Search Results (473)

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

Organoid and spheroid technologies have rapidly become pivotal in thyroid cancer research, offering models that are more physiologically relevant than traditional two-dimensional culture. In the study of papillary and anaplastic thyroid carcinomas, two subtypes that differ both histologically and clinically, three-dimensional (3D) models offer unparalleled insights into tumor biology, therapeutic vulnerabilities, and resistance mechanisms. These models maintain essential tumor characteristics such as cellular diversity, spatial structure, and interactions with the microenvironment, making them extremely valuable for disease modeling and drug testing. This review emphasizes recent progress in the development and use of thyroid cancer organoids and spheroids, focusing on their role in replicating disease features, evaluating targeted therapies, and investigating epithelial–mesenchymal transition (EMT), cancer stem cell behavior, and treatment resistance. Patient-derived organoids have shown potential in capturing individualized drug responses, supporting precision oncology strategies for both differentiated and aggressive subtypes. Additionally, new platforms, such as thyroid organoid-on-a-chip systems, provide dynamic, high-fidelity models for functional studies and assessments of endocrine disruption. Despite ongoing challenges, such as standardization, limited inclusion of immune and stromal components, and culture reproducibility, advancements in microfluidics, biomaterials, and machine learning have enhanced the clinical and translational potential of these systems. Organoids and spheroids are expected to become essential in the future of thyroid cancer research, particularly in bridging the gap between laboratory discoveries and patient-focused therapies. Full article

Electrospun nanofiber membranes (ESNFMs) are exceptional biomaterials for tissue engineering, closely mimicking the native extracellular matrix. However, their inherent fragility poses significant handling, processing, and integration challenges, limiting their widespread application in advanced 3D tissue models and biofabricated devices. This study introduces a novel and on-mat framing technique utilizing extrusion-based printing of a UV-curable biocompatible resin (Biotough D90 MF) to create rigid, integrated support structures directly on chitosan–polyethylene oxide (PEO) ESNFMs. We demonstrate fabrication of these circular frames via precise 3D printing and a simpler manual stamping method, achieving robust mechanical stabilization that enables routine laboratory manipulation without membrane damage. The resulting framed ESNFMs maintain structural integrity during subsequent processing and exhibit excellent biocompatibility in standardized extract assays (116.5 ± 12.2% normalized cellular response with optimized processing) and acceptable performance in direct contact evaluations (up to 78.2 ± 32.4% viability in the optimal configuration). Temporal assessment revealed characteristic cellular adaptation dynamics on nanofiber substrates, emphasizing the importance of extended evaluation periods for accurate biocompatibility determination of three-dimensional scaffolds. This innovative biofabrication approach overcomes critical limitations of previous handling methods, transforming delicate ESNFMs into robust, easy-to-use components for reliable integration into complex cell culture applications, barrier tissue models, and engineered systems. Full article

Natural products demonstrate potent immunomodulatory properties through checkpoint modulation, macrophage polarization, and T cell/natural killer (NK) cell activation. While cancer organoid-immune co-culture platforms enable physiologically relevant modeling of tumor–immune interactions, systematic investigation of natural product immunomodulation in these systems remains entirely unexplored. We conducted a comprehensive literature analysis examining natural products tested in cancer organoids, immunomodulatory mechanisms from traditional models, technical advances in organoid-immune co-cultures, and standardization requirements for clinical translation. Our analysis reveals a critical research gap: no published studies have investigated natural product-mediated immunomodulation using organoid-immune co-culture systems. Even though compounds like curcumin, resveratrol, and medicinal mushroom polysaccharides show extensive immunomodulatory effects in two-dimensional (2D) cultures, and organoid technology achieves high clinical correlation for drug response prediction, all existing organoid studies focus exclusively on direct cytotoxicity. Technical challenges include compound stability, limited matrix penetration requiring substantially higher concentrations than 2D cultures, and maintaining functional immune populations in three-dimensional (3D) systems. The convergence of validated organoid-immune co-culture platforms, Food and Drug Administration (FDA) regulatory support through the Modernization Act 2.0, and extensive natural product knowledge creates unprecedented opportunities. Priority research directions include systematic screening of immunomodulatory natural products in organoid-immune co-cultures, development of 3D-optimized delivery systems, and clinical validation trials. Success requires moving beyond cytotoxicity-focused studies to investigate immunomodulatory mechanisms in physiologically relevant 3D systems, potentially unlocking new precision cancer immunotherapy approaches. Full article

Three-dimensional cell culture systems are relevant in vitro models for studying cellular behavior. In this regard, this present study investigates the interaction between human osteoblast-like cells and 3D-printed scaffolds mimicking physiological and osteoporotic bone structures under simulated microgravity conditions. The objective is to assess the effects of scaffold architecture and dynamic culture conditions on cell adhesion, proliferation, and metabolic activity, with implications for osteoporosis research. Polylactic acid scaffolds with physiological (P) and osteoporotic-like (O) trabecular architectures were 3D-printed by means of fused deposition modeling technology. Morphometric characterization was performed using micro-computed tomography. Human osteoblast-like SAOS-2 and U2OS cells were cultured on the scaffolds under static and dynamic simulated microgravity conditions using a rotary cell culture system (RCCS). Scaffold biocompatibility, cell viability, adhesion, and metabolic activity were evaluated through Bromodeoxyuridine incorporation assays, a water-soluble tetrazolium salt assay, and an enzyme-linked immunosorbent assay of tumor necrosis factor-α secretion. Both scaffold models supported osteoblast-like cell adhesion and growth, with an approximately threefold increase in colonization observed on the high-porosity O scaffolds under dynamic conditions. The dynamic environment facilitated increased surface interaction, amplifying the effects of scaffold architecture on cell behavior. Overall, sustained cell growth and metabolic activity, together with the absence of detectable inflammatory responses, confirmed the biocompatibility of the system. Scaffold microstructure and dynamic culture conditions significantly influence osteoblast-like cell behavior. The combination of 3D-printed scaffolds and a RCCS bioreactor provides a promising platform for studying bone remodeling in osteoporosis and microgravity-induced bone loss. These findings may contribute to the development of advanced in vitro models for biomedical research and potential countermeasures for bone degeneration. 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

Two-dimensional culture systems have been used for a long time in the research field but their disadvantages make it difficult to reproduce the in vivo environment. Three-dimensional culture systems overcome these limitations, simulating the physiological context of an organism, from the molecular level to the cellular, tissue, and organ complexity levels. This review focuses on 3D cellular models, such as spheroids and tumoroids, which reproduce tumor heterogeneity and microenvironments. It also includes 3D cultures of mesenchymal stem cells (MSCs), particularly those derived from teeth. In conclusion, 3D models are profoundly impacting the biomedical field by offering more accurate in vitro platforms for drug development and disease modeling, thereby significantly reducing the reliance on animal testing and leading to the advancement of personalized and regenerative medicine. Full article

Pancreatic adenocarcinoma remains one of the deadliest cancers, with limited treatment options and high chemoresistance. Traditional 2D cell cultures fail to accurately replicate the tumor architecture. Our study introduces three-dimensional (3D) pancreatic adenocarcinoma spheroid models using magnetic aggregation of pancreatic cancer cells and immortalized fibroblasts in either liquid culture medium or embedded in hydrogels. The spheroids’ growth was characterized using optical imaging, while viability was assessed using ATP quantification and flow cytometry. Results demonstrated successful spheroid formation and growth. Further analysis suggested that on one hand, culture in liquid medium and ATP-based viability assessment are practical for initial experiments. On the other hand, hydrogel culture and flow cytometry, although being more resource- and labor-intensive, provided both a more reproducible and detailed viability analysis. Full article

The nervous system maintains homeostasis and coordinated behavior through complex neuronal and glial cells. Traditional models, such as primary rodent neurons and human-induced pluripotent stem cell (hIPSC)-derived neurons, have advanced our understanding of neuronal function and neurotoxic damage; however, they are costly and labor-intensive. SH-SY5Y cells, an immortalized human neuroblastoma cell line, provide a more accessible alternative for studying neuronal processes and neurotoxicity. However, their limited capacity to differentiate into specific neuronal phenotypes remains a challenge. To address this limitation, differentiation protocols using neuronal factors and vitamins have been developed, primarily in two-dimensional (2D) cultures, which reduces physiological relevance. Here, we present a novel three-dimensional (3D) SH-SY5Y model incorporating 2D differentiation protocols to generate cholinergic neurons (ChAT+). This model enhances neurotoxicity studies related to pesticides and mycotoxins. Our protocol produces homogeneous spheroids differentiated into cholinergic neurons using serum restriction and specific factors, maintaining viability and circularity for up to 22 days. Differentiation was validated by immunofluorescence and Western blot by Choline acetyltransferase (ChAT) expression. This scalable and reproducible 3D model provides a valuable in vitro tool for neurotoxicological research, improving physiological relevance and enabling the study of cholinergic neuron differentiation and function. Full article

Multiple myeloma is a hematologic malignancy characterized by the clonal proliferation of plasma cells within the bone marrow. The tumor microenvironment plays a crucial role in multiple myeloma pathogenesis, progression, and drug resistance. Traditional two-dimensional cell culture models have been instrumental in multiple myeloma research. However, they fail to recapitulate the complex in vivo bone marrow microenvironment, leading to limited predictive value for clinical outcomes. Three-dimensional cell culture models emerged as more physiologically relevant systems, offering enhanced insights into multiple myeloma biology. Scaffold-based systems (e.g., hydrogels, collagen, and Matrigel), scaffold-free spheroids, and bioprinted models have been developed to simulate the bone marrow microenvironment, incorporating key components like mesenchymal stromal cells, osteoblasts, endothelial cells, and immune cells. These models enable the functional assessment of cell adhesion-mediated drug resistance, cytokine signaling networks, and hypoxia-induced adaptations, which are often lost in 2D cultures. Moreover, 3D platforms demonstrated improved predictive value in preclinical drug screening, facilitating the evaluation of novel agents and combination therapies in a setting that better mimics the in vivo tumor context. Hence, 3D cultures represent a pivotal step toward bridging the gap between basic myeloma research and translational applications, supporting the development of more effective and patient-specific therapies. Full article

Three-dimensional (3D) in vitro adipocyte models provide physiologically relevant platforms for studying adipogenesis and obesity-related metabolic dysfunction. However, long-term adipocyte culture is often hindered by limited cell–matrix adhesion and spheroid detachment. Previously, we demonstrated that elastin-like polypeptide (ELP)–polyethyleneimine (PEI) coatings functionalized with a trivalent RGD motif enhanced spheroid retention during frequent media changes. The present study investigates the long-term functional consequences of RGD incorporation over a 28-day culture period. 3T3-L1 preadipocytes were seeded, differentiated, and matured on ELP-PEI or ELP-(RGD)₃-PEI coatings. Spheroid morphology, triglyceride content, expression of PPAR-γ, adiponectin, HIF-1α genes, and insulin-stimulated glucose uptake were assessed. Both coatings supported initial spheroid formation, but only ELP-PEI maintained the 3D architecture and supported adipogenic maturation and insulin responsiveness. ELP-(RGD)₃-PEI promoted early retention but led to spheroid disassembly by mid-culture; notably, by day 28, cells reaggregated into abnormally large spheroids with impaired metabolic function, likely due to continued proliferation. These findings highlight the critical role of extracellular matrix-mediated cell–cell versus cell–substrate interactions in maintaining 3D culture fidelity. While RGD enhances adhesion, it disrupts spheroid integrity and compromises adipogenic and metabolic maturation. Taken together, ELP-PEI coatings offer a more conducive microenvironment for long-term 3D adipocyte culture and hold promise for modeling obesity-associated metabolic dysfunction in vitro. Full article

Organoids are three-dimensional (3D) structures that mimic the architecture and functionality of human organs, providing a novel approach to study diseases such as colorectal cancer (CRC). This review aims to explore the impact of organoids on understanding CRC and their potential use in exploring therapeutic outcomes. Colorectal cancer, characterized by the transformation of colonic epithelial cells into adenomas and carcinomas, remains one of the top causes of cancer-related morbidity and mortality worldwide. Traditional two-dimensional (2D) cell cultures fail to replicate the tumor microenvironment in an effective manner, which highlights the need for advanced 3D models. Organoids preserve the genetic and phenotypic properties of the original tumors, allowing for improved disease modeling, drug screening, and personalized medicine applications. When using patient-derived organoids (PDOs), researchers can gain insights into CRC initiation, progression, and treatment outcome. Ultimately, organoids represent an encouraging platform for improving therapeutic strategies for CRC, potentially leading to better patient outcomes through tailored treatment approaches. 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

Microfluidic devices offer well-defined physical environments that are suitable for effective cell seeding and in vitro three-dimensional (3D) cell culture experiments. These platforms have been employed to model in vivo conditions for studying mechanical forces, cell–extracellular matrix (ECM) interactions, and to elucidate transport mechanisms in 3D tissue-like structures, such as tumor and lymph node organoids. Studies have shown that fluid flow behavior in microfluidic slides (µ-slides) directly influences shear stress, which has emerged as a key factor affecting cell proliferation and differentiation. This study investigates fluid flow in the porous channel of a µ-slide using computational fluid dynamics (CFD) techniques to analyze the impact of perfusion flow rate and porous properties on resulting shear stresses. The model of the µ-slide filled with a permeable biomaterial is considered. Porous media fluid flow in the channel is characterized by adding a momentum loss term to the standard Navier–Stokes equations, with a physiological range of permeability values. Numerical simulations are conducted to obtain data and contour plots of the filtration velocity and flow-induced shear stress distributions within the device channel. The filtration flow is subsequently measured by performing protein perfusions into the slide embedded with native human-derived ECM, while the flow rate is controlled using a syringe pump. The relationships between inlet flow rate and shear stress, as well as filtration flow and ECM permeability, are analyzed. The findings provide insights into the impact of shear stress, informing the optimization of perfusion conditions for studying tissues and cells under fluid flow. 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