The effects of fine particulate matter (SRM 2786) on three different 3D lung models exposed at the air-liquid interface - A comparative study.

3D cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to animal experiments for hazard and risk assessment of inhaled compounds. This study compares cocultures composed of either Calu-3, A549 or HBEC3-KT lung epithelial cells, cultured together with THP-1-derived macrophages and EA.hy926 endothelial cells, in terms of barrier capacity and responses to a standard reference sample of fine particulate matter (SRM 2786). High-content imaging analysis revealed a similar cellular composition between the different cell models. The 3D cell cultures with Calu-3 cells showed the greatest barrier capacity, as measured by transepithelial electrical resistance and permeability to Na-fluorescein. Mucus production was detected in 3D cell cultures based on Calu-3 and A549 cells. Exposure to SRM 2786 at ALI increased cytokine release and expression of genes associated with inflammation and xenobiotic metabolism. Moreover, the presence of THP-1-derived macrophages was central to the cytokine responses in all cell models. While the different 3D cell culture models produced qualitatively similar responses, more pronounced pro-inflammatory responses were observed in the basolateral compartment of the A549 and HBEC3-KT models compared to the Calu-3 model, likely due to their reduced barrier capacity and lower retention of secreted mediators in the apical compartment.

Application of microphysiological systems for nonclinical evaluation of cell therapies.

Microphysiological systems (MPS) are gaining broader application in the pharmaceutical industry but have primarily been leveraged in early discovery toxicology and pharmacology studies with small molecules. The adoption of MPS offers a promising avenue to reduce animal use, improve in-vitro-to-in-vivo translation of pharmacokinetics/pharmacodynamics and toxicity correlation, and provide mechanistic understanding of model species suitability. While MPS have demonstrated utility in these areas with small molecules and biologics, cell therapeutic MPS models in drug development have not been fully explored, let alone validated. Distinguishing features of MPS, including long-term viability and physiologically relevant expression of functional enzymes, receptors, and pharmacological targets make them attractive tools for nonclinical characterization. However, there is currently limited published evidence of MPS being utilized to study the disposition, metabolism, pharmacology, and toxicity profiles of cell therapies. This review provides an industry perspective on the nonclinical application of MPS on cell therapies, first with a focus on oncology applications followed by examples in regenerative medicine.

Microphysiological systems (MPS) are advanced cell models, applied in the pharmaceutical industry to characterize novel therapies. While their application in studies of small molecule therapies has been very successful, the use of these models to study cell therapies has been limited. Cell therapies consist of cells and are living drugs, often with complex biological mechanisms of action, which can be very challenging to study. However, MPS have several features that make them attractive for studying cell therapies, including possibilities for longer-term studies and the ability to mimic physiologically relevant biological functions. MPS can mimic complex biological systems and processes, as such, the adoption of MPS offers a promising avenue to reduce the use of animals in the characterization of novel therapies. This review provides an industry perspective on current challenges and highlights opportunities for using MPS in the development of cell therapies.

Single-cell RNA sequencing reveals 2D cytokine assay can model atopic dermatitis more accurately than immune-competent 3D setup.

Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.

Applications of 3D Bioprinting Technology to Brain Cells and Brain Tumor Models: Special Emphasis to Glioblastoma.

Primary brain tumor is one of the most fatal diseases. The most malignant type among them, glioblastoma (GBM), has low survival rates. Standard treatments reduce the life quality of patients due to serious side effects. Tumor aggressiveness and the unique structure of the brain render the removal of tumors and the development of new therapies challenging. To elucidate the characteristics of brain tumors and examine their response to drugs, realistic systems that mimic the tumor environment and cellular crosstalk are desperately needed. In the past decade, 3D GBM models have been presented as excellent platforms as they allowed the investigation of the phenotypes of GBM and testing innovative therapeutic strategies. In that scope, 3D bioprinting technology offers utilities such as fabricating realistic 3D bioprinted structures in a layer-by-layer manner and precisely controlled deposition of materials and cells, and they can be integrated with other technologies like the microfluidics approach. This Review covers studies that investigated 3D bioprinted brain tumor models, especially GBM using 3D bioprinting techniques and essential parameters that affect the result and quality of the study like frequently used cells, the type and physical characteristics of hydrogel, bioprinting conditions, cross-linking methods, and characterization techniques.

Understanding the Impact of Polyunsaturated Fatty Acids on Age-Related Macular Degeneration: A Review.

Age-related Macular Degeneration (AMD) is a multifactorial ocular pathology that destroys the photoreceptors of the macula. Two forms are distinguished, dry and wet AMD, with different pathophysiological mechanisms. Although treatments were shown to be effective in wet AMD, they remain a heavy burden for patients and caregivers, resulting in a lack of patient compliance. For dry AMD, no real effective treatment is available in Europe. It is, therefore, essential to look for new approaches. Recently, the use of long-chain and very long-chain polyunsaturated fatty acids was identified as an interesting new therapeutic alternative. Indeed, the levels of these fatty acids, core components of photoreceptors, are significantly decreased in AMD patients. To better understand this pathology and to evaluate the efficacy of various molecules, in vitro and in vivo models reproducing the mechanisms of both types of AMD were developed. This article reviews the anatomy and the physiological aging of the retina and summarizes the clinical aspects, pathophysiological mechanisms of AMD and potential treatment strategies. In vitro and in vivo models of AMD are also presented. Finally, this manuscript focuses on the application of omega-3 fatty acids for the prevention and treatment of both types of AMD.

A miniaturized multicellular platform to mimic the 3D structure of the alveolar-capillary barrier.

Several diseases affect the alveoli, and the efficacy of medical treatments and pharmaceutical therapies is hampered by the lack of pre-clinical models able to recreate in vitro the diseases. Microfluidic devices, mimicking the key structural and compositional features of the alveoli, offer several advantages to medium and high-throughput analysis of new candidate therapies. Here, we developed an alveolus-on-a-chip recapitulating the microanatomy of the physiological tissue by including the epithelium, the fibrous interstitial layer and the capillary endothelium. A PDMS device was obtained assembling a top layer and a bottom layer obtained by replica molding. A polycaprolactone/gelatin (PCL-Gel) electrospun membrane was included within the two layers supporting the seeding of 3 cell phenotypes. Epithelial cells were grown on a fibroblast-laden collagen hydrogel located on the top side of the PCL-Gel mats while endothelial cells were seeded on the basolateral side of the membrane. The innovative design of the microfluidic device allows to replicate both cell-cell and cell-extracellular matrix interactions according to the in vivo cell arrangement along with the establishment of physiologically relevant air-liquid interface conditions. Indeed, high cell viability was confirmed for up to 10 days and the formation of a tight endothelial and epithelial barrier was assessed by immunofluorescence assays.

Decades Long Involvement of THP-1 Cells as a Model for Macrophage Research: A Comprehensive Review.

Over the years, researchers have endeavored to identify dependable and reproducible in vitro models for examining macrophage behavior under controlled conditions. The THP-1 cell line has become a significant and widely employed tool in macrophage research within these models. Originating from the peripheral blood of individuals with acute monocytic leuke-mia, this human monocytic cell line can undergo transformation into macrophage-like cells, closely mirroring primary human macrophages when exposed to stimulants. Macrophages play a vital role in the innate immune system, actively regulating inflammation, responding to infec-tions, and maintaining tissue homeostasis. A comprehensive understanding of macrophage bi-ology and function is crucial for gaining insights into immunological responses, tissue healing, and the pathogenesis of diseases such as viral infections, autoimmune disorders, and neoplastic conditions. This review aims to thoroughly evaluate and emphasize the extensive history of THP-1 cells as a model for macrophage research. Additionally, it will delve into the significance of THP-1 cells in advancing our comprehension of macrophage biology and their invaluable contributions to diverse scientific domains.

Characterization and optimization of variability in a human colonic epithelium culture model.

Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem-cell-based cultures makes development of useful models a challenge; the stochastic nature of stem-cell differentiation interferes with the ability to build and validate reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut® Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling cell passage number reduces variability and maximizes physiological relevance of the model. In a case study where passage number was optimized, distinct cytokine responses were observed among four human donors, indicating that biological variability can be detected in cell cultures originating from diverse human sources. These findings highlight key considerations for designing assays that can be applied to additional primary-cell derived systems, as well as establish utility of the RepliGut® Planar platform for robust development of human-predictive drug-response assays.

Animal models are frequently used as tools for studying gastrointestinal (GI) disease, but they poorly replicate the complexities of the human gut limiting the clinical translation of new therapeutics in development. Human stem cell derived models can better recapitulate human GI physiology, but the inherent dynamic nature of stem cells introduces variability in culture performance. We identified sources of variability in the primary stem-cell derived RepliGut® Planar model to develop robust and reliable assays that can improve preclinical therapeutic development for GI disease. Analysis of barrier formation, gene expression, and cytokine responses demonstrated that controlling cell passage number reduces variability and maximizes physiological relevance of the model. These findings highlight key assay design considerations that can be applied to additional primary-cell derived systems. Availability of reliable and physiologically relevant cell-based models can reduce animal testing, improve research accuracy, and make new treatments more relevant and effective for patients.

More than a number: Incorporating the aged phenotype to improve in vitro and in vivo modeling of neurodegenerative disease.

Age is the number one risk factor for developing a neurodegenerative disease (ND), such as Alzheimer's disease (AD) or Parkinson's disease (PD). With our rapidly ageing world population, there will be an increased burden of ND and need for disease-modifying treatments. Currently, however, translation of research from bench to bedside in NDs is poor. This may be due, at least in part, to the failure to account for the potential effect of ageing in preclinical modelling of NDs. While ageing can impact upon physiological response in multiple ways, only a limited number of preclinical studies of ND have incorporated ageing as a factor of interest. Here, we evaluate the aged phenotype and highlight the critical, but unmet, need to incorporate aspects of this phenotype into both the in vitro and in vivo models used in ND research. Given technological advances in the field over the past several years, we discuss how these could be harnessed to create novel models of ND that more readily incorporate aspects of the aged phenotype. This includes a recently described in vitro panel of ageing markers, which could help lead to more standardised models and improve reproducibility across studies. Importantly, we cannot assume that young cells or animals yield the same responses as seen in the context of ageing; thus, an improved understanding of the biology of ageing, and how to appropriately incorporate this into the modelling of ND, will ensure the best chance for successful translation of new therapies to the aged patient.

A mechanobiological model of bone metastasis reveals that mechanical stimulation inhibits the pro-osteolytic effects of breast cancer cells.

Bone is highly susceptible to cancer metastasis, and both tumor and bone cells enable tumor invasion through a "vicious cycle" of biochemical signaling. Tumor metastasis into bone also alters biophysical cues to both tumor and bone cells, which are highly sensitive to their mechanical environment. However, the mechanobiological feedback between these cells that perpetuate this cycle has not been studied. Here, we develop highly advanced in vitro and computational models to provide an advanced understanding of how tumor growth is regulated by the synergistic influence of tumor-bone cell signaling and mechanobiological cues. In particular, we develop a multicellular healthy and metastatic bone model that can account for physiological mechanical signals within a custom bioreactor. These models successfully recapitulated mineralization, mechanobiological responses, osteolysis, and metastatic activity. Ultimately, we demonstrate that mechanical stimulus provided protective effects against tumor-induced osteolysis, confirming the importance of mechanobiological factors in bone metastasis development.

Vascularized tumor models for the evaluation of drug delivery systems: a paradigm shift.

As the conversion rate of preclinical studies for cancer treatment is low, user-friendly models that mimic the pathological microenvironment and drug intake with high throughput are scarce. Animal models are key, but an alternative to reduce their use would be valuable. Vascularized tumor-on-chip models combine great versatility with scalable throughput and are easy to use. Several strategies to integrate both tumor and vascular compartments have been developed, but few have been used to assess drug delivery. Permeability, intra/extravasation, and free drug circulation are often evaluated, but imperfectly recapitulate the processes at stake. Indeed, tumor targeting and chemoresistance bypass must be investigated to design promising cancer therapeutics. In vitro models that would help the development of drug delivery systems (DDS) are thus needed. They would allow selecting good candidates before animal studies based on rational criteria such as drug accumulation, diffusion in the tumor, and potency, as well as absence of side damage. In this review, we focus on vascularized tumor models. First, we detail their fabrication, and especially the materials, cell types, and coculture used. Then, the different strategies of vascularization are described along with their classical applications in intra/extravasation or free drug assessment. Finally, current trends in DDS for cancer are discussed with an overview of the current efforts in the domain.

Steering cell orientation through light-based spatiotemporal modulation of the mechanical environment.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved,in vitrotechniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3Din vitromodel systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

Modeling PAH Mixture Interactions in a Human In Vitro Organotypic Respiratory Model.

One of the most significant challenges in human health risk assessment is to evaluate hazards from exposure to environmental chemical mixtures. Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous contaminants typically found as mixtures in gaseous and particulate phases in ambient air pollution associated with petrochemicals from Superfund sites and the burning of fossil fuels. However, little is understood about how PAHs in mixtures contribute to toxicity in lung cells. To investigate mixture interactions and component additivity from environmentally relevant PAHs, two synthetic mixtures were created from PAHs identified in passive air samplers at a legacy creosote site impacted by wildfires. The primary human bronchial epithelial cells differentiated at the air-liquid interface were treated with PAH mixtures at environmentally relevant proportions and evaluated for the differential expression of transcriptional biomarkers related to xenobiotic metabolism, oxidative stress response, barrier integrity, and DNA damage response. Component additivity was evaluated across all endpoints using two independent action (IA) models with and without the scaling of components by toxic equivalence factors. Both IA models exhibited trends that were unlike the observed mixture response and generally underestimated the toxicity across dose suggesting the potential for non-additive interactions of components. Overall, this study provides an example of the usefulness of mixture toxicity assessment with the currently available methods while demonstrating the need for more complex yet interpretable mixture response evaluation methods for environmental samples.

Emerging In Vitro and In Vivo Models: Hope for the Better Understanding of Cancer Progression and Treatment.

Various cancer models have been developed to aid the understanding of the underlying mechanisms of tumor development and evaluate the effectiveness of various anticancer drugs in preclinical studies. These models accurately reproduce the critical stages of tumor initiation and development to mimic the tumor microenvironment better. Using these models for target validation, tumor response evaluation, resistance modeling, and toxicity comprehension can significantly enhance the drug development process. Herein, various in vivo or animal models are presented, typically consisting of several mice and in vitro models ranging in complexity from transwell models to spheroids and CRISPR-Cas9 technologies. While in vitro models have been used for decades and dominate the early stages of drug development, they are still limited primary to simplistic tests based on testing on a single cell type cultivated in Petri dishes. Recent advancements in developing new cancer therapies necessitate the generation of complicated animal models that accurately mimic the tumor's complexity and microenvironment. Mice make effective tumor models as they are affordable, have a short reproductive cycle, exhibit rapid tumor growth, and are simple to manipulate genetically. Human cancer mouse models are crucial to understanding the neoplastic process and basic and clinical research improvements. The following review summarizes different in vitro and in vivo metastasis models, their advantages and disadvantages, and their ability to serve as a model for cancer research.

Human In Vitro Models of Neuroenergetics and Neurometabolic Disturbances: Current Advances and Clinical Perspectives.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.

Revolutionizing Dental Implant Research: A Systematic Review on Three-Dimensional In Vitro Models.

Dental implants have been clinically used for almost five decades with high success rates. In vitro research models used in implant dentistry are limited to two-dimensional experiments, which are reproducible and well adapted to evaluate a single parameter but do not reproduce the complexity of clinical settings. On the contrary, the in vivo research models using animals offer similar histological and anatomical features to humans, and tissue healing can be close to a clinical situation, but those models are usually accompanied with ethical concerns, and their outcomes could not be extrapolated to humans because of interspecies variabilities. This makes the development of novel in vitro models that recapitulate physiological events occurring during dental implant placement of particular interest for current research in dentistry. Also, such models could be challenged by setting a pathological environment (peri-implantitis) to better understand the disease and eventually serve as a platform to evaluate novel treatment modalities. The aim of this systematic literature review was to cover all the in vitro three-dimensional (3D) complex models available for research in implant dentistry. To accomplish this, a comprehensive search of the literature present on Scopus and PubMed databases was done using specific keywords, as well as inclusion/exclusion criteria. Out of 1334 articles found, we have finally included 27 articles in this review with publication dates between 2001 and 2022. In those articles, the 3D models were designed to study tissue-implant interface behavior in bone or gingival tissue. The articles focused on simulating implant integration, evaluating the effect of different conditions on implant integration, or developing an infection model for the implant integration process. The methods used involved implant material and cells organized in a specific 3D structure. The 3D models developed were able to simulate the process of dental implant osseo- and soft tissue integration and lead to results comparable with conventional in vitro and in vivo models. A relatively limited number of articles were obtained, which indicates that this is an emerging field, highly dependent on progresses made in biotechnologies and tissue engineering, and that further investigation is needed to enhance these 3D in vitro models.

Physiologically based kinetic (PBK) modeling as a new approach methodology (NAM) for predicting systemic levels of gut microbial metabolites.

There is a clear need to develop new approach methodologies (NAMs) that combine in vitro and in silico testing to reduce and replace animal use in chemical risk assessment. Physiologically based kinetic (PBK) models are gaining popularity as NAMs in toxico/pharmacokinetics, but their coverage of complex metabolic pathways occurring in the gut are incomplete. Chemical modification of xenobiotics by the gut microbiome plays a critical role in the host response, for example, by prolonging exposure to harmful metabolites, but there is not a comprehensive approach to quantify this impact on human health. There are examples of PBK models that have implemented gut microbial biotransformation of xenobiotics with the gut as a dedicated metabolic compartment. However, the integration of microbial metabolism and parameterization of PBK models is not standardized and has only been applied to a few chemical transformations. A challenge in this area is the measurement of microbial metabolic kinetics, for which different fermentation approaches are used. Without a standardized method to measure gut microbial metabolism ex vivo/in vitro, the kinetic constants obtained will lead to conflicting conclusions drawn from model predictions. Nevertheless, there are specific cases where PBK models accurately predict systemic concentrations of gut microbial metabolites, offering potential solutions to the challenges outlined above. This review focuses on models that integrate gut microbial bioconversions and use ex vivo/in vitro methods to quantify metabolic constants that accurately represent in vivo conditions.

Developmental expression of catecholamine system in the human placenta and rat fetoplacental unit.

Catecholamines norepinephrine and dopamine have been implicated in numerous physiological processes within the central nervous system. Emerging evidence has highlighted the importance of tightly regulated monoamine levels for placental functions and fetal development. However, the complexities of synthesis, release, and regulation of catecholamines in the fetoplacental unit have not been fully unraveled. In this study, we investigated the expression of enzymes and transporters involved in synthesis, degradation, and transport of norepinephrine and dopamine in the human placenta and rat fetoplacental unit. Quantitative PCR and Western blot analyses were performed in early-to-late gestation in humans (first trimester vs. term placenta) and mid-to-late gestation in rats (placenta and fetal brain, intestines, liver, lungs, and heart). In addition, we analyzed the gene expression patterns in isolated primary trophoblast cells from the human placenta and placenta-derived cell lines (HRP-1, BeWo, JEG-3). In both human and rat placentas, the study identifies the presence of only PNMT, COMT, and NET at the mRNA and protein levels, with the expression of PNMT and NET showing gestational age dependency. On the other hand, rat fetal tissues consistently express the catecholamine pathway genes, revealing distinct developmental expression patterns. Lastly, we report significant transcriptional profile variations in different placental cell models, emphasizing the importance of careful model selection for catecholamine metabolism/transport studies. Collectively, integrating findings from humans and rats enhances our understanding of the dynamic regulatory mechanisms that underlie catecholamine dynamics during pregnancy. We identified similar patterns in both species across gestation, suggesting conserved molecular mechanisms and potentially shedding light on shared biological processes influencing placental development.

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.