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin-Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

Can membrane permeability of zwitterionic compounds be predicted by the solubility-diffusion model?

Zwitterions contain both positively and negatively charged functional groups, resulting in an overall net neutral charge. Nevertheless, the membrane permeability of the zwitterionic form of a compound is assumed to be much lower than the permeability of the uncharged neutral form. Although a significant proportion of pharmaceuticals are zwitterionic, it has not been clear so far whether their permeability is dominated by the permeation of the zwitterionic or the neutral form, since neutral fractions are often quite low as compared to the zwitterionic fraction. This complicates the in silico prediction of the permeability of zwitterionic compounds. In this work, we re-evaluated existing in vitro permeability data from literature measured with Caco-2/MDCK cell assays, using more strict exclusion criteria for effects like diffusion limitation by the aqueous boundary layers, paracellular transport, active transport and retention. Using this re-evaluated data set, we show that extracted intrinsic permeabilities of the neutral fraction are well predicted by the solubility-diffusion model (RMSE = 1.21; n = 18) if the permeability of the zwitterionic species is assumed negligible. Our work thus suggests that only the neutral species is relevant for the membrane permeability of zwitterionic compounds, and that membrane permeability of zwitterionic compounds is indeed predictable by the solubility-diffusion model.

Developments and current challenges in the process of cell culture-based seasonal influenza vaccine manufacture in Japan.

Seasonal influenza is an acute respiratory infection primarily caused by influenza A and B viruses, which circulate annually and cause substantial morbidity and mortality worldwide. Annual influenza vaccination is currently the most effective measure for preventing influenza and greatly reduces the risk of disease severity and the incidence of complications and death. Annual seasonal influenza vaccines are traditionally produced in Japan and many other countries using viruses propagated in embryonated chicken eggs. However, at present, the effectiveness of the seasonal influenza vaccines has some significant limitations, partly because of egg-adaptive mutations in the antigenic sites of the influenza virus haemagglutinin, which are caused by the continued evolution of seasonal influenza viruses. To overcome the limitations of egg-based influenza vaccine production, a mammalian cell culture-based influenza vaccine production system has been developed in Japan in the past decade as an alternative to the current production method. In this review, I have summarised the progress in the development of cell-based seasonal influenza vaccines and discussed the technological challenges encountered in the development of influenza vaccines.

Automated Layer Analysis (ALAn): An Image Analysis Tool for the Unbiased Characterization of Mammalian Epithelial Architecture in Culture.

Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo-two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity. Key features • This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion. • ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy. • ALAn code is written in Python3 using the Jupyter Notebook interactive format. • Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland-derived and Caco-2 colon carcinoma cells. • This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.

Exploration of the protective mechanisms of Icariin against cisplatin-induced renal cell damage in canines.

This study delves into the protective mechanisms of Icariin (ICA) against cisplatin-induced damage in Madin-Darby canine kidney (MDCK) cells. Comprising two distinct phases, the investigation initially employed a single-factor randomized design to ascertain the minimal cisplatin concentration eliciting MDCK cell damage, spanning concentrations from 0 to 16 mmol/L. Concurrently, various concentrations of ICA (ranging from 5 to 50 mmol/L) were combined with 1 mmol/L cisplatin to determine the most efficacious treatment concentration. Subsequent investigations utilized four treatment groups: control, 1 mmol/L cisplatin, 1 mmol/L cisplatin + 20 mmol/L ICA, and 1 mmol/L cisplatin + 25 mmol/L ICA, aimed at elucidating ICA's protective mechanisms. Findings from the initial phase underscored a significant reduction in MDCK cell viability with 1 mmol/L cisplatin in comparison to the control (P < 0.01). Notably, the inclusion of 20 and 25 mmol/L ICA substantively ameliorated MDCK cell viability under 1 mmol/L cisplatin (P < 0.01). Moreover, cisplatin administration induced an elevation in inflammatory factors, malondialdehyde (MDA), reactive oxygen species (ROS), and Bax protein levels, while concurrently suppressing superoxide dismutase (SOD), catalase (CAT), and Bcl-2 expression (P < 0.01). Conversely, supplementation of 20 and 25 mmol/L ICA demonstrated a marked increase in mitochondrial membrane potential and levels of SOD, CAT, and Bcl-2 (P < 0.01). These interventions effectively attenuated inflammatory responses and suppressed Bax protein expression (P < 0.05), consequently mitigating cisplatin-induced apoptosis in MDCK cells (P < 0.01). In summary, these findings elucidate the role of ICA in impeding apoptosis in cisplatin-induced MDCK cells by regulating inflammatory responses, oxidative stress, and autophagic protein expression.

Lack of Effect of Antioxidants on Biopharmaceutics Classification System (BCS) Class III Drug Permeability.

The addition of antioxidants to pharmaceutical products is a potential approach to inhibit nitrosamine formation, particularly in solid oral dosage forms like tablets and capsules. The objective was to assess the effect of ten antioxidants on the permeability of four Biopharmaceutics Classification System (BCS) Class III drugs. Bi-directional drug permeability studies in the absence and presence of antioxidants were performed in vitro across MDCK-II monolayers. No antioxidant increased drug permeability, while the positive control sodium lauryl sulfate always increased drug permeability. Results support that any of the ten antioxidants, up to at least 10 mg, can be added to a solid oral dosage form without modulating passive drug intestinal permeability. Additional considerations are also discussed.

Reduction in Interferon-Stimulated Genes Contributes to High-Yield Production of Influenza Virus in Suspension MDCK Cells.

Compared with the traditional vaccine produced in embryonated chicken eggs, cell-based manufacturing represented by the Madin-Darby canine kidney (MDCK) cell line has a larger production scale and reduces the risk of egg shortage in a pandemic. Establishing a culture system that enables high production of the influenza virus is a key issue in influenza vaccine production. Here, a serum-free suspension culture of MDCK (sMDCK) cells was obtained from adherent MDCK (aMDCK) cells by direct adaptation. Viral infection experiments showed that viral yields of influenza A/B virus in sMDCK cells were higher than in aMDCK cells. Transcriptome analysis revealed that numerous interferon-stimulated genes (ISGs) exhibited reduced expression in sMDCK cells. To further clarify the mechanism of high viral production in sMDCK cells, we demonstrated the antiviral role of RIG-I and IFIT3 in MDCK cells by knockdown and overexpression experiments. Furthermore, suppression of the JAK/STAT pathway enhances the viral accumulation in aMDCK cells instead of sMDCK cells, suggesting the reduction in the JAK/STAT pathway and ISGs promotes viral replication in sMDCK cells. Taken together, we elucidate the relationship between the host innate immune response and the high viral productive property of sMDCK cells, which helps optimize cell production processes and supports the production of cell-based influenza vaccines.

MDCK-Adaptive Mutation of A169S Changes Glycosylation Pattern of Hemagglutinin and Enhances MDCK-Based H7N9 Vaccine Virus Production without Loss of Antigenicity and Immunogenicity.

The adaptation of egg-derived H7N9 candidate vaccine virus (CVV) in the mammalian cell line is an approach to developing a high-growth virus strain for the mass production of vaccine manufacturing. The adaptive mutations that occur in hemagglutinin (HA) are critical to the activity and potency of the vaccine virus. Previously, we identified a new mutation of A169S in the HA protein of an MDCK-adapted H7N9 vaccine virus (A/Anhui/2013, RG268); however, whether and how this mutation affects vaccine potency remain to be investigated. In this study, we serially passaged RG268 in MDCK cells and found that the HA titer and the TCID50 of the passaged virus RG268-M5 were 4-fold (HA units/50 µL) and 3.5-fold (log10 TCID50/mL) higher than those of the original CVV. By inspecting tandem MS spectra, we identified a new glycosylation site at N167 near the receptor binding site of the HA protein of RG268-M5. Flow cytometry results revealed that RG268-M5 could efficiently infect MDCK cells and initiate viral protein replication as well as that of RG268. Though the new glycosylation site is in the antigenic epitope of viral HA protein, the HI assay result indicated that the antigenicity of RG268-M5 was similar to RG268. Additionally, immunizing mice with RG268-M5 mixed aluminum hydroxide could induce potent antibody responses against the homologous and heterologous H7N9 viruses in vitro whereas the titers were comparable with those from the RG268 group. These results provide in-depth structural information regarding the effects of site-specific glycosylation on virus properties, which have implications for novel avian influenza vaccine development.

The ABCG2 protein in vitro transports the xenobiotic thiabendazole and increases the appearance of its residues in milk.

Thiabendazole (TBZ) is a broad-spectrum anthelmintic and fungicide used in humans, animals, and agricultural commodities. TBZ residues are present in crops and animal products, including milk, posing a risk to food safety and public health. ABCG2 is a membrane transporter which affects bioavailability and milk secretion of xenobiotics. Therefore, the aim of this work was to characterize the role of ABCG2 in the in vitro transport and secretion into milk of 5-hydroxythiabendazole (5OH-TBZ), the main TBZ metabolite. Using MDCK-II polarized cells transduced with several species variants of ABCG2, we first demonstrated that 5OH-TBZ is efficiently in vitro transported by ABCG2. Subsequently, using Abcg2 knockout mice, we demonstrated that 5OH-TBZ secretion into milk was affected by Abcg2, with a more than 2-fold higher milk concentration and milk to plasma ratio in wild-type mice compared to their Abcg2-/- counterpart.

Enhanced Downstream Processing for a Cell-Based Avian Influenza (H5N1) Vaccine.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Predicting the intrinsic membrane permeability of Caco-2/MDCK cells by the solubility-diffusion model.

Membrane permeability is one of the main determinants for the absorption, distribution, metabolism and excretion of compounds and is therefore of crucial importance for successful drug development. Experiments with artificial phospholipid membranes have shown that the intrinsic membrane permeability (P0) of compounds is well-predicted by the solubility-diffusion model (SDM). However, using the solubility-diffusion model to predict the P0 of biological Caco-2 and MDCK cell membranes has proven unreliable so far. Recent publications revealed that many published P0 extracted from Caco-2 and MDCK experiments are incorrect. In this work, we therefore used a small self-generated set as well as a large revised set of experimental Caco-2 and MDCK data from literature to compare experimental and predicted P0. The P0 extracted from Caco-2 and MDCK experiments were systematically lower than the P0 predicted by the solubility-diffusion model. However, using the following correlation: log P0,Caco-2/MDCK = 0.84 log P0,SDM - 1.85, P0 of biological Caco-2 and MDCK cell membranes was well-predicted by the solubility-diffusion model.

The Screening and Mechanism of Influenza-Virus Sensitive MDCK Cell Lines for Influenza Vaccine Production.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin-Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

Pitfalls in evaluating permeability experiments with Caco-2/MDCK cell monolayers.

When studying the transport of molecules across biological membranes, intrinsic membrane permeability (P0) is more informative than apparent permeability (Papp), because it eliminates external (setup-specific) factors, provides consistency across experiments and mechanistic insight. It is thus an important building block for modeling the total permeability in any given scenario. However, extracting P0 is often difficult, if not impossible, when the membrane is not the dominant transport resistance. In this work, we set out to analyze Papp values measured with Caco-2/MDCK cell monolayers of 69 literature references. We checked the Papp values for a total of 318 different compounds for the extractability of P0, considering possible limitations by aqueous boundary layers, paracellular transport, recovery issues, active transport, a possible proton flux limitation, and sink conditions. Overall, we were able to extract 77 reliable P0 values, which corresponds to about one quarter of the total compounds analyzed, while about half were limited by the diffusion through the aqueous layers. Compared to an existing data set of P0 values published by Avdeef, our approach resulted in a much higher exclusion of compounds. This is a consequence of stricter compound- and reference-specific exclusion criteria, but also because we considered possible concentration-shift effects due to different pH values in the aqueous layers, an effect only recently described in literature. We thus provide a consistent and reliable set of P0, e.g. as a basis for future modeling.

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy.

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes - the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines - the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.

Effects of Aqueous Boundary Layers and Paracellular Transport on the Efflux Ratio as a Measure of Active Transport Across Cell Layers.

The efflux ratio (ER), determined by Caco-2/MDCK assays, is the standard in vitro metric to establish qualitatively whether a compound is a substrate of an efflux transporter. However, others have also enabled the utilisation of this metric quantitatively by deriving a relationship that expresses the ER as a function of the intrinsic membrane permeability of the membrane (P0) as well as the permeability of carrier-mediated efflux (Ppgp). As of yet, Ppgp cannot be measured directly from transport experiments or otherwise, but the ER relationship provides easy access to this value if P0 is known. However, previous derivations of this relationship failed to consider the influence of additional transport resistances such as the aqueous boundary layers (ABLs) and the filter on which the monolayer is grown. Since single fluxes in either direction can be heavily affected by these experimental artefacts, it is crucial to consider the potential impact on the ER. We present a model that includes these factors and show both mathematically and experimentally that this simple ER relationship also holds for the more realistic scenario that does not neglect the ABLs/filter. Furthermore, we also show mathematically how paracellular transport affects the ER, and we experimentally confirm that paracellular dominance reduces the ER to unity and can mask potential efflux.

Automated measurement of transepithelial electrical resistance (TEER) in 96-well transwells using ECIS TEER96: Single and multiple time point assessments.

Transepithelial electrical resistance (TEER) is a widely used technique for quantifying the permeability of epithelial and endothelial cell layers. However, traditional methods of measuring TEER are limited to single timepoint measurements and can subject cells to an altered environment during the measurement. Here, we assessed the validity of TEER measurements by the ECIS TEER96 device, which is designed to take continuous TEER measurements of a cell culture system in a standard laboratory incubator. We found that the instrument accurately measures TEER across TEER values ranging from 10 to 2050 Ω*cm2 and is more accurate than the manual epithelial voltohmmeter electrode at high TEER values. Furthermore, the high-resolution measurements provided by the device allowed for a unique insight into the mechanisms and kinetics of cells in vitro. To demonstrate the continuous measurement capability of the device, we tracked the formation of an MDCKI cell monolayer until TEER plateaued. Furthermore, we treated Caco-2 monolayers with different concentrations of DMSO and the antimicrobial and surfactant compound benzethonium chloride to measure disruptions to barrier integrity. Treatment of both compounds resulted in concentration-dependent loss of barrier integrity. Our results suggest that the ECIS TEER96 device is a reliable and convenient option for measuring TEER in cell cultures and can provide valuable insights into the behavior of cells in vitro. This technology will be especially useful for increasing throughput of drug permeability assays, inflammation studies, and gaining better understanding of disease states in a cell culture system.