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The lack of representative in vitro models recapitulating human tendon (patho)physiology is among the major factors hindering consistent progress in the knowledge‐based development of adequate therapies for tendinopathy.Here, an organotypic 3D tendon‐on‐chip model is designed that allows studying the spatiotemporal dynamics of its cellular and molecular mechanisms.Combining the synergistic effects of a bioactive hydrogel matrix with the biophysical cues of magnetic microfibers directly aligned on the microfluidic chip, it is possible to recreate the anisotropic architecture, cell patterns, and phenotype of tendon intrinsic (core) compartment. When incorporated with vascular‐like vessels emulating the interface between its intrinsic‐extrinsic compartments, crosstalk with endothelial cells are found to drive stromal tenocytes toward a reparative profile. This platform is further used to study adaptive immune cell responses at the onset of tissue inflammation, focusing on interactions between tendon compartment tenocytes and circulating T cells.The proinflammatory signature resulting from this intra/inter‐cellular communication induces the recruitment of T cells into the inflamed core compartment and confirms the involvement of this cellular crosstalk in positive feedback loops leading to the amplification of tendon inflammation.Overall, the developed 3D tendon‐on‐chip provides a powerful new tool enabling mechanistic studies on the pathogenesis of tendinopathy as well as for assessing new therapies. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a lethal disorder characterized by relentless progression of lung fibrosis that causes respiratory failure and early death. Currently, no curative treatments are available, and existing therapies include a limited selection of antifibrotic agents that only slow disease progression. The development of novel therapeutics has been hindered by a limited understanding of the disease's etiology and pathogenesis. A significant challenge in developing new treatments and understanding IPF is the lack of in vitro models that accurately replicate crucial microenvironments. In response, three-dimensional (3D) in vitro models have emerged as powerful tools for replicating organ-level microenvironments seen in vivo. This review summarizes the state of the art in advanced 3D lung models that mimic many physiological and pathological processes observed in IPF. We begin with a brief overview of conventional models, such as 2D cell cultures and animal models, and then explore more advanced 3D models, focusing on lung-on-a-chip systems. We discuss the current challenges and future research opportunities in this field, aiming to advance the understanding of the disease and the development of novel devices to assess the effectiveness of new IPF treatments. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Are Sertoli cells (SCs) from adult Klinefelter men (47,XXY) capable of proliferating in vitro and maintaining their main phenotypical and functional characteristics as do SCs from adult 46,XY patients? SUMMARY ANSWER Isolated SCs from patients with Klinefelter syndrome (KS) can be expanded in vitro while maintaining their characteristics and a stable karyotype, similar to SCs from 46,XY patients. WHAT IS KNOWN ALREADY The mechanism leading to testicular tissue degeneration in KS is still unknown. A few recent studies highlight the main role played by SCs in the physiopathology of the disease, but new study models based on co-culture or testicular organoids are needed to further understand the SC's involvement in the mechanism of testicular degeneration and fibrosis, and to find therapeutical targets. KS SC expansion could be the first step towards developing such in vitro study models. SCs have been isolated from 46,XY men and expanded in vitro while maintaining the expression of phenotypical and functional markers, but propagation of SCs from KS men has not been achieved yet. STUDY DESIGN, SIZE, DURATION Testicular tissue was obtained during a testicular sperm extraction procedure for infertility treatment between 2019 and 2021 from three azoospermic adult KS (47,XXY) men (33±3.6 years old) and from three control patients (46,XY) (36±2 years old) presenting with obstructive azoospermia. SCs isolated from frozen-thawed tissue of KS and 46,XY patients were cultured for 60 days and compared. All patients signed an informed consent according to the ethical board approval of the study protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular biopsies obtained from KS (n = 3) and 46,XY (n = 3) adult patients were slow-frozen. After tissue thawing SCs were isolated using a double-step enzymatic digestion and differential plating, and cultured for 60 days in DMEM medium containing FBS. Analyses were performed at different culture times (passages 5 (P5) and 10 (P10)). Quantification of cells using immunofluorescence (IF) for cell type-specific markers (Sox9, GATA4, ACTA2, INSL3, MAGEA4), SCs characterization using both IF and quantitative real-time PCR for GDNF, BMP4, AR and CLDN11 and cells karyotyping were performed. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate for the first time that a small population of human SCs isolated from frozen-thawed testis of adult KS patients can be expanded in vitro while retaining expression of characteristic markers of SCs and the 47,XXY karyotype, and exhibiting cell-specific functional proteins and gene expression (GDNF, BMP4, AR, and CLDN11) after 60 days in culture. At P10, 83.39 ± 4.2% of cultured cells from KS men and 85.34 ± 4.1% from 46,XY men expressed Sox9, and 88.8 ± 3.9% of KS cells versus 82.9 ± 3.2% of the control cells were positive for GATA4 without any differences between two groups; both Sox9 and GATA4 are typical SC markers. No differences were found between KS and 46,XY SCs in vitro in terms of cells expansion (exponential growth between P1 and P10 with an average cell count of 2.8±1.5×107 versus 3.8±1.2×107 respectively for the KS and control groups at P10). There was no significant statistical difference for functional proteins and genes expressions (GDNF, BMP4, AR, and CLDN11) neither between KS SCs and control SCs nor between P5 and P10. LIMITATIONS, REASONS FOR CAUTION The small number of donor samples is a limitation but it is due to limited availability of tissue for research in KS populations. Although no differences were observed in SCs function in the culture of isolated SCs after 60 days, the possibility of a SCs dysfunction needs to be investigated in more complex 3-dimensional models allowing the establishment of a proper cell organization and further analyses of cell functions and interactions during longer culture periods. WIDER IMPLICATIONS OF THE FINDINGS The demonstration of the possibility to propagate KS SCs in vitro could be useful to build new in vitro models for deciphering testicular cell interactions, determining deficient signalling pathways involved in impaired spermatogenesis, and identifying targets for infertility treatment in KS. As the cell numbers achieved in this study are higher than cell numbers used to develop testicular organoids, we may expect to be able to understand the behaviour and physiopathology of SCs in the disease during the long-term culture of these organoids. Such models could be further applied to understand other causes of deficiencies in seminiferous tubules. STUDY FUNDING/COMPETING INTEREST(S) M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER NCT05997706. [ABSTRACT FROM AUTHOR]

Currently, despite the vast amounts of time and money invested in cancer treatment, cancer remains one of the primary threats to human life. The primary factor contributing to the low treatment efficacy is cancer heterogeneity. The unclear molecular mechanisms underlying tumorigenesis, coupled with the complexity of human physiology, and the inability of animal models to accurately replicate the human tumor microenvironment, pose significant hurdles in the development of novel cancer therapies. Tumor-microenvironment-on-chip (TMOC) represents a research platform that integrates three-dimensional cell culture with microfluidic systems, simulating the essential components and physiological traits of the in vivo tumor microenvironment. It offers a dynamic setting within the chip system to study tumor progression, potentially heralding a breakthrough in cancer research. In this review, we will summarize the current advancements in this platform, encompassing various types of TMOCs and their applications in different types of cancer. From our perspective, the TMOC platform necessitates enhanced integration with tissue engineering techniques and microphysiological environments before it can evolve into a more refined preclinical model for cancer research. [ABSTRACT FROM AUTHOR]

Introduction: Within oncology research, there is a high effort for new approaches to prevent and treat cancer as a life-threatening disease. Specific plant species that adapt to harsh conditions may possess unique properties that may be utilized in the management of cancer. Hypothesis: Chokeberry fruit is rich in secondary metabolites with anti-cancer activities potentially useful in cancer prevention and treatment. Aims of the study and Methods: Based on mentioned hypothesis, the main goal of our study was to evaluate the antitumor effects of dietary administered Aronia melanocarpa L. fruit peels (in two concentrations of 0.3 and 3% [w/w]) in the therapeutic syngeneic 4T1 mouse adenocarcinoma model, the chemopreventive model of chemically induced mammary carcinogenesis in rats, a cell antioxidant assay, and robust in vitro analyses using MCF-7 and MDA-MB-231 cancer cells. Results: The dominant metabolites in the A. melanocarpa fruit peel extract tested were phenolic derivatives classified as anthocyanins and procyanidins. In a therapeutic model, aronia significantly reduced the volume of 4T1 tumors at both higher and lower doses. In the same tumors, we noted a significant dose-dependent decrease in the mitotic activity index compared to the control. In the chemopreventive model, the expression of Bax was significantly increased by aronia at both doses. Additionally, aronia decreased Bcl-2 and VEGF levels, increasing the Bax/Bcl-2 ratio compared to the control group. The cytoplasmic expression of caspase-3 was significantly enhanced when aronia was administered at a higher dosage, in contrast to both the control group and the aronia group treated with a lower dosage. Furthermore, the higher dosage of aronia exhibited a significant reduction in the expression of the tumor stem cell marker CD133 compared to the control group. In addition, the examination of aronia's epigenetic impact on tumor tissue through in vivo analyses revealed significant alterations in histone chemical modifications, specifically H3K4m3 and H3K9m3, miRNAs expression (miR155, miR210, and miR34a) and methylation status of tumor suppressor genes (PTEN and TIMP3). In vitro studies utilizing a methanolic extract of A.melanocarpa demonstrated significant anti-cancer properties in the MCF-7 and MDA-MB-231 cell lines. Various analyses, including Resazurin, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential, were conducted in this regard. Additionally, the aronia extract enhanced the responsiveness to epirubicin in both cancer cell lines. Conclusion: This study is the first to analyze the antitumor effect of A. melanocarpa in selected models of experimental breast carcinoma in vivo and in vitro. The utilization of the antitumor effects of aronia in clinical practice is still minimal and requires precise and long-term clinical evaluations. Individualized cancer-type profiling and patient stratification are crucial for effectively implementing plant nutraceuticals within targeted anti-cancer strategies in clinical oncology. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, immune dysregulation, and systemic fibrosis. Research on SSc has been hindered largely by lack of relevant models to study the progressive nature of the disease and to recapitulate the cell plasticity that is observed in this disease context. Generation of models for fibrotic disease using pluripotent stem cells is important for recapitulating the heterogeneity of the fibrotic tissue and are a potential platform for screening anti-fibrotic drugs. We previously reported a novel in-vitro model for fibrosis using induced pluripotent stem cell-derived mesenchymal cells (iSCAR). Here we report the generation of a "scar-like phenotype" when iPSC derived mesenchymal cells are cultured on hydrogel that mimicks a wound healing/scarring response (iSCAR). First, we performed RNA sequencing (RNA-seq) based transcriptome profiling of iSCAR culture at 48 h and 13 days to characterize early and latestage scarring phenotypes. The next generation RNA-seq of these iSCAR culture at different timepoints detected expression 92% of early "scar associated" genes and 85% late "scar associated" genes, respectively. Comparative transcriptomic analysis of a gene level SSc compendium matrix to the iSCAR wound associated model revealed genes common in both data sets. Early scar formation genes showed biological processes of hypoxia (27.5%), vascular development (13.7%) and glycolysis (27.5), while late scar formation showed genes associated with senescence (22.6%). Next we show the effects of two different antifibrotic compounds to validate the utility of the model as a screening tool to study early and late-stagelate-stage fibrosis. An autotaxin inhibitor was used to validate the iSCAR late stage fibrotic model (iSCAR-T) and an antifibrotic tool screening compound of unknown mechanism (EX00015097) was used to study and validate both early (iSCAR-P) and late-stage (iSCAR-T) fibrosis in the iSCAR model. [ABSTRACT FROM AUTHOR]

More than two decades ago, the advent of Nanotechnology has marked the onset of a new and critical field in science and technology, highlighting the importance of multidisciplinary approaches to assess and model the potential human hazard of newly developed advanced materials in the nanoscale, the nanomaterials (NMs). Nanotechnology is, by definition, a multidisciplinary field, that integrates knowledge and techniques from physics, chemistry, biology, materials science, and engineering to manipulate matter at the nanoscale, defined as anything comprised between 1 and 100 nm. The emergence of nanotechnology has undoubtedly led to significant innovations in many fields, from medical diagnostics and targeted drug delivery systems to advanced materials and energy solutions. However, the unique properties of nanomaterials, such as the increased surface to volume ratio, which provides increased reactivity and hence the ability to penetrate biological barriers, have been also considered as potential risk factors for unforeseen toxicological effects, stimulating the scientific community to investigate to which extent this new field of applications could pose a risk to human health and the environment. [ABSTRACT FROM AUTHOR]

Introduction: This article provides an overview of several experimental models, including in vivo, genetics, chemical, knock-in, knock-out, electrical, in vitro, and optogenetics models, that have been employed to investigate epileptogenesis. The present review introduces a novel categorization of these models, taking into account the fact that the most recent classification that gained widespread acceptance was established by Fisher in 1989. A significant number of such models have become virtually outdated. Objective: This paper specifically examines the models that have contributed to the investigation of partial seizures, generalized seizures, and status epilepticus. Discussion: A description is provided of the primary features associated with the processes that produce and regulate the symptoms of various epileptogenesis models. Numerous experimental epilepsy models in animals have made substantial contributions to the investigation of particular brain regions that are capable of inducing seizures. Experimental models of epilepsy have also enabled the investigation of the therapeutic mechanisms of anti-epileptic medications. Typically, animals are selected for the development and study of experimental animal models of epilepsy based on the specific form of epilepsy being investigated. Conclusions: Currently, it is established that specific animal species can undergo epileptic seizures that resemble those described in humans. Nevertheless, it is crucial to acknowledge that a comprehensive assessment of all forms of human epilepsy has not been feasible. However, these experimental models, both those derived from channelopathies and others, have provided a limited comprehension of the fundamental mechanisms of this disease. [ABSTRACT FROM AUTHOR]

Investigating the complex interactions between microbiota and immunity is crucial for a fruitful understanding progress of human health and disease. This review assesses animal models, next-generation in vitro models, and in silico approaches that are used to decipher the microbiome-immunity axis, evaluating their strengths and limitations. While animal models provide a comprehensive biological context, they also raise ethical and practical concerns. Conversely, modern in vitro models reduce animal involvement but require specific costs and materials. When considering the environmental impact of these models, in silico approaches emerge as promising for resource reduction, but they require robust experimental validation and ongoing refinement. Their potential is significant, paving the way for a more sustainable and ethical future in microbiome-immunity research. [ABSTRACT FROM AUTHOR]

Candida species primarily exist as harmless commensals in the gastrointestinal tract of warm-blooded animals. However, they can also cause life-threatening infections, which are often associated with gut microbial dysbiosis. Identifying the microbial actors that restrict Candida to commensalism remains a significant challenge. In vitro models could enable a mechanistic study of the interactions between Candida and simulated colon microbiomes. Therefore, this study aimed to elucidate the spatial and temporal colonization kinetics of specific Candida , including C. albicans, C. tropicalis , and C. parapsilosis , and their relative Nakaseomyces glabratus , by using an adapted SHIME® model, simulating the ileum, and proximal and distal colons. We monitored fungal and bacterial colonization kinetics under conditions of eubiosis (commensal lifestyle) and antibiotic-induced dysbiosis (pathogenic lifestyle). Our findings highlighted the variability in the colonization potential of Candida species across different intestinal regions. The ileum compartment proved to be the most favourable environment for C. albicans and C. parapsilosis under conditions of eubiosis. Antibiotic-induced dysbiosis resulted in resurgence of opportunistic Candida species, especially C. tropicalis and C. albicans. Future research should focus on identifying specific bacterial species influencing Candida colonization resistance and explore the long-term effects of antibiotics on the mycobiome and bacteriome. [ABSTRACT FROM AUTHOR]

Background: Endometrial cancer (EC) is a prevalent gynecological malignancy globally, with a rising incidence trend. While classic androgens have been implicated with EC risk, the role of their 11-oxygenated metabolites is poorly understood. Here, we studied 11-oxyandrogen formation from steroid precursors in EC for the first time. Methods: We performed in vitro studies on a panel of four EC cell lines of varying differentiation degree and molecular subtype and a control cell line of normal endometrium to assess 11-oxyandrogen formation from steroid precursors. We also characterized the transcriptomic effects of dihydrotestosterone (DHT) and 11-keto-DHT on Ishikawa and RL95-2. Key molecular players in 11-oxyandrogen metabolism and action were explored in endometrial tumors using public transcriptomic datasets. Results: We discovered that within endometrial tumors, the formation of 11-oxyandrogens does not occur from classic androgen precursors. However, we observed distinct regulatory mechanisms at a pre-receptor level in normal endometrium compared to cancerous tissue, and between low- and highgrade tumors. Specifically, in vitro models of low-grade EC formed higher levels of bioactive 11-keto-testosterone from 11-oxyandrogen precursors compared to models of noncancerous endometrium and high-grade, TP53-mutated EC. Moreover, the potent androgen, DHT and its 11-keto homologue induced mild transcriptomic effects on androgen receptor (AR)-expressing EC model, Ishikawa. Finally, using public transcriptomic datasets, we found HSD11B2 and SRD5A2, coding for key enzymes in steroid metabolism, to be associated with better disease-specific survival, whereas higher intra-tumoral AR expression correlated with lower recurrence in TP53-wt tumors. Conclusions: The intra-tumoral metabolism of 11-oxyandrogen precursors is characteristic for low-grade EC of non-TP53-alt molecular subtypes. Our findings support further exploration of circulating 11-oxyandrogens as prognostic biomarkers in EC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Among all of the materials used in tissue engineering in order to develop bioequivalents, collagen shows to be the most promising due to its superb biocompatibility and biodegradability, thus becoming one of the most widely used materials for scaffold production. However, current imaging techniques of the cells within collagen scaffolds have several limitations, which lead to an urgent need for novel methods of visualization. In this work, we have obtained groups of collagen scaffolds and selected the contrasting agents in order to study pores and patterns of cell growth in a non-disruptive manner via X-ray computed microtomography (micro-CT). After the comparison of multiple contrast agents, a 3% aqueous phosphotungstic acid solution in distilled water was identified as the most effective amongst the media, requiring 24 h of incubation. The differences in intensity values between collagen fibers, pores, and masses of cells allow for the accurate segmentation needed for further analysis. Moreover, the presented protocol allows visualization of porous collagen scaffolds under aqueous conditions, which is crucial for the multimodal study of the native structure of samples. [ABSTRACT FROM AUTHOR]

Introduction: Dysregulated neutrophil function plays a significant role in the pathology of infections, cancer, cardiovascular diseases, and autoimmune disorders. Neutrophil activity is influenced by various cell populations, including macrophages, which are crucial regulators. However, the exact role of human macrophages in controlling neutrophil function remains unclear due to a scarcity of studies utilizing human cells in physiologically relevant models. Methods: We adapted our "Infection-on-a-Chip" microfluidic device to incorporate macrophages within the collagen extracellular matrix, allowing for the study of interactions between human neutrophils and macrophages in a context that mimics in vivo conditions. The integration of THP-1 macrophages was optimized and their effect on the endothelial lumen was characterized, focusing on permeability and structural integrity. The device was then employed to examine the influence of macrophages on neutrophil response to infection with the bacterial pathogen Pseudomonas aeruginosa. Results: Integration of THP-1 macrophages into the microfluidic device was successfully optimized, showing no increase in endothelial permeability or structural damage. The presence of macrophages was found to significantly reduce neutrophil transendothelial migration in response to Pseudomonas aeruginosa infection. Conclusions: Our findings highlight the regulatory role of macrophages in modulating neutrophil responses, suggesting potential therapeutic targets to control neutrophil function in various diseases. The modified microfluidic platform offers a valuable tool for mechanistic studies into macrophage-neutrophil interactions in disease contexts. [ABSTRACT FROM AUTHOR]

Nanomedicine has the potential to increase the biostability of drugs to treat retinal diseases, improving their performance and decreasing the required number of intravitreal injections. However, accurate pharmacokinetic studies of these nanoparticle-drug conjugates, nanoparticle motion across the vitreous humour and interaction with the retinal cell layers still need to be investigated. Existing nanoparticle tracking techniques require fluorescent labels, which can impact cytotoxicity, nanoparticles' motion, protein interactions, and cell internalization. In this study, a real-time label-free tracking technology, for single nanoparticles in an optical microscope based on the optical phenomena of caustics, was used to characterise the diffusion of nanoparticles in agar-hyaluronic acid hydrogels, previously validated as vitreous humour substitutes for in vitro models. The results demonstrated that the diffusion of nanoparticles through these hydrogels was heterogeneous, and that nanoparticle size had an important role in nanoparticle distribution across and within in vitro vitreous substitutes. These findings suggest that nanoparticle diameter is a critical parameter for designing novel therapeutics for retinal diseases. Moreover, nanoparticle charge did not affect nanoparticle diffusion or distribution in these synthetic hydrogels. The use of caustics in optical microscopy has been demonstrated to be a reproducible, inexpensive technique for screening novel therapeutics in eye in vitro models. [ABSTRACT FROM AUTHOR]

Periodontitis is a chronic inflammatory disease associated with dysbiosis in subgingival plaque biofilm, characterised by damage to the periodontal tissues, eventually leading to tooth loss. Hence, the pathophysiology of periodontitis and interaction between subgingival plaque and host tissue under various environmental cues are central to the pathogenesis of periodontitis. Therefore, engineering biofilm models that mimic in vivo pathophysiology is crucial to obtaining a clear insight into the pathology and developing targeted therapeutic methods. In this review, we provide a comprehensive overview of the engineering strategies employed of modelling oral biofilms focusing on surface attachment, fluid microenvironment, gas environment, shear force, microbial-host interaction and offer insights into the ongoing challenges and future perspectives, which will enable the development of novel physiological relevant models for oral biofilms. [ABSTRACT FROM AUTHOR]

The lung is a highly mechanical organ as it is exposed to approximately 109 strain cycles, (where strain is the length change of tissue structure per unit initial length), with an approximately 4% amplitude change during quiet tidal breathing or 107 strain cycles at a 25% amplitude during heavy exercises, sighs, and deep inspirations. These mechanical indices have been reported to become aberrant in lung diseases such as acute respiratory distress syndrome (ARDS), pulmonary hypertension, bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), and asthma. Through recent innovations, various in vitro systems/bioreactors used to mimic the lung's mechanical strain have been developed. Among these, the Flexcell tension system which is composed of bioreactors that utilize a variety of programs in vitro to apply static and cyclic strain on different cell-types established as 2D monolayer cultures or cell-embedded 3D hydrogel models, has enabled the assessment of the response of different cells such as fibroblasts to the lung's mechanical strain in health and disease. Fibroblasts are the main effector cells responsible for the production of extracellular matrix (ECM) proteins to repair and maintain tissue homeostasis and are implicated in the excessive deposition of matrix proteins that leads to lung fibrosis. In this review, we summarise, studies that have used the Flexcell tension bioreactor to assess effects of the mechanical lung on the structure, function, and phenotype of lung fibroblasts in homeostatic conditions and abnormal environments associated with lung injury and disease. We show that these studies have revealed that different strain conditions regulate fibroblast proliferation, ECM protein production, and inflammation in normal repair and the diseased lung. [ABSTRACT FROM AUTHOR]

Nephrotoxicity stands as one of the most limiting effects in the development and validation of new drugs. The kidney, among the organs evaluated in toxicity assessments, has a higher susceptibility, with nephrotoxic potential frequently evading detection until late in clinical trials. Traditional cell culture, which has been widely used for decades, does not recapitulate the structure and complexity of the native tissue, which can affect cell function, and the response to cytotoxins does not resemble what occurs in the kidney. In the current study, we aimed to address these challenges by creating in vitro kidney models that faithfully biomimic the dynamics of the renal proximal tubule, using the well-established RPTEC/TERT1 cell line. For doing so, two models were developed, one recreating tubule-like structures (2.5D model) and the other using microfluidic technology (kidney-on-a-chip). The 2.5D model allowed tubular structures to be generated in the absence of hydrogels, and the kidney-on-a-chip model allowed shear stress to be applied to the cell culture, which is a physiological stimulus in the renal tissue. After characterization of both models, different nephrotoxic compounds such as cisplatin, tacrolimus, and daunorubicin were used to study cell responses after treatment. The developed models in our study could be a valuable tool for pre-clinical nephrotoxic testing of drugs and new compounds. [ABSTRACT FROM AUTHOR]

Drug‐carrying nanoparticles are a promising strategy to deliver therapeutics into the brain, but their translation requires better characterization of interactions between nanomaterials and endothelial cells of the blood–brain barrier (BBB). Here, we use a library of 18 layer‐by‐layer electrostatically assembled nanoparticles (NPs) to independently assess the impact of NP core and surface materials on in vitro uptake, transport, and intracellular trafficking in brain endothelial cells. We demonstrate that NP core stiffness determines the magnitude of transport, while surface chemistry directs intracellular trafficking. Finally, we demonstrate that these factors similarly dictate in vivo BBB transport using intravital imaging through cranial windows in mice. We identify that hyaluronic acid surface chemistry increases transport across the BBB in vivo, and flow conditions are necessary to replicate this finding in vitro. Taken together, these findings highlight the importance of assay geometry, cell biology, and fluid flow in developing nanocarriers for delivery to the brain. [ABSTRACT FROM AUTHOR]

Aims This study aimed to assess the pharmacokinetic/pharmacodynamic (PK/PD) targets of danofloxacin to minimize the risk of selecting resistant Pasteurella multocida mutants and to identify the mechanisms underlying their resistance in an in vitro dynamic model, attaining the optimum dosing regimen of danofloxacin to improve its clinical efficacy based on the mutant selection window (MSW) hypothesis. Methods and Results Danofloxacin at seven dosing regimens and 5 days of treatment were simulated to quantify the bactericidal kinetics and enrichment of resistant mutants upon continuous antibiotic exposure. The magnitudes of PK/PD targets associated with different efficacies were determined in the model. The 24 h area under the concentration–time curve (AUC) to minimum inhibitory concentration (MIC) ratios (AUC24h/MIC) of danofloxacin associated with bacteriostatic, bactericidal and eradication effects against P. multocida were 34, 52, and 64 h. This translates to average danofloxacin concentrations (Cav) over 24 h being 1.42, 2.17, and 2.67 times the MIC, respectively. An AUC/MIC-dependent antibacterial efficacy and AUC/mutant prevention concentration (MPC)-dependent enrichment of P. multocida mutants in which maximum losses in danofloxacin susceptibility occurred at a simulated AUC24h/MIC ratio of 72 h (i.e. Cav of three times the MIC). The overexpression of efflux pumps (acrAB-tolC) and their regulatory genes (marA, soxS , and ramA) was associated with reduced susceptibility in danofloxacin-exposed P. multocida. The AUC24h/MPC ratio of 19 h (i.e. Cav of 0.8 times the MPC) was determined to be the minimum mutant prevention target value for the selection of resistant P. multocida mutants. Conclusions The emergence of P. multocida resistance to danofloxacin exhibited a concentration-dependent pattern and was consistent with the MSW hypothesis. The current clinical dosing regimen of danofloxacin (2.5 mg kg−1) may have a risk of treatment failure due to inducible fluoroquinolone resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: Pediatric high-grade gliomas are aggressive and deadly brain tumors that arise in children and are notoriously difficult to cure. Researchers require accurate methods, or "model systems," to better understand these tumors. Model systems include culturing cancer cells in a dish, observing how cancer cells grow in an animal brain, and growing cells in self-organizing 3D cultures called organoids, which more accurately resemble the brain. In this paper, we describe the molecular changes that these cancers acquire that cause them to be so aggressive, the development of past and present model systems, highlight the newest model systems being developed, and offer suggestions regarding how these model systems can be used in the future to develop better therapies for patients. Further, we outline the benefits and limitations of each model system to better guide researchers in the design of their experiments. Developing a variety of model systems that adequately and faithfully represent how tumors exist, progress, recur, and respond to treatment in a patient is absolutely essential to the eventual development of effective therapeutics. Therefore, investing in the continued development of these model systems is paramount to improving patient outcomes. Pediatric high-grade gliomas (pHGG) are malignant and usually fatal central nervous system (CNS) WHO Grade 4 tumors. The majority of pHGG consist of diffuse midline gliomas (DMG), H3.3 or H3.1 K27 altered, or diffuse hemispheric gliomas (DHG) (H3.3 G34-mutant). Due to diffuse tumor infiltration of eloquent brain areas, especially for DMG, surgery has often been limited and chemotherapy has not been effective, leaving fractionated radiation to the involved field as the current standard of care. pHGG has only been classified as molecularly distinct from adult HGG since 2012 through Next-Generation sequencing approaches, which have shown pHGG to be epigenetically regulated and specific tumor sub-types to be representative of dysregulated differentiating cells. To translate discovery research into novel therapies, improved pre-clinical models that more adequately represent the tumor biology of pHGG are required. This review will summarize the molecular characteristics of different pHGG sub-types, with a specific focus on histone K27M mutations and the dysregulated gene expression profiles arising from these mutations. Current and emerging pre-clinical models for pHGG will be discussed, including commonly used patient-derived cell lines and in vivo modeling techniques, encompassing patient-derived xenograft murine models and genetically engineered mouse models (GEMMs). Lastly, emerging techniques to model CNS tumors within a human brain environment using brain organoids through co-culture will be explored. As models that more reliably represent pHGG continue to be developed, targetable biological and genetic vulnerabilities in the disease will be more rapidly identified, leading to better treatments and improved clinical outcomes. [ABSTRACT FROM AUTHOR]

Kidney disease is a global health crisis affecting more than 850 million people worldwide. In the United States, annual Medicare expenditures for kidney disease and organ failure exceed $81 billion. Efforts to develop targeted therapeutics are limited by a poor understanding of the molecular mechanisms underlying human kidney disease onset and progression. Additionally, 90% of drug candidates fail in human clinical trials, often due to toxicity and efficacy not accurately predicted in animal models. The advent of ex vivo kidney models, such as those engineered from induced pluripotent stem (iPS) cells and organ-on-a-chip (organ-chip) systems, has garnered considerable interest owing to their ability to more accurately model tissue development and patient-specific responses and drug toxicity. This review describes recent advances in developing kidney organoids and organ-chips by harnessing iPS cell biology to model human-specific kidney functions and disease states. We also discuss challenges that must be overcome to realize the potential of organoids and organ-chips as dynamic and functional conduits of the human kidney. Achieving these technological advances could revolutionize personalized medicine applications and therapeutic discovery for kidney disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/β-catenin signaling and inhibition of the TGF-β pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/β-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories. [ABSTRACT FROM AUTHOR]

Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) are two common respiratory tract pathogens often associated with acute exacerbations in Chronic Obstructive Pulmonary Disease (COPD) as well as with otitis media (OM) in children. Although there is evidence that these pathogens can adopt persistence mechanisms such as biofilm formation, the precise means through which they contribute to disease severity and chronicity remains incompletely understood, posing challenges for their effective eradication. The identification of potential vaccine candidates frequently entails the characterization of the host-pathogen interplay in vitro even though this approach is limited by the fact that conventional models do not permit long term bacterial infections. In the present work, by using air-liquid-interface (ALI) human airway in vitro models, we aimed to recreate COPD-related persistent bacterial infections. In particular, we explored an alternative use of the ALI system consisting in the assembly of an inverted epithelium grown on the basal part of a transwell membrane with the aim to enable the functionality of natural defense mechanisms such as mucociliary clearance and cellular extrusion that are usually hampered during conventional ALI infection experiments. The inversion of the epithelium did not affect tissue differentiation and considerably delayed NTHi or Mcat infection progression, allowing one to monitor host-pathogen interactions for up to three weeks.Notably, the use of thesemodels, coupledwith confocal and transmission electron microscopy, revealed unique features associated with NTHi and Mcat infection, highlighting persistence strategies including the formation of intracellular bacterial communities (IBCs) and surface-associated biofilm-like structures. Overall, this study demonstrates the possibility to perform long term host-pathogen investigations in vitro with the aim to define persistence mechanisms adopted by respiratory pathogens and individuate potential new vaccine targets. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Organ-on-a-chip technology is attracting growing interest across various domains as a crucial platform for drug screening and testing and is set to play a significant role in precision medicine research. Lymph nodes, being intricately structured organs essential for the body's adaptive immune responses to antigens and foreign particles, are pivotal in assessing the immunotoxicity of novel pharmaceuticals. Significant progress has been made in research on the structure and function of the lymphatic system. However, there is still an urgent need to develop prospective tools and techniques to delve deeper into its role in various diseases' pathological and physiological processes and to develop corresponding immunotherapeutic therapies. Organ chips can accurately reproduce the specific functional areas in lymph nodes to better simulate the complex microstructure of lymph nodes and the interactions between different immune cells, which is convenient for studying specific biological processes. This paper reviews existing lymph node chips and their design approaches. It discusses the applications of the above systems in modeling immune cell motility, cell–cell interactions, vaccine responses, drug testing, and cancer research. Finally, we summarize the challenges that current research faces in terms of structure, cell source, and extracellular matrix simulation of lymph nodes, and we provide an outlook on the future direction of integrated immune system chips. [ABSTRACT FROM AUTHOR]

The treatment of Parkinson's disease has been moving into the focus of pharmaceutical development. Yet, the necessity for reliable model systems in the development phase has made research challenging and in vivo models necessary. We have established reliable, reproducible in vitro model systems to evaluate the binding and transport of dopamine-loaded PLGA nanoparticles for the treatment of Parkinson's disease and put the results in context with comparable in vivo results. The in vitro models have provided similar results concerning the usability of the investigated nanoparticles as the previously used in vivo models and thus provide a good alternative in line with the 3R principles in pharmaceutical research. [ABSTRACT FROM AUTHOR]

The alteration in the neural circuits of both central and peripheral nervous systems is closely related to the onset of neurodegenerative disorders (NDDs). Despite significant research efforts, the knowledge regarding NDD pathological processes, and the development of efficacious drugs are still limited due to the inability to access and reproduce the components of the nervous system and its intricate microenvironment. 2D culture systems are too simplistic to accurately represent the more complex and dynamic situation of cells in vivo and have therefore been surpassed by 3D systems. However, both models suffer from various limitations that can be overcome by employing two innovative technologies: organ‐on‐chip and 3D printing. In this review, an overview of the advantages and shortcomings of both microfluidic platforms and extracellular matrix‐like biomaterials will be given. Then, the combination of microfluidics and hydrogels as a new synergistic approach to study neural disorders by analyzing the latest advances in 3D brain‐on‐chip for neurodegenerative research will be explored. [ABSTRACT FROM AUTHOR]

Mimicking tissue‐oriented organization in vitro has been extensively studied in recent years, using both natural and synthetic materials in combination with external magnetic fields to establish anisotropic conditions. Here, a new combination between magneto‐responsive anisometric PEG microgels and collagen hydrogels is explored to establish anisotropic in vitro models. Different sizes of PEG microgels are tested to assess the impact of both width and aspect ratio on the formation and alignment of collagen hydrogels. Results show that the key properties of collagen hydrogels, regarding fibrillogenesis, rheological properties, and fiber diameter are kept consistent upon the combination with PEG microgels. Furthermore, partial collagen fiber alignment is observed when larger (width 10 µm) PEG microgels are employed and magnetically aligned. In vitro studies show cell alignment within the anisotropic collagen hydrogels from the first day in culture. Interestingly, PEG microgels with higher width and length tend to induce less hydrogel contraction even after 7 days in culture. The results demonstrate the ability to establish a 3D unidirectional collagen hydrogel by magnetically aligning anisometric microgels during the gelation process, which can be promising for different tissue engineering applications. [ABSTRACT FROM AUTHOR]

Organoids have emerged as a powerful platform for studying complex biological processes and diseases in vitro. However, most studies have focused on individual organoids, overlooking the inter‐organ interactions in vivo and limiting the physiological relevance of the models. To address this limitation, the development of a multi‐organoid system has gained considerable attention. This system aims to recapitulate inter‐organ communication and enable the study of complex physiological processes. This review provides a comprehensive overview of the recent advancements in organoid engineering and the emerging strategies for constructing a multi‐organoid system. First, we highlight the critical mechanical, structural, and biochemical factors involved in designing suitable materials for the growth of different organoids. Additionally, we discuss the incorporation of dynamic culture environments to enhance organoid culture and enable inter‐organoid communication. Furthermore, we explore techniques for manipulating organoid morphogenesis and spatial positioning of organoids to establish effective inter‐organoid communication networks. We summarize the achievements in utilizing organoids to recapitulate inter‐organ communication in vitro, including assembloids and microfluidic multi‐organoid platforms. Lastly, we discuss the existing challenges and opportunities in developing a multi‐organoid system from its technical bottlenecks in scalability to its applications toward complex human diseases. [ABSTRACT FROM AUTHOR]

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-1β release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1β 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. [ABSTRACT FROM AUTHOR]

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources. [ABSTRACT FROM AUTHOR]

Cancer is a leading cause of death worldwide and results in nearly 10 million deaths each year. The global economic burden of cancer from 2020 to 2050 is estimated to be USD 25.2 trillion. The spread of cancer to distant organs through metastasis is the leading cause of death due to cancer. However, as of today, there is no cure for metastasis. Tissue engineering is a promising field for regenerative medicine that is likely to be able to provide rehabilitation procedures to patients who have undergone surgeries, such as mastectomy and other reconstructive procedures. Another important use of tissue engineering has emerged recently that involves the development of realistic and robust in vitro models of cancer metastasis, to aid in drug discovery and new metastasis therapeutics, as well as evaluate cancer biology at metastasis. This review covers the current studies in developing tissue-engineered metastasis structures. This article reports recent developments in in vitro models for breast, prostate, colon, and pancreatic cancer. The review also identifies challenges and opportunities in the use of tissue engineering toward new, clinically relevant therapies that aim to reduce the cancer burden. [ABSTRACT FROM AUTHOR]

Isolated airway smooth muscle has been extensively investigated since 1840 to understand the pharmacology of airway diseases. There has often been poor predictability from murine experiments to drugs evaluated in patients with asthma or chronic obstructive pulmonary disease (COPD). However, the use of isolated human airways represents a sensible strategy to optimise the development of innovative molecules for the treatment of respiratory diseases. This review aims to provide updated evidence on the current uses of isolated human airways in validated in vitro methods to investigate drugs in development for the treatment of chronic obstructive respiratory disorders. This review also provides historical notes on the pioneering pharmacological research on isolated human airway tissues, the key differences between human and animal airways, as well as the pivotal differences between human medium bronchi and small airways. Experiments carried out with isolated human bronchial tissues in vitro and ex vivo replicate many of the main anatomical, pathophysiological, mechanical and immunological characteristics of patients with asthma or COPD. In vitro models of asthma and COPD using isolated human airways can provide information that is directly translatable into humans with obstructive lung diseases. Regardless of the technique used to investigate drugs for the treatment of chronic obstructive respiratory disorders (i.e., isolated organ bath systems, videomicroscopy and wire myography), the most limiting factors to produce high‐quality and repeatable data remain closely tied to the manual skills of the researcher conducting experiments and the availability of suitable tissue. [ABSTRACT FROM AUTHOR]

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included. [ABSTRACT FROM AUTHOR]

The article explores the role of tunneling nanotubes (TNTs) in human diseases, focusing on their molecular profiles and functions from 2D cell cultures to complex tissues. Topics include their involvement in tumor growth and metastasis, their contribution to neurodegenerative diseases, and their potential as therapeutic targets.

Infectious diseases are increasingly recognized as a major threat worldwide due to the rise of antimicrobial resistance and the emergence of novel pathogens. In vitro models that can adequately mimic in vivo gastrointestinal physiology are in high demand to elucidate mechanisms behind pathogen infectivity, and to aid the design of effective preventive and therapeutic interventions. There exists a trade‐off between simple and high throughput models and those that are more complex and physiologically relevant. The complexity of the model used shall be guided by the biological question to be addressed. This review provides an overview of the structure and function of the intestine and the models that are developed to emulate this. Conventional models are discussed in addition to emerging models which employ engineering principles to equip them with necessary advanced monitoring capabilities for intestinal host‐pathogen interrogation. Limitations of current models and future perspectives on the field are presented. [ABSTRACT FROM AUTHOR]

Mycotoxins are secondary metabolites produced by filamentous fungi associated with a variety of acute and chronic foodborne diseases. Current toxicology studies mainly rely on monolayer cell cultures and animal models, which are undeniably affected by several limitations. To bridge the gap between the current in vitro toxicology approach and the in vivo predictability of the data, we here investigated the cytotoxic effects induced by the mycotoxins sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) on different 2D and 3D cell cultures. We focused on human tumours (neuroblastoma SH-SY5Y cells and epithelial breast cancer MDA-MB-213 cells) and healthy cells (bone marrow-derived mesenchymal stem cells, BM-MSC, and umbilical vein endothelial cells, HUVECs). The cytotoxicity of STE, OTA, and PAT was determined after 24, 48 and 72 h of exposure using an ATP assay in both culture models. Three-dimensional spheroids' morphology was also analysed using the MATLAB-based open source software AnaSP 1.4 version. Our results highlight how each cell line and different culture models showed specific sensitivities, reinforcing the importance of using more complex models for toxicology studies and a multiple cell line approach for an improved and more comprehensive risk assessment. [ABSTRACT FROM AUTHOR]

One of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC. [ABSTRACT FROM AUTHOR]

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P  < 0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions. [ABSTRACT FROM AUTHOR]

Skin plays crucial roles in the human body: besides protecting the organism from external threats, it acts as a thermal regulator, is responsible for the sense of touch, hosts microbial communities (the skin microbiota) involved in preventing the invasion of foreign pathogens, contains immunocompetent cells that maintain a healthy immunogenic/tolerogenic balance, and is a suitable route for drug administration. In the skin, four defense levels can be identified: besides the physical, chemical, and immune barriers that are inherent to the tissue, the skin microbiota (i.e., the numerous microorganisms living on the skin surface) provides an additional barrier. Studying the skin barrier function or the effects of drugs or cosmetic agents on human skin is a difficult task since snapshot evidence can only be obtained using bioptic samples where dynamic processes cannot properly be followed. To overcome these limitations, many different in vitro models of human skin have been developed that are characterized by diverse levels of complexity in terms of chemical, structural, and cellular composition. The aim of this review is to summarize and discuss the advantages and disadvantages of the different human skin models so far available and to underline how the insertion of a proper microbiota would positively impact an in vitro human skin model in an attempt to better mimic conditions in vivo. [ABSTRACT FROM AUTHOR]

Fibrosis is a common feature of cardiovascular diseases and targets multiple organs, such as the heart and vessels. Endothelial to mesenchymal transition is a complex, vital process that occurs during embryonic formation and plays a crucial role in cardiac development. It is also a fundamental process implicated in cardiac fibrosis and repair, but also in other organs. Indeed, in numerous cardiovascular diseases, the endothelial-to-mesenchymal transition has been shown to be involved in the generation of fibroblasts that are able to produce extracellular matrix proteins such as type I collagen. This massive deposition results in tissue stiffening and organ dysfunction. To advance our understanding of this process for the development of new specific diagnostic and therapeutic strategies, it is essential to develop relevant cellular and animal models of this process. In this review, our aim was to gain an in-depth insight into existing in vitro and in vivo models of endothelial to mesenchymal transition in cardiovascular diseases with a focus on cardiac fibrosis. We discuss important parameters impacting endothelial to mesenchymal transition, and we give perspectives for the development of relevant models to decipher the underlying mechanisms and ultimately find new treatments specific to fibrosis happening in cardiovascular diseases. [ABSTRACT FROM AUTHOR]

There is increasing interest in using magnesium (Mg) alloy orthopedic devices because of their mechanical properties and bioresorption potential. Concerns related to their rapid degradation have been issued by developing biodegradable micro- and nanostructured coatings to enhance corrosion resistance and limit the release of hydrogen during degradation. This systematic review based on four databases (PubMed®, Embase, Web of Science™ and ScienceDirect®) aims to present state-of-the-art strategies, approaches and materials used to address the critical factors currently impeding the utilization of Mg alloy devices. Forty studies were selected according to PRISMA guidelines and specific PECO criteria. Risk of bias assessment was conducted using OHAT and SYRCLE tools for in vitro and in vivo studies, respectively. Despite limitations associated with identified bias, the review provides a comprehensive analysis of preclinical in vitro and in vivo studies focused on manufacturing and application of Mg alloys in orthopedics. This attests to the continuous evolution of research related to Mg alloy modifications (e.g., AZ91, LAE442 and WE43) and micro- and nanocoatings (e.g., MAO and MgF2), which are developed to improve the degradation rate required for long-term mechanical resistance to loading and excellent osseointegration with bone tissue, thereby promoting functional bone regeneration. Further research is required to deeply verify the safety and efficacy of Mg alloys. [ABSTRACT FROM AUTHOR]

Mycotoxins, produced by fungi, frequently occur at different stages in the food supply chain between pre- and postharvest. Globally produced cereal crops are known to be highly susceptible to contamination, thus constituting a major public health concern. Among the encountered mycotoxigenic fungi in cereals, Fusarium spp. are the most frequent and produce both regulated (i.e., T-2 toxin, deoxynivalenol -DON-, zearalenone -ZEA-) and emerging (i.e., enniatins -ENNs-, beauvericin -BEA-) mycotoxins. In this study, we investigated the in vitro cytotoxic effects of regulated and emerging fusariotoxins on HepaRG cells in 2D and 3D models using undifferentiated and differentiated cells. We also studied the impact of ENN B1 and ENN B exposure on gene expression of HepaRG spheroids. Gene expression profiling pinpointed the differentially expressed genes (DEGs) and overall similar pathways were involved in responses to mycotoxin exposure. Complement cascades, metabolism, steroid hormones, bile secretion, and cholesterol pathways were all negatively impacted by both ENNs. For cholesterol biosynthesis, 23/27 genes were significantly down-regulated and could be correlated to a 30% reduction in cholesterol levels. Our results show the impact of ENNs on the cholesterol biosynthesis pathway for the first time. This finding suggests a potential negative effect on human health due to the essential role this pathway plays. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common primary brain tumor, characterized by a remarkable inner complexity and inter-tumor variability. Moreover, it is very aggressive and resistant to conventional treatments, so that it rapidly relapse. Therefore, there is an immediate need for experimental strategies to enhance our comprehension of GBM, aiming to mitigate its economic and social impact. Here, we described different in vivo and in vitro strategies currently used for the study of GBM. First, we gave a brief and general overview of the classical in vivo models, including xenograft mouse and zebrafish models and canine models, offering a wide range of advantages but also presenting a series of strong limitations. Thus, we described in vitro models, starting from more traditional 2D culture models, comparing different approaches, and critically exposing the advantages and disadvantages of using one or the other methods. We also briefly described GBM 2D culture systems that allow recreating multiple cell-cell and cell-extracellular matrix contacts but still do not reflect the complexity of in vivo tumors. We finally described the intricacies of the more novel 3D in vitro models, e.g., spheroids and organoids. These sophisticated models have demonstrated exceptional suitability across a wide spectrum of applications in cancer research, ranging from fundamental scientific inquiries to applications in translational research. Their adaptability and three-dimensional architecture render them invaluable tools, offering new insights and paving the way for advancements in both basic and applied research. [ABSTRACT FROM AUTHOR]

Neurodegeneration is a catastrophic process that develops progressive damage leading to functional and structural loss of the cells of the nervous system and is among the biggest unavoidable problems of our age. Animal models do not reflect the pathophysiology observed in humans due to distinct differences between the neural pathways, gene expression patterns, neuronal plasticity, and other disease-related mechanisms in animals and humans. Classical in vitro cell culture models are also not sufficient for pre-clinical drug testing in reflecting the complex pathophysiology of neurodegenerative diseases. Today, modern, engineered techniques are applied to develop multicellular, intricate in vitro models and to create the closest microenvironment simulating biological, biochemical, and mechanical characteristics of the in vivo degenerating tissue. In THIS review, the capabilities and shortcomings of scaffold-based and scaffold-free techniques, organoids, and microfluidic models that best reflect neurodegeneration in vitro in the biomimetic framework are discussed. [ABSTRACT FROM AUTHOR]

Communication between the maternal endometrium and developing embryo/conceptus is critical to support successful pregnancy to term. Studying the peri-implantation period of pregnancy is critical as this is when most pregnancy loss occurs in cattle. Our current understanding of these interactions is limited, due to the lack of appropriate in vitro models to assess these interactions. The endometrium is a complex and heterogeneous tissue that is regulated in a transcriptional and translational manner throughout the oestrous cycle. While there are in vitro models to study endometrial function, they are static and 2D in nature or explant models and are limited in how well they recapitulate the in vivo endometrium. Recent developments in organoid systems, microfluidic approaches, extracellular matrix biology, and in silico approaches provide a new opportunity to develop in vitro systems that better model the in vivo scenario. This will allow us to investigate in a more high-throughput manner the fundamental molecular interactions that are required for successful pregnancy in cattle. The majority of pregnancy loss in cattle (and other mammals) occurs in the peri-implantation period. The contribution of endometrial dysfunction to this loss remains elusive. Furthering our understanding the function/dysfunction of the endometrium is difficult given the lack of in vitro models that appropriately recapitulate the in vivo scenario or allow high-throughput testing of conceptus–maternal interactions. Here we review the current state of the art of in vitro and in silico model development, which will allow us to test the molecular interactions required for pregnancy success in cattle and other mammalian species. Created with BioRender.com. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma treatment has progressed through the use of immune checkpoint inhibitors, significantly boosting patient survival rates. However, many tumors do not respond to these drugs or develop resistance, partly due to the tumor microenvironment influencing cancer cell growth and immune cell behavior. To gain insights into therapy outcomes, researchers have focused on developing cell-based ex vivo models of melanomas. These models include tumor organoids, engineered tissues, and microfluidic devices designed to replicate melanoma in its natural skin environment. Yet, integrating the tumor microenvironment and immune factors remains challenging. This review explores the creation of in vitro 3D models for normal skin and melanoma, focusing on methods to include immune components. These models hold promise for testing immunotherapies and uncovering resistance mechanisms. By faithfully replicating the tumor microenvironment and its interactions with the immune system, these models can enhance our understanding of immunotherapy resistance and ultimately improve personalized melanoma treatment. Melanoma is responsible for the majority of skin cancer-related fatalities. Immune checkpoint inhibitor (ICI) treatments have revolutionized the management of the disease by significantly increasing patient survival rates. However, a considerable number of tumors treated with these drugs fail to respond or may develop resistance over time. Tumor growth and its response to therapies are critically influenced by the tumor microenvironment (TME); it directly supports cancer cell growth and influences the behavior of surrounding immune cells, which can become tumor-permissive, thereby rendering immunotherapies ineffective. Ex vivo modeling of melanomas and their response to treatment could significantly advance our understanding and predictions of therapy outcomes. Efforts have been directed toward developing reliable models that accurately mimic melanoma in its appropriate tissue environment, including tumor organoids, bioprinted tissue constructs, and microfluidic devices. However, incorporating and modeling the melanoma TME and immune component remains a significant challenge. Here, we review recent literature regarding the generation of in vitro 3D models of normal skin and melanoma and the approaches used to incorporate the immune compartment in such models. We discuss how these constructs could be combined and used to test immunotherapies and elucidate treatment resistance mechanisms. The development of 3D in vitro melanoma models that faithfully replicate the complexity of the TME and its interaction with the immune system will provide us with the technical tools to better understand ICI resistance and increase its efficacy, thereby improving personalized melanoma therapy. [ABSTRACT FROM AUTHOR]

In vitro platforms such as bioreactors and microfluidic devices are commonly designed to engineer tissue models as well as to replicate the crosstalk between cells and microorganisms hosted in the human body. These systems promote nutrient supply and waste removal through culture medium recirculation; consequently, they intrinsically expose cellular structures to shear stress, be it a desired mechanical stimulus to drive the cell fate or a potential inhibitor for the model maturation. Assessing the impact of shear stress on cellular or microbial cultures thus represents a crucial step to define proper environmental conditions for in vitro models. In this light, the aim of this study was to develop a millifluidic device enabling to generate fully controlled shear stress profiles for quantitatively probing its influence on tissue or bacterial models, overcoming the limitations of previous reports proposing similar devices. Relying on this millifluidic tool, we present a systematic methodology to test how adherent cellular structures react to shear forces, which was applied to the case of microbial biofilms as a proof of concept. The results obtained suggest our approach as a suitable testbench to evaluate culture conditions in terms of shear stress faced by cells or microorganisms. [ABSTRACT FROM AUTHOR]