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Objective: The objective of this study was to compare in vitro the different biomechanical properties of 2 balloon-expandable covered stents, VBX (Viabahn VBX, W.L. Gore & Associates, Flagstaff, AZ) and iCover (iVascular, Barcelona, Spain), as bridging stents in fenestrated aortic endoprostheses., Methods: Three biomechanical tests were performed to evaluate a total of 12 stents (6 VBX 6 × 59 mm and 6 iCover 6 × 57m). First, a 3-point bending test was performed with a calibrated dynamometer to evaluate the bending strength of the 40% and 150% deformation of the system in its crimped state and the force necessary to deform the expanded stent by 40% with respect to its initial diameter. Then the stents were expanded to their nominal diameter inside a 6-mm-diameter handmade fenestration and flared up to 8 and 10 mm. The whole set was fixed in the dynamometer to carry out the pullout-force (perpendicular dislocation) and shear-stress-force (axial dislocation) tests. The resulting forces were recorded via the force transducer. Load, deformation, and displacement data were recorded in newtons. The results of each stent test were examined under the microscope, and both stent types were compared., Results: Higher forces were required to bend the crimped VBX (16.24 N [IQR, 13.87-17.47 N] for the iCover and 19.01 N [IQR, 16.01-19.50 N] for VBX; P < 0.001), but lower forces were necessary for the expanded VBX compared with the iCover (0.36 N [IQR, 0.1-0.49 N] and 1.01 N [IQR, 0.97-1.09 N], respectively; p = 0.004). Pullout forces were 3.56 N (IQR, 3-4.1 N) and 7.07 N (IQR, 6.86-7.28 N) for the 8-mm flare and 7.29 N (IQR, 6.13-8.45 N) and 14 N (IQR, 12.05-15.95 N) for the 10-mm flare for the iCover and VBX, respectively ( P = 0.333 for both comparisons). The shear-stress forces needed to dislocate the 6-mm iCover and VBX axially with a 10-mm flare to 50%, 100%, and 200% were 0.88, 1.61, and 3.55 N for the iCover and 0.71, 1.43, and 2.51 N for the VBX ( P = 0.343, P = 0.486, and P = 0.486, respectively)., Conclusion: After evaluating the stents under in vitro conditions, the VBX and iCover stents showed similar results in terms of biomechanical properties, which demonstrates their competence in in vitro conditions. Further in vitro comparisons with other stent grafts are required., Clinical Impact: The BECS used in FEVAR / BEVAR must ensure some properties to avoid the most frequent complications derived from the failure of these bridging stents (stenosis, thrombosis, stent integrity, dislodgement or endoleaks) that represent the most common cause for reintervention. This paper adds to the limited data available, information to the clinician after comparing some properties of two BECS used in these techniques that demonstrate their competence in in-vitro conditions., Competing Interests: Declaration of Conflicting InterestsGaspar Mestres received speaker fees from W.L. Gore & Associates. Vincent Riambau received consulting and speaker fees from iVascular and research grants from W.L. Gore & Associates and iVascular.

The present study tends to evaluate the possible potential of bio-active Morroniside (MOR), against alloxan (ALX)-induced genotoxicity and hyperglycaemia. In silico prediction revealed the interaction of MOR with Poly (ADP-ribose) polymerase (PARP) protein which corroborated well with experimental in vitro L6 cell line and in vivo mice models. Data revealed the efficacy of MOR in the selective activation of PARP protein and modulating other stress proteins NF-κB, and TNF-α to initiate protective potential against ALX-induced genotoxicity and hyperglycaemia. Further, the strong interaction of MOR with CT-DNA (calf thymus DNA) analyzed through CD spectroscopy, UV-Vis study and ITC data revealed the concerted action of bio-factors involved in inhibiting chromosomal aberration and micronucleus formation associated with DNA damage. Finally, MOR does not play any role in microbial growth inhibition which often occurs due to hyperglycemic dysbiosis. Thus, from the overall findings, we may conclude that MOR could be a potential drug candidate for the therapeutic management of induced-hyperglycaemia and genotoxicity.Communicated by Ramaswamy H. Sarma.

Modern drug discovery is driven by high demand in the pharmaceutical industry to test growing libraries of compounds against potential targets. High-throughput screening (HTS) is characterized by fully automated experimentation that leverages robotic liquid handling systems, analytical techniques, and advanced computing and statistics, including the recent integration of artificial intelligence. To align with this trend, it is crucial to develop and implement new HTS platforms that offer improved predictivity and physiological relevance. In recent years, microphysiological systems, commonly known as organ-on-chip (OoC) systems, have progressed from a theoretical concept to a powerful alternative to conventional in vitro and animal models. High-throughput OoC (HT-OoC) systems could represent the disruptive technology sought by pharmaceutical companies to address their enormous research and development (R&D) expenses. In this study, we provide a brief overview of commercial products utilizing modern HT-OoC systems in drug discovery and development. Additionally, we discuss recent trends in R&D aimed at industrialization.

We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia, and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like ciliation patterns. De-differentiated EOECs, formed 10 days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 × 106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1 month after air-liquid interface introduction, >40% and >20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary ciliation rates to >60% in REOEC monolayers with diffuse ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

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-κB 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)-1β and IL-18 upon a stimulation with LPS, leading to the conversion of IL-18, but not IL-1β, 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Takenouchi, Masujin, Ikeda, Haraguchi, Suzuki, Uenishi, Onda and Kokuho.)

Physiologically relevant in vitro models of the human outer retina are required to better elucidate the complex interplay of retinal tissue layers and investigate their role in retinal degenerative disorders. Materials currently used to mimic the function of Bruch's membrane fail to replicate a range of important structural, mechanical, and biochemical properties. Here, we detail the fabrication of a surface-functionalized, fibrous collagen I membrane. We demonstrate its ability to better replicate a range of important material properties akin to the function of human Bruch's membrane when compared with a commonly utilized synthetic polyethylene terephthalate alternative. We further reveal the ability of this membrane to support the culture of the ARPE-19 cell line, as well as human pluripotent stem cell-derived RPE-like cells and human umbilical vein endothelial cells. This material could provide greater physiological relevance to the native Bruch's membrane than current synthetic materials and further improve the outcomes of in vitro outer retinal models.

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., Competing Interests: The authors declare no conflict of interest.

Treatment strategies for progressive intervertebral-disc degeneration often alleviate pain and other symptoms. With the goal of developing strategies to promote the regeneration of the nucleus pulposus (NP), the present study tried to identify the biological effects of hydrostatic (HP) and osmotic pressures on NP cells. The study hypothesis was that a repetitive regimen of cyclic HP followed by constant HP in high-osmolality medium would increase anabolic molecules in NP cells. Bovine NP cells/clusters were enclosed within semi-permeable membrane pouches and incubated under a regimen of cyclic HP for 2 d followed by constant HP for 1 d, repeated 6 times over 18 d. NP cells showed a significantly increased expression of anabolic genes over time: aggrecan, chondroitin sulfate N-acetylgalactosaminyltransferase 1, hyaluronan synthase 2, collagen type 2 (p < 0.05). In addition, the expression of catabolic or degenerative genes (matrix metalloproteinase 13, collagen type 1) and cellular characteristic genes (proliferating cell nucleic antigen, E-cadherin) was suppressed. The amount of sulfated glycosaminoglycan increased significantly at day 18 compared to day 3 (p < 0.01). Immunostaining revealed deposition of extracellular-matrix molecules and localization of other specific molecules corresponding to their genetic expression. An improved understanding of how cells respond to physicochemical stresses will help to better treat the degenerating disc using either cell- or gene-based therapies as well as other potential matrix-enhancing therapies. Efforts to apply these tissue-engineering and regenerative-medicine strategies will need to consider these important physicochemical stresses that may have a major impact on the survivability of such treatments.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150 µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3 weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3 weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Neonatal abstinence syndrome (NAS) refers to cadre of withdrawal manifestations in infants born to mothers who used illicit and licit substances during pregnancy. The increasing prevalence of NAS has been largely due to the maternal use of opioids during pregnancy. NAS contributes to increased morbidity and long-term disability in surviving infants. Clinically, oral opioid therapies for opioid exposure have been a standard treatment with morphine (MO) being the most commonly used medication. Recently, a non-opioid agent, clonidine (CD) has also been used with potentially favorable short- and long-term outcomes in infants. However, data regarding the cellular and molecular effects of these treatments on the developing brain is still lacking due to a lack of a reliable animal model that targets the neonatal brain. To address this gap in knowledge we determined the effects of MO or CD on the cell death of neonatal cortical explant cultures that were exposed to oxycodone (OXY) in utero. Sprague Dawley rats were randomized and implanted with programmable infusion pumps before mating to receive either the OXY (dose increasing from 1.21–1.90 mg/kg/day to a maximum dose of 2.86–3.49 mg/kg/day) or normal saline (NS) throughout pregnancy and until one week after delivery. Male and female rat pups were sacrificed on postnatal day 4, and the prefrontal cortex (PFC) and hippocampus (HC) were dissected and treated with MO (0.10–1.00 µM) or CD (1.20–120.00 µM) in culture media. After 5 days of treatment the explants were labeled with propidium iodide to detect cell death. Dead cells were analyzed and counted under fluorescence microscopy. In explants from the PFC, cell death was greater in those prenatally exposed to OXY and postnatally treated with MO (OXY/MO) (736.8 ± 76.5) compared to OXY/CD (620.9 ± 75.0; p = 0.005). In the HC explants, mean cell death counts were not significantly different between groups regardless of prenatal exposure or postnatal treatment (p = 0.19). The PFC is vital in controlling higher-order executive functions such as behavioral flexibility, learning and working memory. Therefore, our finding is consistent with executive function problems in children with prenatal opioid exposure.

The development of 3D scaffold-based models would represent a great step forward in cancer research, offering the possibility of predicting the potential in vivo response to targeted anticancer or anti-angiogenic therapies. As regards, 3D in vitro models require proper materials, which faithfully recapitulated extracellular matrix (ECM) properties, adequate cell lines, and an efficient vascular network. The aim of this work is to investigate the possible realization of an in vitro 3D scaffold-based model of adipose tissue, by incorporating decellularized 3D plant structures within the scaffold. In particular, in order to obtain an adipose matrix capable of mimicking the composition of the adipose tissue, methacrylated gelatin (GelMA), UV photo-crosslinkable, was selected. Decellularized fennel, wild fennel and, dill leaves have been incorporated into the GelMA hydrogel before crosslinking, to mimic a 3D channel network. All leaves showed a loss of pigmentation after the decellularization with channel dimensions ranging from 100 to 500 µm up to 3 μm, comparable with those of human microcirculation (5–10 µm). The photo-crosslinking process was not affected by the embedded plant structures in GelMA hydrogels. In fact, the weight variation test, performed on hydrogels with or without decellularized leaves showed a weight loss in the first 96 h, followed by a stability plateau up to 5 weeks. No cytotoxic effects were detected comparing the three prepared GelMA/D-leaf structures; moreover, the ability of the samples to stimulate differentiation of 3T3-L1 preadipocytes in mature adipocytes was investigated, and cells were able to grow and proliferate in the structure, colonizing the entire microenvironment and starting to differentiate. The developed GelMA hydrogels mimicked adipose tissue together with the incorporated plant structures seem to be an adequate solution to ensure an efficient vascular system for a 3D in vitro model. The obtained results showed the potentiality of the innovative proposed approach to mimic the tumoral microenvironment in 3D scaffold-based models.

In oral and maxillofacial bone reconstruction, autografts from the iliac crest represent the gold standard due to their superior clinical performance, compared to autografts derived from other extraoral regions. Thus, the aim of our study was to identify putative differences between osteoblasts derived from alveolar (hOB-A) and iliac crest (hOB-IC) bone of the same donor (nine donors) by means of their molecular properties in 2D and 3D culture. We thereby focused on the gene expression of biomarkers involved in osteogenic differentiation, matrix formation and osteoclast modulation. Furthermore, we examined the transcriptional response to Vit.D3 in hOB-A and hOB-IC. Our results revealed different modulation modes of the biomarker expression in osteoblasts, namely cell origin/bone entity-dependent, and culture configuration- and/or time-dependent modulations. SEMA3A, SPP1, BGLAP and PHEX demonstrated the strongest dependence on cell origin. With respect to Vit.D3-effects, BGLAP, SPP1 and ALPL displayed the highest Vit.D3-responsiveness. In this context we demonstrated that the transcriptional Vit.D3-response concerning SPP1 and ALPL in human osteoblasts depended on the cell origin. The results indicate a higher bone remodeling activity of iliac crest than alveolar osteoblasts and support the growing evidence that a high osteoclast activity at the host-/donor bone interface may support graft integration.

Heterothermy, as a temperature-dependent physiological continuum, may affect host-pathogen interactions through modulation of immune responses. Here, we evaluated proliferation and functional performance of a macrophage cell line established from the greater mouse-eared (Myotis myotis) bat at 8, 17.5, and 37°C to simulate body temperatures during hibernation, daily torpor and euthermia. Macrophages were also frozen to −20°C and then examined for their ability to proliferate in the immediate post-thaw period. We show that bat macrophages can proliferate at lower temperatures, though their growth rate is significantly slower than at 37°C. The cells differed in their shape, size and ability to attach to the plate surface at both lower temperatures, being spheroidal and free in suspension at 8°C and epithelial-like, spindle-shaped and/or spheroidal at 17.5°C. While phagocytosis at temperatures of 8 and 17.5°C amounted to 85.8 and 83.1% of the activity observed at 37°C, respectively, full phagocytic activity was restored within minutes of translocation into a higher temperature. Bat-derived macrophages were also able to withstand temperatures of −20°C in a cryoprotectant-free cultivation medium and, in the immediate post-thaw period, became viable and were able to proliferate. Our in vitro data enhance understanding of macrophage biology.

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

Various in vitro models using primary and established 2- and 3-dimensional cultures, multicellular tumor spheroids, standardized tumor slice cultures, tumor organoids, and microfluidic systems obtained from tumor lesions/biopsies of head and neck cancer (HNC) have been employed for exploring and monitoring treatment options. All of these in vitro models are to a different degree able to capture the diversity of tumors, recapitulate the disease genetically, histologically, and functionally and retain their tumorigenic potential upon xenotransplantation. The models were used for the characterization of the malignant features of the tumors and for in vitro screens of drugs approved for the treatment of HNC, including chemotherapy and radiotherapy as well as recently developed targeted therapies and immunotherapies, or for novel treatments not yet licensed for these tumor entities. The implementation of the best suitable model will enlarge our knowledge of the oncogenic properties of HNC, expand the drug repertoire and help to develop individually tailored treatment strategies resulting in the translation of these findings into the clinic. This review summarizes the different approaches using preclinical in vitro systems with their advantages and disadvantages and their implementation as preclinical platforms to predict disease course, evaluate biomarkers and test therapy efficacy.

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.

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.

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.

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.

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.

In developed countries, the aging of the population and the associated increase in age-related diseases are causing major unresolved medical, social, and environmental matters. Therefore, research on aging has become one of the most important and urgent issues in life sciences. If the molecular mechanisms of the onset and progression of neurodegenerative diseases are elucidated, we can expect to develop disease-modifying methods to prevent neurodegeneration itself. Since the discovery of induced pluripotent stem cells (iPSCs), there has been an explosion of disease models using disease-specific iPSCs derived from patient-derived somatic cells. By inducing the differentiation of iPSCs into neurons, disease models that reflect the patient-derived pathology can be reproduced in culture dishes, and are playing an active role in elucidating new pathological mechanisms and as a platform for new drug discovery. At the same time, however, we are faced with a new problem: how to recapitulate aging in culture dishes. It has been pointed out that cells differentiated from pluripotent stem cells are juvenile, retain embryonic traits, and may not be fully mature. Therefore, attempts are being made to induce cell maturation, senescence, and stress signals through culture conditions. It has also been reported that direct conversion of fibroblasts into neurons can reproduce human neurons with an aged phenotype. Here, we outline some state-of-the-art insights into models of neuronal aging in vitro. New frontiers in which stem cells and methods for inducing differentiation of tissue regeneration can be applied to aging research are just now approaching, and we need to keep a close eye on them. These models are forefront and intended to advance our knowledge of the molecular mechanisms of aging and contribute to the development of novel therapies for human neurodegenerative diseases associated with aging. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) is an essential structure for the maintenance of brain homeostasis. Alterations to the BBB are linked with a myriad of pathological conditions and play a significant role in the onset and evolution of neurodegenerative diseases, including Alzheimer's disease. Thus, a deeper understanding of the BBB's structure and function is mandatory for a better knowledge of neurodegenerative disorders and the development of effective therapies. Because studying the BBB in vivo imposes overwhelming difficulties, the in vitro approach remains the main possible way of research. With many in vitro BBB models having been developed over the last years, the main aim of this review is to systematically present the most relevant designs used in neurological research. In the first part of the article, the physiological and structural–functional parameters of the human BBB are detailed. Subsequently, available BBB models are presented in a comparative approach, highlighting their advantages and limitations. Finally, the new perspectives related to the study of Alzheimer's disease with the help of novel devices that mimic the in vivo human BBB milieu gives the paper significant originality. [ABSTRACT FROM AUTHOR]

Alcohol-associated liver disease (ALD) is an intricate disease that results in a broad spectrum of liver damage. The presentation of ALD can include simple steatosis, steatohepatitis, liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). Effective prevention and treatment strategies are urgently required for ALD patients. In previous decades, numerous rodent models were established to investigate the mechanisms of alcohol-associated liver disease and explore therapeutic targets. This review provides a summary of the latest developments in rodent models, including those that involve EtOH administration, which will help us to understand the characteristics and causes of ALD at different stages. In addition, we discuss the pathogenesis of ALD and summarize the existing in vitro models. We analyse the pros and cons of these models and their translational relevance and summarize the insights that have been gained regarding the mechanisms of alcoholic liver injury. [ABSTRACT FROM AUTHOR]

The evidence shows that there is an associated relationship between hepatosteatosis and insulin resistance. While some existing genetic induction animal and patient models challenge this relationship, indicating that hepatosteatosis is dissociated from insulin resistance. However, the molecular mechanisms of this dissociation remain poorly understood due to a lack of available, reliable, and simplistic setup models. Currently, we used primary rat hepatocytes (rHPCs), cocultured with rat hepatic stellate cells (HSC-T6) or human foreskin fibroblast cells (HFF-1) in stimulation with high insulin and glucose, to develop a model of steatosis charactered as dissociated lipid accumulation from insulin resistance. Oil-Red staining significantly showed intracellular lipid accumulated in the developed model. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c) and elongase of very-long-chain fatty acids 6 (ELOVL6), key genes responsible for lipogenesis, were detected and obviously increased in this model. Inversely, the insulin resistance related genes expression included phosphoenolpyruvate carboxykinase 1 (PCK1), pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), and glucose-6-phosphatase (G6pase) were decreased, suggesting a dissociation relationship between steatosis and insulin resistance in the developed model. As well, the drug metabolism of this developed model was investigated and showed up-regulation of cytochrome P450 3A (CYP3A) and down-regulation of cytochrome P450 2E1 (CYP2E1) and cytochrome P450 1A2 (CYP1A2). Taken together, those results demonstrate that the in vitro model of dissociated steatosis from insulin resistance was successfully created by our cocultured cells in high insulin and glucose medium, which will be a potential model for investigating the mechanism of insulin resistance dissociated steatosis, and discovering a novel drug for its treatment. [ABSTRACT FROM AUTHOR]

The pharmaceutical research sector has been facing the challenge of neurotherapeutics development and its inherited high-risk and high-failure-rate nature for decades. This hurdle is partly attributable to the presence of brain barriers, considered both as obstacles and opportunities for the entry of drug substances. The blood–cerebrospinal fluid (CSF) barrier (BCSFB), an under-studied brain barrier site compared to the blood–brain barrier (BBB), can be considered a potential therapeutic target to improve the delivery of CNS therapeutics and provide brain protection measures. Therefore, leveraging robust and authentic in vitro models of the BCSFB can diminish the time and effort spent on unproductive or redundant development activities by a preliminary assessment of the desired physiochemical behavior of an agent toward this barrier. To this end, the current review summarizes the efforts and progresses made to this research area with a notable focus on the attribution of these models and applied techniques to the pharmaceutical sector and the development of neuropharmacological therapeutics and diagnostics. A survey of available in vitro models, with their advantages and limitations and cell lines in hand will be provided, followed by highlighting the potential applications of such models in the (neuro)therapeutics discovery and development pipelines. [ABSTRACT FROM AUTHOR]

Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFβ/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration. [ABSTRACT FROM AUTHOR]

There is a growing interest to understand if and how the gut microbiome is causally linked to the pathogenesis and/or progression of diseases. While in vitro cell line models are commonly used for studying specific aspects of the host–microbe interaction, gnotobiotic murine models are considered the preferred platform for studying causality in microbiome research. Nevertheless, findings from animal studies provide limited opportunity for delineating various areas of interest to the human gut microbiome research. Gut-on-chips are biomimetics recapitulating intestinal physiology which enable investigation of bidirectional effects of the host and microbiome. We posit that they could advance causal and ecological gut microbiome research in three major areas: (i) diet–microbiome and drug–microbiome interaction; (ii) microbiome-targeted therapeutics pharmacoecology; and (iii) mechanistic studies of gut microbiome and microbiome-targeted intervention in extraintestinal pathologies. Gnotobiotic murine models are currently the cornerstone of causal gut microbiome research. Nevertheless, they provide limited opportunity for delineating various areas of interest to the human gut microbiome research. Current in vitro microbial ecology and host–microbe coculture methods also have limited capacity for studying the ecological bidirectional interaction of host and gut microbiome. Gut-microbiome-on-chips provide physiological and ecological advantages over other in vitro methods, allowing for monitoring microbial growth and ecological dynamic as well as host–microbiome interactions while adjusting and measuring the ecologically relevant parameters (e.g. , pH and O 2 level) in real time. Gut-microbiome-on-chips could be connected with other chips to model gut–liver, gut–lung, or gut–brain axes. Gut-microbiome-on-chip could expand microbiome research in three major areas: (i) diet–microbiome and drug–microbiome interactions; (ii) pharmacoecology of microbiome-targeted therapeutics; and (iii) mechanistic studies of gut microbiome and microbiome-targeted intervention in extraintestinal pathologies within the context of a personalized and precision medicine framework. [ABSTRACT FROM AUTHOR]

Recent reports of adverse health effects (e.g., capsular contracture, lymphoma) linked to the absence or presence of texture on soft-tissue implants (e.g., breast implants) suggest surface topography may have pathological impact(s). We propose that surface texture influences the transfer of displacements, experienced by an implant undergoing micromotion, to surrounding interfacial extracellular matrix, which in turn impacts the activity of the resident cells and is based on degree of tissue integration. We hypothesize that transfer of displacements due to micromotion promotes interstitial fluid movement that imposes hydrodynamic stresses (pressures, shear stresses) on cells residing in the interfacial tissues and impacts their activity. To address this, we developed a computer simulation to approximate hydrodynamic stresses in the interstitial environment of saturated poroelastic tissues (model soft-tissue implantation sites) generated from oscillatory implant micromotion as a function of the magnitude of translational displacement, direction of motion, degree of tissue integration, and surface roughness of the implant. Highly integrated implants were predicted to generate the highest fluid shear stresses within model tissues, with oscillatory fluid shear stresses up to 80 dyn/cm2 for a 20-μm displacement. Notably, application of oscillatory 80 dyn/cm2 shear stress to cultured human fibroblasts elicited cell death after 20 h compared to cells maintained under static conditions or exposed to 80 dyn/cm2 steady, unidirectional shear. These results indicate that oscillatory interstitial fluid stresses generated by micromotion of an integrated implant may influence the activity of the surrounding cells and play a role in the body's fibrotic response to textured soft-tissue implants. [ABSTRACT FROM AUTHOR]

Apple pomace (AP) contains biomolecules that induce changes in intestinal fermentation of monogastrics with positive expected health effects. The weaning of piglets can induce economic losses due to intestinal disturbances; new weaning strategies are, thus, welcome. The purpose of this study was to test the effect of AP on fermentation products by using baby-SPIME, an in vitro multi-compartment model dedicated to piglet weaning. A comparison was done on short chain fatty acid (SCFA) ratio and the microbiota induced in bioreactors between a control culture medium vs. an AP culture medium. The results of 2 preliminary runs showed that AP medium increased the molar ratio of propionate (p = 0.021) and decreased the molar ratio of butyrate (p = 0.009). Moreover, this medium increased the cumulative relative abundance of Prevotella sp. and Akkermansia sp. in bioreactors. AP could promote an ecosystem enriched with bacteria known as next-generation probiotics (NGP)—likely influencing the energy metabolism of piglets by their fermentation metabolites. AP could be used as a dietary strategy to influence bacterial changes in the intestine by stimulating the growth of bacteria identified as NGP. [ABSTRACT FROM AUTHOR]

For decades, insight into fundamental principles of human biology and disease has been obtained primarily by experiments in animal models. While this has allowed researchers to understand many human biological processes in great detail, some developmental and disease mechanisms have proven difficult to study due to inherent species differences. The advent of organoid technology more than 10 years ago has established laboratory-grown organ tissues as an additional model system to recapitulate human-specific aspects of biology. The use of human 3D organoids, as well as other advances in single-cell technologies, has revealed unprecedented insights into human biology and disease mechanisms, especially those that distinguish humans from other species. This review highlights novel advances in organoid biology with a focus on how organoid technology has generated a better understanding of human-specific processes in development and disease. This review highlights novel advances in organoid biology with a focus on how organoid technology has generated a better understanding of human-specific processes in development and disease. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) are the most common bacterial sexually transmitted infections (STIs) worldwide. The primary site of infection for both bacteria is the epithelium of the endocervix in women and the urethra in men; both can also infect the rectum, pharynx and conjunctiva. Ct/Ng co-infections are more common than expected by chance, suggesting Ct/Ng interactions increase susceptibility and/or transmissibility. To date, studies have largely focused on each pathogen individually and models exploring co-infection are limited. We aimed to determine if Ng co-infection influences chlamydial infection and development and we hypothesized that Ng-infected cells are more susceptible to chlamydial infection than uninfected cells. To address this hypothesis, we established an in vitro model of Ct/Ng coinfection in cultured human cervical epithelial cells. Our data show that Ng co-infection elicits an anti-chlamydial effect by reducing chlamydial infection, inclusion size, and subsequent infectivity. Notably, the anti-chlamydial effect is dependent on Ng viability but not extracellular nutrient depletion or pH modulation. Though this finding is not consistent with our hypothesis, it provides evidence that interaction of these bacteria in vitro influences chlamydial infection and development. This Ct/Ng co-infection model, established in an epithelial cell line, will facilitate further exploration into the pathogenic interplay between Ct and Ng. [ABSTRACT FROM AUTHOR]

Summary: This study aimed to elucidate the information mechanism of advanced glycation end products (AGEs) in sturgeon fillets, and its inhibition by polyphenol. The results revealed that LTVH fillets (processed using low temperature vacuum heating method, LTVH) did not have lower values of AGEs than those treated using TC method (a typical thermal procedure using boiling water at 100 °C). The addition of tea polyphenol, on the other hand, drastically reduced the AGEs levels in LTVH fillets. An in vitro model including sturgeon myofibrillar protein and glucose was employed in this work to evaluate the inhibition of AGEs by tea polyphenol. The results showed that free Arginine was susceptible to interaction with glucose, which caused the development of AGEs. However, the addition of tea polyphenol competed with glucose for binding to free Arginine, leading to a substantial decrease in free Arginine concentration and an increase of free amino loss rate, resulting in the inhibition of AGEs generation. The LTVH technique, in combination with tea polyphenol, can be used to prevent the production of AGEs on fillets during the thermal treatment. [ABSTRACT FROM AUTHOR]

The hollow fiber cartridge has the advantages of good semi-permeability, high surface area to volume ratio, convenient operation, and so on. Its application in chemical analysis, drug in vitro experiment, hemodialysis, and other fields has been deeply studied. This paper introduces the basic structure of hollow fiber cartridge, compares the advantages and disadvantages of a hollow fiber infection model constructed by a hollow fiber cartridge with traditional static model and animal infection model and introduces its application in drug effects, mechanism of drug resistance, and evaluation of combined drug regimen. The principle and application of hollow fiber bioreactors for cell culture and hollow fiber dialyzer for dialysis and filtration were discussed. The hollow fiber cartridge, whether used in drug experiments, artificial liver, artificial kidney, etc., has achieved controllable experimental operation and efficient and accurate experimental results, and will provide more convenience and support for drug development and clinical research in the future. [ABSTRACT FROM AUTHOR]

Background: Neuroimmune communication plays an important role in allergic inflammation, but the neuroimmune regulation of allergic rhinitis remains unclear. Objective: The goal of this study was to investigate the role of CD4-positive T lymphocyte (CD4+ T cells) adhesion to D-U87 neuron-like cells in mediating allergic rhinitis CD4+ T cell differentiation. Methods: D-U87 neuron-like cells were derived from the human glioblastoma U87 cell line. CD4+ T cells were isolated from human peripheral blood using a magnetic separation technique. In vitro coculture of D-U87 neuron-like cells and CD4+ T cells was established. The number of adherent CD4+ T cells was counted using a fluorescence microscope. The percentages of CD4+IFNγ+ and CD4+IL4+ T cells and the levels of IFNγ and IL4 cytokines in the supernatant were measured by flow cytometry. Results: The results showed that the number of adherent CD4+ T cells in patients with allergic rhinitis was significantly higher than that in healthy controls. In allergic rhinitis, the percentage of CD4+IL4+ T cells was significantly increased in the adherent group compared with that in the nonadherent group. Moreover, blocking ICAM1 and E-selectin decreased the number of adherent CD4+ T cells and the percentage of CD4+IL4+ T cells in allergic rhinitis. Conclusion: Adhesion contributes to CD4+IL4+ T cell differentiation in the in vitro coculture system of D-U87 neuron-like cells and allergic rhinitis CD4+ T cells, which may provide new insights into therapeutic strategies for allergic rhinitis. [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]

Introduction: To study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells. Methods: We compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs. Results and Discussion: Supplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations. Conclusion: We conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models. [ABSTRACT FROM AUTHOR]

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis. Synopsis: The kinetics and biological function of epigenetic reprogramming during mammalian germ cell development remain poorly understood. Here, single‐cell analysis of X‐chromosome activation changes during primordial germ cell‐like cell (PGCLC) differentiation uncovers hallmarks of female germ cell development. Time‐resolved tracing of X‐chromosome dynamics during PGCLC specification from embryonic stem cells shows heterogeneous and moderate X‐inactivation compared to somatic cell lineages.Germ cell‐competent epiblast cells show heterogeneous and incomplete X‐inactivation in vivo.RNA expression profiling highlights uncoupling of X‐inactivation and PGCLC fate acquisition.Subsequent X‐chromosome reactivation occurs step‐wise through prophase I of meiosis.PGCLCs with deficient X‐inactivation or premature X‐reactivation are abnormal and have reduced meiotic potential. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) is a cellular and physical barrier with a crucial role in homeostasis of the brain extracellular environment. It controls the imports of nutrients to the brain and exports toxins and pathogens. Dysregulation of the blood–brain barrier increases permeability and contributes to pathologies, including Alzheimer's disease, epilepsy, and ischemia. It remains unclear how a dysregulated BBB contributes to these different syndromes. Initial studies on the role of the BBB in neurological disorders and also techniques to permit the entry of therapeutic molecules were made in animals. This review examines progress in the use of human models of the BBB, more relevant to human neurological disorders. In recent years, the functionality and complexity of in vitro BBB models have increased. Initial efforts consisted of static transwell cultures of brain endothelial cells. Human cell models based on microfluidics or organoids derived from human-derived induced pluripotent stem cells have become more realistic and perform better. We consider the architecture of different model generations as well as the cell types used in their fabrication. Finally, we discuss optimal models to study neurodegenerative diseases, brain glioma, epilepsies, transmigration of peripheral immune cells, and brain entry of neurotrophic viruses and metastatic cancer cells. [ABSTRACT FROM AUTHOR]

The general anticoagulant evaluation requires high expense equipment, reagents, and space. Therefore, not all laboratories can perform research related to anticoagulant. In this study, we propose a novel simple method "in vitro thrombus-growth model" that can evaluate anticoagulant ability by measuring weight. The in vitro thrombus-growth model is prepared by creating a "growthclot" with citrate plasma, calcium chloride, and thrombin, and then pouring new citrate plasma onto it. The prepared growth-clots were increased in volume in citrated human plasma, including surpluses calcium chloride, which was released slowly, leading to clot coagulation around the plasma. As a result of evaluating the anticoagulant ability of direct thrombin inhibitor using this in vitro thrombus-growth model, it was confirmed that clot growth was suppressed in a concentrationdependent manner. Therefore, this thrombus-growth model is useful as a primary anticoagulant test that can to discover compounds with anticoagulant activity perform in any laboratory. [ABSTRACT FROM AUTHOR]

Simple Summary: The skeletal muscle of younger adults has a remarkable regenerative capacity, which substantially declines with age. Despite many interspecies differences, animals have been used to study new treatments to promote muscle regeneration in humans. This study reports a novel human experimental model using human skeletal muscle tissue of older adults that was extracted during surgical procedures. We describe an optimal procedure for maintaining human skeletal muscle tissue under experimental conditions for 11 days. This experimental model allows the investigation of resident macrophages and stem cells, which mediate muscle regeneration. Findings from studies of muscle regeneration can significantly contribute to the treatment of age-related loss of skeletal muscle mass, which may predispose older adults to severe morbidities. We established a human experimental model using excised skeletal muscle tissues from reconstructive surgeries in eight older adults. Muscle samples from each participant were preserved immediately or maintained in agarose medium for the following 5, 9, or 11 days. Immunofluorescence analyses of the structural proteins, actin and desmin, confirmed the integrity of muscle fibers over 11 days of maintenance. Similarly, the numbers of CD80-positive M1 and CD163-positive M2 macrophages were stable over 11 days in vitro. However, the numbers of PAX7-positive satellite cells and MYOD-positive myoblasts changed in opposite ways, suggesting that satellite cells partially differentiated in vitro. Further experiments revealed that stimulation with unsaturated fatty acid C18[2]c (linoleic acid) increased resident M1 macrophages and satellite cells specifically. Thus, the use of human skeletal muscle tissue in vitro provides a direct experimental approach to study the regulation of muscle tissue regeneration by macrophages and stem cells and their responses to therapeutic compounds. [ABSTRACT FROM AUTHOR]

The human blood–brain barrier (BBB) represents the interface of microvasculature and the central nervous system, regulating the transport of nutrients and protecting the brain from external threats. To gain a deeper understanding of (patho)physiological processes affecting the BBB, sophisticated models mimicking the in vivo situation are required. Currently, most in vitro models are cultivated on stiff, semipermeable, and non-biodegradable Transwell® membrane inserts, not adequately mimicking the complexity of the extracellular environment of the native human BBB. To overcome these disadvantages, we developed three-dimensional electrospun scaffolds resembling the natural structure of the human extracellular matrix. The polymer fibers of the scaffold imitate collagen fibrils of the human basement membrane, exhibiting excellent wettability and biomechanical properties, thus facilitating cell adhesion, proliferation, and migration. Cultivation of human induced pluripotent stem cells (hiPSCs) on these scaffolds enabled the development of a physiological BBB phenotype monitored via the formation of tight junctions and validated by the paracellular permeability of sodium fluorescein, further accentuating the non-linearity of TEER and barrier permeability. The novel in vitro model of the BBB forms a tight endothelial barrier, offering a platform to study barrier functions in a (patho)physiologically relevant context. [ABSTRACT FROM AUTHOR]

Background: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). Methods: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. Results: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. Conclusion: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing. [ABSTRACT FROM AUTHOR]

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS). [ABSTRACT FROM AUTHOR]

Bioengineered autologous skin substitutes (BASS) technology is an emerging field for skin burn therapy. However, further studies on BASS characterization, viability against standard procedures for wound healing, and protocol optimization are necessary for the improvement of BASS technology for clinical use. The aim of this study is to evaluate the effect of common antiseptics for clinical use in BASS, focusing on cell viability, inflammatory cytokine pattern, and epithelium and skin barrier integrity, in order to establish the most adequate treatment for wound care after BASS grafting. Human keratinocytes (hKT) and dermal fibroblasts (hDF) were isolated from foreskin samples and integrated into hyaluronic acid-based BASS. The following antiseptics were applied every 48 h: ethanol (70%), chlorhexidine digluconate (1%), sodium hypochlorite (0.02%), povidone iodine (100 mg/mL), and polyhexanide (0.1%), during a follow-up of 16 days. Sodium hypochlorite was the only treatment that showed a high cell viability percentage throughout the evaluation time compared to other antiseptic treatments, as well as a similar cytokine secretion pattern as control BASS. No significant differences were found regarding epidermal barrier function. These findings point towards sodium hypochlorite being the least aggressive antiseptic treatment for BASS post-transplantation wound care. [ABSTRACT FROM AUTHOR]

The 24-hour (24 h) post-transfusion survival of donor red blood cells (RBCs) is an important marker of transfusion efficacy. Nonetheless, within that period, donated RBCs may encounter challenges able to evoke rapid stress-responses. The aim of the present study was to assess the effect of exposure to plasma and body temperature upon stored RBCs under recipient-mimicking conditions in vitro from the first hours "post-transfusion" up to 24 h. For this purpose, packed RBCs from seven leukoreduced CPD/SAGM units were reconstituted with plasma of twenty-seven healthy individuals and incubated for 24 h at 37oC. Three units were additionally used to examine stress-responses in 3-hour intervals post mixing with plasma (n = 5) until 24 h. All experiments were performed in shortly-, medium-, and long-stored RBCs. Hemolysis, redox, morphology, membrane protein binding and vesiculation parameters were assessed. Even though spontaneous hemolysis was minimal post-reconstitution, it presented a time-dependent increase. A similar time-course profile was evident for the concentration of procoagulant extracellular vesicles and the osmotic fragility (shortly-stored RBCs). On the contrary, mechanical fragility and reactive oxygen species accumulation were characterized by increases in medium-stored RBCs, evident even from the first hours in the recipient-mimicking environment. Finally, exposure to plasma resulted in rapid improvement of morphology, especially in medium-stored RBCs. Overall, some RBC properties vary significantly during the first 24 h post-mixing, at levels different from both the storage ones and the standard end-of-24 h. Such findings may be useful for understanding the performance of RBCs and their possible clinical effects −especially on susceptible recipients− during the first hours post-transfusion. [ABSTRACT FROM AUTHOR]

Franklin R Amthor, Christianne E Strang Department of Psychology, The University of Alabama at Birmingham, Birmingham, AL, 35294-1170, USACorrespondence: Franklin R AmthorDepartment of Psychology, The University of Alabama at Birmingham, 1300 University Blvd, Birmingham, AL, 35294-1170, USATel +1 (205) 934-2694Email amthorfr@uab.eduPurpose: Electrical stimulation of the human central nervous system via surface electrodes has been used for both learning enhancement and the amelioration of neurodegenerative or psychiatric disorders. However, data are sparse on how such electrical stimulation affects neural circuits at the cellular level. This study assessed the effects of tACS-like currents at 10 Hz on On-center retinal ganglion cell responsiveness, using the rabbit retina eyecup preparation as a model for central nervous system effects.Methods: We made extracellular recordings of light-evoked spike responses in different classes of On-center retinal ganglion cells before, during and after brief applications of 1 microampere alternating currents using single electrodes and microelectrode arrays.Results: tACS-like currents (tACS) of 1 microampere produced effects on On-center ganglion cell response profiles immediately after initiation or cessation of tACS, without driving phase-locked firing in the absence of light stimuli. tACS affected the initial transient responses to light stimulation for all cells, sustained response components (if any) more strongly for sustained cells, and the center-surround balance more strongly for transient cells.Conclusion: tACS sculpted light-evoked responses that lasted for one or more hours after cessation of current without, itself, directly inducing significant firing changes. Functionally, tACS effects could result in effects on contrast thresholds for both broad classes of cells, but because tACs differentially affects the center-surround balance of transient On-center cells, there may be greater effects on the spatial resolution and gain. The isolated retina appears to be a useful model to understand tACS actions at the neuronal level.Keywords: retina, tACS mechanisms, CNS, in vitro model, neural coding, neuro-modulation