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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.

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.

International audience; Drug permeation across the cornea remains a major challenge due to its unique and complex anatomy and physiology. Static barriers such as the different layers of the cornea, as well as dynamic aspects such as the constant renewal of the tear film and the presence of the mucin layer together with efflux pumps, all present unique challenges for effective ophthalmic drug delivery. To overcome some of the current ophthalmic drug limitations, the identification and testing of novel drug formulations such as liposomes, nanoemulsions, and nanoparticles began to be considered and widely explored. In the early stages of corneal drug development reliable in vitro and ex vivo alternatives, are required, to be in line with the principles of the 3Rs (Replacement, Reduction, and Refinement), with such methods being in addition faster and more ethical alternatives to in vivo studies. The ocular field remains limited to a handful of predictive models for ophthalmic drug permeation. In vitro cell culture models are increasingly used when it comes to transcorneal permeation studies. Ex vivo models using excised animal tissue such as porcine eyes are the model of choice to study corneal permeation and promising advancements have been reported over the years. Interspecies characteristics must be considered in detail when using such models. This review updates the current knowledge about in vitro and ex vivo corneal permeability models and evaluates their advantages and limitations.

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.

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.

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.

The blood–brain barrier (BBB) is a highly sophisticated system with the ability to regulate compounds transporting through the barrier and reaching the central nervous system (CNS). The BBB protects the CNS from toxins and pathogens but can cause major issues when developing novel therapeutics to treat neurological disorders. PLGA nanoparticles have been developed to successfully encapsulate large hydrophilic compounds for drug delivery. Within this paper, we discuss the encapsulation of a model compound Fitc-dextran, a large molecular weight (70 kDa), hydrophilic compound, with over 60% encapsulation efficiency (EE) within a PLGA nanoparticle (NP). The NP surface was chemically modified with DAS peptide, a ligand that we designed which has an affinity for nicotinic receptors, specifically alpha 7 nicotinic receptors, found on the surface of brain endothelial cells. The attachment of DAS transports the NP across the BBB by receptor-mediated transcytosis (RMT). Assessment of the delivery efficacy of the DAS-conjugated Fitc-dextran-loaded PLGA NP was studied in vitro using our optimal triculture in vitro BBB model, which successfully replicates the in vivo BBB environment, producing high TEER (≥230 ) and high expression of ZO1 protein. Utilising our optimal BBB model, we successfully transported fourteen times the concentration of DAS-Fitc-dextran-PLGA NP compared to non-conjugated Fitc-dextran-PLGA NP. Our novel in vitro model is a viable method of high-throughput screening of potential therapeutic delivery systems to the CNS, such as our receptor-targeted DAS ligand-conjugated NP, whereby only lead therapeutic compounds will progress to in vivo studies.

This thesis focused on the fabrication of kidney proximal tubule models. A coaxial printing system allowed the robust and straightforward fabrication of coiled perfusable microfibers, replicating the kidney proximal convoluted tubules. The formed alginate-gelatin microfibers offer a complex tubular shape in which the cells exhibit mature markers, such as functional transporters and polarized monolayers. Moreover, this model has proven to support both healthy and cystinotic proximal tubule cell lines and allows for mechanistically studying tubulopathies. Though distinctive 3D-printing strategies offer opportunities to mimic the structure of the convoluted proximal tubule, printing resolution remains restricted for soft hydrogel materials. Therefore, a unique method was studied, whereby post-printing treatment induced resolution enhancement of methacrylated hyaluronic acid / gelatin methacryloyl (HAMA/GelMA) based hydrogels. This shrinking technique opens a wide application to create higher resolutions for 3D printing. However, we also found that the polycationic shrinking reagents used were relatively toxic for our cells of interest. Therefore, more research is needed to prevent the direct exposure of cells to the shrinking reagents. Using melt electrowriting, scaffolds were fabricated that are self-supportive, yet small-sized and highly porous. These scaffolds enable direct access to the basolateral and luminal cell sides to facilitate solute exchange with vasculature in immediate proximity, which is critical for functional proximal tubule constructs. The polymer applied, polycaprolactone, is used to prepare biomaterials that generally show good biocompatibility and is compatible with implantation in the future. Kidney proximal tubule cells and glomerular endothelial cells form polarized monolayers in our tubules, form their own extracellular matrix, and show functionality for important kidney transporters. In our preliminary results we showed initial work with induced pluripotent stem cell-derived kidney organoids, which is very promising for the development of implantable proximal tubule grafts. The aim was to develop vascularized kidney tubules for studying the clearance of protein bound uremic toxins, while gaining knowledge for the development of implantable kidney tubules. The developed models allow for studying transepithelial secretion of uremic toxins, an overarching application for these constructs. This will further advance our understanding of the kidney secretion processes and aid in studying interventions to main proximal tubule function in chronic kidney disease conditions in vitro. Furthermore, while fundamental by nature, our research deliverables enhance mechanistic insight in additive manufacturing and kidney development processes. Altogether, our findings will further advance the field of kidney engineering, while working towards kidney replacement therapies.

This thesis focused on the fabrication of kidney proximal tubule models. A coaxial printing system allowed the robust and straightforward fabrication of coiled perfusable microfibers, replicating the kidney proximal convoluted tubules. The formed alginate-gelatin microfibers offer a complex tubular shape in which the cells exhibit mature markers, such as functional transporters and polarized monolayers. Moreover, this model has proven to support both healthy and cystinotic proximal tubule cell lines and allows for mechanistically studying tubulopathies. Though distinctive 3D-printing strategies offer opportunities to mimic the structure of the convoluted proximal tubule, printing resolution remains restricted for soft hydrogel materials. Therefore, a unique method was studied, whereby post-printing treatment induced resolution enhancement of methacrylated hyaluronic acid / gelatin methacryloyl (HAMA/GelMA) based hydrogels. This shrinking technique opens a wide application to create higher resolutions for 3D printing. However, we also found that the polycationic shrinking reagents used were relatively toxic for our cells of interest. Therefore, more research is needed to prevent the direct exposure of cells to the shrinking reagents. Using melt electrowriting, scaffolds were fabricated that are self-supportive, yet small-sized and highly porous. These scaffolds enable direct access to the basolateral and luminal cell sides to facilitate solute exchange with vasculature in immediate proximity, which is critical for functional proximal tubule constructs. The polymer applied, polycaprolactone, is used to prepare biomaterials that generally show good biocompatibility and is compatible with implantation in the future. Kidney proximal tubule cells and glomerular endothelial cells form polarized monolayers in our tubules, form their own extracellular matrix, and show functionality for important kidney transporters. In our preliminary results we showed initial work with induced pluripotent stem cell-derived kidney organoids, which is very promising for the development of implantable proximal tubule grafts. The aim was to develop vascularized kidney tubules for studying the clearance of protein bound uremic toxins, while gaining knowledge for the development of implantable kidney tubules. The developed models allow for studying transepithelial secretion of uremic toxins, an overarching application for these constructs. This will further advance our understanding of the kidney secretion processes and aid in studying interventions to main proximal tubule function in chronic kidney disease conditions in vitro. Furthermore, while fundamental by nature, our research deliverables enhance mechanistic insight in additive manufacturing and kidney development processes. Altogether, our findings will further advance the field of kidney engineering, while working towards kidney replacement therapies.

This thesis focused on the fabrication of kidney proximal tubule models. A coaxial printing system allowed the robust and straightforward fabrication of coiled perfusable microfibers, replicating the kidney proximal convoluted tubules. The formed alginate-gelatin microfibers offer a complex tubular shape in which the cells exhibit mature markers, such as functional transporters and polarized monolayers. Moreover, this model has proven to support both healthy and cystinotic proximal tubule cell lines and allows for mechanistically studying tubulopathies. Though distinctive 3D-printing strategies offer opportunities to mimic the structure of the convoluted proximal tubule, printing resolution remains restricted for soft hydrogel materials. Therefore, a unique method was studied, whereby post-printing treatment induced resolution enhancement of methacrylated hyaluronic acid / gelatin methacryloyl (HAMA/GelMA) based hydrogels. This shrinking technique opens a wide application to create higher resolutions for 3D printing. However, we also found that the polycationic shrinking reagents used were relatively toxic for our cells of interest. Therefore, more research is needed to prevent the direct exposure of cells to the shrinking reagents. Using melt electrowriting, scaffolds were fabricated that are self-supportive, yet small-sized and highly porous. These scaffolds enable direct access to the basolateral and luminal cell sides to facilitate solute exchange with vasculature in immediate proximity, which is critical for functional proximal tubule constructs. The polymer applied, polycaprolactone, is used to prepare biomaterials that generally show good biocompatibility and is compatible with implantation in the future. Kidney proximal tubule cells and glomerular endothelial cells form polarized monolayers in our tubules, form their own extracellular matrix, and show functionality for important kidney transporters. In our preliminary results we showed initial work with induced pluripotent stem cell-derived kidney organoids, which is very promising for the development of implantable proximal tubule grafts. The aim was to develop vascularized kidney tubules for studying the clearance of protein bound uremic toxins, while gaining knowledge for the development of implantable kidney tubules. The developed models allow for studying transepithelial secretion of uremic toxins, an overarching application for these constructs. This will further advance our understanding of the kidney secretion processes and aid in studying interventions to main proximal tubule function in chronic kidney disease conditions in vitro. Furthermore, while fundamental by nature, our research deliverables enhance mechanistic insight in additive manufacturing and kidney development processes. Altogether, our findings will further advance the field of kidney engineering, while working towards kidney replacement therapies.

Alphaherpesviruses infect humans and most animals. They can cause severe morbidity and mortality. The pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that can infect most mammals. The PRV persists in the host by establishing a latent infection, and stressful stimuli can induce the latent viruses to reactivate and cause recurrent diseases. The current strategies of antiviral drug therapy and vaccine immunization are ineffective in eliminating these viruses from the infected host. Moreover, overspecialized and complex models are also a major obstacle to the elucidation of the mechanisms involved in the latency and reactivation of the PRV. Here, we present a streamlined model of the latent infection and reactivation of the PRV. A latent infection established in N2a cells infected with the PRV at a low multiplicity of infection (MOI) and maintained at 42 °C. The latent PRV was reactivated when the infected cells were transferred to 37 °C for 12 to 72 h. When the above process was repeated with a UL54-deleted PRV mutant, it was observed that the UL54 deletion did not affect viral latency. However, viral reactivation was limited and delayed. This study establishes a powerful and streamlined model to simulate PRV latency and reveals the potential role of temperature in PRV reactivation and disease. Meanwhile, the key role of the early gene UL54 in the latency and reactivation of PRV was initially elucidated.

The blood–brain barrier (BBB) is a functional interface that provides selective permeability, protection from toxic substances, transport of nutrients, and clearance of brain metabolites. Additionally, BBB disruption has been shown to play a role in many neurodegenerative conditions and diseases. Therefore, the aim of this study was to establish a functional, convenient, and efficient in vitro co-cultured BBB model that can be used for several physiological conditions related to BBB disruption. Mouse brain-derived endothelial (bEnd.3) and astrocyte (C8-D1A) cells were co-cultured on transwell membranes to establish an intact and functional in vitro model. The co-cultured model and its effects on different neurological diseases and stress conditions, including Alzheimer’s disease (AD), neuroinflammation, and obesity, have been examined by transendothelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran, and tight junction protein analyses. Scanning electron microscope images showed evidence of astrocyte end-feet processes passing through the membrane of the transwell. Moreover, the co-cultured model showed effective barrier properties in the TEER, FITC, and solvent persistence and leakage tests when compared to the mono-cultured model. Additionally, the immunoblot results showed that the expression of tight junction proteins such as zonula occludens-1 (ZO-1), claudin-5, and occludin-1 was enhanced in the co-culture. Lastly, under disease conditions, the BBB structural and functional integrity was decreased. The present study demonstrated that the co-cultured in vitro model mimicked the BBB’s structural and functional integrity and, under disease conditions, the co-cultured model showed similar BBB damages. Therefore, the present in vitro BBB model can be used as a convenient and efficient experimental tool to investigate a wide range of BBB-related pathological and physiological studies.

BackgroundWe have previously described a tri-culture of neurons, astrocytes, and microglia that accurately mimics thein vivoneuroinflammatory response (both neurotoxic and neuroprotective) to a wide range of neuroinflammatory stimuli. Electrophysiological activity is an essential measure of neuronal function, which is influenced by neuroinflammation. Microelectrode array (MEA) technology is a versatile tool to non-invasively studyin vitroneural networks by simultaneously recording electrophysiological activity from multiple sites. In this study, we used extracellular recordings to determine the effect of microglia on neural network formation and stability in primary cortical cultures and monitor the changes in neural activity in response to neuroinflammatory stimuli.MethodsPrimary neonatal rat cortical tri-cultures of neurons, astrocytes, and microglia or co-cultures of neurons and astrocytes were cultured on custom MEAs and the neural activity was monitored for 21 daysin vitroto assess culture maturation and network formation. Quantification of synaptic puncta and averaged spike waveforms were used to determine the difference in excitatory to inhibitory neuron ratio (E/I ratio) of the neurons in tri- and co-cultures. The electrophysiological response to lipopolysaccharide (LPS) treatment of both culture types were compared.ResultsThe tri- and co-culture showed minimal difference in electrophysiological markers of neural network formation and stability with the exception of a significant increase in spike frequency in the tri-culture at later timepoints (DIV 17 and 21). Additionally, there was no significant difference in the density of either post-synaptic or excitatory pre-synaptic puncta between the culture types. However, characterization of the average spike waveforms revealed that the tri-culture had an E/I ratio much closer to that found in the rat cortex. Finally, only the tri-culture displayed a significant decrease in both the number of active channels and spike frequency following LPS exposure.ConclusionsThis study demonstrates that the microglia in the tri-culture do not disrupt neural network formation and stability as quantified using extracellular recordings and may be a better representation of thein vivocortex due to the closer E/I ratio than more traditional isolated neuron and neuron-astrocyte co-cultures. Additionally, the tri-culture is better able to mimic the neuroinflammatory response to LPS, which was quantified via changes in neural electrophysiological activity.

Bovine coronavirus (BCoV) is one of the major viral pathogens of cattle, responsible for economic losses and causing a substantial impact on animal welfare. Several in vitro 2D models have been used to investigate BCoV infection and its pathogenesis. However, 3D enteroids are likely to be a better model with which to investigate host–pathogen interactions. This study established bovine enteroids as an in vitro replication system for BCoV, and we compared the expression of selected genes during the BCoV infection of the enteroids with the expression previously described in HCT-8 cells. The enteroids were successfully established from bovine ileum and permissive to BCoV, as shown by a seven-fold increase in viral RNA after 72 h. Immunostaining of differentiation markers showed a mixed population of differentiated cells. Gene expression ratios at 72 h showed that pro-inflammatory responses such as IL-8 and IL-1A remained unchanged in response to BCoV infection. Expression of other immune genes, including CXCL-3, MMP13, and TNF-α, was significantly downregulated. This study shows that the bovine enteroids had a differentiated cell population and were permissive to BCoV. Further studies are necessary for a comparative analysis to determine whether enteroids are suitable in vitro models to study host responses during BCoV infection.

This study presents a multilayer in vitro human skin platform to quantitatively relate predicted spatial time–temperature history with measured tissue injury response. This information is needed to elucidate high-temperature, short-duration burn injury kinetics and enables determination of relevant input parameters for computational models to facilitate treatment planning. Multilayer in vitro skin platforms were constructed using human dermal keratinocytes and fibroblasts embedded in collagen I hydrogels. After three seconds of contact with a 50–100 °C burn tip, ablation, cell death, apoptosis, and HSP70 expression were spatially measured using immunofluorescence confocal microscopy. Finite element modeling was performed using the measured thermal characteristics of skin platforms to determine the temperature distribution within platforms over time. The process coefficients for the Arrhenius thermal injury model describing tissue ablation and cell death were determined such that the predictions calculated from the time–temperature histories fit the experimental burn results. The activation energy for thermal collagen ablation and cell death was found to be significantly lower for short-duration, high-temperature burns than those found for long-duration, low-temperature burns. Analysis of results suggests that different injury mechanisms dominate at higher temperatures, necessitating burn research in the temperature ranges of interest and demonstrating the practicality of the proposed skin platform for this purpose.

Background Adipogenesis, glycosaminoglycan hyaluronan (HA) production, inflammation, and fibrosis are the main pathogenic mechanisms responsible for Graves’ orbitopathy (GO). We hypothesized that d...

Objective To assess the actual xylitol content in sugar-free chewing gums available in a market of Saudi Arabia and investigate its effect on acid production and pH change in vitro. Materials and methods: A total of 29 different brands of xylitol-containing sugar-free chewing gums were collected from five major grocery stores in Saudi Arabia. Xylitol was extracted and its concentration was determined using the D-Sorbitol/Xylitol Enzymatic Kit (Megazyme; Bray, Wicklow, Ireland). The pH of the extracts and the amount of acid production for each product was measured after 15-minute and 30-minute incubation with Streptococcus mutans. Descriptive analysis, concentrations, and one-way analysis of variance (ANOVA) with the least significant difference (LSD) as multiple comparisons were performed. Results: The xylitol content in grams was clearly stated on the labels of 16 products, while it was stated in percentages on the labels of ten products. The mean pH of most of the tested products was 5.857 ± 0.096. Significant differences in pH were recorded among 20 products (p ≤ 0.05). Highly significant differences in pH (p = 0.001) were observed in five of the products. Conclusion: The results of this study indicate, using an in vitro model, that xylitol can significantly affect salivary pH. The actual xylitol content in most brands of chewing gums currently available in the markets of Saudi Arabia is less than the concentration recommended for prevention of caries. Accurate information with proper labeling is required to enable dental professionals to take correct and informed decisions about recommending the use of these products.

In vitro engineering of human articular cartilage (AC) is a regenerative medicine challenge. The main objective of this study was the development of a repeatable scaffold-free in vitro model of cho...

In order to achieve a high sample throughput, permeation experiments are often carried out using 96-well sandwich plates. Even though agitation is regarded as important, permeation studies in 96-well format are often carried out without agitation since orbital shaking, the most common agitation method for 96-well plates, has been reported to create difficulties (e.g., well-to-well cross-talk), and high cost and low availability limits the use of other agitation techniques (e.g., magnetic stirring). This study investigates how orbital shaking and magnetic stirring affect the apparent permeability of model compounds with different water-solubilities (methylene blue, carbamazepine, and albendazole) using a novel 96-well sandwich plate comprising a cellulose-hydrate membrane (PermeaPlain (R) plate). Orbital shaking was found less efficient than magnetic stirring in terms of homogeneously distributing a small volume of dye within the donor compartment. Furthermore, in terms of achieving maximum trans-barrier flux, magnetic stirring was found a more effective agitation method than orbital shaking. Obviously, with orbital shaking the medium in the bottom compartment of the sandwich plates never was mixed in-phase. The impact of insufficient mixing on permeation was found strongest with the most lipophilic compound, which correlates with literature reports that the contribution of the unstirred water layer towards the overall resistance of the barrier is most expressed in case of lipophilic drugs. Finally, it was tested how different liquid volumes in the bottom compartment of the plates affect the well-to-well cross-talk during permeation experiments under orbital shaking. This study revealed that 250-300 mu L should be used in the bottom compartment of the sandwich plates to reduce well-to-well cross-talk when using orbital shaking for agitation. (C) 2021 Published by Elsevier Inc. on behalf of American Pharmacists Association.

Christianne E Strang, Franklin R Amthor 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, Birmingham, AL, 35294-1170, USA, Tel +1 205 934-2694, Fax +1 205 975-6110, Email amthorfr@uab.eduPurpose: Transcranial alternating current stimulation (tACS) is used as a brain stimulation mechanism to enhance learning, ameliorate some psychiatric disorders, and modify behavior. This study assessed the effects of near threshold tACS-like currents on Off-center and On-Off retinal ganglion cell responsiveness in the rabbit retina eyecup preparation as a model for central nervous system effects.Materials and Methods: We made extracellular recordings in the isolated rabbit eyecup preparation using single electrodes and microelectrode arrays to measure light-evoked spike responses in different classes of Off-center and On-Off retinal ganglion cells before, during, and after brief applications of alternating currents of 1– 2 microamperes, at frequencies of 10, 20, 30, and 40 Hz.Results: tACS application sculpted the light-evoked response profiles without directly driving spiking activity of the 20 Off-center and On-Off ganglion cells we recorded from. During tACS application, Off responses were significantly enhanced for 6 cells and significantly suppressed for 14 cells, but after tACS application, Off responses were significantly enhanced for 7 cells and suppressed for 12 cells. The Off responses of the remaining two cells returned to baseline. On responses were less affected during and after tACS.Conclusion: tACS sculpts Off-center and On-Off retinal ganglion cell responsiveness. The dissimilarity of effects in different cells within the same class and the differential effects on the On and Off components of the light response within the same cell are consistent with the hypothesis that tACS acts at threshold on amacrine cells in the inner plexiform layer.Keywords: retina, tACS mechanisms, CNS, in vitro model, neural coding, neuromodulation

Tissue-resident macrophages are of vital importance as they preserve tissue homeostasis in all mammalian organs. Nevertheless, appropriate cell culture models are still limited. Here, we propose a ...

This study aimed to develop valid in vitro models for preclinical evaluation of proliferative and anti-inflammatory effects of human allogeneic serum eye drops for dry eye disease (DED) treatment. A DED wound healing model was developed by analyzing the influence of coating and serum concentrations on human corneal epithelial (HCE-T) wound closure. Further, intralaboratory variance, freeze–thaw cycle effects, donor variability and stability assays were conducted. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were used to induce the gene expression of matrix metalloproteinase 9 (MMP9), cyclooxygenase 2 (COX2), transforming growth factor-β (TGFβ) and IL-1β. MMP9 induction was optimized using a design-of-experiments (DoE) approach and applied to examine serum under static and dynamic conditions. MMP9 protein expression was analyzed by ELISA. The DED wound healing model detected proliferative effects of serum down to 1% with a small intralaboratory variance. Serum stability was shown over six months, donor variance could be detected, and freeze–thaw cycle effects did not affect wound closure. Serum decreased MMP9 expression on the gene and protein levels. The induction method was successfully optimized using DoE modeling and transferred to a dynamic setting mimicking tear film fluidics. The DED wound healing and inflammatory DED model present useful in vitro models for the preclinical evaluation of allogeneic serum eye drops without the use of animal experiments.

Healing of burn injury is a complex process that often leads to the development of functional and aesthetic complications. To study skin regeneration in more detail, organotypic skin models, such as full skin equivalents (FSEs) generated from dermal matrices, can be used. Here, FSEs were generated using de-epidermalized dermis (DED) and collagen matrices MatriDerm® and Mucomaix®. Our aim was to validate the MatriDerm- and Mucomaix-based FSEs for the use as in vitro models of wound healing. Therefore, we first characterized the FSEs in terms of skin development and cell proliferation. Proper dermal and epidermal morphogenesis was established in all FSEs and was comparable to ex vivo human skin models. Extension of culture time improved the organization of the epidermal layers and the basement membrane in MatriDerm-based FSE but resulted in rapid degradation of the Mucomaix-based FSE. After applying a standardized burn injury to the models, re-epithelization occurred in the DED- and MatriDerm-based FSEs at 2 weeks after injury, similar to ex vivo human skin. High levels of pro-inflammatory cytokines were present in the culture media of all models, but no significant differences were observed between models. We anticipate that these animal-free in vitro models can facilitate research on skin regeneration and can be used to test therapeutic interventions in a preclinical setting to improve wound healing.

Hintergrund: Arthrose ist eine chronische und degenerative Gelenkerkrankung, die sich durch Degeneration des Knorpels auszeichnet. Während meiner Arbeit wollte ich einen neuen Ansatz zur Modellierung von Knorpel und auf die Reaktion von Medikamenten entwickeln. Das Differenzierungsprotokoll musste dafür optimiert werden, ebenso wie der 3D Organ-on-a-Chip-Ansatz, um weitere Erkenntnisse über Osteoarthritis und individuelle Unterschiede zu gewinnen. Relevanz: Obwohl die Inzidenz von Arthrose sehr hoch ist, ist die Wirksamkeit von Arthrosebehandlungen derzeit gering. Wir haben den Organ-on-a-Chip-Ansatz genutzt, um weitere Erkenntnisse über Arthrose zu gewinnen und Medikamente speziell für Patient*innen zu testen, um das Potential der personalisierten Medizin bei der Behandlung von Arthrose besser zu verstehen. Ziel: Ziel der Arbeit war es, herauszufinden, welche verschiedenen Quellen (Hoffa-Fettkörper und abdominelles Fettgewebe) mesenchymaler Stammzellen, die durch Genexpression mittels qPCR und Morphologie charakterisiert werden können, erforscht werden können, um das robusteste Modell zur Differenzierung in Knorpelzellen zu definieren. Die Arbeit zielte darauf ab, die Unterschiede in der Expression von CD-Markern, Biomarkern und morphologischen Profil von Knorpelzellen im Vergleich zu in vitro differenzierten mesenchymalen Stammzellen darzustellen. Es wurde untersucht, wie sich die Genexpression nach einer entzündlichen Stimulation und mit Medikamenten verändert. Methoden: Nach dreiwöchiger Differenzierung in die chondrogene Richtung wurden die mesenchymalen Stammzellen zehn Tage lang in einen Chip eingesetzt, und mit verschiedenen Medikamenten in zwei Chips pro Patient*in behandelt. Nach der Entnahme der Zellen wurde mRNA isoliert, cDNA generiert und eine qPCR-Analyse durchgeführt. Ergebnisse: Die Ergebnisse zeigen, dass es Unterschiede zwischen den mesenchymalen Stammzellen aus abdominellem Fettgewebe und dem Hoffa-Fettkörper in Bezug auf Morphologie und der Genexpression gibt und dass diese Zellen geeignet sind, sich in chondrogener Richtung zu differenzieren. Betrachtet man die Morphologie der Zellen, so wurden Unterschiede zwischen 2D und 3D festgestellt. Die CD-Marker wurden unterschiedlich exprimiert. Die Biomarker wurden in regenerative, degenerative und Zytokine + Wachstumsfaktoren kategorisiert, und die biochemische Entzündung zeigte, dass die degenerativen Marker und Zytokine im Gegensatz zur nicht entzündeten Umgebung hochreguliert waren. Diskussion: Die in dieser Arbeit vorgestellten Daten legen nahe, dass mesenchymale Stammzellen aus Fettgewebe und Hoffa-Fettpolster die Fähigkeit haben, in vitro eine Chondrogenese zu induzieren und durch die hinzugefügte biochemische Entzündung die Pathologie der Osteoarthritis zu imitieren. Diese Ergebnisse unterstützen das Konzept, dass mesenchymale Stammzellen aus abdominellem Fettgewebe und Hoffa-Fettkörper in Kombination mit dem Organ-on-a-Chip-Ansatz die Knorpelbildung induzieren können. Sie können möglicherweise in Verfahren ohne erforderliche Knorpelbiopsie eingesetzt werden und uns dadurch Einblicke in die individuelle genetische Prädisposition von Osteoarthritis geben. Background: Osteoarthritis, typified by degeneration of joint tissue, is a chronic and degenerative joint disease. During my thesis I aimed to generate a new approach to model cartilage and its response to medications. Therefore, a differentiation protocol of mesenchymal stem cells (MSC) into cartilage cells had to be optimized as well as the 3D organ-on-a-chip approach to gain further knowledge of osteoarthritis and individual differences. Relevance: Even though osteoarthritis has an enormous incidence, right now treatments of osteoarthritis display low efficacy. We have used the organ-on-a-chip-approach to gain further knowledge of osteoarthritis and to test medications specifically for patient to gain a better understanding of the potential of personalized medicine in treating with osteoarthritis. Aim: The aim of this thesis was to find out what different sources (Hoffa Fat Pad and Adipose tissue) of MSCs, which can be characterized by gene expression using qPCR and morphology, can be explored to define the most robust model to differentiate into cartilage cells. The thesis aimed to compare the differences in CD markers, candidate gene expression and morphologic profiling between cartilage cells in comparison to in vitro chondrogenic differentiated MSCs. In addition, it was evaluated how gene expression changed after an inflammatory challenge with medications compared to untreated chondrocytes. Methods: After three weeks of differentiation towards chondrogenic direction, MSCs were loaded in an organ-on-a-chip- device for ten days. The cells were treated with different medications in two chips per patient. After the cells were harvested off the chip mRNA isolation, cDNA generation and qPCR analysis was performed. Results: The results show differences between the Adipose and Hoffa Fat Pad MSCs in morphology and gene expression status and that Adipose and Hoffa Fat Pad MSCs are both suitable cells to differentiate into the chondrogenic direction. Looking at the morphology of the cells, differences were seen between 2D vs. 3D. The CD markers were differently expressed when comparing between the different cell types. The biomarkers were categorized into regenerative, degenerative and cytokines + growth factors and the biochemical inflammation showed that the degenerative markers and cytokines were upregulated in contrast to the non-inflamed environment. Discussion: The data presented in this thesis suggests that MSCs from Adipose and Hoffa Fat Pad MSCs could have the ability to induce chondrogenesis in vitro and mimic through the added biochemically inflammation the pathology of osteoarthritis. These findings support the concept that the Adipose and Hoffa Fat Pad MSCs, when combined with the organ-an-on-a-chip-approach, can potentially be used in procedures without required cartilage biopsy and give us insights into individual genetic predisposition for osteoarthritis.

PurposeThis 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.MethodshAFCs from spinal trauma patients were stimulated with TNF-alpha or IL-1 beta. 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 mu M) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model.ResultsIL-1ss stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 mu M cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-alpha and IL-1 beta 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.ConclusionCxb can inhibit PGE-2 production in hAFCs in an IL-1 beta-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., European Spine Journal, 32 (6), ISSN:0940-6719, ISSN:1432-0932, ISSN:001991155

PURPOSE: The purpose of this study was to compare 3 foam dressings to (1) determine the biomechanical performance of existing negative pressure wound therapy (NPWT) foams and (2) to determine if a test foam is possibly suitable as an antimicrobial “white” foam alternative for use in NPWT. DESIGN: A comparison of mechanical performance of 3 foams used for vacuum-assisted NPWT. SUBJECTS AND SETTING: Preclinical laboratory study using an in vitro model. METHODS: The performance of a “white” foam (polyvinyl alcohol [PVA]), an antimicrobial “black” foam (polyurethane [PU]), and an antimicrobial white foam alternative (test PVA) were tested and compared using 3 mechanically relevant criteria. First, the fluid removal rate was measured for 72 hours. Next, the pressure input was compared to the pressure directly beneath the center of the foam. Finally, the spread of negative pressure beneath the foam was measured and compared. RESULTS: Significant differences were found in fluid removal rates; specifically, the PU foam removed fluids faster than the PVA and test PVA foams, and the currently available PVA foams performed similarly. Both the PU and test PVA foams were able to transmit the negative pressure through the center of the dressing, while the typical PVA foam began failing at 140 mm Hg, with 50% of the samples failing at 200 mm Hg. All PU replicate foams evenly distributed the pressure, while 47% to 60% of the test PVA foams and 7% of the typical PVA foams distributed pressures evenly. CONCLUSIONS: Study findings suggest that the test PVA foam does not mechanically interfere with NPWT and performs equivalently to currently used foams. These results suggest that the test PVA may be modified and incorporated into a vacuum-assisted NPWT device. In addition, the methods employed in these experiments provide a reproducible means to compare biomechanical compatibility of various NPWT foams, dressings, and subdrape devices.

Animal testing is often criticized due to ethical issues and complicated translation of the results obtained to the clinical stage of drug development. Existing alternative models for nanopharmaceutical testing still have many limitations and do not significantly decrease the number of animals used. We propose a simple, bioinspired in vitro model for nanopharmaceutical drug testing based on the decellularized spinach leaf's vasculature. This system is similar to human arterioles and capillaries in terms of diameter (300-10 μm) and branching. The model has proven its suitability to access the maneuverability of magnetic nanoparticles, particularly those composed of Fe3O4. Moreover, the thrombosis has been recreated in the model's vasculature. We have tested and compared the effects of both a single-chain urokinase plasminogen activator (scuPA) and a magnetically controlled nanocomposite prepared by heparin-mediated cross-linking of scuPA with Fe3O4 nanoparticles. Compositions were tested both in static and flow conditions.

Fibrinolysis can be abnormally activated in several critical care settings but it's often misdiagnosed by standard laboratory tests. Although rotational thromboelastometry can assess the whole coagulative process, its ability to detect fibrinolysis has been questioned. Aim of this study was to investigate the ability of thromboelastometry in detecting induced fibrinolysis in an in-vitro model. Whole blood samples were taken from 18 healthy volunteers. Each sample was split and added with increasing urokinase concentrations till to reach 0, 50, 75 and 100 IU/ml. Thromboelastometry tests, extem and aptem, were performed on the obtained samples. If significant lysis at 50 IU/ml was recorded, also 10, 25 and 35 IU/ml drug concentrations were tested. No lytic effects were detected in 10 IU/ml samples. Lysis variables were the most sensitive in detecting fibrinolysis even at 25 IU/ml (P

European Union's Horizon 2020 research and innovation programme H2020 “NEWDEAL” (grant agreement No. 720905)

Ischemic Stroke is a multifaceted occurrence, caused by the blockage of blood flow/oxygen into the brain, triggering cell damage and neuronal death. Stroke remains one of the major causes of death and adult disability worldwide, whereas acute ischemic stroke (AIS) represents 71% of stroke events. Time is the most important factor for the outcome of these stroke patients: the quicker the restoration of the blood flow, the better the outcomes. However, despite the increase in survival rates, neurological outcomes must be improved, especially in terms of independence/motor function. The combination of reperfusion and brain protective medicine is the future of stroke therapy. Hence, research has been focusing on the study of possible cytoprotective molecules that can reduce brain damage and/or reverse apoptosis in the brain. Despite the huge number of drugs studied, very few reached clinical practice yet. Thus, the establishment of more accurate in vitro preclinical models is essential for the success of future therapies. Brain spheroids, which are a recent development, represent a very valuable asset within this research field. In the present work, we aim to optimize a combination of brain spheroids with oxygen and glucose deprivation (OGD) as an in vitro model for stroke. Through morphological, viability and molecular tests, we show that this model recapitulates the human brain during ischemic stroke. Moreover, we tested three drugs considered protective (BDNF, TTR and MK801) using this model. Then, we established a practical, straightforward method to evaluate the drug effects on the spheroids after OGD, using only morphological parameters and fluorescent probes. Lastly, immunostaining optimization was performed using control and OGD spheroids. Overall, this method could represent an innovative technique for high throughput screening (HTS) assays. The next step would be to apply the model hereby described in the screening of new promising compounds, and understand how different cell populations can be affected; however, further optimizations are still needed. All in all, we contributed with an innovative tool, with which we can screen new compounds or repurpose approved ones, in larger screening assays. Acidentes Vasculares Cerebrais (AVCs) são ocorrências multifacetadas, causadas pelo bloqueio da corrente sanguínea/oxigénio para o cérebro, instigando dano celular e morte neuronal. Os AVCs continuam a ser uma das principais causas de morte e de incapacidade no mundo. Os AVCs isquémicos representam 71% dos eventos. O tempo é o principal fator determinante do desenlace do evento: quanto mais rapidamente for restaurado o fluxo sanguíneo, melhor o resultado para o paciente. Porém, apesar do aumento nas taxas de sobrevivência, o resultado neurológico deve ser melhorado, especialmente no que toca à independência/função motora. A combinação entre reperfusão e fármacos que protejam o cérebro será o próximo desenvolvimento na terapia dos AVCs. Assim, a investigação tem-se focado no estudo de possíveis moléculas citoprotetoras que reduzam o dano cerebral e/ou revertam a apoptose no cérebro. Apesar do grande número de moléculas estudadas, muito poucas são já utilizadas na medicina. O estabelecimento de modelos in vitro mais precisos é essencial para o sucesso de terapias futuras. Os esferóides cerebrais representam um desenvolvimento recente, constituindo um recurso valioso para esta investigação. Neste trabalho, pretendemos otimizar uma combinação entre esferóides cerebrais e privação de oxigénio e glucose (PGO), como modelo de AVC in vitro. Através de testes morfológicos, de viabilidade e moleculares, demonstrámos que este é um modelo que recapitula o que ocorre no cérebro humano durante o AVC. Para além disso, testámos três moléculas consideradas protectoras (BDNF, TTR e MK801) utilizando este modelo. Depois, estabelecemos um método prático e simples para avaliar o efeito destes fármacos nos esferóides após PGO, utilizando apenas parâmetros morfológicos e sondas fluorescentes. Por último, foi feita uma otimização do processo de imunocoloração, utilizando esferóides de controlo e PGO. Este método poderá ser uma técnica inovadora para rastreio de fármacos em larga escala. No futuro, após otimizações adicionais, seria importante estender a utilização do modelo aqui desenvolvido à testagem de outros compostos promissores, e entender como as diferentes populações celulares são afetadas. Em suma, com este trabalho, contribuímos com uma ferramenta inovadora, com a qual podemos fazer triagens de larga escala de novos compostos, ou repropor alguns já testados. Mestrado em Bioquímica

Low-dose methotrexate (MTX) is a standard therapy for rheumatoid arthritis due to its low cost and efficacy. Despite these benefits, MTX has been reported to cause chronic drug-induced liver injury, namely liver fibrosis. The hallmark of liver fibrosis is excessive scarring of liver tissue, triggered by hepatocellular injury and subsequent activation of hepatic stellate cells (HSCs). However, little is known about the precise mechanisms through which MTX causes hepatocellular damage and activates HSCs. Here, we investigated the mechanisms leading to hepatocyte injury in HepaRG and used immortalized stellate cells (hTERT-HSC) to elucidate the mechanisms leading to HSC activation by exposing mono- and co-cultures of HepaRG and hTERT-HSC to MTX. The results showed that at least two mechanisms are involved in MTX-induced toxicity in HepaRG: (i) oxidative stress through depletion of glutathione (GSH) and (ii) impairment of cellular respiration in a GSH-independent manner. Furthermore, we measured increased levels of endoplasmic reticulum (ER) stress in activated HSC following MTX treatment. In conclusion, we established a human-relevant in vitro model to gain mechanistical insights into MTX-induced hepatotoxicity, linked oxidative stress in HepaRG to a GSH-dependent and -independent pathway, and hypothesize that not only oxidative stress in hepatocytes but also ER stress in HSCs contribute to MTX-induced activation of HSCs.

Mezenhimski hondrosarkom (MCS) je agresiven podtip hondrosarkoma, ki prizadene predvsem otroke in mladostnike. Pred odkritjem genske fuzije HEY1-NCoA2 v večini MCS, je njegova diagnoza predstavljala izziv zaradi histopatoloških podobnosti s številnimi drugimi primitivnimi sarkomi, ki se predstavljajo z morfologijo majhnih modrih in okroglih malignih celic. S trenutnimi pristopi zdravljenja, je bila mediana preživetja brez napredovanja bolezni ocenjena na 57 mesecev. Dodatni lastnosti MCS sta tudi visoka zunajskeletna pojavnost, in visoka stopnja poznih ponovitev. Kirurška resekcija MCS pa je pokazala izboljšano stopnjo preživetja kot zdravljenje s konvencionalno kemoterapijo. V tej študiji smo odkrili potencialno terapevtsko sredstvo za zdravljenju mezenhimskega hondrosarkoma (MCS), ki ga poganja genska fuzija HEY-NCoA2 in je že na voljo v kliniki, imatinib mezilat. Pokazali smo potencial kombiniranja bioloških in genomskih funkcionalnih asociacijskih omrežij za boljše prepoznavanje nadomestnih tarč, saj se trenutno genske fuzije HEY1-NCoA2 še ne more direktno ciljati. Prikazujemo tudi utemeljitev strategije ponovne uporabe licenčnih zdravil za zdravljenje MCS, potek dela te študije pa bi lahko uporabili za študije drugih redkih rakov, ki jih poganja le ena genska fuzija. Takšna strategija tudi podpira trenutna prizadevanja iskanj boljših načinov zdravljenja redkih rakov, ki večinoma nimajo predkliničnih modelov, so zato premalo raziskani in za katere je razvoj novih zdravil nepraktičen. Poleg tega pa lahko tudi odkrije potencialne rezistence na trenutno uporabljenje kemoterapije, za zadravljenje tovrstnih rakov. Navsezadnje, rezultati našega dela predstavljajo podlago za ponovno klinično oceno učinkovitosti imatiniba za zdravljenje MCS, ki ga poganja HEY1-NCoA2 genska fuzija. Mesenchymal chondrosarcoma (MCS) is an aggressive subtype of chondrosarcoma, mainly affecting children and young adults. Before the discovery of a recurrent HEY1-NCoA2 gene fusion in most MCS patient samples, MCS diagnosis presented a challenge due to histopathological similarities to many other small blue round cell neoplasms of primitive sarcomas. With current treatments, the median event-free survival is 57 months. MCS characteristics are high extraskeletal occurrence and a late recurrence rate. Surgical resection shows improved survival rates compared to treatment with conventional chemotherapy. This study identified a potential therapeutic agent to treat HEY-NCoA2 gene fusion-driven mesenchymal chondrosarcoma (MCS), which is already available in the clinic, imatinib mesylate. Our findings highlighted the potential of combining biological and genome-wide functional association network frameworks to facilitate the identification of proxy druggable targets of MCS since HEY1-NCoA2 gene fusion cannot yet be directly targeted. Therefore we show the rationale to utilize the repurposing of licensed drugs strategy for the treatment of MCS and the workflow of this study could be applied to other rare single fusion-driven tumors. This strategy also supports current efforts for reinvigorating therapy development in underresearched rare cancers, without preclinical models and in which de novo drug development is, in the majority of cases, impractical. Additionally, it can also identify the potential resistance of these tumors to currently used chemotherapy to treat them. Lastly, this work rationally justifies a re-evaluation of licensed multikinase inhibitor imatinib to treat HEY1-NCoA2 fusion-driven MCS in a clinical trial.

The formation of pharmacobezoars from suspensions of spray-dried amorphous solid dispersions (SD-ASDs) of new chemical entities (NCEs) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) represents a non-compound related adverse effect in preclinical oral toxicity studies in rodents. Whereas the contribution of the insolubility of the carrier polymer to this process taking place in the acidic environment of the rodent stomach is conclusive, unawareness of the extent of in vivo pharmacobezoar formation is adverse. In order to evaluate the risk of pharmacobezoar formation before in vivo administration, we subsequently introduce an in vitro model to assess the agglomeration potential of solid dispersions. To verify that the pharmacobezoar formation potential can be assessed based on the observed agglomeration potential, we conducted a sequence of experiments with two HPMC-AS-based SD-ASD formulations. In vitro, we found their different in vivo pharmacobezoar formation potential reflected by a significantly increased agglomerated mass of formulation 1 per day compared to formulation 2. In order to find an approach to reduce the agglomeration potential of solid dispersion from suspensions, we further applied the model to investigate the impact of the viscosity of the vehicle used to prepare suspensions on agglomerate formation.

Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 106-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four passages. Infected cells displayed typical features of equine sarcoid cells, including hyperproliferation, and loss of contact inhibition. Neither multiple passaging nor storage negatively affected cell hyperproliferation, viral DNA replication, and gene transcription, suggestive for infection-mediated cell immortalization. Intriguingly, extracellular vesicles released by BPV1-infected fibroblasts contained viral DNA that was most abundant in the fractions enriched for apoptotic bodies and exosomes. This viral DNA is likely taken up by non-infected fibroblasts. We conclude that equine primary fibroblasts stably infected with BPV1 and BPV2 virions constitute a valuable near-natural model for the study of yet unexplored mechanisms underlying the pathobiology of BPV1/2-induced sarcoids.

Diabetic neuropathy is a well-known complication of diabetes. It has been recently confirmed that hyperglycemia-induced toxicity participates in multiple cellular pathways that are typical for neural deterioration. Nampt/PBEF/visfatin is a novel endogenous ligand, which some studies have shown its neuroprotective effects on neurodegenerative disease. Therefore, we hypothesized that visfatin might prevent high glucose (HG)-induced neurotoxicity via the inhibition of apoptosis, autophagy, and reactive oxygen species (ROS) responses properly. In this study, Pheochromocytoma Cell Line 12 (PC12) cells were exposed to both HG concentrations (50, 75, 100,125, 150 mM) and visfatin (50, 100, 150 ng/ml) in different time-points to determine the optimum time and dose of glucose and visfatin. To investigate the effects of visfatin on HG-induced damage in PC12 diabetic neuropathy model, we examined ROS response, apoptosis, and autophagy by using ROS detection kit, flow cytometry, and Real-time PCR/western blot, respectively. We determined that HG concentration significantly increased ROS level and apoptosis of diabetic PC12 cells. However, visfatin treatment significantly decreased ROS production (P < 0.05) and apoptosis of diabetic PC12 cells (P < 0.0001). Beclin-1 mRNA level (P < 0.05) and Lc3-II protein level (P < 0.05) showed that autophagy pathway is impaired by HG concentrations. We concluded that visfatin could sufficiently decrease neural damage caused by ROS production and apoptosis under HG-induced toxicity.

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.

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inflammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p

Heart failure is a major health risk, and with limited availability of donor organs, there is an increasing need for developing cardiac assist devices (CADs). Mock circulatory loops (MCL) are an important in-vitro test platform for CAD’s performance assessment and optimisation. The MCL is a lumped parameter model constructed out of hydraulic and mechanical components aiming to simulate the native cardiovascular system (CVS) as closely as possible. Further development merged MCLs and numerical circulatory models to improve flexibility and accuracy of the system; commonly known as hybrid MCLs. A total of 128 MCLs were identified in a literature research until 25 September 2020. It was found that the complexity of the MCLs rose over the years, recent MCLs are not only capable of mimicking the healthy and pathological conditions, but also implemented cerebral, renal and coronary circulations and autoregulatory responses. Moreover, the development of anatomical models made flow visualisation studies possible. Mechanical MCLs showed excellent controllability and repeatability, however, often the CVS was overly simplified or lacked autoregulatory responses. In numerical MCLs the CVS is represented with a higher order of lumped parameters compared to mechanical test rigs, however, complex physiological aspects are often simplified. In hybrid MCLs complex physiological aspects are implemented in the hydraulic part of the system, whilst the numerical model represents parts of the CVS that are too difficult to represent by mechanical components per se. This review aims to describe the advances, limitations and future directions of the three types of MCLs.

The neural circuits of the central nervous system are the regulatory pathways for feeling, motion control, learning, and memory, and their dysfunction is closely related to various neurodegenerative diseases. Despite the growing demand for the unraveling of the physiology and functional connectivity of the neural circuits, their fundamental investigation is hampered because of the inability to access the components of neural circuits and the complex microenvironment. As an alternative approach, in vitro human neural circuits show principles of in vivo human neuronal circuit function. They allow access to the cellular compartment and permit real-time monitoring of neural circuits. In this review, we summarize recent advances in reconstituted in vitro neural circuits using engineering techniques. To this end, we provide an overview of the fabrication techniques and methods for stimulation and measurement of in vitro neural circuits. Subsequently, representative examples of in vitro neural circuits are reviewed with a particular focus on the recapitulation of structures and functions observed in vivo, and we summarize their application in the study of various brain diseases. We believe that the in vitro neural circuits can help neuroscience and the neuropharmacology. Statement of significance Despite the growing demand to unravel the physiology and functional connectivity of the neural circuits, the studies on the in vivo neural circuits are frequently limited due to the poor accessibility. Furthermore, single neuron-based analysis has an inherent limitation in that it does not reflect the full spectrum of the neural circuit physiology. As an alternative approach, in vitro engineered neural circuit models have arisen because they can recapitulate the structural and functional characteristics of in vivo neural circuits. These in vitro neural circuits allow the mimicking of dysregulation of the neural circuits, including neurodegenerative diseases and traumatic brain injury. Emerging in vitro engineered neural circuits will provide a better understanding of the (patho-)physiology of neural circuits.

Recent research on Gut-Brain Axis (GBA) has suggested that the gut luminal environment, including the dietary components and commensal microbiota, could affect behavior, emotion, and cognitive abilities in the brain. Various signaling pathways exist between the gut and the brain, and several studies have implied exosomes could mediate the communication. Current research on GBA has relied heavily on animal models, making the research challenging. Recent advances in organ-on-a-chip technology could be a solution for GBA research. In present work, we have developed a modular microfluidic chip, where gut epithelial and brain endothelial cells were co-cultured to form the gut epithelial barrier and the Blood-Brain Barrier (BBB) interconnected via microfluidic channels. These modules can be easily assembled and disassembled, and cell barriers were well formed when co-cultured under appropriate fluidic flow. Responses to microbial byproducts were consistent with previously known observations of interaction between gut epithelium and BBB. We observed the transport of fluorescently labeled exosomes across the gut barrier towards the BBB. Our results suggest this model can be used as a novel in vitro model of GBA for studying the interaction between the gut and the brain.

Obesity is associated with the development of liver disease and its progression to hepatocellular carcinoma. This link may be attributed to adipocytokines such as visfatin and resistin which have been shown to promote liver cancer incidence and progression. Studies have yet to determine the role of visfatin and resistin in liver cancer, specifically in the context of obesity. The objective of this study was to investigate the effect of neutralizing visfatin and resistin in obese (OB) or normal weight (NW) sera to determine the contribution of these proteins in obesity-induced invasive liver cancer. Sera from OB or NW males was used to determine the efficacy of neutralizing visfatin and resistin to reduce the obesity-induced liver cancer phenotype. HepG2 and SNU-449 cells were exposed to OB and NW sera ± antibodies for visfatin or resistin. The neutralizing antibodies differentially suppressed invasion, reactive oxygen species production, and matrix metalloproteinase-9 secretion. These changes corresponded with a decrease in phosphorylated extracellular signal-regulated kinases and protein kinase B in HepG2 cells, but differences were not observed in CAP1 or β-catenin. In conclusion, visfatin and resistin have differential roles in obesity-associated liver cancer and may be potential targets to reverse the impact of obesity on liver cancer progression.

Drug-induced liver toxicity remains a major cause of drug withdrawal from animal testing and human clinical trials. A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. Previously reported fluid-based systems have some advantages for hepatocyte culture, but have insufficient liver-specific functions because they simply involve moving conventional hepatocyte cultures from a dish into a fluid-based system. Importantly, these cultures have no liver tissue-specific structures that construct liver-specific cellular polarities, such as apical, basolateral, and basal faces. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions. Impact statement A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions.

SPIONs are composed of Fe3O4 or gamma Fe2O3 cores and a biocompatible shell from Dextran, PLA, PEG, Chitosan, or PVA. SPIONs have many important applications in medicine and biology such as cell sorting, drug carrier, magnetic hyperthermia, and magnetic resonance imaging (MRI). This study aims to check the ability of copolymer PLA-PEG coated Fe3O4 ferromagnetic nanosystems (Fe3O4@PLA-PEG) produced for MRI application. The results showed that these nanoparticles had non-toxicity on BT-474 and Sarcoma 180 cell lines. Evaluation on T2 imaging mode revealed that Fe3O4@PLA-PEG nanoparticles were capable of enhancing the MRI image contrast in different conditions, including water, cell lysates solution, and even inside the cells. The report demonstrates that Fe3O4@PLA-PEG nanoparticles have a high potential application in MRI for clinical diagnosis.

Purpose The purpose of this study is to test the concept of a relatively low cost but biocompatible customized surgical guide printing method using a new composite material for the FDM process to support accurate virtual model reconstruction in CT. Design/methodology/approach Current additive manufacturing printed surgical guides have problems of scanning artifacts or low computed tomography (CT) values for virtual model reconstruction in CT-assisted surgical operations. These tools always face difficulties in precise positioning due to the effect of human soft tissues and manually made unstable landmarks. To solve this problem, this paper proposes a modified material, polyetheretherketone powder mixed with barium sulfate powder, for printing customized surgical guides with relatively low cost to support a synchronized scanning strategy, for the accurate reconstruction of human tissues and in vitro models. Findings A set of benchmarking experiments and clinical simulation cases were conducted. The results showed that the proposed solution can be used to print surgical guides to form stable and clear CT graphs for three-dimensional digital model reconstruction. Human tissues and in vitro models can be accurately reconstructed using clear CT graphs without any scanning artifacts or difficulties in image segmentation for virtual model reconstruction, thus facilitating accurate operation guidance and positioning. Originality/value This method has wide application potential for printing modular or customized surgical guides with low cost and reusability, especially for surgical operations using CT-assisted navigation systems in underdeveloped regions where medical device costs are a critical issue.

The in vitro release of theophylline from an extended-release dosage form was studied under different hydrodynamic conditions in a United States Pharmacopoeial (USP) dissolution system II and a bespoke in vitro tubular model of the human colon, the Dynamic Colon Model (DCM). Five biorelevant motility patterns extracted from in vivo data were applied to the DCM, mimicking the human proximal colon under baseline conditions and following stimulation using polyethylene glycol or maltose; these represent the lower and upper bounds of motility normally expected in vivo. In the USPII, tablet dissolution was affected by changing hydrodynamic conditions at different agitation speeds of 25, 50 and 100 rpm. Applying different motility patterns in the DCM affected the dissolution profiles produced, with theophylline release at 24 h ranging from 56.74 ± 2.00% (baseline) to 96.74 ± 9.63% (maltose-stimulated). The concentration profiles of theophylline were markedly localized when measured at different segments of the DCM tube, highlighting the importance of a segmented lumen in intestine models and in generating spatial information to support simple temporal dissolution profiles. The results suggested that the shear stresses invoked by the unstimulated, healthy adult human colon may be lower than those in the USPII at 25 rpm and thus insufficient to achieve total release of a therapeutic compound from a hydroxyethyl cellulose matrix. When operated under stimulated conditions, drug release in the DCM was between that achieved at 25 and 50 rpm in the USPII.

Many diseases of the central nervous system are age-associated and do not directly result from genetic mutations. These include late-onset neurodegenerative diseases (NDDs), which represent a challenge for biomedical research and drug development due to the impossibility to access to viable human brain specimens. Advancements in reprogramming technologies have allowed to obtain neurons from induced pluripotent stem cells (iPSCs) or directly from somatic cells (iNs), leading to the generation of better models to understand the molecular mechanisms and design of new drugs. Nevertheless, iPSC technology faces some limitations due to reprogramming-associated cellular rejuvenation which resets the aging hallmarks of donor cells. Given the prominent role of aging for the development and manifestation of late-onset NDDs, this suggests that this approach is not the most suitable to accurately model age-related diseases. Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows the possibility to generate patient-derived neurons that maintain aging and epigenetic signatures of the donor. This aspect may be advantageous for investigating the role of aging in neurodegeneration and for finely dissecting underlying pathological mechanisms. Here, we will compare iPSC and iN models as regards the aging status and explore how this difference is reported to affect the phenotype of NDD in vitro models.