Search Results (695)

Recent studies have demonstrated that the coumarin derivative 4-Methylumbelliferone (4MU) has an antidiabetic effect in rodent models. 4MU is known to decrease the availability of hyaluronan (HA) substrates and inhibit the activity of different HA synthases. Nevertheless, it has been observed that 4MU may also affect cellular metabolism. In this study, we utilize the rat insulinoma beta cell line (INS-1E) cultured in both two-dimensional (2D) and three-dimensional (3D) experimental settings (pseudo islets), as an in vitro model to study beta cell functionality. For the first time, we observed that treating INS1E cells with 4MU results in improved insulin secretion. Additionally, we discovered that 4MU treatment elicited morphological changes from multilayer to monolayer conditions, along with a varied distribution of insulin granules and cell adhesion properties. Notably, we found that insulin secretion is not correlated with HA production. The same result was observed in co-culture experiments involving INS-1E cells and stromal vascular fraction (SVF) from adipose tissue. These experiments aim to investigate the effects of 4MU on beta cells in the context of its potential use in early-stage type 1 diabetes and in enhancing islet transplantation outcomes. Full article

Pancreatic cancer (PC) is an aggressive malignancy with a poor prognosis, requiring innovative approaches to evaluate new therapies. Considering the high activity of unsymmetrical bisacridines (UAs) in PC monolayer cultures, we employed multicellular tumor spheroids (MCTS) to assess whether UAs retain pro-apoptotic activity under more physiologically relevant conditions. Ultra-low attachment plates were used to form spheroids from three PC cell lines (Panc-1, MIA PaCa-2, and AsPC-1) with different genotypes and phenotypes. The effects of UA derivatives (C-2028, C-2045, and C-2053) were evaluated using microscopy and flow cytometry (7-AAD for viability and annexin V-FITC/PI for membrane integrity). UAs altered the morphology of the spheroids and reduced their growth. Notably, Panc-1 spheroids exhibited compromised integrity. The increase in 7-AAD⁺ cells confirmed diminished cell viability, and annexin V-FITC assays showed apoptosis as the dominant death pathway. Interestingly, the exact derivative was most active against a given cell line regardless of culture conditions. These results confirm that UAs maintain anticancer activity in 3D cultures and induce apoptosis, with varying efficacy across different cell lines. This underscores the value of diverse cellular models in compound evaluation and supports UAs as promising candidates for pancreatic cancer therapy. Full article

This work focuses on the effectiveness of removing ammonium from real municipal wastewater using synthetic faujasite (FAU-type) and β (BEA-type) zeolites and a commercial β (BEA-type) sample. The results demonstrated that synthetic samples presented enhanced performance on ammonium removal in comparison with commercial zeolite due to higher Al content and larger specific surface area, promoting better accessibility to active adsorption sites of the adsorbents. Synthetic FAU-type and BEA-type zeolites achieved a maximum adsorption capacity of 28.87 and 12.62 mg·g⁻¹, respectively, outperforming commercial BEA-type zeolite (6.50 mg·g⁻¹). Adsorption assays, associated with kinetic studies and adsorption isotherms, were better fitted using the pseudo-second order model and the Langmuir model, respectively, suggesting that chemisorption, involving ion exchange, and monolayer formation at the zeolite surface, was the main mechanism involved in the NH₄⁺ adsorption process. After ammonium adsorption, the NH₄⁺-loaded zeolite samples were used to stimulate the growth of tomato seedlings; the results revealed a change in the biomass production for seedlings grown in vitro, especially when the BEA_C_NH₄ sample was employed, leading to a 15% increase in the fresh mass in comparison with the control sample. In contrast, the excess of ammonium adsorbed over the BEA_S_NH₄ and FAU_NH₄ samples probably caused a toxic effect on seedling growth. The elemental analysis results supported the hypothesis that the presence of NH₄⁺-loaded zeolite into the culture medium was important for the release of nitrogen. The obtained results show then that the investigated zeolites are promising both as efficient adsorbents to mitigate the environmental impact of ammonium-contaminated water bodies and as nitrogen-rich fertilizers. Full article

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

The application of cellular spheroids in bone tissue engineering research has gained significant interest in the last decade. Compared to monolayer cell cultures, the 3D architecture allows for more physiological cell–cell and cell–extracellular matrix (ECM) interactions that make cellular spheroids a suitable model system to investigate the bone ECM in vitro. The use of 3D model systems requires fine-tuning of the experimental methods used to study cell morphology, ECM deposition and mineralization, and cell–ECM interactions. In this study, we use a construct made of MC3T3-E1 cellular spheroids encapsulated in an alginate hydrogel to study and characterize the deposited ECM. Spheroid shape and structure were evaluated using confocal microscopy. The deposited collagenous ECM was characterized using Second Harmonic Imaging Microscopy (SHIM), quantitative hydroxyproline (HYP) assay, and Transmission Electron Microscopy (TEM). The use of hydrogel constructs enabled easy handling and imaging of the samples, while also helping to preserve the spheroid’s stability by preventing cells from adhering to the culture dish surface. We used a non-modified alginate hydrogel that did not facilitate cell attachment and therefore functioned as an inert encapsulating scaffold. Constructs were cultured for up to 4 weeks. SHIM, HYP assay, and TEM confirmed the deposition of a collagenous matrix. We demonstrated that alginate-encapsulated bone spheroids are a convenient and promising model for studying the bone ECM in vitro. Full article

Accurate in vitro models of intestinal permeability are essential for predicting oral drug absorption. Standard models like Caco-2 cells have well-known limitations, including lack of segment-specific physiology, but are widely used. Emerging models such as organoid-derived monolayers and microphysiological systems (MPS) offer enhanced physiological relevance but require comparative validation. We performed a head-to-head evaluation of Caco-2 cells, human jejunal (J2) and duodenal (D109) enteroid-derived cells, and EpiIntestinal^TM tissues cultured on either static Transwell and flow-based MPS platforms. We assessed tissue morphology, barrier function (TEER, dextran leakage), and permeability of three model small molecules (caffeine, propranolol, and indomethacin), integrating the data into a physiologically based gut absorption model (PECAT) to predict human oral bioavailability. J2 and D109 cells demonstrated more physiologically relevant morphology and higher TEER than Caco-2 cells, while the EpiIntestinal^TM model exhibited thicker and more uneven tissue structures with lower TEER and higher passive permeability. MPS cultures offered modest improvements in epithelial architecture but introduced greater variability, especially with enteroid-derived cells. Predictions of human fraction absorbed (F_abs) were most accurate when using static Caco-2 data with segment-specific corrections based on enteroid-derived values, highlighting the utility of combining traditional and advanced in vitro gut models to optimize predictive performance for F_abs. While MPS and enteroid-based systems provide physiological advantages, standard static models remain robust and predictive when used with in silico modeling. Our findings support the need for further refinement of enteroid-MPS integration and advocate for standardized benchmarking across gut model systems to improve translational relevance in drug development and regulatory reviews. Full article

Impedance-based cellular assays allow determination of biological functions of cell populations in real-time by measuring electrical impedance. As compared to end-point assays, such as trans-epithelial electrical resistance assays, for example, they enable fast, non-invasive, and easy detection of cell kinetics—their growth, attachment, and interaction can be monitored over time. In our experiment, Caco-2 cells were cultured on E-plates 16. Next, fully differentiated cells were treated with either TNF-α or 3,4-dihydroxy-L-phenylalanine (L-DOPA). We aimed to verify the possibility of real-time testing of the viability, monolayer formation, and integrity (i.e., the presence of a functional and polarized monolayer) of Caco-2 cells by the xCELLigence real-time cell analyzer (RTCA) S16 system (Agilent Technologies). Full article

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication, gaining recognition as tumor biomarkers and promising therapeutic targets. As the study of EVs advances, it has become increasingly clear that the cellular context in which they are produced significantly influences their composition and function. Traditional two-dimensional in vitro models are being progressively replaced by more advanced three-dimensional systems, such as tumor spheroids and organoids. These 3D models are particularly valuable in cancer research, providing a more accurate representation of the complex cellular and molecular heterogeneity that characterizes tumors, better mimicking the in vivo microenvironment compared to standard monolayer cultures. This review explores the role of EVs derived from tumor spheroids and organoids in key oncogenic processes, including tumor growth, metastasis, and interactions within the tumor microenvironment. We highlight how EVs contribute to the spread of cancer cells, affecting surrounding tissues, and promote immune evasion, which poses significant challenges in cancer therapy. Full article

Background/Objectives: Rho-associated coiled-coil-containing protein kinase inhibitors (ROCKis) have now become known as modulators of corneal endothelial wound repair and cell survival. However, evidence remains fragmented across laboratory and clinical reports. We performed a systematic review to synthesize preclinical and clinical data on ROCKis in corneal disease, assess their efficacy and safety, and identify research gaps. Methods: We searched PubMed, Web of Science, Scopus, and Google Scholar (until May 2025) for English-language original studies evaluating ROCKis in corneal models or patients. Inclusion criteria encompassed in vitro, ex vivo, in vivo, and clinical trials reporting functional outcomes (endothelial cell density, wound closure, visual acuity). Results: Thirty-one studies met criteria: 14 preclinical studies and 17 clinical studies. Preclinical models (rabbit, porcine, human explants) uniformly showed ROCKis (Y-27632, Ripasudil, Netarsudil, H-1152) accelerate corneal endothelial cell proliferation, migration, and restoration of a hexagonal monolayer with improved barrier and pump function over days to weeks. In 17 clinical investigations, topical Ripasudil or Netarsudil and cultured cell injections achieved significant corneal thinning, endothelial cell density and central corneal thickness changes, and visual acuity improvements (≥2 lines) with minimal adverse events. Overall bias was moderate in non-randomized studies and low in the RCTs. Conclusions: ROCKis demonstrate consistent pro-regenerative effects on corneal endothelium in multiple models and show promising clinical efficacy in Fuchs endothelial dystrophy and pseudophakic endothelial failure. Future work should explore novel delivery systems and larger controlled trials to optimize dosing, safety, and long-term outcomes. Full article

Compelling clinical evidence strongly indicates that anti-angiogenesis therapeutics including Bevacizumab, a humanised anti-VEGF mAb, can alleviate the resistance to immunotherapy. We explored the direct modulation of Bevacizumab on endothelial cell (EC) immune response including surface expression of adhesion and MHC molecules and EC-elicited proliferation of immune cells under inflammatory conditions. Flow cytometry showed that addition of VEGF inhibited TNF-α stimulation of expression of ICAM-1 and VCAM-1 on HUVECs, whereas Bevacizumab enhanced this TNF-α-stimulated expression. The presence of MHC Class I on HUVECs was decreased by VEGF and increased by TNF-α, respectively. Bevacizumab reversed VEGF downregulation and promoted TNF-α upregulation of MHC class I expression, suggesting that anti-VEGF treatment can boost the endothelial immunological reaction, a prerequisite for immune cell trafficking. Functionally, real-time monitoring of the proliferation of human PBMCs co-cultured on HUVEC monolayers over 3 days showed opposing effects on the proliferation of PBMCs between VEGF and TNF-α. Consistently, Bevacizumab antagonised VEGF suppression and sensitized TNF-α activation of PBMC growth over the time course. In line with these findings, Bevacizumab increased the surface expression of CD69 on VEGF-treated T cells collected from PBMCs after 3-day co-cultures with HUVECs. Furthermore, the proliferation of CD3+, CD8+ and CD4+ T cells was promoted via Bevacizumab. Collectively, this study demonstrates that targeting VEGF can enhance the immune response of ECs required for T cell recruitment. Our findings provide insights to a deeper understanding of increased vascular inflammatory response conferred by anti-VEGF treatment in addition to inhibiting angiogenesis, which supports its favourable dual role in the positive immunological synergism with immunotherapy. Full article

Glioblastoma (GBM) is the most aggressive form of malignant gliomas, with the eicosanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) playing a pivotal role in its progression via the COX-2/prostaglandin E2/4 axis. COX-2 upregulations in tumor cells induces a pro-inflammatory tumor microenvironment (TME), affecting the behavior of invading bone marrow-derived macrophages (Mϕ) and brain-resident microglia (MG) through unclear autocrine and paracrine mechanisms. Using CRISPR/Cas9 technology, we generated COX-2 knockout U87 glioblastoma cells. In spheroids and in vivo xenografts, this resulted in a significant inhibition of tumorigenic properties, while not observed in standard adherent monolayer culture. Here, the knockout induced a G1 cell cycle arrest in adherent cells, accompanied by increased ROS, mitochondrial activity, and cytochrome c-mediated apoptosis. In spheroids and xenograft models, COX-2 knockout led to notable growth delays and increased cell death, characterized by features of both apoptosis and autophagy. Interestingly, these effects were partially reversed in subcutaneous xenografts after co-culture with Mϕ, while co-culture with MG enhanced the growth-suppressive effects. In an orthotopic model, COX-2 knockout tumors displayed reduced proliferation (fewer Ki-67 positive cells), increased numbers of GFAP-positive astrocytes, and signs of membrane blebbing. These findings highlight the potential of COX-2 knockout and suppression as a therapeutic strategy in GBM, particularly when combined with suppression of infiltrating macrophages and stabilization of resident microglia populations to enhance anti-tumor effects. Full article

Background/Objectives: Three-dimensional (3D) cell culture models more accurately simulate the in vivo tissue environments as compared to conventional two-dimensional (2D) monolayer cultures. Among these, spheroid cultures are particularly valuable for pharmaceutical research, as they allow for the study of tumor growth, drug responses, and cell–cell interactions in a physiologically relevant manner. Superhydrophobic surfaces (SHSs) have shown a promise in enhancing spheroid formation by reducing cell–substrate adhesion and promoting cell–cell aggregation. This study aims to evaluate the effectiveness of two different SHS coatings (SHS1: fluorinated; SHS2: silicone-based) in generating co-culture spheroids composed of non-tumoral fibroblasts (3T3) and tumoral epidermoid carcinoma cells (A431), thereby mimicking aspects of the tumor microenvironment. Methods: Co-cultures of 3T3 and A431 cells were seeded at varying ratios onto SHS1 and SHS2 substrates to assess their ability to support 3D spheroid formation. Spheroids were characterized by measurements of circularity and size distribution, viability through live/dead staining, and surface topography using 3D profilometry. Results: Spheroid formation was significantly influenced by both the surface properties and the fibroblast-to-carcinoma cell ratio. The fluorinated SHS1 surface facilitated superior cell viability and promoted the formation of well-rounded, uniform spheroids. In contrast, the silicone-based SHS2 surface resulted in less defined spheroidal structures and lower overall viability. Profilometry confirmed more consistent and compact 3D architectures on SHS1. Conclusions: This study demonstrates that SHS1, a fluorinated superhydrophobic coating, is more effective than SHS2 in supporting the formation of viable and structurally coherent 3D co-culture spheroids. These findings underscore the potential of SHS1 as a low-cost, tunable platform for developing in vitro cancer models and advancing the study of tumor–stroma interactions. Full article

The brain actively obtains nutrients through various transporters on brain microvessel endothelial cells (BMECs). Major facilitator superfamily domain–containing protein 2a (MFSD2A) serves as a key transporter of docosahexaenoic acid (DHA) at the blood–brain barrier (BBB) and is exclusively expressed in BMECs. Although brain pericytes (PCs) regulate MFSD2A expression in BMECs, the underlying mechanism remains unclear. To determine whether PDGF-BB/PDGFRβ signaling between endothelial cells (ECs) and PCs affects MFSD2A protein expression and plasma membrane localization in ECs, we examined the impact of AG1296 (a PDGF receptor inhibitor) and Pdgfrb-knockdown PCs on a non-contact coculture BBB model comprising the primary cultures of rat brain ECs and PCs. The effects of PCs on MFSD2A expression, localization, and brain endothelial DHA uptake was assessed using Western blot, immunofluorescence staining, and [¹⁴C]DHA uptake by ECs, respectively. In ECs cocultured with PCs, MFSD2A expression and plasma membrane localization were significantly higher than in EC monolayers. Moreover, conditioned medium derived from PCs failed to enhance MFSD2A expression. The increased expression and membrane localization of MFSD2A were inhibited by AG1296 and Pdgfrb-knockdown PCs. Furthermore, PCs significantly increased [¹⁴C]DHA uptake by ECs. These findings suggest that PCs enhance MFSD2A expression and plasma membrane localization in ECs through PDGF-BB/PDGFRβ signaling. Full article

Background: Intestinal epithelial barrier (IEB) dysfunction is related to multiple gastrointestinal disorders, notably inflammatory bowel disease (IBD). Betulinic acid (BA), a compound derived from birch bark, has demonstrated potential therapeutic benefits in IBD. Nevertheless, the impact of BA on IEB function has not been fully elucidated. Methods: The current study aimed to explore the potential underlying mechanisms of BA in dextran sodium sulfate (DSS)-induced IBD in mice and co-culture models involving Caco-2/HT29-MTX-E12 cell monolayers or mouse intestinal organoids (IOs) in conjunction with macrophages stimulated by lipopolysaccharide (LPS). Results: In vivo, BA treatment significantly improved body weight and colon length, alleviated disease activity index (DAI) scores, and reduced colonic histopathological injury in IBD mice. In vitro, BA reduced the flux of FITC-dextran; increased the TEER; and decreased the production of IL-6, IL-1β, and TNF-α while increasing IL-10 mRNA levels. Additionally, BA enhanced IEB formation by upregulating ZO-1, occludin (OCLN), and claudin-1 (CLDN1). Molecular docking studies revealed significant docking scores and interactions between BA and PPAR-γ. Moreover, BA significantly upregulated PPAR-γ protein expression, decreased NF-κB and MLC2 phosphorylation, and reduced MLCK protein expression. However, this effect was reversed by GW9662, an effective PPAR-γ antagonist. Conclusions: The findings reveal that BA mitigates IBD by safeguarding the intestinal barrier against dysfunction. This effect may be attributed to its ability to suppress inflammation and enhance the expression of tight junction proteins by modulating the PPAR-γ/NF-κB signaling pathway. Full article

Chemotherapy resistance is a major obstacle in the treatment of ovarian cancer, often resulting in disease recurrence and poor prognosis for patients. A key contributor to this resistance is the overexpression of ATP-binding cassette (ABC) transporters, including breast cancer resistance protein (BCRP/ABCG2), which actively effluxes chemotherapeutic agents such as topotecan (TOP) or mitoxantrone (MIT), limiting their intracellular accumulation and efficacy. This study investigated the potential of elacridar (GG918), a potent dual P-gp and BCRP inhibitor, to overcome drug resistance in ovarian cancer cell lines. Both TOP-sensitive and TOP-resistant ovarian cancer cells were grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroid models to better mimic the tumor microenvironment. The expression of the ABCG2 gene was quantified via qPCR and BCRP protein levels were assessed by western blotting and immunofluorescence. Drug response was evaluated using MTT viability assays, while BCRP transporter activity was examined using flow cytometry and microscopic assessment of the intracellular retention of BCRP fluorescent substrates (Hoechst 33342 and MIT). In both 2D and 3D cultures, elacridar effectively inhibited BCRP function and significantly enhanced sensitivity to TOP. These findings suggest that elacridar can inhibit BCRP-mediated drug resistance in ovarian cancer cell models. Full article