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Introduction: Mechanical thrombectomy becomes more complex when the occlusion occurs in a tortuous cerebral anatomy, increasing the puncture to reperfusion time and the number of attempts for clot removal. Therefore, an understanding of stent retriever performance in these locations is necessary to increase the efficiency and safety of the procedure. An in vitro investigation into the effects of occlusion site tortuosity, blood clot hematocrit, and device geometry was conducted to identify their individual influence on stent retriever removal forces. Methods: Embolus analogs were used to create occlusions in a mock circulatory flow loop, and in vitro mechanical thrombectomies were performed in arterial models of increasing tortuosity. The stent retriever removal forces of Solitaire Platinum and EmboTrap II devices were recorded through each geometry with and without embolus analogs present. Similar experiments were also conducted with Solitaire stent retrievers of varying lengths and diameters and 0, 25, and 50% hematocrit embolus analogs. Results: The removal force increased as model tortuosity increased for both the Solitaire Platinum and EmboTrap II stent retriever devices. The average removal forces in the simplest geometry with the Solitaire Platinum and EmboTrap II were 0.24 ± 0.01 N and 0.37 ± 0.02 N, respectively, and increased to 1.2 ± 0.08 N and 1.6 ± 0.17 N, respectively, in the most complex geometry. Slight increases in removal force were found with 0% hematocrit embolus analogs, however, no statistical significance between removal force and EA hematocrit was observed. A comparison between stent retriever removal forces between devices of different diameters also proved to be significant (p < 0.01), while forces between devices of varying lengths were not (p > 0.05). Conclusion: Benchtop mechanical thrombectomies performed with commercial stent retrievers of varying geometry showed that device removal forces increase with increasing model tortuosity, clot hematocrit does not play a significant role in device removal force, and that a stent retriever's diameter has a greater impact on removal forces compared to its length. These results provide an improved understanding of the overall forces involved in mechanical thrombectomy and can be used to develop safer and more effective stent retrievers for the most difficult cases. [ABSTRACT FROM AUTHOR]

Background: Heart failure (HF) is a condition characterized by the heart's inability to meet the body's demands, resulting in various complications. Two primary types of HF exist, namely HF with preserved left ventricular ejection fraction (LVEF) and HF reduced with LVEF. The progression of HF involves compensatory mechanisms such as cardiac hypertrophy, fibrosis, and alterations in gene expression. Pressure overload and volume overload are common etiologies of HF, with pressure overload often stemming from conditions like hypertension, leading to left ventricular hypertrophy and fibrosis. In contrast, volume overload can arise from chronic valvular regurgitant disease, also inducing left ventricular hypertrophy. Main body: In vitro cell culture techniques serve as vital tools in studying HF pathophysiology, allowing researchers to investigate cellular responses and potential therapeutic targets. Additionally, biomarkers, measurable biological characteristics, play a crucial role in diagnosing and predicting HF. Some notable biomarkers include adrenomedullin, B-type natriuretic peptide, copeptin, galectin-3, interleukin-6, matrix metalloproteinases (MMPs), midregional pro-atrial natriuretic peptide, myostatin, procollagen type I C-terminal propeptide, procollagen type III N-terminal propeptide and tissue inhibitors of metalloproteinases (TIMPs). These biomarkers aid in HF diagnosis, assessing its severity, and monitoring treatment response, contributing to a deeper understanding of the disease and potentially leading to improved management strategies and outcomes. Conclusions: This review provides comprehensive insights into various in vivo models of HF, commonly utilized cell lines in HF research, and pivotal biomarkers with diagnostic relevance for HF. By synthesizing this information, researchers gain valuable resources to further explore HF pathogenesis, identify novel therapeutic targets, and enhance diagnostic and prognostic approaches. [ABSTRACT FROM AUTHOR]

Introduction: Reprogramming of human-induced pluripotent stem cells (iPSCs) and their differentiation into specific cell types, such as induced sensory-like neurons (iSNs), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, the development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving the survival and quality of iSNs. Methods: iSNs were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-b-D-arabinofuranosyl-cytosine hydrochloride (AraC) treatment, magnetic-activated cell sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively. Results: Passaging of cells during differentiation did not increase the iSN-to- total-cell-count ratio, and MACS of immature iSNs led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10µM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSNs shortly after seeding also helped to reduce non-iSN cells. Conclusion: In direct comparison with other methods, treatment with 10µM FdU for 24 h after differentiation shows promise for improving iSN culture purity, which could benefit downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of thismethod and enhance its reproducibility and applicability.Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC- based research. [ABSTRACT FROM AUTHOR]

Since the global restrictions on antibiotics in poultry systems, there has been a growing demand for natural and sustainable feed additives for disease prevention and poultry nutrition. This study evaluated the effects of incorporating sugarcane bagasse (SCB) lignin into broiler chicken diets. The performance of the chickens, including body weight, feed intake, and mortality, as well as intestinal histomorphometry, and cecum content pH, microbiota, and volatile fatty acids were assessed. In addition, we also aimed to validate an in vitro gastrointestinal tract (GIT) model developed by Carvalho et al. (2023). One hundred and eight 1-day-old Ross 308 chicks were randomly and equally divided into two groups. The first group was fed a basal diet (BD group), while the second group was fed a basal diet supplemented with 1% (w/w) SCB lignin (BD + SCB lignin group) for 36 days. The in vivo conditions of the chicken GIT were replicated in an in vitro model. In the in vivo study, SCB lignin increased cecum acetate and butyrate levels while reducing Bifidobacterium and Enterobacteriaceae, without affecting productivity (body weight, feed intake, and mortality). The in vitro assessment reflected microbiota trends observed in vivo, although without statistical significance. The divergence in organic acid production between the in vivo and in vitro conditions likely resulted from issues with inoculum preparation. This study demonstrates that SCB lignin incorporation positively influences cecal microbiota composition without impacting the animals' productivity and physiology, suggesting its potential as a functional feed additive. For a more reliable in vitro model, adjustments in inoculum preparation are necessary. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is the predominant pathogen isolated in diabetic foot infections. Recently, the skin commensal bacterium, Helcococcus kunzii, was found to modulate the virulence of this pathogen in an in vivo model. This study aims to elucidate the molecular mechanisms underlying the interaction between these two bacterial species, using a proteomic approach. Results: Our results reveal that H. kunzii can coexist and proliferate alongside S. aureus in a Chronic Wound Media (CWM), thereby mimicking an in vitro chronic wound environment. We noted that the secreted proteome of H. kunzii induced a transcriptional effect on S. aureus virulence, resulting in a decrease in the expression level of agrA, a gene involved in quorum sensing. The observed effect could be ascribed to specific proteins secreted by H. kunzii including polysaccharide deacetylase, peptidoglycan DD-metalloendopeptidase, glyceraldehyde-3-phosphate dehydrogenase, trypsin-like peptidase, and an extracellular solute-binding protein. These proteins potentially interact with the agr system, influencing S. aureus virulence. Additionally, the virulence of S. aureus was notably affected by modifications in iron-related pathways and components of cell wall architecture in the presence of H. kunzii. Furthermore, the overall metabolism of S. aureus was reduced when cocultured with H. kunzii. Conclusion: Future research will focus on elucidating the role of these excreted factors in modulating virulence. [ABSTRACT FROM AUTHOR]

• The OphtalMimic device offers a precise simulation of human ocular conditions for improved ophthalmic drug product evaluation. • A novel hydrogel-based hybrid membrane closely replicates the human cornea's water content and morphology. • The device successfully differentiated between the drainage profiles of various ophthalmic formulations. • The system demonstrated its potential as a reliable platform for the high-throughput screening of ophthalmic drugs. • This advancement aligns with ethical mandates to reduce animal testing and accelerate the development of safer and more effective ophthalmic drugs. Envisaging to improve the evaluation of ophthalmic drug products while minimizing the need for animal testing, our group developed the OphthalMimic device, a 3D-printed device that incorporates an artificial lacrimal flow, a cul-de-sac area, a moving eyelid, and a surface that interacts effectively with ophthalmic formulations, thereby providing a close representation of human ocular conditions. An important application of such a device would be its use as a platform for dissolution/release tests that closely mimic in vivo conditions. However, the surface that artificially simulates the cornea should have a higher resistance (10 min) than the previously described polymeric films (5 min). For this key assay upgrade, we describe the process of obtaining and thoroughly characterizing a hydrogel-based hybrid membrane to be used as a platform base to simulate the cornea artificially. Also, the OphthalMimic device suffered design improvements to fit the new membrane and incorporate the moving eyelid. The results confirmed the successful synthesis of the hydrogel components. The membrane's water content (86.25 ± 0.35 %) closely mirrored the human cornea (72 to 85 %). Furthermore, morphological analysis supported the membrane's comparability to the natural cornea. Finally, the performance of different formulations was analysed, demonstrating that the device could differentiate their drainage profile through the viscosity of PLX 14 (79 ± 5 %), PLX 16 (72 ± 4 %), and PLX 20 (57 ± 14 %), and mucoadhesion of PLXCS0.5 (69 ± 1 %), PLX16CS1.0 (65 ± 3 %), PLX16CS1.25 (67 ± 3 %), and the solution (97 ± 8 %). In conclusion, using the hydrogel-based hybrid membrane in the OphthalMimic device represents a significant advancement in the field of ophthalmic drug evaluation, providing a valuable platform for dissolution/release tests. Such a platform aligns with the ethical mandate to reduce animal testing and promises to accelerate the development of safer and more effective ophthalmic drugs. [ABSTRACT FROM AUTHOR]

Critically‐sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D‐printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D‐printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3‐E1 osteoblast (dMC3T3‐OB) and differentiated RAW264.7 osteoclast cells with 3D‐printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3‐OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast‐like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO‐based nanocomposites containing higher amounts of nHA have better interactions with osteoblast‐like and osteoclast‐like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair. [ABSTRACT FROM AUTHOR]

Introduction: Vaccination is one of the most effective infection prevention strategies. Viruses with high mutation rates -such as influenza-escape vaccine-induced immunity and represent significant challenges to vaccine design. Influenza vaccine strain selection is based on circulating strains and immunogenicity testing in animal models with limited predictive outcomes for vaccine effectiveness in humans. Methods: We developed a human in vitro vaccination model using human tonsil tissue explants cultured in 3D perfusion bioreactors to be utilized as a platform to test and improve vaccines. Results: Tonsils cultured in bioreactors showed higher viability, metabolic activity, and more robust immune responses than those in static cultures. The in vitro vaccination system responded to various premanufactured vaccines, protein antigens, and antigen combinations. In particular, a multivalent in vitro immunization with three phylogenetically distant H3N2 influenza strains showed evidence for broader B cell activation and induced higher antibody cross-reactivity than combinations with more related strains. Moreover, we demonstrate the capacity of our in vitro model to generate de novo humoral immune responses to a model antigen. Discussion: Perfusion-cultured tonsil tissue may be a valuable human in vitro model for immunology research with potential application in vaccine candidate selection. [ABSTRACT FROM AUTHOR]

Prostate cancer is the most common cancer in over 50% of countries and the third most common malignancy worldwide. The tumor microenvironment includes the non‐cancerous cells and components present in the tumor, including the molecules they produce and release. Prostate cancer proliferation, angiogenesis, metastasis, and drug resistance are closely associated with the tumor microenvironment. With the continuous development of in vitro tumor models, they have gradually become important tools for recapitulating in vivo parental tumors and studying the reciprocal interactions between tumors and their microenvironment. In this perspective, we describe the significant in vitro models of prostate cancer, analyze the research results on incorporating the tumor microenvironment into these models, and compare their advantages and disadvantages. Furthermore, we highlight the future developmental direction of in vitro prostate cancer model research according to the hotspots of in vitro model research in other cancer types to facilitate precision medicine in prostate cancer. [ABSTRACT FROM AUTHOR]

Red river hogs (RRHs) (Potamochoerus porcus), a wild species of Suidae living in Africa with a major distribution in the Guinean and Congolian forests, are natural reservoirs of African swine fever virus (ASFV) and typically are asymptomatic. Since blood and tissue macrophages of suid animals are target cell lineages of ASFV, RRH-derived macrophages are expected to play an important role in suppressing disease development in infected individuals. In the present study, we successfully isolated RRH-derived blood macrophages using co-culture techniques of RRH blood cells with porcine kidney-derived feeder cells and immortalized them by transferring SV40 large T antigen and porcine telomerase reverse transcriptase genes. The newly established macrophage cell line of the RRH-derived blood cell origin (RZJ/IBM) exhibited an Iba1-, CD172a-, and CD203a-positive typical macrophage-like phenotype and up-regulated the phosphorylation of nuclear factor-kB p65 subunit and p38 mitogen-activated protein kinase in response to the bacterial cell wall components, lipopolysaccharide (LPS) and muramyl dipeptide. In addition, RZJ/IBM cells produced the precursor form of interleukin (IL)-1b and IL-18 upon a stimulation with LPS, leading to the conversion of IL-18, but not IL-1b, into the mature form. Time-lapse live cell imaging with pHrodo dye-conjugated Escherichia coli BioParticles demonstrated the phagocytotic activity of RZJ/IBM cells. It is important to note that RZJ/IBM cells are clearly susceptible to ASFV infection and support viral replication in vitro. Therefore, the RZJ/IBM cell line provides a unique model for investigating the pathogenesis of ASFV. [ABSTRACT FROM AUTHOR]

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases. [ABSTRACT FROM AUTHOR]

BACKGROUND AND OBJECTIVE: Ultrasound-guided interventions (such as biopsies) of unclear lesions are indicated if microcirculatory changes indicate possible malignant lesions. These place high demands on the ultrasound device used. In order to potentially reduce the often associated high technical effort, the wireless ultrasound device Vscan AirTM was examined as a possible ultrasound device for the intervention biopsy. METHODS: As part of an advanced training course on Computertomographie- and ultrasound-guided biopsy and ablation procedures, participants were asked about the image quality of the handheld device used by means of questionnaires. Various lesions were evaluated at a depth of 1.0 to 5.0 cm in an in vitro liver model. The image quality was evaluated independently before, during and after the intervention. The rating scale contained values from 0 (no assessment possible) to 5 (maximum high image quality). A high-end device was used as a reference. RESULTS: A total of 11 participants took part in the study (n = 4 male [36.4%], n = 7 female [63.6%]). A total of five tumor like lesions at different depths (1 cm, 2 cm, 3 cm, 4 cm, >4 cm) were assessed separately. In all cases, an adequate biopsy of the target lesion (1 cm in length, core filling 5 mm) was successful. From a depth of 3 cm, the image quality of the mobile device increasingly decreased, but the image quality of the high-end system was still not impaired. Compared to the high-end device, there was a highly significant difference in image quality from a depth of 3 cm (p < 0.01). Assessment by inexperienced examiners using a handheld device was adequately possible. CONCLUSIONS: Mobile interventional ultrasound represents a potential alternative for the biopsy of unclear tumorous lesions with microcirculatory disorders with limited depth localization. [ABSTRACT FROM AUTHOR]

First trimester placental development comprises some of the most critical yet understudied events that impact fetal development. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence offspring throughout their lives. Thus, a paradigm to study early placental development is necessary, and this has spurred on the pursuit of new in vitro model systems that recapitulate specific aspects of placentation. One of the most complex and translationally valid models to arise are organoids, three-dimensional structures comprising multiple differentiated cell types that originate from a common progenitor population. Trophoblasts are the progenitor cells of the placenta, serving as the proliferative base for placental development. Recent advances have enabled the derivation of organoids from primary tissue, yet access to first trimester human samples is ethically constrained; derivation from established trophoblast stem cell lines is an alternative source. Organoids have already proven useful in generating insights into molecular events that underlie trophoblast differentiation, with the identification of new cell subtypes that are primed to differentiate down different paths. In this review, (1) we recap early pregnancy development events, (2) provide an overview of the cellular complexity of the placenta, (3) discuss the generation of organoids from tissue versus cellular sources, (4) highlight the value of translational animal models, and (5) focus on the complexities of the molecular regulation of trophoblast organoid development, differentiation, and function. [ABSTRACT FROM AUTHOR]

Pollution by microplastics and nanoplastics (MNPs) raises concerns, not only regarding their environmental effects, but also their potential impact on human health by internalization via the small intestine. However, the detailed pathways of MNP internalization and their toxicities to the human intestine have not sufficiently been understood, thus, further investigations are required. This work aimed to understand the behavior of MNPs, using in vitro human intestine models, tri-culture models composed of enterocyte Caco-2 cells, goblet-like HT29-MTX-E12 cells, and microfold cells (M cells) induced by the lymphoblast cell line Raji B. Three sizes (50, 100, and 500 nm) of polystyrene (PS) particles were exposed as MNPs on the culture model, and size-dependent translocation of the MNPs and the contributions of each cell were clarified, emphasizing the significance of the tri-culture model. In addition, potential concerns of MNPs were suggested when they invaded the circulatory system of the human body. [ABSTRACT FROM AUTHOR]

Simple Summary: This review aims to improve our understanding of gastrointestinal tract cancer and the side effects of cancer treatment by using advanced in vitro systems. Traditional models like cell cultures and animal studies provide valuable insights but have limitations in replicating the complexity of human disease and raise ethical concerns. By focusing on bioreactor-based in vitro systems, which can mimic the physical and chemical environment of the gastrointestinal tract, this study aims to provide more accurate models for studying cancer and its treatment side effects. These advancements could lead to better insights into disease mechanisms, potentially improving treatment strategies and benefiting the broader research community. Human diseases are multifaceted, starting with alterations at the cellular level, damaging organs and their functions, and disturbing interactions and immune responses. In vitro systems offer clarity and standardisation, which are crucial for effectively modelling disease. These models aim not to replicate every disease aspect but to dissect specific ones with precision. Controlled environments allow researchers to isolate key variables, eliminate confounding factors and elucidate disease mechanisms more clearly. Technological progress has rapidly advanced model systems. Initially, 2D cell culture models explored fundamental cell interactions. The transition to 3D cell cultures and organoids enabled more life-like tissue architecture and enhanced intercellular interactions. Advanced bioreactor-based devices now recreate the physicochemical environments of specific organs, simulating features like perfusion and the gastrointestinal tract's mucus layer, enhancing physiological relevance. These systems have been simplified and adapted for high-throughput research, marking significant progress. This review focuses on in vitro systems for modelling gastrointestinal tract cancer and the side effects of cancer treatment. While cell cultures and in vivo models are invaluable, our main emphasis is on bioreactor-based in vitro modelling systems that include the gut microbiome. [ABSTRACT FROM AUTHOR]

Obesity-related chronic low-grade inflammation plays a central role in the development of insulin resistance. Macrophages are key players in adipose tissue homeostasis, and their phenotypic shift from the anti-inflammatory or alternatively activated (M2) form to the pro-inflammatory, classically activated (M1) form is a hallmark of insulin resistance. However, adipose tissue macrophages (ATMs) have been identified as a distinct subpopulation of macrophages in several recent studies. These ATMs, described as metabolically activated macrophages (MMe), differ from M1 and are primarily found in the adipose tissue of obese individuals. In our study, we developed an in vitro model of MMe macrophages to establish a simple and reproducible system to understand their characteristics and role in the pathophysiology of insulin resistance. We examined their characteristics such as inflammatory patterns, surface markers, and metabolic features, and compared them with M1 and M2 macrophages. We found that a cell line-based in vitro model effectively mirrors the characteristics of ATMs, highlighting distinct inflammatory phenotypes, metabolism, surface markers, altered lysosomal activity, and ER stress akin to macrophages in vivo. This model captures the subtle distinctions between MMe and M1, and can be effectively used to study several features of macrophage–adipose interactions of therapeutic importance. [ABSTRACT FROM AUTHOR]

The vascular system, essential for human physiology, is vital for transporting nutrients, oxygen, and waste. Since vascular structures are involved in various disease pathogeneses and exhibit different morphologies depending on the organ, researchers have endeavored to develop organ-specific vascular models. While animal models possess sophisticated vascular morphologies, they exhibit significant discrepancies from human tissues due to species differences, which limits their applicability. To overcome the limitations arising from these discrepancies and the oversimplification of 2D dish cultures, microphysiological systems (MPS) have emerged as a promising alternative. These systems more accurately mimic the human microenvironment by incorporating cell interactions, physical stimuli, and extracellular matrix components, thus facilitating enhanced tissue differentiation and functionality. Importantly, MPS often utilize human-derived cells, greatly reducing disparities between model and patient responses. This review focuses on recent advancements in MPS, particularly in modeling the human organ-specific vascular system, and discusses their potential in biological adaptation. [ABSTRACT FROM AUTHOR]

Toxicological assessment of chemicals is crucial for safeguarding human health and the environment. However, traditional animal experiments are associated with ethical, technical, and predictive limitations in assessing the toxicity of chemicals to the skin. With the recent development of bioengineering and tissue engineering, three‐dimensional (3D) skin models have been commonly used as an alternative for toxicological studies. The skin consists of the subcutaneous, dermis, and epidermis. All these layers have crucial functions such as physical and biological protection and thermoregulation. The epidermis is the shallowest layer protecting against external substances and media. Because the skin is the first contact point for many substances, this organ is very significant for assessing local toxicity following skin exposure. According to the classification of the United Nations Global Harmonized System, skin irritation is a major potentially hazardous characteristic of chemicals, and this characteristic must be accurately assessed and classified for enhancing chemical safety management and preventing and reducing chemical accidents. This review discusses the research progress of 3D skin models and introduces their application in assessing chemical skin irritation. Assessing chemical toxicity is essential for maintaining the safety of the human health and environment. Traditional animal experiments face ethical, technical, and predictive limitations, the application of alternative models becomes critical. This review focuses on and summarizes the research progress of 3D skin models and their application in the assessment of chemical‐induced skin irritation, providing a basis for the development of in vitro models and their application to skin irritation. [ABSTRACT FROM AUTHOR]

Abstract Background Heart failure (HF) is a condition characterized by the heart’s inability to meet the body’s demands, resulting in various complications. Two primary types of HF exist, namely HF with preserved left ventricular ejection fraction (LVEF) and HF reduced with LVEF. The progression of HF involves compensatory mechanisms such as cardiac hypertrophy, fibrosis, and alterations in gene expression. Pressure overload and volume overload are common etiologies of HF, with pressure overload often stemming from conditions like hypertension, leading to left ventricular hypertrophy and fibrosis. In contrast, volume overload can arise from chronic valvular regurgitant disease, also inducing left ventricular hypertrophy. Main body In vitro cell culture techniques serve as vital tools in studying HF pathophysiology, allowing researchers to investigate cellular responses and potential therapeutic targets. Additionally, biomarkers, measurable biological characteristics, play a crucial role in diagnosing and predicting HF. Some notable biomarkers include adrenomedullin, B-type natriuretic peptide, copeptin, galectin-3, interleukin-6, matrix metalloproteinases (MMPs), midregional pro-atrial natriuretic peptide, myostatin, procollagen type I C-terminal propeptide, procollagen type III N-terminal propeptide and tissue inhibitors of metalloproteinases (TIMPs). These biomarkers aid in HF diagnosis, assessing its severity, and monitoring treatment response, contributing to a deeper understanding of the disease and potentially leading to improved management strategies and outcomes. Conclusions This review provides comprehensive insights into various in vivo models of HF, commonly utilized cell lines in HF research, and pivotal biomarkers with diagnostic relevance for HF. By synthesizing this information, researchers gain valuable resources to further explore HF pathogenesis, identify novel therapeutic targets, and enhance diagnostic and prognostic approaches.

Abstract Background Staphylococcus aureus is the predominant pathogen isolated in diabetic foot infections. Recently, the skin commensal bacterium, Helcococcus kunzii, was found to modulate the virulence of this pathogen in an in vivo model. This study aims to elucidate the molecular mechanisms underlying the interaction between these two bacterial species, using a proteomic approach. Results Our results reveal that H. kunzii can coexist and proliferate alongside S. aureus in a Chronic Wound Media (CWM), thereby mimicking an in vitro chronic wound environment. We noted that the secreted proteome of H. kunzii induced a transcriptional effect on S. aureus virulence, resulting in a decrease in the expression level of agrA, a gene involved in quorum sensing. The observed effect could be ascribed to specific proteins secreted by H. kunzii including polysaccharide deacetylase, peptidoglycan DD-metalloendopeptidase, glyceraldehyde-3-phosphate dehydrogenase, trypsin-like peptidase, and an extracellular solute-binding protein. These proteins potentially interact with the agr system, influencing S. aureus virulence. Additionally, the virulence of S. aureus was notably affected by modifications in iron-related pathways and components of cell wall architecture in the presence of H. kunzii. Furthermore, the overall metabolism of S. aureus was reduced when cocultured with H. kunzii. Conclusion Future research will focus on elucidating the role of these excreted factors in modulating virulence.

Cell encapsulation into spherical microparticles is a promising bioengineering tool in many fields, including 3D cancer modelling and pre-clinical drug discovery. Cancer microencapsulation models can more accurately reflect the complex solid tumour microenvironment than 2D cell culture and therefore would improve drug discovery efforts. However, these microcapsules, typically in the range of 1 – 5000 µm in diameter, must be carefully designed and amenable to high-throughput production. This review therefore aims to outline important considerations in the design of cancer cell microencapsulation models for drug discovery applications and examine current techniques to produce these. Extrusion (dripping) droplet generation and emulsion-based techniques are highlighted and their suitability to high-throughput drug screening in terms of tumour physiology and ease of scale up is evaluated.

Generation of cardiomyocytes from human pluripotent stem cells (hPSCs) is of high interest for disease modelling and regenerative medicine. hPSCs can provide an unlimited source of patient-specific cardiomyocytes that are otherwise difficult to obtain from individuals. Moreover, the low proliferation rate of adult cardiomyocytes and low viability ex vivo limits the quantity of study material. Most protocols for the differentiation of cardiomyocytes from hPSCs are based on the temporal modulation of the Wnt pathway. However, during the initial stage of GSK-3 inhibition, a substantial number of cells are lost due to detachment. In this study, we aimed to increase the efficiency of generating cardiomyocytes from hPSCs. We identified cell death as a detrimental factor during this initial stage of in vitro cardiomyocyte differentiation. Through pharmacological targeting of different types of cell death, we discovered that ferroptosis was the main cell death type during the first 48 h of the in vitro differentiation procedure. Inhibiting ferroptosis using ferrostatin-1 during cardiomyocyte differentiation resulted in increased robustness and cell yield.

First trimester placental development comprises some of the most critical yet understudied events that impact fetal development. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence offspring throughout their lives. Thus, a paradigm to study early placental development is necessary, and this has spurred on the pursuit of new in vitro model systems that recapitulate specific aspects of placentation. One of the most complex and translationally valid models to arise are organoids, three-dimensional structures comprising multiple differentiated cell types that originate from a common progenitor population. Trophoblasts are the progenitor cells of the placenta, serving as the proliferative base for placental development. Recent advances have enabled the derivation of organoids from primary tissue, yet access to first trimester human samples is ethically constrained; derivation from established trophoblast stem cell lines is an alternative source. Organoids have already proven useful in generating insights into molecular events that underlie trophoblast differentiation, with the identification of new cell subtypes that are primed to differentiate down different paths. In this review, (1) we recap early pregnancy development events, (2) provide an overview of the cellular complexity of the placenta, (3) discuss the generation of organoids from tissue versus cellular sources, (4) highlight the value of translational animal models, and (5) focus on the complexities of the molecular regulation of trophoblast organoid development, differentiation, and function.

Abstract Background Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients’ BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. Methods Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. Results Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. Conclusions Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.

Abstract Background This study aimed to engineer and optimise a dysbiotic biofilm model to develop in vitro root caries for investigating microbial modulation strategies. The model involved growing complex biofilms from a saliva inoculum collected from four volunteers using two strategies. In the first strategy (“pre-treatment strategy”), bovine root slabs were used, and two natural compounds were incorporated at time 0 of the 10-day biofilm experiment, which included sucrose cycles mimicking the cariogenic environment. In the second strategy (“post-treatment strategy”), mature biofilms were grown in a modified Calgary biofilm device coated with collagen and hydroxyapatite for 7 days and then were exposed to the same natural compounds. The metatranscriptome of each biofilm was then determined and analysed. Collagenase activity was examined, and the biofilms and dentine were imaged using confocal and scanning electron microscopy (SEM). Mineral loss and lesion formation were confirmed through micro-computed tomography (μ-CT). Results The pH confirmed the cariogenic condition. In the metatranscriptome, we achieved a biofilm compositional complexity, showing a great diversity of the metabolically active microbiome in both pre- and post-treatment strategies, including reads mapped to microorganisms other than bacteria, such as archaea and viruses. Carbohydrate esterases had increased expression in the post-treated biofilms and in samples without sugar cycles, while glucosyltransferases were highly expressed in the presence of sucrose cycles. Enrichment for functions related to nitrogen compound metabolism and organic cyclic component metabolism in groups without sucrose compared to the sucrose-treated group. Pre-treatment of the roots with cranberry reduced microbial viability and gelatinase (but not collagenase) activity (p

Abstract Hepatocellular carcinoma (HCC) is among the most common malignancies worldwide and is characterized by high rates of morbidity and mortality, posing a serious threat to human health. Interventional embolization therapy is the main treatment against middle- and late-stage liver cancer, but its efficacy is limited by the performance of embolism, hence the new embolic materials have provided hope to the inoperable patients. Especially, hydrogel materials with high embolization strength, appropriate viscosity, reliable security and multifunctionality are widely used as embolic materials, and can improve the efficacy of interventional therapy. In this review, we have described the status of research on hydrogels and challenges in the field of HCC therapy. First, various preparation methods of hydrogels through different cross-linking methods are introduced, then the functions of hydrogels related to HCC are summarized, including different HCC therapies, various imaging techniques, in vitro 3D models, and the shortcomings and prospects of the proposed applications are discussed in relation to HCC. We hope that this review is informative for readers interested in multifunctional hydrogels and will help researchers develop more novel embolic materials for interventional therapy of HCC. Graphical Abstract

We have developed an in vitro model for the evaluation of potential anticoccidial properties of novel compounds aimed to control chicken coccidiosis, a costly disease for the poultry industry. This disease is caused by protozoan parasites of the genus Eimeria (Apicomplexa), and it is mainly controlled by chemoprophylaxis with ionophores and chemical anticoccidials; however, there is an overall agreement about the limitation of these traditional drugs and the need to improve current methods of control. Anticoccidial activities of novel compounds is currently evaluated by expensive experiments that involve large numbers of chickens. The use of our in vitro model for the pre-screening of essential oils led to a reduction of 67% of the chickens used in the in vivo trials for validation. Eimeria parasites can only complete their life cycle in their animal host, therefore chickens are required for their propagation as they cannot be propagated in vitro. In this study, we describe how further optimisation of this in vitro model by miniaturisation can have an additional impact in reduction of the number of chickens used for the generation of parasite stocks for provision of the in vitro model. We have estimated that the use of one chicken could support the evaluation of 10 compounds with a 96-well plate format versus only two compounds with a 24-well plate format, which means an 80% reduction in chicken use. In this study we have proved that the miniaturisation into a 96-well plate format perfectly mimics the invasion and replication observed before in the 24-well plate format. In addition, the 96-well plate format has allowed the simultaneous pre-screening of higher numbers of anticoccidial drugs at different concentrations following streamlined protocols in a more cost-effective way, factors that are beneficial for a wider uptake of the model by other researchers investigating anticoccidial compounds.

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Background: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. Methods: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. Results: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. Conclusions: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model. [ABSTRACT FROM AUTHOR]

Background: This study aimed to engineer and optimise a dysbiotic biofilm model to develop in vitro root caries for investigating microbial modulation strategies. The model involved growing complex biofilms from a saliva inoculum collected from four volunteers using two strategies. In the first strategy ("pre-treatment strategy"), bovine root slabs were used, and two natural compounds were incorporated at time 0 of the 10-day biofilm experiment, which included sucrose cycles mimicking the cariogenic environment. In the second strategy ("post-treatment strategy"), mature biofilms were grown in a modified Calgary biofilm device coated with collagen and hydroxyapatite for 7 days and then were exposed to the same natural compounds. The metatranscriptome of each biofilm was then determined and analysed. Collagenase activity was examined, and the biofilms and dentine were imaged using confocal and scanning electron microscopy (SEM). Mineral loss and lesion formation were confirmed through micro-computed tomography (μ-CT). Results: The pH confirmed the cariogenic condition. In the metatranscriptome, we achieved a biofilm compositional complexity, showing a great diversity of the metabolically active microbiome in both pre- and post-treatment strategies, including reads mapped to microorganisms other than bacteria, such as archaea and viruses. Carbohydrate esterases had increased expression in the post-treated biofilms and in samples without sugar cycles, while glucosyltransferases were highly expressed in the presence of sucrose cycles. Enrichment for functions related to nitrogen compound metabolism and organic cyclic component metabolism in groups without sucrose compared to the sucrose-treated group. Pre-treatment of the roots with cranberry reduced microbial viability and gelatinase (but not collagenase) activity (p < 0.05). SEM images showed the complexity of biofilms was maintained, with a thick extracellular polysaccharides layer. Conclusions: This root caries model was optimized to produce complex cariogenic biofilms and root caries-like lesions, and could be used to test microbial modulation in vitro. Pre-treatments before biofilm development and cariogenic challenges were more effective than post-treatments. The clinical significance lies in the potential to apply the findings to develop varnish products for post-professional tooth prophylaxis, aiming at implementing a strategy for dysbiosis reversal in translational research. 2cRgxuoMbmgt6TZPkgExXQ Video Abstract [ABSTRACT FROM AUTHOR]

Aim: This study aims to investigate the potential neuroprotective effects of fibroblast growth factor-21 (FGF21) on dorsal root ganglion (DRG) neurons under high glucose (HG) conditions, mimicking diabetic neuropathy. Specifically, we hypothesize that FGF21 enhances cell viability and reduces glucose-induced neuronal death via the activation of the phosphatidyl-inositol-3-kinase (PI3K)/AKT signaling pathway. Materials and Methods: DRG neurons were cultured from 1-day-old to 2-day-old Wistar rats and exposed to HG concentrations to simulate diabetic conditions. Various concentrations of FGF21 were administered to the DRG neurons. Cell viability was assessed using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), a widely used enzymatic method for determining cellular metabolic activity. The involvement of the PI3K/AKT signaling pathway in mediating the effects of FGF21 was also examined through biochemical assays and pathway inhibitors. Results: Administration of FGF21 to DRG neurons exposed to HG conditions significantly protected cell viability and reduced glucose-induced neuronal death (p<0.05). The protective effects of FGF21 were found to be dose-dependent, with higher concentrations showing more pronounced benefits. Furthermore, the activation of the PI3K/AKT signaling pathway was confirmed to play a crucial role in the neuroprotective mechanism of FGF21, as inhibition of this pathway attenuated the protective effects. Conclusion: This study demonstrates for the first time that FGF21 has a neuroprotective effect on DRG neuron survival in a diabetic neuropathy model. By activating the PI3K/AKT signaling pathway, FGF21 helps maintain cell viability and reduces glucoserelated neuronal death. These findings provide a promising basis for the development of new therapeutic strategies for the treatment of diabetic neuropathy, leveraging the neuroprotective properties of FGF21. [ABSTRACT FROM AUTHOR]

The present study aimed to generate 3D spheroid cultures from three rainbow trout (RTE, RTK and RTS) cell lines by simple and reproducible methods and evaluated them for the propagation of fish viral pathogen for the first time. The 3D spheroids were generated in the present study by scaffold-free method using 3D-orbital shaker. The growth of spheroids was documented on each day until 15 days and their viability was assessed by using AO/EB and Hoechst 33258 fluorescent staining under bright field microscope. The successfully generated RTK spheroids (RTKs) from the RTK cells were further used for the propagation of SJNNV virus in direct comparison of viral growth with conventional 2D monolayer culture of RTK cell line. Immunofluorescent staining confirms the expression of SJNNV major capsid protein in infected RTK spheroid earlier than the RTK monolayer. The samples collected from both RTK spheroid and RTK monolayer at different days of post infection was confirmed by RT-PCR and their viral copy numbers found high in RTK spheroid while quantified by qRT-PCR. Further, SJNNV major capsid protein was confirmed by western blot and ELISA. The viral load obtained using RTK spheroid was found to be high when compared to the monolayer and this study suggests that the spheroid generated from RTK cells is more suitable in vitro tool that can be used for propagation of fish viral pathogens in large-scale for production of whole virus vaccine. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a novel biomimetic in vitro extraluminal migration model to observe the migration of bacteria along indwelling urinary catheters within the urethra and assess the efficacy of a prototype chlorhexidine diacetate (CHX) coating to prevent this migration. The in vitro urethra model utilised chromogenic agar. A catheter was inserted into each in vitro urethra. One side of the urethra was then inoculated with bacteria to replicate a contaminated urethral meatus. The models were then incubated for 30 days (d), with the migration distance recorded each day. Four indwelling catheter types were used to validate the in vitro urethra model and methodology. Using the biomimetic in vitro urethra model, E. coli and S. aureus migrated the entire length of a control catheter within 24–48 h (h). In the presence of a prototype CHX coating, full migration of the channel was prevented for 30 d. The results of this study support the hypothesis that catheter-associated urinary tract infections (CAUTIs) could be prevented by targeting catheter-mediated extraluminal microbial migration from outside of the urinary tract into the bladder. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can a co-culture of three cell types mimic the in vivo layers of the uterine wall? SUMMARY ANSWER Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo. WHAT IS KNOWN ALREADY We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells. STUDY DESIGN, SIZE, DURATION Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min. PARTICIPANTS/MATERIALS, SETTING, METHODS The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm. MAIN RESULTS AND THE ROLE OF CHANCE We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P -value < 0.05) when the length of exposure was increased from 10 to 30 min. Furthermore, the inner layers of ESC and MSMC, which are not in direct contact with the applied shearing fluid, also increased their F-actin polymerization. LARGE SCALE DATA N/A. LIMITATIONS, RESONS FOR CAUTION The mixed seeding co-culture model was exposed to steady WSS of one magnitude, whereas the uterus is a dynamic organ with intra-uterine peristaltic fluid motions that vary in vivo with different time-dependent magnitude. Further in vitro studies may explore the response to peristaltic WSS or other physical and/or hormonal perturbations that may mimic the spectrum of pathophysiological aspects. WIDER IMPLICATIONS OF THE FINDINGS Numerous in vitro models were developed in order to mimic the human endometrium and endometrium–myometrium interface (EMI) region. The present co-culture models seem to be the first constructed from EEC, ESC, and MSMC on a collagen-coated synthetic membrane. These multi-cell in vitro models better represent the complex in vivo anatomy of the EMI region. The mixed seeding multi-cell in vitro model may easily be implemented in controlled studies of uterine function in reproduction and the pathogenesis of diseases. STUDY FINDING/COMPETING INTEREST(S) This study was supported in part by Tel Aviv University funds. All authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases remain as the most common cause of death worldwide. To reveal the underlying mechanisms in varying cardiovascular diseases, in vitro models with cells and supportive biomaterial can be designed to recapitulate the essential components of human heart. In this study, we analyzed whether 3D co-culture of cardiomyocytes (CM) with vascular network and with adipose tissue-derived mesenchymal stem/stromal cells (ASC) can support CM functionality. CM were cultured with either endothelial cells (EC) and ASC or with only ASC in hydrazide-modified gelatin and oxidized gellan gum hybrid hydrogel to form cardiovascular multiculture and myocardial co-culture, respectively. We studied functional characteristics of CM in two different cellular set-ups and analyzed vascular network formation, cellular morphology and orientation. The results showed that gellan gum-gelatin hydrogel supports formation of two different cellular networks and functional CM. We detected formation of a modest vascular network in cardiovascular multiculture and extensive ASC-derived alpha smooth muscle actin -positive cellular network in multi- and co-culture. iPSC-CM showed elongated morphology, partly aligned orientation with the formed networks and presented normal calcium transients, beating rates, and contraction and relaxation behavior in both setups. These 3D cardiac models provide promising platforms to study (patho) physiological mechanisms of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Asbestos fibres have been considered an environmental hazard for decades. However, little is known about the attempts of circulating immune cells to counteract their toxicity. We addressed the early effects of fibre‐released soluble factors (i.e. heavy metals) in naïve immune cells, circulating immediately below the alveolar/endothelial cell layer. By comparison, the direct fibre effects on endotheliocytes were also studied since these cells are known to sustain inflammatory processes. The three mineral fibres analysed showed that mainly chrysotile (CHR) and erionite (ERI) were able to release toxic metals in extracellular media respect to crocidolite (CRO), during the first 24 h. Nevertheless, all three fibres were able to induce oxidative stress and genotoxic damage in indirectly challenged naïve THP‐1 monocytes (separated by a membrane). Conversely, only CHR‐released metal ions induced apoptosis, NF‐κB activation, cytokines and CD163 gene overexpression, indicating a differentiation towards the M0 macrophage phenotype. On the other hand, all three mineral fibres in direct contact with HECV endothelial cells showed cytotoxic, genotoxic and apoptotic effects, cytokines and ICAM‐I overexpression, indicating the ability of these cells to promote an inflammatory environment in the lung independently from the type of inhaled fibre. Our study highlights the different cellular responses to mineral fibres resulting from both the nature of the cells and their function, but also from the chemical–physical characteristics of the fibres. In conclusion, CHR represented the main pro‐inflammatory trigger, able to recruit and activate circulating naïve monocytes, through its released metals, already in the first 24 h after inhalation. The early effects of mineral fibre‐released heavy metals in THP‐1 cells, mimicking the immune cells in the alveolar capillaries, were investigated, and by comparison, also the direct fibre effects on the endothelial cell layer. The study highlights that the cellular responses result from the nature of the cells, their function and from the fibre chemical–physical characteristics. CHR represented the main pro‐inflammatory trigger, able to recruit and activate circulating monocytes, through its released metals, already at 24 h after inhalation. [ABSTRACT FROM AUTHOR]

In vitro models of the pancreas can aid in developing therapies for pancreatic diseases. Nonetheless, most pancreatic tissue engineering is limited to insulin‐secreting β‐cells or pancreatic adenocarcinoma models. Combining all essential tissue components, including exocrine, endocrine, and blood vasculature, is crucial to recapitulate native tissue organization. In this study, extrusion‐based 3D bioprinting to create pancreatic tissue constructs containing both endocrine and exocrine compartments is exploited. Mouse pluripotent stem cell‐derived pancreatic progenitors, pancreatic endothelial cells, and mesenchymal stem cells are bioprinted. During postprinting cultivation, the cells differentiated into exocrine and endocrine lineages, resulting in vascularized pancreatic tissue‐like constructs with multiple cell types. However, the bioprinted constructs contracted significantly postprinting, hindering control of cell positioning and shape preservation. Therefore, 2 strategies to reduce the contraction and deformation of the bioprinted constructs are developed. These bioprinting techniques and biomaterial combinations allow us to investigate the influence of construct design and cellular composition on pancreatic cell fate. The results reveal that increased construct stiffness and endothelial component presence significantly promoted endocrine while suppressing exocrine differentiation. Overall, a novel strategy for pancreatic tissue engineering that advances and holds promise for pancreas disease and development modeling, as well as pharmaceutical testing is demonstrated. [ABSTRACT FROM AUTHOR]

Background: Retrograde Drilling (RD) is a surgical technique employed for osteochondral lesions of the talus (OCLTs) to reach the subchondral bone lesion from behind, thus preserving cartilage integrity. The aim of the present pilot study was to set up an in vitro model of OCLTs to evaluate the regenerative potential of biological approaches that could be associated with the RD technique. Methods: For this purpose, an OCLT was created in human osteochondral specimens, to try to mimic the RD technique, and to compare the regenerative potential of two biological treatments. For this purpose, three groups of treatments were performed in vitro: (1) no treatment (empty defect); (2) autologous bone graft (ABG); (3) hyaluronic membrane enriched with autologous bone marrow cells. Tissue viability; production of Collagen I and II, Vascular Endothelial Growth Factor, and Aggrecan; and histological and microCT evaluations were performed after 30 days of culture in normal culture conditions. Results: It was observed that Group 3 showed the highest viability, and Group 2 showed the highest protein production. From a histological and microtomographic point of view, it was possible to appreciate the structure of the morcellized bone with which the defect of Group 2 was filled, while it was not yet possible to observe the deposition of mineralized tissue in Group 3. Conclusions: To conclude, this pilot study shows the feasibility of an alternative in vitro model to evaluate and compare the regenerative potential of two biological scaffolds, trying to mimic the RD technique as much as possible. The tissues remained vital for up to 4 weeks and both ABG and hyaluronic acid-based scaffolds stimulated the release of proteins linked to regenerative processes in comparison to the empty defect group. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is among the most common malignancies worldwide and is characterized by high rates of morbidity and mortality, posing a serious threat to human health. Interventional embolization therapy is the main treatment against middle- and late-stage liver cancer, but its efficacy is limited by the performance of embolism, hence the new embolic materials have provided hope to the inoperable patients. Especially, hydrogel materials with high embolization strength, appropriate viscosity, reliable security and multifunctionality are widely used as embolic materials, and can improve the efficacy of interventional therapy. In this review, we have described the status of research on hydrogels and challenges in the field of HCC therapy. First, various preparation methods of hydrogels through different cross-linking methods are introduced, then the functions of hydrogels related to HCC are summarized, including different HCC therapies, various imaging techniques, in vitro 3D models, and the shortcomings and prospects of the proposed applications are discussed in relation to HCC. We hope that this review is informative for readers interested in multifunctional hydrogels and will help researchers develop more novel embolic materials for interventional therapy of HCC. [ABSTRACT FROM AUTHOR]

Nowadays, dietary supplements are a permanent part of our diet. Using various simulated in vitro digestive models, the bioavailability of dietary supplement ingredients has also been investigated. In most cases, static models are used instead of dynamic ones. This article focuses on the division of applications of in vitro methods, such as assessing the quality of dietary supplements (in chemical and pharmaceutical form), the impact of diet on the assessment of the bioavailability of product ingredients, the impact of supplement ingredients on the state of intestinal microflora, and the development of new products using various encapsulation methods. The review included publications from 2000 to 2024 showing the use of in vitro methods in dietary supplements containing polysaccharides, proteins, elements, vitamins, and bioactive substances, as well as probiotic and prebiotic products. The impact of components in dietary supplements on the human digestive tract and their degree of bioaccessibility were determined through the use of in vitro methods. The application of in vitro methods has also become an effective tool for designing new forms of dietary supplements in order to increase the availability and durability of labile ingredients in these products. [ABSTRACT FROM AUTHOR]

Keloids are a common connective tissue disorder with an ill-understood etiopathogenesis and no effective treatment. This is exacerbated because of the absence of an animal model. Patient-derived primary keloid cells are insufficient as they age through passaging and have a limited supply. Therefore, there is an unmet need for development of a cellular model that can consistently and faithfully represent keloid's pathognomic features. In view of this, we developed keloid-derived immortalized fibroblast (KDIF) cell lines from primary keloid fibroblasts (PKF) by transfecting the human telomerase reverse transcriptase (hTERT) gene. The TERT gene encodes the catalytic subunit of the telomerase enzyme, which is responsible for maintaining the cellular replicative potential (cellular immortalization). Primary fibroblasts from keloid-specific lesional (peripheral, middle, and top) as well as extralesional sites were isolated and evaluated for cell line development and comparative cellular characteristics by employing qRT-PCR and immunofluorescence staining. Moreover, the immortalized behavior of KDIF cell lines was evaluated by comparing with cutaneous fibrosarcoma and dermatofibrosarcoma protuberans cell lines. Stable KDIF cell lines with elevated expression of hTERT exhibited the cellular characteristics of site-specific keloid fibroblasts. Histochemical staining for bgalactosidase revealed a significantly lower number of β-gal-positive cells in all three KDIF cell lines compared with that in PKFs. The cell growth curve pattern was studied over 10 passages for all three KDIF cell lines and was compared with the control groups. The results showed that all three KDIF cell lines grew significantly faster and obtained a fast growing characteristic as compared to primary keloid and normal fibroblasts. Phenotypic behavior in growth potential is an indication of hTERT-mediated immortalized transformation. Cell migration analysis revealed that the top and middle KDIF cell lines exhibited similar migration trend as site-specific PKFs. Notably, peripheral KDIF cell line showed significantly enhanced cell migration in comparison to the primary peripheral fibroblasts. All KDIF cell lines expressed Collagen I protein as a keloid-associated fibrotic marker. Functional testing with triamcinolone inhibited cell migration in KDIF. ATCC short tandem repeat profiling validated the KDIF as keloid representative cell line. In summary, we provide the first novel KDIF cell lines. These cell lines overcome the limitations related to primary cell passaging and tissue supply due to immortalized features and present an accessible and consistent experimental model for keloid research. [ABSTRACT FROM AUTHOR]

The murine cell line MH-22A was originally established at least 50 years ago from a non-metastasizing tumor induced by 3-methylcholanthrene in C3HA mice and further adapted for in vitro growth. MH-22A cells grow predominantly epithelioid and they lead to the formation of transplantable tumors when injected subcutaneously into mice. In silico translation of genome data of this nearly triploid cell line to the human genome has recently characterized MH-22A as a suitable model for hepatoblastoma or early hepatocellular carcinoma. Here we established a multi-loci short tandem repeat signature for this cell line that allows quick and reliable cell line authentication. [ABSTRACT FROM AUTHOR]

The formation of pathogenic multispecies biofilms in the human oral cavity can lead to implant-associated infections, which may ultimately result in implant failure. These infections are neither easily detected nor readily treated. Due to high complexity of oral biofilms, detailed mechanisms of the bacterial dysbiotic shift are not yet even fully understood. In order to study oral biofilms in more detail and develop prevention strategies to fight implant-associated infections, in vitro biofilm models are sorely needed. In this study, we adapted an in vitro biofilm flow chamber model to include miniaturized transparent 3D-printed flow chambers with integrated optical pH sensors – thereby enabling the microscopic evaluation of biofilm growth as well as the monitoring of acidification in close proximity. Two different 3D printing materials were initially characterized with respect to their biocompatibility and surface topography. The functionality of the optically accessible miniaturized flow chambers was then tested using five-species biofilms (featuring the species Streptococcus oralis, Veillonella dispar, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis) and compared to biofilm growth on titanium specimens in the established flow chamber model. As confirmed by live/dead staining and fluorescence in situ hybridization via confocal laser scanning microscopy, the flow chamber setup proved to be suitable for growing reproducible oral biofilms under flow conditions while continuously monitoring biofilm pH. Therefore, the system is suitable for future research use with respect to biofilm dysbiosis and also has great potential for further parallelization and adaptation to achieve higher throughput as well as include additional optical sensors or sample materials.

IntroductionMechanical thrombectomy becomes more complex when the occlusion occurs in a tortuous cerebral anatomy, increasing the puncture to reperfusion time and the number of attempts for clot removal. Therefore, an understanding of stent retriever performance in these locations is necessary to increase the efficiency and safety of the procedure. An in vitro investigation into the effects of occlusion site tortuosity, blood clot hematocrit, and device geometry was conducted to identify their individual influence on stent retriever removal forces.MethodsEmbolus analogs were used to create occlusions in a mock circulatory flow loop, and in vitro mechanical thrombectomies were performed in arterial models of increasing tortuosity. The stent retriever removal forces of Solitaire Platinum and EmboTrap II devices were recorded through each geometry with and without embolus analogs present. Similar experiments were also conducted with Solitaire stent retrievers of varying lengths and diameters and 0, 25, and 50% hematocrit embolus analogs.ResultsThe removal force increased as model tortuosity increased for both the Solitaire Platinum and EmboTrap II stent retriever devices. The average removal forces in the simplest geometry with the Solitaire Platinum and EmboTrap II were 0.24 ± 0.01 N and 0.37 ± 0.02 N, respectively, and increased to 1.2 ± 0.08 N and 1.6 ± 0.17 N, respectively, in the most complex geometry. Slight increases in removal force were found with 0% hematocrit embolus analogs, however, no statistical significance between removal force and EA hematocrit was observed. A comparison between stent retriever removal forces between devices of different diameters also proved to be significant (p 0.05).ConclusionBenchtop mechanical thrombectomies performed with commercial stent retrievers of varying geometry showed that device removal forces increase with increasing model tortuosity, clot hematocrit does not play a significant role in device removal force, and that a stent retriever’s diameter has a greater impact on removal forces compared to its length. These results provide an improved understanding of the overall forces involved in mechanical thrombectomy and can be used to develop safer and more effective stent retrievers for the most difficult cases.

Abstract Prostate cancer is the most common cancer in over 50% of countries and the third most common malignancy worldwide. The tumor microenvironment includes the non‐cancerous cells and components present in the tumor, including the molecules they produce and release. Prostate cancer proliferation, angiogenesis, metastasis, and drug resistance are closely associated with the tumor microenvironment. With the continuous development of in vitro tumor models, they have gradually become important tools for recapitulating in vivo parental tumors and studying the reciprocal interactions between tumors and their microenvironment. In this perspective, we describe the significant in vitro models of prostate cancer, analyze the research results on incorporating the tumor microenvironment into these models, and compare their advantages and disadvantages. Furthermore, we highlight the future developmental direction of in vitro prostate cancer model research according to the hotspots of in vitro model research in other cancer types to facilitate precision medicine in prostate cancer.

IntroductionReprogramming of human-induced pluripotent stem cells (iPSCs) and their differentiation into specific cell types, such as induced sensory-like neurons (iSNs), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, the development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving the survival and quality of iSNs.MethodsiSNs were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-β-D-arabinofuranosyl-cytosine hydrochloride (AraC) treatment, magnetic-activated cell sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively.ResultsPassaging of cells during differentiation did not increase the iSN-to-total-cell-count ratio, and MACS of immature iSNs led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10 μM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSNs shortly after seeding also helped to reduce non-iSN cells.ConclusionIn direct comparison with other methods, treatment with 10 μM FdU for 24 h after differentiation shows promise for improving iSN culture purity, which could benefit downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of this method and enhance its reproducibility and applicability. Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC-based research.

IntroductionVaccination is one of the most effective infection prevention strategies. Viruses with high mutation rates -such as influenza- escape vaccine-induced immunity and represent significant challenges to vaccine design. Influenza vaccine strain selection is based on circulating strains and immunogenicity testing in animal models with limited predictive outcomes for vaccine effectiveness in humans.MethodsWe developed a human in vitro vaccination model using human tonsil tissue explants cultured in 3D perfusion bioreactors to be utilized as a platform to test and improve vaccines.ResultsTonsils cultured in bioreactors showed higher viability, metabolic activity, and more robust immune responses than those in static cultures. The in vitro vaccination system responded to various premanufactured vaccines, protein antigens, and antigen combinations. In particular, a multivalent in vitro immunization with three phylogenetically distant H3N2 influenza strains showed evidence for broader B cell activation and induced higher antibody cross-reactivity than combinations with more related strains. Moreover, we demonstrate the capacity of our in vitro model to generate de novo humoral immune responses to a model antigen.DiscussionPerfusion-cultured tonsil tissue may be a valuable human in vitro model for immunology research with potential application in vaccine candidate selection.

ABSTRACT Central venous catheters (CVCs) are widely used for intravenous medication administration. However, biofilm formation along the catheter surface is the main most important cause of catheter-related bloodstream infections. Nowadays, several antimicrobial-coated catheters are available to prevent biofilm development. In this study, we introduced a new dynamic in vitro model to evaluate the antimicrobial activity against bacterial biofilms on CVCs. Rifampicin–minocycline-coated catheters and control catheters without antimicrobial component were assembled into the model to test the antimicrobial activity on external surface and internal surface. After 1 h irrigation of Staphylococcus epidermidis or Staphylococcus aureus preculture and 23 h irrigation of Trypticase Soy Broth, the viable adherent organism was collected and counted. The enumeration results showed that the number of bacteria attached to antibacterial catheter was significantly less than that of the control catheter, both on external surface (P < 0.05) and internal surface (P < 0.05). The results were further confirmed by the scanning electron microscopy. In conclusion, the dynamic in vitro model can be applied to evaluate the antimicrobial activity against bacterial biofilms grown on the external and internal surfaces of CVCs used in clinical practice.IMPORTANCEFor the first time, a new dynamic in vitro model was constructed to evaluate the antimicrobial activity against bacterial biofilms on central venous catheters (CVCs) on both external surface and internal surface. This model could be applied to evaluate the antimicrobial activity against bacterial biofilms not only on CVCs but also other types of catheters.