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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.

A chicken gastrointestinal tract (GIT) simulation model was developed to help predict the potential effects of feed additives supplementation on chicken’ microbiota. The chemical and enzymatic conditions for oral, gastric, intestinal, and cecum fermentation phases were designed to closely resemble the chicken GIT conditions. For cecum fermentation, the inoculum was obtained from the cecal contents of 18 38-day broiler chickens. The impact of inoculum preservation on bacteria viability was assessed by comparing two methods of preservation with fresh inoculum: (1) 5% dimethyl sulfoxide (DMSO) at −80 °C and (2) 30% glycerol at −20 °C. The fermentation with fresh and frozen (DMSO method) inoculums was performed and compared using standard chicken feed (SCF) and SCF with 1% fructooligosaccharides (FOS), and inoculum control (IC) condition without feed matrix was used as a baseline. Inoculum’s viability was assessed throughout 90 days of storage by culture media platting, while bacterial growth and metabolites production during fermentation was evaluated by quantitative polymerase chain reaction (qPCR), high-performance liquid chromatography (HPLC), and total ammonia nitrogen quantification. The DMSO method was shown to be the most suitable for cecal inoculum storage. Higher growth of beneficial cecal bacteria for fresh inoculum was observed in SCF while for frozen inoculum, was the SCF + FOS condition. Also, frozen inoculum had lower activity of butyrate producers and proteolytic bacteria, showing different fermentation profiles. The GIT model developed showed to be useful to test the effect of feed additives supplementation.

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-α, 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.

The changing climate and agricultural practices continue to drive the increased prevalence and levels of both regulated and emerging mycotoxins in feed and food crops. This poses a considerable challenge for the agricultural sector in developing and developed countries as there is growing evidence that the emerging contaminants are capable of inducing a wide range of adverse health effects in livestock. This study evaluated and compared the capacity of commercially available mycotoxin binders to reduce the bioavailability of eight important emerging mycotoxins commonly found in livestock feeds. A novel in vitro model simulating the gastrointestinal track (GIT) of a monogastric animal was developed. Then, the efficiency of ten commercial binders with multi-mycotoxin binding claims were evaluated using the developed in vitro GIT model. Whist all the ten products demonstrated the capacity to simultaneously decrease the levels of more than one emerging mycotoxins, only one product (a mixed silicate) was able to considerably reduce the concentrations of all eight emerging mycotoxins simultaneously under the simulated in vitro conditions. This study highlights, for the first time, a significant gap in emerging mycotoxin mitigation through the deployment of binding agents and identifies the need for the development of novel mitigation strategies for this important group of mycotoxins.

Propolis, owing to its antibacterial and anti-inflammatory properties, acts as a cariostatic agent, capable of preventing the accumulation of dental plaque and inhibiting inflammation. The anti-inflammatory properties of propolis are attributed to caffeic acid phenethyl ester (CAPE), which is present in European propolis. The objective of the conducted study was to assess the anti-inflammatory effects of the Polish ethanolic extract of propolis (EEP) and isolated CAPE on stimulated with LPS and IFN-α, as well as the combination of LPS and IFN-α. The cytotoxicity of the tested compounds was determined using the MTT assay. The concentrations of specific cytokines released by the HGF-1 cell line following treatment with EEP (25–50 µg/mL) or CAPE (25–50 µg/mL) were assessed in the culture supernatant. In the tested concentrations, both CAPE and EEP did not exert cytotoxic effects. Our results demonstrate that CAPE reduces TNF-α and IL-6 in contrast to EEP. Propolis seems effective in stimulating HGF-1 to release IL-6 and IL-8. A statistically significant difference was observed for IL-8 in HGF-1 stimulated by LPS+IFN-α and treated EEP at a concentration of 50 µg/mL (p = 0.021201). Moreover, we observed that CAPE demonstrates a stronger interaction with IL-8 compared to EEP, especially when CAPE was administered at a concentration of 50 µg/mL after LPS + IFN-α stimulation (p = 0.0005). Analysis of the phenolic profile performed by high-performance liquid chromatography allowed identification and quantification in the EEP sample of six phenolic acids, five flavonoids, and one aromatic ester—CAPE. Propolis and its compound—CAPE—exhibit immunomodulatory properties that influence the inflammatory process. Further studies may contribute to explaining the immunomodulatory action of EEP and CAPE and bring comprehensive conclusions. [ABSTRACT FROM AUTHOR]

Amyotrophic Lateral Sclerosis (ALS) is one of the commonest neurodegenerative diseases of adult-onset, which is characterized by the progressive death of motor neurons in the cerebral cortex, brain stem and spinal cord. The dysfunction and death of motor neurons lead to the progressive muscle weakness, atrophy, fasciculations, spasticity and ultimately the whole paralysis of body. Despite the identification of several genetic mutations associated with the pathogenesis of ALS, including mutations in chromosome 9 open reading frame 72 leading to the abnormal expansion of GGGGCC repeat sequence, TAR DNA-binding protein 43, fused in sarcoma/translocated in liposarcoma, copper/zinc superoxide dismutase 1 (SOD1) and TANK-binding kinase 1, the exact mechanisms underlying the specific degeneration of motor neurons that causes ALS remain incompletely understood. At present, since the transgenic model expressed SOD1 mutants was established, multiple in vitro models of ALS have been developed for studying the pathology, pathophysiology and pathogenesis of ALS as well as searching the effective neurotherapeutics. This review reviewed the details of present established in vitro models used in studying the pathology, pathophysiology and pathogenesis of ALS. Meanwhile, we also discussed the advantages, disadvantages, cost and availability of each models. [ABSTRACT FROM AUTHOR]

We investigated three calcium silicate-based sealers with respect to their chemical characterization, cytotoxicity, and attachment to RAW264.7 cells. BioRoot RCS (BR), Bio-C Sealer (BC), and Sealer Plus BC (SPBC) were assessed using Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence spectroscopy (XRF), and energy-dispersive X-ray spectroscopy (EDX) (n = 4) for elemental characterization, and using scanning electron microscopy (SEM) to evaluate cell morphology and adhesion. Cytotoxicity was determined at different dilutions (1:1, 1:2, and 1:5) using the succinate dehydrogenase activity (MTT assay). Statistical analysis was performed for normal distribution using the Shapiro–Wilk test and for homoscedasticity using Levene's test, and one-way ANOVA, Tukey's/Dunnett's post hoc tests for cell viability and XRF (α = 0.05). Calcium silicate hydrate and calcium hydroxide were detected by FTIR in all groups. EDX detected a higher calcium content for BR and SPBC and aluminum only in the premixed sealers. XRF detected the highest calcium release in BR (p < 0.05). The surface morphology showed irregular precipitates for all the sealers. SPBC at a 1:2 dilution resulted in the lowest cell viability compared to BR (p < 0.05) and BC (p < 0.05). The calcium silicate-based sealers produced a statistically significant reduction in cellular viability at a 1:1 dilution compared to the control group (p < 0.0001). All the sealers maintained viability above 70%. [ABSTRACT FROM AUTHOR]

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization, and different degrees of mineralization where the intrafibrillar and extrafibrillar minerals are intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement are demonstrated. Then, this cell‐free in vitro system is explored as a realistic, functional model for the in situ investigation of matrix‐controlled mineralization processes. Combined Raman and electron microscopy indicate that glycosaminoglycans (GAGs) play a more prominent role than generally assumed in the matrix–mineral interactions. The experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral. [ABSTRACT FROM AUTHOR]

NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here, we investigate the potential for the tetrahydrouridine component of NDec to act as an inhibitor or substrate of key concentrative nucleoside transporters (CNT1–3) and equilibrative nucleoside transporters (ENT1–2). Nucleoside transporter inhibition and tetrahydrouridine accumulation assays were performed using Madin-Darby canine kidney strain II (MDCKII) cells overexpressing human CNT1, CNT2, CNT3, ENT1, and ENT2 transporters. Results showed that tetrahydrouridine did not influence CNT- or ENT-mediated uridine/adenosine accumulation in MDCKII cells at the concentrations tested (25 and 250 µM). Accumulation of tetrahydrouridine in MDCKII cells was initially shown to be mediated by CNT3 and ENT2. However, while time- and concentration-dependence experiments showed active accumulation of tetrahydrouridine in CNT3-expressing cells, allowing for estimation of K m (3,140 µM) and V max (1,600 pmol/mg protein/min), accumulation of tetrahydrouridine was not observed in ENT2-expressing cells. Potent CNT3 inhibitors are a class of drugs not generally prescribed to patients with SCD, except in certain specific circumstances. These data suggest that NDec can be administered safely with drugs that act as substrates and inhibitors of the nucleoside transporters included in this study. [ABSTRACT FROM AUTHOR]

Lip vermilion is unique and can be distinguished from the adjacent skin and oral mucosa. However, because of the lack of appropriate evaluation tools, skin and/or oral mucosa substitutes such as in vitro vermilion epithelial models have been used for lip product testing. We aimed to develop and characterize a lip vermilion epithelium reconstruction model (LVERM) using skin and oral keratinocytes. LVERM was manufactured by co-culturing primary skin and oral keratinocytes, using a device that allowed the separation of cell seeding, and created an intercalated cell-free zone, referred to as the vermilion part. After removing the device, LVERM construction was completed in 8 days, in a submerged condition. Subsequently, they were placed in an air–liquid interface for 7 days. To determine the epithelial characteristics of LVERM, keratin 2e (KRT2) and small proline-rich protein 3 (SPRR3) expression patterns were examined. The in vivo expression profiles of KRT2 and SPRR3 genes in vermilion were also examined. We found that a continuous multi-layered epithelium was generated in the LVERM that exhibited ortho- and para-keratinization in the skin and oral mucosa parts, respectively. Although an intermediate keratinization pattern was observed in the vermilion part, KRT2 and SPRR3 were co-expressed in the suprabasal layer, consistent with the expression pattern of a single vermilion epithelial model. Clustering analysis revealed that KRT2 and SPRR3 gene expression in vermilion was location-dependent within the sample. Therefore, LVERM can be used as an evaluation tool for lip products and has great importance in innovative approaches for cosmetic testing. [ABSTRACT FROM AUTHOR]

The development of brain in vitro models requires the application of novel biocompatible materials and biopolymers as scaffolds for controllable and effective cell growth and functioning. The "ideal" brain in vitro model should demonstrate the principal features of brain plasticity like synaptic transmission and remodeling, neurogenesis and angiogenesis, and changes in the metabolism associated with the establishment of new intercellular connections. Therefore, the extracellular scaffolds that are helpful in the establishment and maintenance of local microenvironments supporting brain plasticity mechanisms are of critical importance. In this review, we will focus on some carbohydrate metabolites—lactate, pyruvate, oxaloacetate, malate—that greatly contribute to the regulation of cell-to-cell communications and metabolic plasticity of brain cells and on some resorbable biopolymers that may reproduce the local microenvironment enriched in particular cell metabolites. [ABSTRACT FROM AUTHOR]

It is well known that pelvic organ prolapse (POP) significantly reduces the quality of life of affected women and in many cases requires corrective surgery. Aim of the study was to compare the immune response against titanized versus non-titanized meshes, especially macrophage polarization and immune checkpoint association. For this, we analyzed 644 POP surgeries, which were performed between 2017 and 2022, in our department. Four of them needed revision surgery caused by erosion. We analyzed the influx of CD68 & CD163 positive macrophages and the expression of immune checkpoint molecules PD-L1 and PD1 in these 4 patients. We identified a large number of CD68 and CD163 positive macrophages and additionally a PD-L1 expression of these cells. Based on the in-vivo results, we isolated monocytes and co-cultivated monocytes with different mesh material covered with or without fibroblasts. We identified a significantly enhanced macrophage activation and PD-L1 expression in macrophages surrounding non-titanized polypropylene mesh material. Encapsulation of the material by fibroblasts was crucial for that. Specifically, CD68-positive macrophages are upregulated (p < 0.001), co-expressing PD-L1 (p < 0.001) in monocytes co-cultivated with non-titanized polypropylene meshes. Monocytes co-cultivated with titanized polypropylene meshes showed significantly lower expression of CD163 (p = 0.027) and PD-L1 (p = 0.022). In conclusion, our in vitro data suggest that the titanium coating leads to a decreased polarization of macrophages and to a decreased immune response compared to non-titanized meshes. This could be an indication for the increased incidence of erosion of the non-titanized meshes, which is a severe complication of this procedure and requires revision surgery. Pelvic organ prolapse is a well-known problem for women and often requires corrective surgery. Polypropylene meshes are often used, which differ in their coating (titanized vs. non-titanized). A severe side effect of these surgeries is mesh erosion, due to onset of inflammation, which requires revision surgery. We examined all erosion cases (4 of 644 patients) with implanted nontitanium-coated meshes by immunohistochemistry and found upregulation of macrophage polarization (as markers CD68 and CD163) and increased expression of the immune checkpoint molecules PD-L1 and PD1. This suggests inflammatory processes and an enhanced immune response. In addition, we set up an in vitro experiment to investigate whether coating plays a role. Here, we demonstrated that the non-titanized meshes elicited a significantly higher immune response in comparison to titanized meshes, which could lead to the higher erosion rate of the non-titanized meshes. Our results highlight the benefit of titanized meshes, which should lead to a lower revision surgery rate and thus improved patient outcome. [Display omitted] [ABSTRACT FROM AUTHOR]

Irrigation and debridement using an irrigation solution is a fundamental step during the surgical treatment of both acute and chronic periprosthetic joint infection (PJI). However, there is no consensus on the optimal solution, nor is there sufficient evidence on the optimal irrigation time and combination of solutions. Therefore, it is necessary to determine which solution or combination of solutions is most efficacious against biofilm, as well as the optimal irrigation time. We conducted an experimental in vitro model by inoculating stainless steel discs with ATCC strains of methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and a clinical strain of Staphylococcus epidermidis. The discs were all irrigated with commonly used antiseptic solutions (10% and 3% povidone iodine, hydrogen peroxide, 3% acetic acid, and Bactisure™) for 1 min, 3 min, and 5 min and their combinations for 9 min (3 min each) vs. sterile saline as a positive control. We evaluated the reduction in biofilm based on colony-forming unit (cfu) counts and in combination assays, also based on cell viability and scanning electron microscopy. All antiseptics alone reduced more than 90% of cfu counts after 1 min of irrigation; the worst results were for hydrogen peroxide and 3% acetic acid. When solutions were sequentially combined, the best results were observed for all those starting with acetic acid, in terms of both reduction of log cfu/mL counts and viable cells. We consider that a combination of antiseptic solutions, particularly that comprising the sequence acetic acid + povidone iodine + hydrogen peroxide, would be the best option for chemical debridement during PJI surgery. [ABSTRACT FROM AUTHOR]

Background: Appropriate load distribution among the supporting elements is essential for the long-term success of implant-assisted removable partial dentures; however, there is little information available on load distribution. Purpose: This study aimed to evaluate the effect of implant location on load distribution in implant-assisted removable partial dentures by reviewing in vitro models and finite-element analysis studies. Materials and methods: English-language studies which examined the load distribution of implant-assisted removable partial dentures and were published between January 2001 and October 2022 were extracted from PubMed, ScienceDirect, and Scopus online databases, and manual searching. Two reviewers selected the articles based on the predetermined inclusion and exclusion criteria, followed by data extraction and analysis. Results: Forty-seven studies were selected after evaluating the titles and abstracts of 264 articles; two were identified manually. After screening the text, 12 studies were included: six in vitro model experiments and six finite-element analysis studies. All included studies used a mandibular free-end missing model (Kennedy Class I or II). The influence of implant location on load distribution to the abutment tooth, implant, and mucosa under the denture base was summarized in three cases: implant at the premolar, first molar, and second molar region. Due to differences in the measurement method of load distribution and loading condition to the denture, the results differed among the studies. Conclusions: The implant location in implant-assisted removable partial dentures can affect load distribution to the supporting elements, such as the abutment tooth, implant, and mucosa under the denture base. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of a citrus extract rich in citrus flavonoids on intestinal metabolic responses in subjects with features of metabolic syndrome, in an in vitro colon fermentation system (TIM-2) and fecal samples obtained from human subjects in an in vivo trial. In the TIM-2 system inoculated with fecal samples of volunteers with features of metabolic syndrome, continuous citrus extract supplementation (500 mg/day) resulted in increased cumulative short-chain fatty acid (SCFA) levels compared to the control condition, which was mainly due to increased production of butyrate, acetate, and valerate. In human volunteers, 12 weeks of daily supplementation with 500 mg citrus extract resulted in a significant shift in the SCFA profile towards more butyrate (p = 0.022) compared to the placebo group. Furthermore, there was a trend towards a reduction in fecal calprotectin levels, a marker for intestinal inflammation, compared to the placebo (p = 0.058). Together, these results suggest that citrus extract intake may have a positive effect on intestinal metabolic responses and through this, on host health in subjects with features of metabolic syndrome. Further research is needed to provide more insight into the potential underlying mechanisms and to study effects on clinical parameters. [ABSTRACT FROM AUTHOR]

The intestinal epithelial Caco-2 cell monolayer is a well-established in vitro model useful for predicting intestinal drug absorption in humans. Coculture models of Caco-2 and goblet-cell-like HT29-MTX cells have been developed to overcome the lack of a mucus layer; however, those models are much leakier compared to the intestinal epithelium. Here, we developed a partially laminated culture model where HT29-MTX cells were superimposed onto a Caco-2 monolayer to overcome this issue. A morphological study showed that the piled HT29-MTX cells were voluntarily incorporated into the Caco-2 monolayer, and mucus production was confirmed via periodic acid-Schiff and mucin protein 2 staining. Permeability was evaluated in terms of transepithelial electrical resistance (TEER) and the apparent permeability of paracellular markers with different molecular sizes. The partially laminated model maintained the high barrier function of the Caco-2 monolayer, whose permeability appeared adjustable according to the HT29-MTX/Caco-2 cell ratio. In contrast, the coculture models showed abnormally high permeability of those markers, correlated with low TEER. Thus, the partially laminated model enabled in vitro recapitulation of effective mucosal barrier function. Consequently, this novel model may be useful as an in vitro high-throughput evaluation system for enteral mucosal permeability and mucus-penetrating efficiency of drugs and nanocarriers. [ABSTRACT FROM AUTHOR]

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air–liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems. [ABSTRACT FROM AUTHOR]

A chicken gastrointestinal tract (GIT) simulation model was developed to help predict the potential effects of feed additives supplementation on chicken' microbiota. The chemical and enzymatic conditions for oral, gastric, intestinal, and cecum fermentation phases were designed to closely resemble the chicken GIT conditions. For cecum fermentation, the inoculum was obtained from the cecal contents of 18 38-day broiler chickens. The impact of inoculum preservation on bacteria viability was assessed by comparing two methods of preservation with fresh inoculum: (1) 5% dimethyl sulfoxide (DMSO) at −80 °C and (2) 30% glycerol at −20 °C. The fermentation with fresh and frozen (DMSO method) inoculums was performed and compared using standard chicken feed (SCF) and SCF with 1% fructooligosaccharides (FOS), and inoculum control (IC) condition without feed matrix was used as a baseline. Inoculum's viability was assessed throughout 90 days of storage by culture media platting, while bacterial growth and metabolites production during fermentation was evaluated by quantitative polymerase chain reaction (qPCR), high-performance liquid chromatography (HPLC), and total ammonia nitrogen quantification. The DMSO method was shown to be the most suitable for cecal inoculum storage. Higher growth of beneficial cecal bacteria for fresh inoculum was observed in SCF while for frozen inoculum, was the SCF + FOS condition. Also, frozen inoculum had lower activity of butyrate producers and proteolytic bacteria, showing different fermentation profiles. The GIT model developed showed to be useful to test the effect of feed additives supplementation. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is a major health problem that affects millions of persons worldwide every year among all age groups, mainly young children, and elderly persons. It is the leading cause of death for children under the age of 16 and is highly correlated with a variety of neuronal disorders, such as epilepsy, and neurodegenerative disease, such as Alzheimer's disease or amyotrophic lateral sclerosis. Over the past few decades, our comprehension of the molecular pathway of TBI has improved, yet despite being a major public health issue, there is currently no U.S. Food and Drug Administration?approved treatment for TBI, and a gap remains between these advances and their application to the clinical treatment of TBI. One of the major hurdles for pushing TBI research forward is the accessibility of TBI models and tools. Most of the TBI models require costume-made, complex, and expensive equipment, which often requires special knowledge to operate. In this study, we present a modular, three-dimensional printed TBI induction device, which induces, by the pulse of a pressure shock, a TBI-like injury on any standard cell-culture tool. Moreover, we demonstrate that our device can be used on multiple systems and cell types and can induce repetitive TBIs, which is very common in clinical TBI. Further, we demonstrate that our platform can recapitulate the hallmarks of TBI, which include cell death, decrease in neuronal functionality, axonal swelling (for neurons), and increase permeability (for endothelium). In addition, in view of the continued discussion on the need, benefits, and ethics of the use of animals in scientific research, this in vitro, high-throughput platform will make TBI research more accessible to other labs that prefer to avoid the use of animals yet are interested in this field. We believe that this will enable us to push the field forward and facilitate/accelerate the availability of novel treatments.

BackgroundInteractions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required.MethodsPrimary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared.ResultsThe tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture.ConclusionsThe tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena.

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.

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.

Organoids are in vitro organ-like structures that faithfully recapitulate many characteristics of a specific organ. During the past decades, major progress has been accomplished in establishing three-dimensional (3D) culture systems toward stem cell-derived organoids. As a significant technological breakthrough, these amazing 3D organoid constructs bridge the conventional 2D in vitro models and in vivo animal models and provide an unprecedented opportunity to investigate the complexities of veterinary diseases ranging from their pathogenesis to the prevention, therapy, or even future organ replacement strategies. In this review, we briefly discuss several definitions used in organoid research and highlight the currently known achievements in modeling veterinary diseases, including infectious and inflammatory diseases, cancers, and metabolic diseases. The applications of organoid technology in veterinary disease modeling are still in their infancy stage but the future is promising.

Background: Cell culture is the proliferation of a cell population in vitro by isolating from the original tissue or growing from existing ones. One essential source is the monkey kidney cell cultures which have an essential role in biomedical study. This is due to the significant homology between the human and macaque genomes making these useful for cultivating human viruses, especially enteroviruses, and growing vaccines. Methods: This study developed cell cultures derived from the kidney of Macaca fascicularis (Mf) and validated its gene expression. Results: The primary cultures were successfully subcultured up to six passages, grew as monolayers, and exhibited epithelial‐like morphology. The cultured cells remained heterogeneous in phenotype and they expressed CD155 and CD46 as viral receptors, cell morphology (CD24, endosialin, and vWF), proliferation, also apoptosis markers (Ki67 and p53). Conclusions: These results indicated that the cell cultures can be used as in vitro model cells for vaccine development and bioactive compound. [ABSTRACT FROM AUTHOR]

Variability in supply, paucity of donors and cellular instability under in vitro conditions have limited the application of primary human hepatocytes (PHHs) to hepatotoxicity testing. Therefore, alternative sources have been sought for functional liver cells. Many of the earlier in vitro hepatotoxicity studies were carried out using hepatoma-derived cell lines. These cell lines have overcome some of the limitations of PHHs with regard to phenotypic stability and availability; however, they suffer from their own inherent limitations, such as the lack of drug-metabolizing functionality, which renders them inadequate for situations where toxic metabolite formation of the parent drug occurs. In the last decade we have witnessed a burgeoning interest of the research community in using hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) as in vitro hepatotoxicity models. HLCs offer the perspective of a defined and renewable supply of functional hepatocytes; more importantly, HLCs maintain their original donor genotype and afford donor diversity, thus opening new avenues to patient-specific toxicity testing. In this review, we first introduce various in vitro hepatotoxicity models, then focus on HLCs and their application in hepatotoxicity studies, and finally offer some perspectives on future developments of the field. [ABSTRACT FROM AUTHOR]

For supersaturating formulations of BCS-II compounds, which by definition have high intestinal permeability, a closed USP apparatus does not provide the necessary absorptive conditions during dissolution. To address this, an artificial gut simulator (AGS) has been constructed consisting of a 2.5 mL donor compartment in which a hollow fiber-based absorption module is suspended. Drug from donor diffuses across the hollow fiber membrane to be absorbed by the continuously flowing intraluminal receiver fluid. The membrane surface area and intraluminal fluid flow rate are tuned to obtain the physiologically observed absorption rate constant for a weakly basic, poorly water-soluble model compound, ketoconazole (KTZ). Supersaturated solutions of KTZ were generated in the donor in pH 6.5 phosphate buffer by the pH-shift method in the absence (closed system, control) and presence (open system, biorelevant) of an optimally or suboptimally tuned absorption module. Drug concentrations in the donor and intraluminal fluids were determined by in-line UV spectroscopy. The presence of an absorptive sink reduced the supersaturated solution's crystallization propensity, more so in the case of the optimally tuned AGS. This study demonstrates the significance of simulating absorption of drug at a physiological rate during dissolution studies, especially to predict the performance of formulations of BCS-II drugs. [Display omitted] [ABSTRACT FROM AUTHOR]

Simple Summary: The word "model" can be used with different meanings and different contexts, like a model student, clay models or a model railway. In some cases, the context can clarify exactly what is meant by "model", but sometimes several meanings of model can be present in one area. For instance, with reference to cancer research, there can be ambiguity for what is meant by model. This paper reviews the use of the word model as related to cancer research and within the specific area of the microenvironment that surrounds a cancer tumour. The review grouped different definitions of model into four categories (model organisms, in vitro models, mathematical models and computational models) and explored what is meant in each case, mentioning the advantages and disadvantages of the different models Next, a quantitative investigation of the scientific publications listed in the database of the United States National Library of Medicine was performed by counting the frequencies of use of these terms, as well as the components of the microenvironments and the organs modelled with these techniques. The Oxford English Dictionary includes 17 definitions for the word "model" as a noun and another 11 as a verb. Therefore, context is necessary to understand the meaning of the word model. For instance, "model railways" refer to replicas of railways and trains at a smaller scale and a "model student" refers to an exemplary individual. In some cases, a specific context, like cancer research, may not be sufficient to provide one specific meaning for model. Even if the context is narrowed, specifically, to research related to the tumour microenvironment, "model" can be understood in a wide variety of ways, from an animal model to a mathematical expression. This paper presents a review of different "models" of the tumour microenvironment, as grouped by different definitions of the word into four categories: model organisms, in vitro models, mathematical models and computational models. Then, the frequencies of different meanings of the word "model" related to the tumour microenvironment are measured from numbers of entries in the MEDLINE database of the United States National Library of Medicine at the National Institutes of Health. The frequencies of the main components of the microenvironment and the organ-related cancers modelled are also assessed quantitatively with specific keywords. Whilst animal models, particularly xenografts and mouse models, are the most commonly used "models", the number of these entries has been slowly decreasing. Mathematical models, as well as prognostic and risk models, follow in frequency, and these have been growing in use. [ABSTRACT FROM AUTHOR]

The development of microbiome-targeted strategies is limited by individual differences in gut microbiome composition and metabolic responses to interventions. In vitro models that can replicate this variation allow us to conduct pre-clinical studies and assess efficacy. This study describes the exposure of 16 individual fecal microbiota samples to 5 different fibers using an in vitro system for the anaerobic cultivation of bacteria. The individual microbiota differed in composition and metabolite profiles (short-chain fatty acids and branched-chain fatty acids) after incubation with the fibers. Furthermore, microbiota composition after fiber incubation was significantly different between subjects with good intestinal health and subjects with Inflammatory Bowel Disease (IBD). α-diversity was differently affected by dietary fibers; for example, exposure to psyllium resulted in increased diversity in the healthy group and in decreased diversity in the IBD group. Instead, the functional metabolic profile did not differ between the two groups. Finally, the combination of all fibers, tested on the microbiota from IBD subjects, resulted in stronger overall effects on both microbiota composition and metabolite production compared to the single fibers. These results confirm that incubation with dietary fiber results in different compositional and functional effects on individual microbiota and that in vitro models represent successful tools for studying individual fiber effects. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies nowadays. The available chemo- and immunotherapies are often ineffective in treating PDAC due to its immunosuppressive and highly desmoplastic tumor immune microenvironment (TIME), which is hardly reproduced in the existing preclinical models. The PDAC TIME results from a peculiar spatial organization between different cell types. For this reason, developing new human models recapitulating the tissue organization and cell heterogeneity of PDAC is highly desirable. We developed human 3D heterocellular tumor spheroids of PDAC formed by cancer cells, endothelial cells, pancreatic stellate cells (PSC), and monocytes. As a control, we formed spheroids using immortalized epithelial pancreatic ductal cells (non-cancerous spheroids) with cellular heterogeneity similar to the tumor spheroids. Normal spheroids containing endothelial cells formed a complex 3D endothelial network significantly compromised in tumor spheroids. Monocyte/macrophages within the 4-culture tumor spheroids were characterized by a higher expression of CD163, CD206, PD-L1, and CD40 than those in the non-cancerous spheroids suggesting their differentiation towards an immunosuppressive phenotype. The heterocellular tumor spheroids presented a hypoxic core populated with PSC and monocytes/macrophages. The 4-culture tumor spheroids were characterized by spatial proximity of PSC and monocytes to the endothelial cells and a cytokine signature with increased concentrations of CXCL10, CCL2, and IL-6, which have been observed in PDAC patients and associated with poor survival. Further, 4-culture tumor spheroids decreased the concentrations of T-cell chemoattracting cytokines, i.e., CCL4, CCL5, and CXCL9, when compared with the non-cancerous spheroids, revealing a critical immunosuppressive feature of the different types of cells forming the tumor spheroids. Our results showed that the 4-culture tumor spheroids better resembled some critical features of patients' PDAC TIME than monoculture tumor spheroids. Using the proposed human 3D spheroid model for therapy testing at the preclinical stage may reveal pitfalls of chemo- and immunotherapies to help the development of better anti-tumor therapies. [ABSTRACT FROM AUTHOR]

Introduction: Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, has been a global threat to human beings for several decades. Treating tuberculosis has become more difficult as the prevalence of drug-resistant tuberculosis has increased globally. Evidence suggests that the comprehensive landscape of resistance mechanisms in MTB is ambiguous. More importantly, little is known regarding the series of events connected to resistance mechanisms in MTB before exposure to anti-TB drugs, during exposure to the drugs, and finally, when the MTB becomes resistant after exposure, upon analyses of its genome. Methods: We used the wild-type strain of MTB (H37Rv) in an in vitro model for generating induced resistance using a sub-inhibitory concentration of isoniazid, and the generated resistance-associated variants (RAVs) were identified using the whole genome sequencing method. Results: The detection of an inhA promoter mutation (fabG1-15C>T), which results in increased production of InhA protein, was found to be a major mechanism for developing resistance to isoniazid in the first place. We observed adaptation of MTB resistance mechanisms in high isoniazid stress by alteration and abolishment of KatG due to the detection of katG S315N, the common region of mutation that confers isoniazid resistance, along with katG K414N, katG N138S, and katG A162E. Furthermore, we detected the ahpC-72C>T and ahpC 21C>A mutations, but further investigation is needed to determine their role in compensating for the loss of KatG activity. Discussion: This suggests that increased InhA production is themainmechanism where there are low levels of isoniazid, whereas the alteration of KatG was found to be utilized in mycobacterium with a high concentration of isoniazid. Our work demonstrates that this in vitro approach of generating induced resistance could provide clinically relevant information after the fabG1-15C>T mutation, which is the common mutation found in clinical isolates. Moreover, other mutations detected in this work can also be found in clinical isolates. These findingsmay shed light on the impact of isoniazid in generating RAV and the resistance mechanism scenario that mycobacterium used under various isoniazid-pressuring conditions. More research is needed to understand better the role of RAV and mechanical resistance events within the mycobacterium genome in promoting a promising drug prediction platform that could lead to the right treatment for patients with MDR-TB and XDR-TB. [ABSTRACT FROM AUTHOR]

The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird (Turdus merula) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories. [ABSTRACT FROM AUTHOR]

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The chronic lesion has become a major biological difficulty. Using nanoparticles as drug delivery systems is remarkable nowadays. The unique properties of chitosan in combination with epidermal growth factor (EGF) can accelerate the wound‐healing process. In this study, Chitosan‐EGF (CS‐EGF) nanoparticles were manipulated and characterized by Transmission Electron Microscopy (TEM) microscopy, dynamic light scattering (DLS) method, and Fourier Transform Infrared Spectroscopy (FTIR) radiation. The antibacterial effect was estimated by minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) methods and the proliferation assay was measured on the HFF‐1 (human fibroblast cell line). Then, migration assay was accomplished and the gene expression analysis for transforming growth factor‐beta (TGF‐β), vascular endothelial growth factor (VEGF), and platelets‐derived growth factor (PDGF) was manipulated by the real‐time‐PCR method. The obtained results were considered statistically significant with P < 0.05. Obtained results illustrated no toxic effect on the HFF‐1 cell line treated with CS‐EGF. In cellular proliferation and migration assays, CS‐EGF nanoparticles demonstrated 2‐folds higher than the control. In the duration of 72 h of the experiment, and concentration of 10 µM, 90 ±10% of cells were migrated and the whole scratch was covered by fibroblasts. The real‐time‐PCR analysis also showed 7.5, 4.5‐, and 7‐fold upregulation of all TGF‐β, VEGF, and PDGF genes in comparison with the control group. This study confirmed that using chitosan nanoparticles as a carrier for EGF can accelerate cellular remodelling and annihilate bacterial infection in the process of treatment. As CS‐EGF nanoparticles in the acceleration of the skin remodelling process showed promising results, subsequent studies might be useful. [ABSTRACT FROM AUTHOR]

The transition in the field of bone tissue engineering from bone regeneration to in vitro models has come with the challenge of recreating a dense and anisotropic bone‐like extracellular matrix (ECM). Although the mechanism by which bone ECM gains its structure is not fully understood, mechanical loading and curvature have been identified as potential contributors. Here, guided by computational simulations, we evaluated cell and bone‐like tissue growth and organization in a concave channel with and without directional fluid flow stimulation. Human mesenchymal stromal cells were seeded on donut‐shaped silk fibroin scaffolds and osteogenically stimulated for 42 days statically or in a flow perfusion bioreactor. After 14, 28, and 42 days, constructs were investigated for cell and tissue growth and organization. As a result, directional fluid flow was able to improve organic tissue growth but not organization. Cells tended to orient in the tangential direction of the channel, possibly attributed to its curvature. Based on our results, we suggest that organic ECM production but not anisotropy can be stimulated through the application of fluid flow. With this study, an initial attempt in three‐dimensions was made to improve the resemblance of in vitro produced bone‐like ECM to the physiological bone ECM. [ABSTRACT FROM AUTHOR]

In vitro models for culturing complex microbial communities are progressively being used to study the effects of different factors on the modeling of in vitro-cultured microorganisms. In previous work, we validated a 3D in vitro model of the human gut microbiota based on electrospun gelatin scaffolds covered with mucins. The aim of this study was to evaluate the effect of Bacillus cereus, a pathogen responsible for food poisoning diseases in humans, on the gut microbiota grown in the model. Real-time quantitative PCR and 16S ribosomal RNA-gene sequencing were performed to obtain information on microbiota composition after introducing B. cereus ATCC 14579 vegetative cells or culture supernatants. The adhesion of B. cereus to intestinal mucins was also tested. The presence of B. cereus induced important modifications in the intestinal communities. Notably, levels of Proteobacteria (particularly Escherichia coli), Lactobacillus, and Akkermansia were reduced, while abundances of Bifidobacterium and Mitsuokella increased. In addition, B. cereus was able to adhere to mucins. The results obtained from our in vitro model stress the hypothesis that B. cereus is able to colonize the intestinal mucosa by stably adhering to mucins and impacting intestinal microbial communities as an additional pathogenetic mechanism during gastrointestinal infection. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is caused by a wide range of physical events and can induce an even larger spectrum of short- to long-term pathophysiologies. Neuroscientists have relied on animal models to understand the relationship between mechanical damages and functional alterations of neural cells. These in vivo and animal-based in vitro models represent important approaches to mimic traumas on whole brains or organized brain structures but are not fully representative of pathologies occurring after traumas on human brain parenchyma. To overcome these limitations and to establish a more accurate and comprehensive model of human TBI, we engineered an in vitro platform to induce injuries via the controlled projection of a small drop of liquid onto a 3D neural tissue engineered from human iPS cells. With this platform, biological mechanisms involved in neural cellular injury are recorded through electrophysiology measurements, quantification of biomarkers released, and two imaging methods [confocal laser scanning microscope (CLSM) and optical projection tomography (OPT)]. The results showed drastic changes in tissue electrophysiological activities and significant releases of glial and neuronal biomarkers. Tissue imaging allowed us to reconstruct the injured area spatially in 3D after staining it with specific nuclear dyes and to determine TBI resulting in cell death. In future experiments, we seek tomonitor the effects of TBI-induced injuries over a prolonged time and at a higher temporal resolution to better understand the subtleties of the biomarker release kinetics and the cell recovery phases. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. Methods: hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. Results: IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. Conclusion: Cxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM. [ABSTRACT FROM AUTHOR]

The hypothesis of the potential impact of the sex of red blood cell (RBC) concentrate (RCC) donors, as well as the sex of the recipients, on the clinical outcome, is still under evaluation. Here, we have evaluated the sex impact on RBC properties using in vitro transfusion models. Using a "flask model", RBCs from RCCs (representing the donor)—at different storage lengths—were incubated in a sex-matched and sex-mismatched manner with fresh frozen plasma pools (representing the recipient) at 37 °C, with 5% of CO2 up to 48 h. Standard blood parameters, hemolysis, intracellular ATP, extracellular glucose and lactate were quantified during incubation. Additionally, a "plate model", coupling hemolysis analysis and morphological study, was carried out in similar conditions in 96-well plates. In both models, RBCs from both sexes hemolyzed significantly less in female-derived plasma. No metabolic or morphological differences were observed between sex-matched and -mismatched conditions, even though ATP was higher in female-derived RBCs during incubations. Female plasma reduced hemolysis of female- as well as male-derived RBCs, which may be related to a sex-dependent plasma composition and/or sex-related intrinsic RBC properties. [ABSTRACT FROM AUTHOR]

The complex, dynamic environment of the human lower gastrointestinal tract is colonized by hundreds of bacterial species that impact health and performance. Ex vivo study of the functional interactions between microbial community members in conditions representative of those in the gut is an ongoing challenge. We have developed an in vitro 40-plex platform that provides an oxygen gradient to support simultaneous maintenance of microaerobic and anaerobic microbes from the gut microbiome that can aid in rapid characterization of microbial interactions and direct comparison of individual microbiome samples. In this report, we demonstrate that the platform more closely maintained the microbial diversity and composition of human donor fecal microbiome samples than strict anaerobic conditions. The oxygen gradient established in the platform allowed the stratification and subsequent sampling of diverse microbial subpopulations that colonize microaerobic and anaerobic micro-environments. With the ability to run forty samples in parallel, the platform has the potential to be used as a rapid screening tool to understand how the gut microbiome responds to environmental perturbations such as toxic compound exposure, dietary changes, or pharmaceutical treatments. [ABSTRACT FROM AUTHOR]

ObjectivesTo assess the validity of a bench-top model of an optical tympanometry device to diagnose in vitro model of middle ear effusion (MEE).Methods and materialsWe illuminated an in vitro model of ear canal and tympanic membrane with broadband light and relayed remitted light to a spectrometer system. We then used our proprietary algorithm to extract spectral features that, together with our logistic regression classifiers, led us to calculate a set of simplified indices related to different middle ear states. Our model included a glass vial covered with a porcine submucosa (representing the tympanic membrane) and filled with air, water, or milk solution (representing different MEE), and a set of cover-glass slips filled with either blood (representing erythema) or cerumen. By interchanging fluid types and cover-glass slips, we made measurements on combinations corresponding to normal healthy ear and purulent or serous MEE.ResultsEach simulated condition had a distinct spectral profile, which was then employed by our algorithm to discriminate clean and cerumen-covered purulent and serous MEE. Two logistic purulent and serous MEE classifiers correctly classified all in vitro middle ear states with 100% accuracy assessed by leave-one-out and k-fold cross validation.ConclusionsThis proof-of-concept in vitro study addressed an unmet need by introducing a device that easily and accurately can assess middle ear effusion. Future in vivo studies aimed at collecting data from clinical settings are warranted to further elucidate the validity of the technology in diagnosing pediatric acute otitis media.

One route of human exposure to environmental chemicals is oral uptake. This is primarily true for chemicals that may leach from food packaging materials, such as bisphenols and phthalate esters. Upon ingestion, these compounds are transported along the intestinal tract, from where they can be taken up into the blood stream or distributed to mucosal sites. At mucosal sites, mucosal immune cells and in the blood stream peripheral immune cells may be exposed to these chemicals potentially modulating immune cell functions. In the present study, we investigated the impact of three common bisphenols and two phthalate esters on mucosal-associated invariant T (MAIT) cells in vitro, a frequent immune cell type in the intestinal mucosae and peripheral blood of humans. All compounds were non-cytotoxic at the chosen concentrations. MAIT cell activation was only slightly affected as seen by flow cytometric analysis. Phthalate esters did not affect MAIT cell gene expression, while bisphenol-exposure induced significant changes. Transcriptional changes occurred in ∼ 25 % of genes for BPA, ∼ 22 % for BPF and ∼ 8 % for BPS. All bisphenols down-modulated expression of CCND2, CCL20, GZMB and IRF4, indicating an effect on MAIT cell effector function. Further, BPA and BPF showed a high overlap in modulated genes involved in cellular stress response, activation signaling and effector function suggesting that BPF may not be safe substitute for BPA.

Traumatic brain injuries are serious clinical incidents associated with some of the poorest outcomes in neurological practice. Coupled with the limited regenerative capacity of the brain, this has significant implications for patients, carers, and healthcare systems, and the requirement for life-long care in some cases. Clinical treatment currently focuses on limiting the initial neural damage with long-term care/support from multidisciplinary teams. Therapies targeting neuroprotection and neural regeneration are not currently available but are the focus of intensive research. Biomaterial-based interventions are gaining popularity for a range of applications including biomolecule and drug delivery, and to function as cellular scaffolds. Experimental investigations into the development of such novel therapeutics for traumatic brain injury will be critically underpinned by the availability of appropriate high throughput, facile, ethically viable, and pathomimetic biological model systems. This represents a significant challenge for researchers given the pathological complexity of traumatic brain injury. Specifically, there is a concerted post-injury response mounted by multiple neural cell types which includes microglial activation and astroglial scarring with the expression of a range of growth inhibitory molecules and cytokines in the lesion environment. Here, we review common models used for the study of traumatic brain injury (ranging from live animal models to in vitro systems), focusing on penetrating traumatic brain injury models. We discuss their relative advantages and drawbacks for the developmental testing of biomaterial-based therapies.

With obesity and its comorbidities continuing to rise, we urgently need to improve our understanding of what mechanisms trigger the white adipose tissue to become dysfunctional in response to over-feeding. The recent invent of 3D culturing models has produced several noteworthy protocols for differentiating unilocular adipocytes in vitro, promising to revolutionize the obesity research field by providing more representative adipose tissue models for such mechanistic studies. In parallel, these 3D models provide important insights to how profoundly the microenvironment influences adipocyte differentiation and morphology. This commentary highlights some of the most recent 3D models, including human unilocular vascularized adipocyte spheroids (HUVASs), developed by our lab. We discuss recent developments in the field, provide further insights to the importance of the microvasculature for adipocyte maturation, and summarize what challenges remain to be solved before we can achieve a culture model that fully recapitulates all aspects of human white adipocyte biology in vitro. Taken together, the commentary highlights important recent advances regarding 3D adipocyte culturing and underlines the many advantages these models provide over traditional 2D cultures, with the aim of convincing more laboratories to switch to 3D models.