Corrigendum to Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment.

This corrects the article DOI: 10.14573/altex.2212081.

Minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA).

Standard information reporting helps to ensure that assay conditions and data are consistently reported and to facilitate inter-laboratory comparisons. Here, we present recommendations on minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA). The TEER assay is extensively used to evaluate the health of an epithelial/endothelial cell culture model and as an indicator of the potential toxicity of a test substance. This publication is the result of an international collaboration─called the RespTox (Respiratory Toxicity) Collaborative─through which twelve laboratories shared their protocols for assessing the barrier function of respiratory epithelial cells using the TEER assay following exposure to substances. The protocols from each laboratory were reviewed to identify general steps for performing the TEER assay, interlaboratory differences between steps, the rationale for differences, whether these differences impact results or cross-laboratory comparisons between TEER measurements. While the MIRTA recommendations are focused on respiratory epithelial cell systems, these recommendations can be adapted for other cell systems that form barriers. The use of these recommendations will support data transparency and reproducibility, reduce challenges in data interpretation, enable cross-laboratory comparisons, help assess study quality, and facilitate the incorporation of the TEER assay into national and international testing guidance.

Can TK-TD modelling bridge the gap between in vitro and in vivo mammalian toxicity data?

Repeated dietary dose testing is used to assess longer term toxicity of chemicals, such as pesticides, to mammals. However, the internal pesticide concentration varies significantly as feeding rate relative to body size fluctuates over time. Toxicokinetic-toxicodynamic (TK-TD) models can estimate internal toxicant concentration over time and link this directly to observed effects on endpoints such as the growth rate of laboratory rats. Using TK-TD models it is therefore possible to predict the effects that would result from a constant internal concentration of a pesticide. This presents the possibility of comparison with data from in vitro experiments, potentially facilitating quantitative in vitro to in vivo extrapolation (QIVIVE). We used in vivo TK-TD models to identify relevant internal concentrations and then estimated the experimental conditions required to replicate these in cultured cells, using in vitro TK models. Cell population growth was measured, with a view to extrapolating through time and comparing effect sizes with in vivo predictions. However, observed cell proliferation was not significantly affected by the tested concentrations of any of the five pesticides in this study and so extrapolation was not possible. In light of this negative result, we highlight areas for future work towards QIVIVE of graded sublethal effects in mammals. The most pressing objective is improving the accuracy of in vivo TK predictions, which could be achieved with dietary dosing in TK studies.

Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies.

Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the "in vitro mammalian cell micronucleus test" (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line (p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.

A drug-incorporated-microparticle-eggshell-membrane-scaffold (DIMES) dressing: A novel biomaterial for localised wound regeneration.

Chronic wounds affect millions of people annually and have emotional and financial implications in addition to health issues. The current treatment for chronic wounds involves the repeated use of bandages and drugs such as antibiotics over an extended period. A cost-effective and convenient solution for wound healing is the development of drug-incorporated bandages. This study aimed to develop a biocompatible bandage made of drug-incorporated poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) and eggshell membrane (ESM) for cornea wound healing. ESM has desirable properties for wound healing and can be isolated from eggshells using acetic acid or ethylenediaminetetraacetic acid (EDTA) protocols. Fluorescein isothiocyanate-labelled Bovine Serum Albumin (FITC-BSA) was used as a model drug, and the PLGA MPs were fabricated using a solvent extraction method. The MPs were successfully attached to the fibrous layer of the ESM using NaOH. The surface features of the ESM samples containing MPs were studied using a field emission scanning electron microscope (FESEM) and compared with blank ESM images. The findings indicated that the MPs were attached to the ESM fibres and had similar shapes and sizes as the control MPs. The fibre diameters of the MPs samples were assessed using Fiji-ImageJ software, and no significant changes were observed compared to the blank ESM. The surface roughness, Ra values, of the MPs incorporated ESM samples were evaluated and compared to the blank ESM, and no significant changes were found. Fourier transform infrared (FTIR) spectroscopy was used to analyse the chemical Composition of the bandage, and the spectra showed that the FBM were effectively incorporated into the ESM. The FTIR spectra identified the major peaks of the natural ESM and the PLGA polymer in the bandage. The bandage was transparent but had a reduced visibility in the waterproof test card method. The bandage achieved sustained drug release up to 10 days and was found to be biocompatible and non-toxic in a chorioallantoic membrane (CAM) assay. Overall, the drug-incorporated PLGA MPs-ESM bandage has great potential for treating chronic wounds.

Profiling alveolar macrophage responses to inhaled compounds using in vitro high content image analysis.

One of the main hurdles in the development of new inhaled medicines is the frequent observation of foamy macrophage (FM) responses in non-clinical studies in experimental animals, which raises safety concerns and hinders progress into clinical trials. We have investigated the potential of a novel multi-parameter high content image analysis (HCIA) assay as an in vitro safety screening tool to predict drug induced FM. Rat (NR8383) and human U937-derived alveolar macrophages were exposed in vitro to a panel of model compounds with different biological activity, including inhaled bronchodilators, inhaled corticosteroids (ICS), phospholipidosis inducers and proapoptotic agents. An HCIA was utilized to produce drug-induced cell response profiles based on individual cell health, morphology and lipid content parameters. The profiles of both rat and human macrophage cell lines differentiated between cell responses to marketed inhaled drugs and compounds known to induce phospholipidosis and apoptosis. Hierarchical clustering of the aggregated data allowed identification of distinct cell profiles in response to exposure to phospholipidosis and apoptosis inducers. Additionally, in NR8383 cell responses formed two distinct clusters, associated with increased vacuolation with or without lipid accumulation. U937 cells presented a similar trend but appeared less sensitive to drug exposure and presented a narrower range of responses. These results indicate that our multi-parameter HCIA assay is suitable to generate characteristic drug-induced macrophage response profiles, thus enabling differentiation of foamy macrophage phenotypes associated with phospholipidosis and apoptosis. This approach shows great potential as pre-clinical in vitro screening tool for safety assessment of candidate inhaled medicines.

Skin permeation and penetration of mometasone furoate in the presence of emollients: An ex vivo evaluation of clinical application protocols.

Background: Topical corticosteroids (TCS) and emollients are developed independently by the pharmaceutical industry but are often used together in practice. There is potential for the TCS and emollient formulations to interact on the skin surface affecting TCS absorption into the skin. Clinical guidelines acknowledge this issue but lack an evidence base and differ in their recommendations. There is a current clinical need to establish whether the application protocol employed for TCS and emollient products can impact delivery of TCS to the skin. Objectives: To investigate whether the sequence of application of a TCS and emollient and the time between their application can affect TCS skin absorption. Methods: The delivery of mometasone furoate (MF) to ex vivo human skin was evaluated following the application of Elocon cream either 5 or 30 min, before and after three different emollients. Mechanistic explanation of the changes in drug absorption was provided by modelling the skin permeation data and Raman microscopy of mixed Elocon cream and emollient formulations. Results: A circa fivefold difference in MF absorption was observed depending on the emollient and application protocol. Applying Elocon cream at short intervals in relation to Hydromol intensive significantly increased MF absorption regardless of the application protocol. In contrast, applying Elocon cream after Diprobase cream or ointment significantly reduced MF absorption relative to Elocon cream alone or when Elocon cream was applied before these emollients. The changes in drug absorption observed were attributed to the presence of emollients altering Elocon cream formulation performance through different mechanisms, including introduction of penetration enhancing excipients and inducing drug crystallization in the mixed TCS emollient layer on the skin surface. Conclusions: Emollients can affect MF absorption in different ways depending on the emollient and sequence of administration. Using a 30 min gap between product applications may not be sufficient to mitigate emollient effects on TCS absorption.

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment.

New approach methodologies (NAMs) based on human biology enable the assessment of adverse biological effects of pharmaceuticals and other chemicals. Currently, however, it is unclear how NAMs should be used during drug development to improve human safety evaluation. A series of 5 workshops with 13 international experts (regulators, preclinical scientists, and NAMs developers) was conducted to identify feasible NAMs and to discuss how to exploit them in specific safety assessment contexts. Participants generated four "maps" of how NAMs can be exploited in the safety assessment of the liver, respiratory, cardiovascular, and central nervous systems. Each map shows relevant endpoints measured and tools used (e.g., cells, assays, platforms), and highlights gaps where further development and validation of NAMs remains necessary. Each map addresses the fundamental scientific requirements for the safety assessment of that organ system, providing users with guidance on the selection of appropriate NAMs. In addition to generating the maps, participants offered suggestions for encouraging greater NAM adoption within drug development and their inclusion in regulatory guidelines. A specific recommendation was that pharmaceutical companies should be more transparent about how they use NAMs in-house. As well as giving guidance for the four organ systems, the maps provide a template that could be used for additional organ safety testing contexts. Moreover, their conversion to an interactive format would enable users to drill down to the detail necessary to answer specific scientific and regulatory questions.

Polymer Architecture Effects on Poly(N,N-Diethyl Acrylamide)-b-Poly(Ethylene Glycol)-b-Poly(N,N-Diethyl Acrylamide) Thermoreversible Gels and Their Evaluation as a Healthcare Material.

Thermoreversible gels which transition between liquid-like and solid-like states when warmed have enabled significant novel healthcare technologies. Poly(N,N-diethyl acrylamide) (PDEA) is a thermoresponsive polymer which can be used as a trigger to form thermoreversible gels, however its use in these materials is limited and crucial design principles are unknown. Herein ABA copolymers with the structure PDEA-b-poly(ethylene glycol) (PEG)-b-PDEA are synthesized to give four block copolymers with varied molecular weight of PDEA and PEG blocks. Rheometry on solutions of the block copolymers reveals that high molecular weight PEG blocks are required to form thermoreversible gels with predominantly solid-like behavior. Furthermore, small-angle X-ray scattering elucidates clear differences in the nanostructure of the copolymer library which can be linked to distinct rheological behaviors. A thermoreversible gel formulation based on PDEA (20 kDa)-b-PEG (10 kDa)-b-PDEA (20 kDa) is designed by optimizing the polymer concentration and ionic strength. It is found that the gel is mucoadhesive, stable, and non-toxic, as well as giving controlled release of a hydrophobic drug. Overall, this study provides insight into the effect of polymer architecture on the nanostructure and rheology of PDEA-b-PEG-b-PDEA and presents the development of a highly functional thermoreversible gel with high promise for healthcare applications.

High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses.

Introduction: Lung diseases are an increasing global health burden affecting millions of people worldwide. Only a few new inhaled medicines have reached the market in the last 30 years, in part due to foamy alveolar macrophage (FAM) responses observed in pre-clinical rat studies. The induction mechanism and signaling pathways involved in the development of highly vacuolated 'foamy' phenotype is not known. Furthermore, it has not been determined if these observations are adaptive or adverse responses. Aim: To determine if high content image analysis techniques can distinguish between alveolar macrophage activation (LPS/IFN-γ activated and IL-4 activated macrophages) and if this could be applied to understanding the generation of 'foamy' macrophage phenotypes. Methods: NR8383 rat alveolar macrophages were stimulated with a mix of cytokines (LPS/IFN-γ or IL-4) for 24 h. The cells were further exposed to FAM inducing-compounds amiodarone and staurosporine. Following 24 h incubation, phagocytosis and lipid accumulation were measured using flow cytometry and high content image analysis techniques. The alveolar macrophages responses after exposure to cytokines were assessed by evaluation: (i) cell surface and biochemical markers such as: nitric oxide production, arginase-1 activity and MRC-1 receptor expression (ii) cellular morphology (iii) cellular functionality (phagocytic activity and lipids accumulation). Results: Macrophages activated with LPS/IFN-γ showed distinct morphological (increased vacuolation) features and functionality (increased lipidosis, decreased phagocytic activity). Foamy macrophage phenotypes induced by amiodarone also displayed characteristics of proinflammatory macrophages (significantly increased nitric oxide production, increased vacuolation and lipidosis and decreased phagocytosis). In contrast, staurosporine treatment resulted in increased NO production, as well as arginase-1 activity. Conclusion: High content image analysis was able to determine distinct differences in morphology between non-activated and LPS/IFN-γ activated macrophages, characterized by increased vacuolation and lipidosis. When exposed to compounds that induce a FAM phenotype, healthy non-activated macrophages displayed proinflammatory (amiodarone) or pro-apoptotic (staurosporine) characteristics but these responses were independent of a change in activation status. This technique could be applied in early drug discovery safety assessment to identify immune responses earlier and increase the understanding of alveolar macrophage responses to new molecules challenge in development of new inhalation therapies, which in turn will enhance decision-making in an early safety assessment of novel drug candidates.

The eggshell membrane: A potential biomaterial for corneal wound healing.

The eggshell membrane (ESM) is an abundant resource with innate complex structure and composition provided by nature. With at least 60 million tonnes of hen eggs produced globally per annum, utilisation of this waste resource is highly attractive in positively impacting sustainability worldwide. Given the morphology and mechanical properties of this membrane, it has great potential as a biomaterials for wound dressing. However, to date, no studies have demonstrated nor reported this application. As such, the objective of this investigation was to identify and optimise a reproducible extraction protocol of the ESM and to assess the physical, chemical, mechanical and biological properties of the substrate with a view to use as a wound dressing. ESM samples were isolated by either manual peeling (ESM-strip) or via extraction using acetic acid [ESM-A0.5] or ethylenediaminetetraacetic acid, EDTA [ESM-E0.9]. Energy dispersive X-ray spectroscopy (EDS) confirmed that there were no traces of calcium residues from the extraction process. Fourier transform infrared (FTIR) spectroscopy revealed that the extraction method (acetic acid and EDTA) did not alter the chemical structures of the ESM and also clarified the composition of the fibrous proteins of the ESM. Scanning electron microscopy (SEM) analyses revealed a three-layer composite structure of the ESM: an inner layer as continuous, dense and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane) and the outer layer (outer shell membrane) of larger fibres. Material properties including optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical profiling of the ESM samples were performed and demonstrated suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. Taken together, these results document the development of a novel sustainable biomaterial that may be used for ophthalmic wounds and/or other biomedical therapies.

Co-Incubation with PPARß/δ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery.

Peroxisome proliferator activated receptor beta/delta (PPARß/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARß/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARß/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARß/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L-165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARß/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARß/δ agonists leads to a significant increase in Pdk-4 and Angptl-4 mRNA expression, which is significantly decreased in the presence of PPARß/δ antagonists. Docking using computational chemistry methods indicates that PPARß/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARß/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARß/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARß/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARß/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.

Investigating the Suitability of High Content Image Analysis as a Tool to Assess the Reversibility of Foamy Alveolar Macrophage Phenotypes In Vitro.

Many potential inhaled medicines fail during development due to the induction of a highly vacuolated or "foamy" alveolar macrophage phenotype response in pre-clinical studies. There is limited understanding if this response to an inhaled stimulus is adverse or adaptive, and additionally if it is a transient or irreversible process. The aim of this study was to evaluate whether high content image analysis could distinguish between different drug-induced foamy macrophage phenotypes and to determine the extent of the reversibility of the foamy phenotypes by assessing morphological changes over time. Alveolar-like macrophages derived from the human monocyte cell line U937 were exposed for 24 h to compounds known to induce a foamy macrophage phenotype (amiodarone, staurosporine) and control compounds that are not known to cause a foamy macrophage phenotype in vitro (fluticasone and salbutamol). Following drug stimulation, the cells were rested in drug-free media for the subsequent 24 or 48 h. Cell morphometric parameters (cellular and nuclear area, vacuoles numbers and size) and phospholipid content were determined using high content image analysis. The foamy macrophage recovery was dependent on the mechanism of action of the inducer compound. Amiodarone toxicity was associated with phospholipid accumulation and morphometric changes were reversed when the stimulus was removed from culture environment. Conversely cells were unable to recover from exposure to staurosporine which initiates the apoptosis pathway. This study shows that high content analysis can discriminate between different phenotypes of foamy macrophages and may contribute to better decision making in the process of new drug development.

Comparison of Oral, Intranasal and Aerosol Administration of Amiodarone in Rats as a Model of Pulmonary Phospholipidosis.

'Foamy' alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by differential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.

Predicting Skin Permeability by Means of Computational Approaches: Reliability and Caveats in Pharmaceutical Studies.

The skin is the main barrier between the internal body environment and the external one. The characteristics of this barrier and its properties are able to modify and affect drug delivery and chemical toxicity parameters. Therefore, it is not surprising that permeability of many different compounds has been measured through several in vitro and in vivo techniques. Moreover, many different in silico approaches have been used to identify the correlation between the structure of the permeants and their permeability, to reproduce the skin behavior, and to predict the ability of specific chemicals to permeate this barrier. A significant number of issues, like interlaboratory variability, experimental conditions, data set building rationales, and skin site of origin and hydration, still prevent us from obtaining a definitive predictive skin permeability model. This review wants to show the main advances and the principal approaches in computational methods used to predict this property, to enlighten the main issues that have arisen, and to address the challenges to develop in future research.

Diffusion through the ex vivo vitreal body - Bovine, porcine, and ovine models are poor surrogates for the human vitreous.

The human vitreous humour is a complex gel structure whose composition and physical properties can vary considerably from person to person and also change with age. To date, the viscoelastic properties of the human vitreous gel has not been thoroughly investigated and despite many years of intensive research, an ideal vitreous substitute remains a challenge. Understanding the physical structure and properties of the vitreous is of fundamental and therapeutic interest, providing a clear insight into diffusion and transport of administered ophthalmic drug molecules into the vitreous. A number of mammalian surrogates, mainly bovine, porcine and ovine vitreous humours have been used in the literature as a means of studying ophthalmic drug transport and diffusion. In this study, the mechanical, physical and rheological properties of ovine, porcine, and bovine surrogates were investigated and compared to human vitreous. In addition, a bespoke Franz cell construct was used to compare the diffusion of a model drug (fluorescein) through vitreous samples. Despite the similarity in rheological properties between bovine, porcine and human vitreous samples, diffusion of fluorescein through the different vitreous samples revealed great differences in values of steady-state flux and diffusion coefficient. In addition, a first-generation vitreous mimic, composed of 4.5 mg/mL hyaluronic acid with complex viscosity of 0.3 ±â€¯0.01 Pa has been evaluated and was demonstrated to be a better mimic of the human vitreous than the mammalian samples investigated.

Influence of surface geometry on the culture of human cell lines: A comparative study using flat, round-bottom and v-shaped 96 well plates.

In vitro cell based models have been invaluable tools for studying cell behaviour and for investigating drug disposition, toxicity and potential adverse effects of administered drugs. Within this drug discovery pipeline, the ability to assess and prioritise candidate compounds as soon as possible offers a distinct advantage. However, the ability to apply this approach to a cell culture study is limited by the need to provide an accurate, in vitro-like, microenvironment in conjunction with a low cost and high-throughput screening (HTS) methodology. Although the geometry and/or alignment of cells has been reported to have a profound influence on cell growth and differentiation, only a handful of studies have directly compared the growth of a single cell line on different shaped multiwell plates the most commonly used substrate for HTS, in vitro, studies. Herein, the impact of various surface geometries (flat, round and v-shaped 96 well plates), as well as fixed volume growth media and fixed growth surface area have been investigated on the characteristics of three commonly used human cell lines in biopharmaceutical research and development, namely ARPE-19 (retinal epithelial), A549 (alveolar epithelial) and Malme-3M (dermal fibroblastic) cells. The effect of the surface curvature on cells was characterised using a combination of a metabolic activity assay (CellTiter AQ/MTS), LDH release profiles (CytoTox ONE) and absolute cell counts (Guava ViaCount), respectively. In addition, cell differentiation and expression of specific marker proteins were determined using flow cytometry. These in vitro results confirmed that surface topography had a significant effect (p < 0.05) on cell activity and morphology. However, although specific marker proteins were expressed on day 1 and 5 of the experiment, no significant differences were seen between the different plate geometries (p < 0.05) at the later time point. Accordingly, these results highlight the impact of substrate geometry on the culture of a cell line and the influence it has on the cells' correct growth and differentiation characteristics. As such, these results provide important implications in many aspects of cell biology the development of a HTS, in vitro, cell based systems to further investigate different aspects of toxicity testing and drug delivery.

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

In Vitro Cell Models for Ophthalmic Drug Development Applications.

Tissue engineering is a rapidly expanding field that aims to establish feasible techniques to fabricate biologically equivalent replacements for diseased and damaged tissues/organs. Emerging from this prospect is the development of in vitro representations of organs for drug toxicity assessment. Due to the ever-increasing interest in ocular drug delivery as a route for administration as well as the rise of new ophthalmic therapeutics, there is a demand for physiologically accurate in vitro models of the eye to assess drug delivery and safety of new ocular medicines. This review summarizes current existing ocular models and highlights the important factors and limitations that need to be considered during their use.

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1.

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.