Novel in vitro evidence on the beneficial effect of quercetin treatment in vascular calcification.

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

Innovative explorations: unveiling the potential of organoids for investigating environmental pollutant exposure.

As the economy rapidly develops, chemicals are widely produced and used. This has exacerbated the problems associated with environmental pollution, raising the need for efficient toxicological evaluation techniques to investigate the toxic effects and mechanisms of toxicity of environmental pollutants. The progress in the techniques of cell culture in three dimensions has resulted in the creation of models that are more relevant in terms of biology and physiology. This enables researchers to study organ development, toxicology, and drug screening. Adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs) can be obtained from various mammalian tissues, including cancerous and healthy tissues. Such stem cells exhibit a significant level of tissue memory and ability to self-assemble. When cultivated in 3D in vitro environments, the resulting organoids demonstrate a remarkable capacity to recapitulate the cellular composition and function of organs in vivo. Recently, many tumors' tissue-derived organoids have been widely used in research on tumor pathogenesis, drug development, precision medicine, and other fields, including those derived from colon cancer, cholangiocarcinoma, liver cancer, and gastric cancer. However, the application of organoid models for evaluating the toxicity of environmental pollutants is still in its infancy. This review introduces the characteristics of the toxicity responses of organoid models upon exposure to pollutants from the perspectives of organoid characteristics, tissue types, and their applications in toxicology; discusses the feasibility of using organoid models in evaluating the toxicity of pollutants; and provides a reference for future toxicological studies on environmental pollutants based on organoid models.

In vitroanalysis of insoluble salt formation mechanism associated with Mg corrosion-variations depending on the diffusion environment in model tissue.

Magnesium (Mg) alloys have attracted attention as biodegradable metals, but the details of their corrosion behavior under biological environment have not been elucidated. Previous studies have suggested that diffusion through blood flow may influence Mg corrosion. Therefore, to understand the degradation behaviors of Mg, we analyzed insoluble salt precipitation associated with Mg corrosion in model tissue with different diffusion rates. A pure Mg specimen was immersed into a model tissue prepared with cell culture medium supplemented by a thickener at a different concentration (0.2%-0.5%) to form the gel. Micro-focus x-ray computed tomography of the gel was performed to observe gas cavity formation around the specimen. The insoluble salt layer formed on the specimen surface were analyzed by scanning electron microscopy with energy-dispersive x-ray spectroscopy, and Raman spectroscopy. As results, gas cavity formation was observed for all specimens. At day 7, the gas cavity volume was the highest at 0.5% thickener gel followed by 0.3% thickener gel. The insoluble salts were classified into three types based on their morphology; plate-like, granular-like, and crater-like salts. The crater-like salts were observed to cover 16.8 ± 3.9% of the specimen surface immersed in the 0.5% thickener gel, at the specimen area contacted to the gas cavity. The crater-like salts were composed by Mg hydroxide and carbonate from the deepest to the top layer. In plate-like or granular-like salts, Mg carbonate was formed in the deepest layer, but phosphates and carbonates, mainly containing calcium not Mg, were formed on the surface layer. In conclusion, the increase in the thickener concentration increased the gas cavity volume contacting to the specimen surface, resulting in the increase in precipitation of Mg hydroxide and carbonate, composing crater-like salts. Mg hydroxide and carbonate precipitation suggests the local increase in OH-concentration, which may be attributed to the decrease in diffusion rate.

Novel Correlation between TGF-ß1/-ß3 and Hormone Receptors in the Human Corneal Stroma.

This study investigated the interplay between transforming growth factor beta (TGF-ß1/T1 and TGF-ß3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 106 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERß), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERß, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERß, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-ß isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.

The frontline of alternatives to animal testing: novel in vitro skin model application in drug development and evaluation.

The FDA Modernization Act 2.0 has brought nonclinical drug evaluation into a new era. In vitro models are widely used and play an important role in modern drug development and evaluation, including early candidate drug screening and preclinical drug efficacy and toxicity assessment. Driven by regulatory steering and facilitated by well-defined physiology, novel in vitro skin models are emerging rapidly, becoming the most advanced area in alternative testing research. The revolutionary technologies bring us many in vitro skin models, either laboratory-developed or commercially available, which were all built to emulate the structure of the natural skin to recapitulate the skin's physiological function and particular skin pathology. During the model development, how to achieve balance among complexity, accessibility, capability, and cost-effectiveness remains the core challenge for researchers. This review attempts to introduce the existing in vitro skin models, align them on different dimensions, such as structural complexity, functional maturity, and screening throughput, and provide an update on their current application in various scenarios within the scope of chemical testing and drug development, including testing in genotoxicity, phototoxicity, skin sensitization, corrosion/irritation. Overall, the review will summarize a general strategy for in vitro skin model to enhance future model invention, application, and translation in drug development and evaluation.

Eicosapentaenoic acid reduces the proportion of IL-17A-producing T cells in a 3D psoriatic skin model.

Psoriasis is a skin disease presenting as erythematous lesions with accentuated proliferation of epidermal keratinocytes, infiltration of leukocytes, and dysregulated lipid metabolism. T cells play essential roles in the disease. n-3 polyunsaturated fatty acids are anti-inflammatory metabolites, which exert an immunosuppressive effect on healthy T cells. However, the precise mechanistic processes of n-3 polyunsaturated fatty acids on T cells in psoriasis are still unrevealed. In this study, we aimed to evaluate the action of eicosapentaenoic acid (EPA) on T cells in a psoriatic skin model produced with T cells. A coculture of psoriatic keratinocytes and polarized T cells was prepared using culture media, which was either supplemented with 10 µM EPA or left unsupplemented. Healthy and psoriatic skin substitutes were produced according to the self-assembly method. In the coculture model, EPA reduced the proportion of IL-17A-positive cells, while increasing that of FOXP3-positive cells, suggesting an increase in the polarization of regulatory T cells. In the 3D psoriatic skin model, EPA normalized the proliferation of psoriatic keratinocytes and diminished the levels of IL-17A. The expression of the proteins of the signal transducer and activator of transcription was influenced following EPA supplementation with downregulation of the phosphorylation levels of signal transducer and activator of transcription 3 in the dermis. Finally, the NFκB signaling pathway was modified in the EPA-supplemented substitutes with an increase in Fas amounts. Ultimately, our results suggest that in this psoriatic model, EPA exerts its anti-inflammatory action by decreasing the proportion of IL-17A-producing T cells.

Exploring the use of modified in vitro digestion assays for the evaluation of ritonavir loaded solid lipid-based formulations.

Solid lipid-based formulations (sLBFs) have the potential to increase the oral bioavailability of drugs with poor solubility in water, while counteracting some of the disadvantages of liquid LBFs. The most common experimental set-up to study the performance of LBFs in vitro is the lipolysis assay, during which the LBFs are digested by lipases in an environment mimicking the human small intestine. However, this assay has failed in many cases to correctly predict the performance of LBFs in vivo, highlighting the need for new and improved in vitro assays to evaluate LBFs at the preclinical stage. In this study, the suitability of three different in vitro digestion assays for the evaluation of sLBFs was assessed; the classic one-step intestinal digestion assay, a two-step gastrointestinal digestion assay and a bicompartmental assay permitting the simultaneous monitoring of digestion and permeation of the active pharmaceutical ingredient (API) across an artificial membrane (Lecithin in Dodecane - LiDo). Three sLBFs (M1-M3) with varied composition and ritonavir as model drug were prepared and examined. When comparing the ability of these formulations to keep the drug solubilized in the aqueous phase, all three assays show that M1 performs better, while M3 presents poor performance. However, the classic in vitro intestinal digestion assay fails to provide a clear ranking of the three formulations, something that is more evident when using the two modified and more physiologically relevant assays. Also, the two modified assays provide additional information about the performance of the formulations including the performance in the gastric environment and intestinal flux of the drug. These modified in vitro digestion assays are valuable tools for the development and evaluation of sLBFs to make better informed decisions of which formulations to pursue for in vivo studies.

Effect of antioxidant lycopene on human osteoblasts.

OBJECTIVE: The aim of this in vitro study is to evaluate the effect of antioxidant lycopene on human osteoblasts. MATERIAL AND METHOD: The human osteoblast cell line (CRL-11372) was obtained from the American Type Culture Collection (ATCC Manassas, Va) and grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 mg/ ml) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The effective dose of lycopene was determined by MTT assay and a real-time cell analysis (RTCA) system. Proliferative effects were analyzed by in vitro wound healing model. Gene expressions of type 1 collagen (COL1A1), osteocalcin (OCN), and growth differentiation factor-5 (GDF-5) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) at 72 h. Statistical differences between test groups were analyzed with a one-way ANOVA test. RESULTS: MTT assay showed that the doses between 10-5 and 1 µmol of lycopene had dose-dependent proliferative effects. The doses between 10-5 and 10-1 µmol were most effective at 72 h. Lycopene accelerates the healing rate by increasing osteoblast proliferation. CONCLUSION: Results suggested that lycopene had proliferative effects on human osteoblasts, which may help to increase bone regeneration, and thus, it can be useful in tissue engineering procedures. CLINICAL RELEVANCE: By the help of antioxidants like lycopene capacity, velocity and quality of new bone forming may be increased in periodontal and dental implant treatments.

An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues.

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid-containing engineered microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin, and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT, we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.

Recent progresses in novel in vitro models of primary neurons: A biomaterial perspective.

Central nervous system (CNS) diseases have been a growing threat to the health of humanity, emphasizing the urgent need of exploring the pathogenesis and therapeutic approaches of various CNS diseases. Primary neurons are directly obtained from animals or humans, which have wide applications including disease modeling, mechanism exploration and drug development. However, traditional two-dimensional (2D) monoculture cannot resemble the native microenvironment of CNS. With the increasing understanding of the complexity of the CNS and the remarkable development of novel biomaterials, in vitro models have experienced great innovation from 2D monoculture toward three-dimensional (3D) multicellular culture. The scope of this review includes the progress of various in vitro models of primary neurons in recent years to provide a holistic view of the modalities and applications of primary neuron models and how they have been connected with the revolution of biofabrication techniques. Special attention has been paid to the interaction between primary neurons and biomaterials. First, a brief introduction on the history of CNS modeling and primary neuron culture was conducted. Next, detailed progress in novel in vitro models were discussed ranging from 2D culture, ex vivo model, spheroid, scaffold-based model, 3D bioprinting model, and microfluidic chip. Modalities, applications, advantages, and limitations of the aforementioned models were described separately. Finally, we explored future prospects, providing new insights into how basic science research methodologies have advanced our understanding of the CNS, and highlighted some future directions of primary neuron culture in the next few decades.

Human Platelet Lysate as Alternative of Fetal Bovine Serum for Enhanced Human In Vitro Bone Resorption and Remodeling.

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.

Application of the C3 inhibitor compstatin in a human whole blood model designed for complement research - 20 years of experience and future perspectives.

The complex molecular and cellular biological systems that maintain host homeostasis undergo continuous crosstalk. Complement, a component of innate immunity, is one such system. Initially regarded as a system to protect the host from infection, complement has more recently been shown to have numerous other functions, including involvement in embryonic development, tissue modeling, and repair. Furthermore, the complement system plays a major role in the pathophysiology of many diseases. Through interactions with other plasma cascades, including hemostasis, complement activation leads to the broad host-protective response known as thromboinflammation. Most complement research has been limited to reductionistic models of purified components and cells and their interactions in vitro. However, to study the pathophysiology of complement-driven diseases, including the interaction between the complement system and other inflammatory systems, holistic models demonstrating only minimal interference with complement activity are needed. Here we describe two such models; whole blood anticoagulated with either the thrombin inhibitor lepirudin or the fibrin polymerization peptide blocker GPRP, both of which retain complement activity and preserve the ability of complement to be mutually reactive with other inflammatory systems. For instance, to examine the relative roles of C3 and C5 in complement activation, it is possible to compare the effects of the C3 inhibitor compstatin effects to those of inhibitors of C5 and C5aR1. We also discuss how complement is activated by both pathogen-associated molecular patterns, inducing infectious inflammation caused by organisms such as Gram-negative and Gram-positive bacteria, and by sterile damage-associated molecular patterns, including cholesterol crystals and artificial materials used in clinical medicine. When C3 is inhibited, it is important to determine the mechanism by which inflammation is attenuated, i.e., whether the attenuation derives directly from C3 activation products or via downstream activation of C5, since the mechanism involved may determine the appropriate choice of inhibitor under various conditions. With some exceptions, most inflammatory responses are dependent on C5 and C5aR1; one exception is venous air embolism, in which air bubbles enter the blood circulation and trigger a mainly C3-dependent thromboembolism, with the formation of an active C3 convertase, without a corresponding C5 activation. Under such conditions, an inhibitor of C3 is needed to attenuate the inflammation. Our holistic blood models will be useful for further studies of the inhibition of any complement target, not just C3 or C5. The focus here will be on targeting the critical complement component, activation product, or receptor that is important for the pathophysiology in a variety of disease conditions.

Carcinosinumproduces phenotypic changes of 4T1 tumor cells in vitro

Considering that there are few published studies that specifically address the exclusive use of Carcinosinumin different potencies and, most of them focused on genotypic and clinical effects, the present study was proposed to identify possible phenotypic changes, including viability, expression of HER-2 and metastatic abilities, using 4T1 cells in vitroas a model. Carcinosinum was tested in different homeopathic potencies (12cH; 30cH; 200cH) mechanically prepared using sterile pure water. The time space between preparing the potencies and using them was 24 hours.The final dilutions were inserted into the culture medium in a volume equal to 10%, at the time of cell seeding. The same succussed vehicle used to prepare the drugs (70% ethanol) diluted 1:100 in sterile pure water was used as control. All treated cells were cultured in 25 mL flasks, with cell density of 5 x 105cells/mL. After 24 hours of treatment, cells were analyzed for apoptosis index using Annexin V kit and the Countess® system. The morphology of 4T1 cells was monitored by staining cell smears with hematoxylin-eosin and Giemsa methods. HER-2 expression was assessed by immunocytochemistry and metalloproteinase activity was assessed by zymography. The determination of the cytokine profile was performed using Cytometric Bead Array (CBA). The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. Carcinosinum30cH presented the highest apoptotic index and reduction of MMP-9-Pro expression; Carcinosinum200cH produced the highest positivity for HER-2 and no specific effect was seen after the treatment with Carcinosinum12cH. No change in cytokine expression was seen among treatments. We conclude that Carcinosinum30cH and 200cH can change phenotypic features important totumor development in vitro. The clinical meaning of these data deserves further investigation.

What Do In Vitro and In Vivo Models Tell Us about Anisakiasis? New Tools Still to Be Explored.

Anisakiasis is a zoonosis caused by the ingestion of raw or undercooked seafood infected with third-stage larvae (L3) of the marine nematode Anisakis. Based on L3 localization in human accidental hosts, gastric, intestinal or ectopic (extra-gastrointestinal) anisakiasis can occur, in association with mild to severe symptoms of an allergic nature. Given the increasing consumption of fish worldwide, the European Food Safety Authority declared Anisakis as an emerging pathogen. Despite its importance for public health and economy, the scientific literature is largely characterized by taxonomic, systematic and ecological studies, while investigations on clinical aspects, such as the inflammatory and immune response during anisakiasis, using a proper model that simulates the niche of infection are still very scarce. The aims of this review are to describe the clinical features of anisakiasis, to report the main evidence from the in vivo and in vitro studies carried out to date, highlighting limitations, and to propose future perspectives in the study field of anisakiasis.

A Citrus Fruit Extract High in Polyphenols Beneficially Modulates the Gut Microbiota of Healthy Human Volunteers in a Validated In Vitro Model of the Colon.

The effect of a Citrus Fruit Extract high in the polyphenols hesperidin and naringin (CFE) on modulation of the composition and activity of the gut microbiota was tested in a validated, dynamic in vitro model of the colon (TIM-2). CFE was provided at two doses (250 and 350 mg/day) for 3 days. CFE led to a dose-dependent increase in Roseburia, Eubacterium ramulus, and Bacteroides eggerthii. There was a shift in production of short-chain fatty acids, where acetate production increased on CFE, while butyrate decreased. In overweight and obesity, acetate has been shown to increase fat oxidation when produced in the distal gut, and stimulate secretion of appetite-suppressive neuropeptides. Thus, the data in the in vitro model point towards mechanisms underlying the effects of the polyphenols in CFE with respect to modulation of the gut microbiota, both in composition and activity. These results should be confirmed in a clinical trial.

Metformin alters therapeutic effects in the BALB/c tumor therapy model.

BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.

Sesquiterpenoid bilobalide inhibits gastric carcinoma cell growth and induces apoptosis both in vitro and in vivo models.

Gastric carcinoma is one of the most aggressive types of cancer that ranks fifth among all cancer incidences and third in cancer mortality. As it exhibits a prolonged asymptomatic condition and high recurrence rate, it is a great challenge to treat gastric cancer. Traditional medicine that utilizes herbal phytochemicals to treat various diseases is a potent alternative for current allopathic treatment. Hence, we evaluated the potency of a phytochemical bilobalide for treating gastric cancer in in vitro and in vivo models. Bilobalide, a sesquiterpenoid, is present in the Ginkgo biloba plant that belongs to the family of Ginkgoaceae. The cytotoxicity effect of bilobalide was evaluated in both gastric cancer (AGS) cells and normal gastric epithelial cells. Apoptosis-inducing property of bilobalide against the AGS cell line was analyzed with different fluorescent staining techniques and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and cell cycle analysis was carried out by flow cytometry. The in vivo studies were assessed with N-methyl-N-nitrosourea (MNU)-induced gastric cancer in rats. Serum-specific gastric markers were quantified and histopathological analysis of stomach tissue was performed. The expression of target-signaling molecules was analyzed by a reverse-transcription polymerase chain reaction. The in vitro results proved that bilobalide effectively suppressed the AGS cell growth and induced cell death by nuclear damage and apoptosis induction. The bilobalide treatment effectively arrested the cell cycle of AGS cells via inhibiting the PI3K-signaling pathway. Our in vivo results also confirmed that the bilobalide persuasively inhibited the MNU-induced gastric carcinoma via inhibiting the thioredoxin-fold family proteins and inflammatory markers' expression. Overall, our results authentically prove that bilobalide possesses therapeutic potency to cure gastric carcinoma.

Limitations of an in vitro model of the poultry digestive tract on the evaluation of the catalytic performance of phytases.

BACKGROUND: The study aims to investigate the limitation of a poultry digestive tract model developed by Menezes-Blackburn et al. [J Agric Food Chem 63: 6142-6149 (2015)] on the evaluation of the bioefficacy of phytases. RESULTS: It was confirmed that the in vitro model does not mimic the in vivo situation in the birds sufficiently well to identify the best phytase product under real conditions, or to draw conclusion on the effect of phytate concentration, phytate source or feed composition on the bioefficacy of phytase. Addition of calcium ion (Ca2+ ) up to a concentration of 10 g kg-1 to the feed substrate, for example, did not affect enzymatic phytate dephosphorylation in the in vitro model in contrast to the observation in poultry. CONCLUSION: The in vitro approach was shown to be applicable as a complementary tool in the pre-selection of promising phytase candidates, resulting in a reduction in the number of feeding trials in the initial screening phase. © 2020 The Author. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Achieving delayed release of freeze-dried probiotic strains by extrusion, spheronization and fluid bed coating - evaluated using a three-step in vitro model.

Intake of probiotics is associated with many health benefits, which has generated an interest in formulating viable probiotic supplements. The present study had two aims. The first aim was to achieve gastrointestinal protection and delayed release of viable probiotics by pelletizing and coating freeze-dried probiotic strains, using riboflavin as a marker for release. The second aim was to set up a dynamic three-step in vitro model simulating the conditions in the human gastric, duodenum/jejunum and ileum compartments using physiologically relevant media to evaluate delayed release of the formulations. To simulate lowered bile acid concentrations in the ileum area of the gastrointestinal tract, a novel method using the bile acid sequestrant cholestyramine to lower bile acid concentrations in the small intestinal medium to physiologically relevant levels was attempted. Granulation, extrusion and spheronization was used to develop pellets containing viable probiotics using freeze-dried Lactobacullus reuteri as a model strain. Fluid bed coating the pellets with the pH-sensitive polymers Eudragit S100 or Eudragit FS30D resulted in targeted release in the ileum step of the three-step in vitro model based on release of the marker riboflavin.

ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity.

INTRODUCTION: Pre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. METHODS: Human neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. RESULTS: After 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. DISCUSSION & CONCLUSIONS: Combined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.