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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]

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]

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]

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]

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]

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]

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]

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]

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.

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

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.

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.

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]

Engineered 3D neural tissues made of neurons and glial cells derived from human induced pluripotent stem cells (hiPSC) are among the most promising tools in drug discovery and neurotoxicology. They represent a cheaper, faster, and more ethical alternative to in vivo animal testing that will likely close the gap between in vitro animal models and human clinical trials. Micro-Electrode Array (MEA) technology is known to provide an assessment of compound effects on neural 2D cell cultures and acute tissue preparations by real-time, noninvasive, and long-lasting electrophysiological monitoring of spontaneous and evoked neuronal activity. Nevertheless, the use of engineered 3D neural tissues in combination with MEA biochips still involves series of constraints, such as drastically limited diffusion of oxygen and nutrients within tissues mainly due to the lack of vascularization. Therefore, 3D neural tissues are extremely sensitive to experimental conditions and require an adequately designed interface that provides optimal tissue survival conditions. A well-suited technique to overcome this issue is the combination of the Air-Liquid Interface (ALI) tissue culture method with the MEA technology. We have developed a full 3D neural tissue culture process and a data acquisition system composed of high-end electronics and novel MEA biochips based on porous, flexible, thin-film membranes integrating recording electrodes, named as "Strip-MEA," to allow the maintenance of an ALI around the 3D neural tissues. The main motivation of the porous MEA biochips development was the possibility to monitor and to study the electrical activity of 3D neural tissues under different recording configurations, (i) the Strip-MEA can be placed below a tissue, (ii) or by taking advantage of the ALI, be directly placed on top of the tissue, or finally, (iii) it can be embedded into a larger neural tissue generated by the fusion of two (or more) tissues placed on both sides of the Strip-MEA allowing the recording from its inner part. This paper presents the recording and analyses of spontaneous activity from the three positioning configurations of the Strip-MEAs. Obtained results are discussed with the perspective of developing in vitro models of brain diseases and/or impairment of neural network functioning. [ABSTRACT FROM AUTHOR]

Our previous study investigated the major flavonoids and antioxidant potential of Asian water lily (Nymphaea lotus L., family Nymphaeaceae) stamens and perianth extracts. Quercetin-3-O-rhamnoside (Que-3-Rha) and kaempferol-3-O-galactoside (Kae-3-Gal) were reported as the two most prominent flavonoids found in these extracts. Many flavonoids have been reported on the skin anti-aging effect that are useful for cosmeceutical/phytopharmaceutical application. However, Que-3-Rha and Kae-3-Gal occurring in this medicinal plant have not yet been evaluated for their ability to inhibit skin-aging enzymes. Therefore, this study aimed (1) to assess the enzyme inhibitory activity of Que-3-Rha and Kae-3-Gal, and (2) to conduct molecular modeling of these compounds against critical enzymes involved in skin aging such as collagenase, elastase, and tyrosinase. In vitro enzymatic assays demonstrated that both of the two most prominent flavonoids exhibited moderate to good inhibitory activity toward these enzymes. These experimental findings were supported by molecular docking analysis, which indicated that Que-3-Rha and Kae-3-Gal showed superior binding affinity to the target enzymes compared to the positive controls. Additionally, computational predictions suggested favorable skin permeability and no severe toxicity for both compounds. The results from molecular dynamic (MD) simulation revealed that all the complexes remained stable during the 200 ns MD simulation. Structural analyses and binding free energy calculations also supported the inhibitory potential of these two flavonoids against skin-aging enzymes. In conclusion, this study provides valuable insights into the anti-aging potential of the two major flavonoids occurring in this medicinal plant, paving the way for further development of cosmeceutical/phytopharmaceutical products targeting skin aging. [ABSTRACT FROM AUTHOR]

Oxygen is a prominent element in the well-being of humans, but also responsible for causing tissue damage. The oxygen molecule gets reduced to form a highly reactive oxygen species due to various metabolic events happening in the host. The generated free radicals could react with other biomolecules like protein, lipid, carbohydrate and nucleic acid to impart severe diseases and disorders. Antioxidants could effectively block the oxidation process by neutralizing the produced free radicals. Hence, the present study aims to validate the potent antioxidant activities of Couroupita guianensis Aubl. using goat liver slices that could serve as an in vivo simulated in vitro model. The methanolic extract and the alkaloid fraction of both leaves and flowers of C. guianensis Aubl. were prepared and their enzymatic and non-enzymatic antioxidant activities were evaluated. Hydrogen peroxide was used as a standard free radical throughout the experiments in all the treatment groups. The results have envisaged that the plant has enormous antioxidant activities in oxidatively stressed goat liver slices to combat various oxidative stress-related diseases and disorders. Conclusively, the in vitro model system to explore the antioxidant profiles will eventually mitigate the sacrifice of animals by exactly mimicking the in vivo conditions for research endeavors. [ABSTRACT FROM AUTHOR]

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.

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed.High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions.In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases.

Heart failure (HF) is a syndrome of symptoms and signs caused by cardiac insufficiency and have become a serious global health problem. The aim of this study is to clarify the role and mechanism failure of MIR17HG. We established the in vitro HF model by using H2O2-treated AC-16 and HCM cells, and the reactive oxygen species (ROS) level and natriuretic peptide precursor B (NPPB) expression were also detected. The RNA expression of MIR17HG, miR-153-3p, SIRT1, and NPPB were detected by quantitative reverse transcription PCR while the SIRT1 and NPPB expression were detected by western blot. The binding relationship among MIR17HG, miR-153-3p, and SIRT1 were assessed by dual-luciferase reporter assay and RNA binding protein immunoprecipitation assay. Then, MIR17HG and SIRT1 were overexpressed by lentivirus transfection, and the influence of MIR17HG and SIRT1 on H2O2-induced apoptosis mediated by p53 were evaluated. The results show that MIR17HG and SIRT1 were significantly downregulated, while miR-153-3p was significantly upregulated in HF model. Overexpression of MIR17HG reduced miR-153-3p and alleviated HF, while knockdown of SIRT1 weakened the effects of MIR17HG, suggesting that SIRT1 was the direct target of MIR17HG/miR-153-3p axis. MIR17HG is significantly downregulated in HF model. Our research shows that MIR17HG protects cardiomyocytes from ROS-induced damage via the MIR17HG/miR-153-3p/SIRT1 axis, suggesting that MIR17HG and SIRT1 are potential therapeutic targets in HF.

Cholangiocarcinoma is the second most common malignant tumor of the liver，which is characterized by atypical early symptoms，high malignancy and high mortality. Currently，the overall incidence of cholangiocarcinoma has been increased year by year worldwide and a majority of cholangiocarcinoma patients obtain poor prognosis. Effective diagnosis and treatment strategies are urgently required to change this situation. The establishment of disease models is a hot topic in cancer research. The establishment of experimental models of cholangiocarcinoma contributes to in-depth understanding and study of the incidence，development and drug therapeutic effect of cholangiocarcinoma，which is of significance for early diagnosis and treatment of cholangiocarcinoma.

Tumor necrosis factor alpha (TNF-α)/hypoxia-treated 3T3-L1 adipocytes have been used to model inflamed and insulin-resistant adipose tissue: this study examines gaps in the model. We tested whether modulating TNF-α/hypoxia treatment time could reduce cell death while still inducing inflammation and insulin resistance. Adipocytes were treated with TNF-α (12 h or 24 h) and incubated in a hypoxic chamber for 24 h. To examine maintenance of the phenotype over time, glucose and FBS were added at 24 h post initiation of treatment, and the cells were maintained for an additional 48 h. Untreated adipocytes were used as a control. Viability, insulin resistance, and inflammation were assessed using Live/Dead staining, RT-qPCR, ELISA, and glucose uptake assays. Treatment for 12 h with TNF-α in the presence of hypoxia resulted in an increase in the percentage of live cells compared to 24 h treated cells. Importantly, insulin resistance and inflammation were still induced in the 12 h treated adipocytes: the expression of the insulin sensitive and inflammatory genes was decreased and increased, respectively. In 72 h treated adipocytes, no significant differences were found in the viability, glucose uptake or insulin-sensitive and inflammatory gene expression. This study provides a modified approach to in vitro odeling adipocyte inflammation and insulin resistance.

In the present study, in vitro neuronal cell cultures were exposed to the two most common varieties of EDCs reported in food, phthalates and bisphenol A (BPA). Specifically, Bis (2-ethylhexyl) phthalate (DEHP), Mono-butyl-phthalate (MBP), Di-n-octylphthalate (OP), and Bisphenol A (BPA) were exposed in Neuro-2a neuroblastoma cell line. The enzymatic activity of methionine synthase (MS) was reduced by up to 3-fold in comparison to cultures that were not exposed to EDCs. MS is the key enzyme in the metabolic pathway that has been proposed elsewhere to be related to redox/methylation related disorders.

Advanced three-dimensional (3D) bioprinting technology enables the precise production of complex vascular structures and biomimetic models, driving advancements in tissue engineering and disease mechanism research. At the core of this technology is smart bioink, which is suitable for fabricating biomimetic models that can be vascularised to meet the property requirements of various tissues. Examples of smart bioinks include decellularized extracellular matrix (dECM), photocrosslinkable, reversible, and microgel-based biphasic (MB) bioinks, whose mechanical properties can be tuned through external stimuli. This tuning helps generate high-resolution and complex-shaped vascular networks essential for cell survival and functional maturation. This review explores advanced 3D bioprinting strategies using smart bioinks for spatially controlled and perfusable vascular in vitro models, emphasising the reconstruction of functional vascular structures within 3D bioprinted models. It also discusses challenges and future prospects, suggesting that 3D bioprinted models could serve as alternatives to traditional animal models for disease modelling and drug screening.

ABSTRACTThe prevalence of inflammatory bowel disease (IBD) is rising globally; however, its etiology is still not fully understood. Patient genetics, immune system, and intestinal microbiota are considered critical factors contributing to IBD. Preclinical animal models are crucial to better understand the importance of individual contributing factors. Among these, the dextran sodium sulfate (DSS) colitis model is the most widely used. DSS treatment induces gut inflammation and dysbiosis. However, its exact mode of action remains unclear. To determine whether DSS treatment induces pathogenic changes in the microbiota, we investigated the microbiota-modulating effects of DSS on murine microbiota in vitro. For this purpose, we cultured murine microbiota from the colon in six replicate continuous bioreactors. Three bioreactors were supplemented with 1% DSS and compared with the remaining PBS-treated control bioreactors by means of microbiota taxonomy and functionality. Using metaproteomics, we did not identify significant changes in microbial taxonomy, either at the phylum or genus levels. No differences in the metabolic pathways were observed. Furthermore, the global metabolome and targeted short-chain fatty acid (SCFA) quantification did not reveal any DSS-related changes. DSS had negligible effects on microbial functionality and taxonomy in vitro in the absence of the host environment. Our results underline that the DSS colitis mouse model is a suitable model to study host–microbiota interactions, which may help to understand how intestinal inflammation modulates the microbiota at the taxonomic and functional levels.

Neuroinflammation plays a central role in many neurological disorders, ranging from traumatic brain injuries to neurodegeneration. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. In order to study neuroinflammation and its electrophysiological fingerprints, there is a need for in vitro models that accurately capture the in vivo phenomena. In this study, we employed a new tri-culture of primary rat neurons, astrocytes, and microglia in combination with extracellular electrophysiological recording techniques using multiple electrode arrays (MEAs) to determine the effect of microglia on neural function and the response to neuroinflammatory stimuli. Specifically, we established the tri-culture and its corresponding neuron-astrocyte co-culture (lacking microglia) counterpart on custom MEAs and monitored their electrophysiological activity for 21 days to assess culture maturation and network formation. As a complementary assessment, we quantified synaptic puncta and averaged spike waveforms to determine the difference in excitatory to inhibitory neuron ratio (E/I ratio) of the neurons. The results demonstrate that the microglia in the tri-culture do not disrupt neural network formation and stability and may be a better representation of the in vivo rat cortex due to its more similar E/I ratio as compared to more traditional isolated neuron and neuron-astrocyte co-cultures. In addition, only the tri-culture displayed a significant decrease in both the number of active channels and spike frequency following pro-inflammatory lipopolysaccharide exposure, highlighting the critical role of microglia in capturing electrophysiological manifestations of a representative neuroinflammatory insult. We expect the demonstrated technology to assist in studying various brain disease mechanisms.

Objective: FL058 is a novel beta-lactamase inhibitor with a broad spectrum of activity and a favorable safety profile. The objective of this study was to evaluate pharmacokinetic/pharmacodynamic (PK/PD) relationships for the combination of FL058 and meropenem in an in vitro infection model. Methods: By simulating human concentration-time profiles in the in vitro model, meropenem combined with FL058 when administered 1 g/0.5 g, 1 g/1 g, 2 g/1 g, and 2 g/2 g q8h by 3-h infusion achieved approximately 2- and 4-log10 kill to KPC/OXA-producing Klebsiella pneumoniae and Escherichia coli; the combination therapy could not inhibit NDM-producing K. pneumoniae but could maintain NDM-producing E. coli around a baseline. Results: The PK/PD indexes that best described the bacterial killing from baseline in log10 CFU/mL at 24 h were the percent time of free drug above the minimal inhibitory concentration (MIC) (%fT > MIC, MIC with FL058 at 4 mg/L) for meropenem and the percent time of free drug above 1 mg/L (%fT > 1 mg/L) for FL058. The targets for achieving a static effect and the 1- and 2-log10 kill were 74, 83, and 99 for %fT > MIC of meropenem and 40, 48, and 64 for %fT > 1 mg/L of FL058, respectively. The PK/PD index of %fT > 1 mg/L can provide a basis for evaluating clinical dosing regimens for FL058 combined with meropenem. Conclusion: FL058 combined with meropenem might be a potential treatment for KPC- and/or OXA-48-producing Enterobacterales infection. [ABSTRACT FROM AUTHOR]

Bioinks play a fundamental role in skin bioprinting, dictating the printing fidelity, cell response, and function of bioprinted 3D constructs. However, the range of bioinks that support skin cells' function and aid in the bioprinting of 3D skin equivalents with tailorable properties and customized shapes is still limited. In this study, we describe a bioinspired design strategy for bioengineering double crosslinked pectin-based bioinks that recapitulate the mechanical properties and the presentation of cell-adhesive ligands and protease-sensitive domains of the dermal extracellular matrix, supporting the bioprinting of bilayer 3D skin models. Methacrylate-modified pectin was used as a base biomaterial enabling hydrogel formation via either chain-growth or step-growth photopolymerization and providing independent control over bioink rheology, as well as the mechanical and biochemical cues of cell environment. By tuning the concentrations of crosslinker and polymer in bioink formulation, dermal constructs were bioprinted with a physiologically relevant range of stiffnesses that resulted in strikingly site-specific differences in the morphology and spreading of dermal fibroblasts. We also demonstrated that the developed thiol-ene photo-clickable bioinks allow for the bioprinting of skin models of varying shapes that support dermis and epidermis reconstruction. Overall, the engineered bioinks expand the range of printable biomaterials for the extrusion bioprinting of 3D cell-laden hydrogels and provide a versatile platform to study the impact of material cues on cell fate, offering potential for in vitro skin modeling. [ABSTRACT FROM AUTHOR]

The ethical concerns surrounding the use of animals in cosmetics testing have spurred the demand for alternative test models. Digital manufacturing, often denoted as 3D printing, presents a compelling and cost-effective avenue for fabricating such testing models. The current manuscript proposes a digitally manufactured alternative testing platform that comprises three main components: bioprinted skin model, platform fabrication and electronic printing. For the first time, we assessed bioprinted skin models for the intention for use in cosmetic testing. We employed OECD guidelines to test on the skin model's responsiveness to anti-ageing products and irritant compounds. Our studies showed the efficacy of our bioprinted skin model capable of evaluating cosmetic compounds. An evaluation matrix was used to select the appropriate digital manufacturing technology to fabricate the setup for alternative testing model. Multi-material jetting was selected as the most appropriate additive manufacturing technology to introduce modularity and water-tight designs for the test platform. We have also introduced an electronic component in the test platform fabricated using electronic printing to provide localised heating on the skin model. Overall, this study highlights the potential of digital manufacturing and biomanufacturing to promote ethical and sustainable practices in cosmetic testing. Moreover, the decision matrix showcase in this study will be beneficial for scientists and researchers in application-driven selection on 3D printing technology. [ABSTRACT FROM AUTHOR]

Background: Tranexamic acid (TXA) is an antifibrinolytic agent applied in orthopedic surgery and has been proven to reduce post-surgery infection rates. We previously showed that TXA also had an additional direct antimicrobial effect against planktonic bacteria. Therefore, we aimed to evaluate whether it has a synergistic effect if in combination with antibiotics. Materials and Methods: Three ATCC and seven clinical strains of staphylococci were tested against serial dilutions of vancomycin and gentamicin alone and in combination with TXA at 10 and 50 mg/ml. The standardized microtiter plate method was used. Minimal inhibitory concentrations (MICs) were calculated by standard visualization of well turbidity (the lowest concentration at which complete absence of well bacterial growth was observed by the researcher) and using the automated method (the lowest concentration at which ≥80% reduction in well bacterial growth was measured using a spectrophotometer). Results: Tranexamic acid-10 mg/ml reduced the MIC of vancomycin and gentamicin with both the standard method (V: 1-fold dilution, G: 4-fold dilutions) and the automated turbidity method (vancomycin: 8-fold dilutions, gentamicin: 8-fold dilutions). TXA-50 mg/ml reduced the MIC of gentamicin with both the standard turbidity method (6-fold dilutions) and the automated turbidity method (1-fold dilutions). In contrast, for vancomycin, the MIC remained the same using the standard method, and only a 1-fold dilution was reduced using the automated method. Conclusion: Ours was a proof-of-concept study in which we suggest that TXA may have a synergistic effect when combined with both vancomycin and gentamicin, especially at 10 mg/ml, which is the concentration generally used in clinical practice. [ABSTRACT FROM AUTHOR]

Vascular calcification is a pathological chronic condition characterized by calcium crystal deposition in the vessel wall and is a recurring event in atherosclerosis, chronic kidney disease, and diabetes. The lack of effective therapeutic treatments opened the research to natural products, which have shown promising potential in inhibiting the pathological process in different experimental models. This study investigated the anti-calcifying effects of Quercetin and Berberine extracts on vascular smooth muscle cells (VSMCs) treated with an inorganic phosphate solution for 7 days. Quercetin has shown the highest anti-calcifying activity, as revealed by the intracellular quantitative assay and morphological analysis. Confocal microscopy revealed downregulation of RUNX2, a key marker for calcified phenotype, which was otherwise upregulated in calcified VSMCs. To investigate the anti-inflammatory activity of Quercetin, culture media were subjected to immunometric assays to quantify the levels of IL-6 and TNF-a, and the caspase-1 activity. As expected, calcified VSMCs released a large quantity of inflammatory mediators, significantly decreasing in the presence of Quercetin. In summary, our findings suggest that Quercetin counteracted calcification by attenuating the VSMC pathological phenotypic switch and reducing the inflammatory response. In our opinion, these preliminary in vitro findings could be the starting point for further investigations into the beneficial effects of Quercetin dietary supplementation against vascular calcification. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma is the second most common malignant tumor of the liver, which is characterized by atypical early symptoms, high malignancy and high mortality. Currently, the overall incidence of cholangiocarcinoma has been increased year by year worldwide and a majority of cholangiocarcinoma patients obtain poor prognosis. Effective diagnosis and treatment strategies are urgently required to change this situation. The establishment of disease models is a hot topic in cancer research. The establishment of experimental models of cholangiocarcinoma contributes to in-depth understanding and study of the incidence, development and drug therapeutic effect of cholangiocarcinoma, which is of significance for early diagnosis and treatment of cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Surgery has been the major treatment method for HCC owing to HCC’s poor sensitivity to radiotherapy and chemotherapy. However, its effectiveness is limited by postoperative tumour recurrence and metastasis. Systemic therapy is applied to eliminate postoperative residual tumour cells and improve the survival of patients with advanced HCC. Recently, the emergence of various novel targeted and immunotherapeutic drugs has significantly improved the prognosis of advanced HCC. However, targeted and immunological therapies may not always produce complete and long-lasting anti-tumour responses because of tumour heterogeneity and drug resistance. Traditional and patient-derived cell lines or animal models are used to investigate the drug resistance mechanisms of HCC and identify drugs that could reverse the resistance. This study comprehensively reviewed the established methods and applications of in-vivo and in-vitro HCC drug resistance models to further understand the resistance mechanisms in HCC treatment and provide a model basis for possible individualised therapy.

The activation of ferroptosis is being pursued in cancer research as a strategy to target apoptosis-resistant cells. By contrast, in various diseases that affect the cardiovascular system, kidneys, liver, and central and peripheral nervous systems, attention is directed toward interventions that prevent ferroptotic cell death. Mechanistic insights into both research areas stem largely from studies using cellular in vitro models. However, intervention strategies that show promise in cellular test systems often fail in clinical trials, which raises concerns regarding the predictive validity of the utilized in vitro models.In this study, the human LUHMES cell line, which serves as a model for human dopaminergic neurons, was used to characterize factors influencing the activation of ferroptosis. Erastin and RSL-3 induced cell death that was distinct from apoptosis. Parameters such as the differentiation state of LUHMES cells, cell density, and the number and timing of medium changes were identified as determinants of sensitivity to ferroptosis activation. In differentiated LUHMES cells, interventions at mechanistically divergent sites (iron chelation, coenzyme Q10, peroxidase mimics, or inhibition of 12/15-lipoxygenase) provide almost complete protection from ferroptosis. LUHMES cells allowed the experimental modulation of intracellular iron concentrations and demonstrated a correlation between intracellular iron levels, the rate of lipid peroxidation, as well as the sensitivity of the cells to ferroptotic cell death.These findings underscore the importance of understanding the various factors that influence ferroptosis activation and highlight the need for well-characterized in vitro models to enhance the reliability and predictive value of observations in ferroptosis research, particularly when translating findings into in vivo contexts.