Search Results (592)

The growing demand for alternative meat products is accelerating research into reproductive cell sources for cell-based meat processes, also called cultured meat. Porcine umbilical cord tissue is recognized as an advantageous source of mesenchymal stem cells (MSCs). However, effective decontamination must be achieved without compromising tissue integrity and cell recovery. In this study, we evaluated the decontamination of porcine umbilical cords using plasma-functionalized liquid (PFL) generated by a microwave-driven plasma source. It was applied alone and in combination with ultrasound, with the combined approach demonstrating superior performance. Specifically, the ultrasound–PFL combination treatment reduced the initial microbial load of individual tissue samples, ranging from 4.08 to 7.41 log₁₀ CFU/g, approaching the limit of detection of the applied microbiological assays. Statistical analysis indicated a significant contribution of both PFL and ultrasound to microbial reduction, while mesenchymal stem cell yields (5.4 × 10⁵ cells/g tissue) and cell viability (84%) remained comparable to antibiotic-rinsed controls. Recovered cells retained functional capacity, as demonstrated by successful 3D spheroid formation. These results highlight ultrasound-assisted PFL rinsing as an efficient, long-lasting, and antibiotic-free decontamination strategy without compromising tissue compatibility. This study thereby extends the application of plasma-functionalized liquids and demonstrates the feasibility of sustainable stem cell sourcing. It offers opportunities in cultured meat bioprocessing. Full article

SATB2 (special AT-rich binding protein 2) functions as a chromatin-associated epigenetic regulator that modulates gene expression, in part by serving as a transcriptional cofactor. This study assessed whether SATB2 overexpression is sufficient to promote in vitro transformation of human mesothelial cells and whether SATB2 suppression in mesothelioma cancer stem cell (CSC)–enriched populations is associated with altered chemoresistance. SATB2 expression was high in human malignant pleural mesothelioma (MPM) cell lines but absent in Met5A mesothelial cells. Ectopic SATB2 expression in Met5A cells was associated with acquisition of malignant and stem cell–like phenotypes, including increased expression of stem cell markers and pluripotency-associated factors, as well as anchorage-independent growth in soft agar and spheroid formation in suspension culture. In contrast, Met5A cells transduced with an empty vector did not form colonies or mesospheres. SATB2 overexpression in Met5A cells was also associated with increased motility, migration, and invasion, accompanied by induction of epithelial–mesenchymal transition (EMT)–related transcription factors relative to empty vector controls. Conversely, shRNA-mediated SATB2 knockdown in an MPM cell line attenuated proliferation, EMT-associated features, and CSC-like characteristics. Chromatin immunoprecipitation assays identified SATB2 occupancy at promoter regions of Bcl2, XIAP, KLF4, c-Myc, NANOG, and SOX2, consistent with a role in transcriptional regulation of genes linked to transformation, pluripotency, cell survival, proliferation, and EMT. In CSC-enriched cells, SATB2 inhibition was associated with increased sensitivity to cisplatin and pemetrexed, concomitant with reduced OCT4 and SOX2 expression. Collectively, these findings support SATB2 as a candidate therapeutic target in MPM and suggest that SATB2 suppression may enhance chemotherapy response when combined with standard agents. Full article

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior, drug response, and cell–matrix interactions. Here, we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates, facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation, GelMA preparation, bioprinting, and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research, including studies of metastasis, drug resistance, cancer cell lipid metabolism, and TME-associated hypoxia. Full article

Ex vivo functional testing for multiple myeloma is rapidly evolving, yet no single assay has reached the level of reliability and clinical utility needed for routine decision-making. Existing approaches generally fall into three categories: 2D cultures, 3D models, and dynamic systems. Each contributes valuable but incomplete insight into therapeutic response. Among these, 2D assays remain the most mature, with the most extensive clinical correlations to date, though their simplified architecture limits their ability to reflect the full complexity of the bone marrow microenvironment. However, 3D systems, including spheroids and matrix-based organoids, offer improved preservation of tumor heterogeneity and microenvironmental cues. These platforms show emerging clinical relevance and may hold advantages over traditional 2D formats, and validation efforts are developing. Dynamic systems, including microfluidic models and perfused bone-marrow mimetics, represent the most physiologically ambitious category, yet their technical intricacy and early stage of development have so far limited broad clinical correlation. Altogether, the current landscape highlights substantial progress but lacks an optimal assay. In this review, we take the unique approach of examining published ex vivo tests that have demonstrated a level of clinical correlation. We evaluate their respective formats, strengths and limitations, and discuss considerations for what an ideal future assay may encompass. Full article

Colorectal cancer (CRC) progression and therapy resistance are driven in part by metabolic reprogramming and the persistence of cancer stem-like cells (CSCs). The seven in absentia homolog 2 (SIAH2)/with-no-lysine kinase 1 (WNK1) signaling axis has emerged as a potential regulator of these processes, yet its functional role in CRC metabolism and tumor–stroma crosstalk remains incompletely understood. Integrated analyses of The Cancer Genome Atlas–Colon Adenocarcinoma (TCGA-COAD) and Gene Expression Omnibus (GEO, GSE17538) datasets revealed significant upregulation of SIAH2 and WNK1 in CRC tissues, with strong positive correlations to glycolysis- and hypoxia-associated genes, including PFKP, LDHA, BPGM, ADH1A, ADH1B, and HIF-1α. Single-cell and clinical profiling further demonstrated preferential enrichment of SIAH2 in undifferentiated, stem-like tumor cell populations. Functional studies across multiple CRC cell lines showed that SIAH2 silencing suppressed proliferation, clonogenic growth, tumor sphere formation, and cell-cycle progression, whereas SIAH2 overexpression exerted opposite effects. Seahorse extracellular flux analyses established that SIAH2 promotes glycolytic capacity and metabolic flexibility. At the protein level, SIAH2 regulated glycolytic enzymes and WNK1/hypoxia-inducible factor-1α (HIF-1α) signaling, effects that were amplified by cancer-associated fibroblast (CAF)-derived conditioned medium. CAF exposure enhanced SIAH2 expression, CSC spheroid growth, and resistance to fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy, whereas SIAH2 depletion effectively abrogated these effects. Collectively, these findings identify the SIAH2/WNK1 axis as a central metabolic regulator linking glycolysis, CSC maintenance, and microenvironment-driven therapy resistance in CRC, highlighting its potential as a therapeutic target. Full article

There is a growing demand for plant-derived antioxidants to replace synthetic ones in skincare applications. Phytochemicals are characterized by certain limitations, including poor bioavailability and chemical instability, which affect their industrial exploitation. Tomato peel extract has been used as a source of lycopene, which is renowned for its antioxidant properties. To improve the bioavailability of extracted lycopene, polymeric (poly-lactic-co-glycolic acid) nano-carriers were synthesized by comparing two non-ionic surfactants, polyvinyl alcohol (PVA) and Tween 20. The impact of surfactants has been studied by evaluating: (i) colloidal stability determined by dynamic light scattering; (ii) lycopene retention and bioactivity over time, as measured by spectrophotometric assays; (iii) biological interactions on 2D and 3D keratinocyte and melanocyte cell cultures. It was found that both surfactants enable the formation of stable lycopene-loaded nanoparticles suspensions; however, greater colloidal stability was exhibited by nanoparticles prepared with Tween 20. PVA, on the other hand, provided greater nanoparticle stability in terms of loaded lycopene retention and antioxidant activity. Tween 20 surfactant improves the internalization of lycopene-loaded nanoparticles in human skin spheroids. It was demonstrated that both surfactants provided excellent intracellular antioxidant activity of lycopene. This was observed in keratinocytes, melanocytes, adherent cells, and spheroids, suggesting potential skincare applications. Full article

The recreation of the tumor microenvironment remains a significant challenge in the development of experimental cancer models. The present study constitutes an investigation into the interconnection between tumor, endothelial and stromal cells in heterotypic breast cancer spheroids. The generation of models was achieved through the utilization of MCF7, MDA-MB-231, and SK-BR-3 tumor cell lines, in conjunction with endothelial TIME-RFP cells and either cancer-associated (BrC4f) or normal (BN120f) fibroblasts, within ultra-low attachment plates. It was established that stromal cells, most notably fibroblasts, were conducive to the aggregation of tumor cells into spheroids and the formation of pseudovessels in close proximity to fibroblast bands. In contrast to the more aggressive tumor models MDA-MB-231 and SK-BR-3, microenvironment cells do not influence the migration ability of MCF7 tumor cells. Heterotypic spheroids incorporating CAFs demonstrated a more aggressive and immunosuppressive phenotype. Multiplex immunoassay analysis of cytokines, followed by STRING cluster analysis, was used to identify key processes including angiogenesis, invasion, stem cell maintenance, and immunosuppression. Furthermore, a cluster of cytokines (LIF, SDF-1, HGF, SCGFb) was identified as potentially involved in the regulation of PD-L1 expression by tumor cells. This finding reveals a potential mechanism of immune evasion and suggests new avenues for therapeutic investigation. Full article

Cancer remains one of the most common causes of death worldwide and imposes enormous social and economic burdens. Human tumor-associated NADH oxidase (ENOX2, also known as tNOX) is a cancer cell-specialized NADH oxidase that is expressed on the membranes of cancer cells. In this study, we investigated the potential role of ENOX2 in regulating stemness properties in oral cancer through a combination of in vitro, in vivo, and bioinformatics approaches. We found that ENOX2 physically interacted with the stem cell transcription factor, SOX2, in co-immunoprecipitation experiments. The expression and activity of ENOX2 were elevated in p53-functional SAS and p53-mutated HSC-3 oral cancer cell spheroids compared with their monolayer counterparts. Consistently, SIRT1, a downstream effector modulated by ENOX2 through NAD⁺ generation, was also upregulated in spheroid cultures. Functional studies further established that ENOX2 overexpression significantly enhanced spheroid formation, self-renewal properties, stem cell marker expression, and PKCδ expression, whereas ENOX2 knockdown produced the opposite effects. In xenograft models, ENOX2-overexpressing oral cancer cell spheroids exhibited enhanced tumorigenicity, while ENOX2-silenced spheroids formed significantly smaller tumors. Complementary analyses of public transcriptomic and proteomic datasets revealed elevated ENOX2 expression in human head and neck tumor tissues compared with adjacent normal tissues. Based on these findings and literature-supported correlations, we propose a putative ENOX2-SIRT1-SOX2 regulatory framework that may contribute to the acquisition and maintenance of stem-like properties of oral cancer cells. While the ENOX2–SOX2 interaction was experimentally validated, the roles of SIRT1 and other downstream components are inferred from bioinformatic analyses and prior studies; thus, this axis represents a hypothetical model that warrants further mechanistic investigation. Collectively, our results identify ENOX2 as a potential regulator of oral cancer stemness and provide a conceptual foundation for future studies aimed at elucidating its downstream pathways and clinical relevance in head and neck tumors. Full article

Background/Objectives: Obesity-associated hyperleptinemia has been linked to breast cancer (BC) progression via mechanisms that remain incompletely understood. This study explores the role of leptin and its receptor (LEPR) in facilitating BC cell proliferation, migration, epithelial–mesenchymal transition (EMT), and STAT3 signaling pathway activation. Methods: We analyzed gene expression and survival data from TCGA BRCA dataset. MCF-7 and MDA-MB-231 BC cells were exposed to leptin at 10 ng/mL (lean-associated levels) and 100 ng/mL (elevated levels linked to obesity). MTT assays, colony formation tests, wound-healing and tumor spheroid dissemination experiments evaluated cell proliferation and migration. Immunofluorescence and Western blot analysis assessed changes in EMT markers and cytoskeletal alterations, while Western blotting and qPCR assessed STAT3 and NCOA1 expression and activation levels. Results: Elevated LEPR expression was linked with unfavorable prognosis in BC patients. Higher doses of leptin (100 ng/mL) significantly enhanced cellular proliferation rates and migratory capabilities, in both cell lines, and promoted EMT characteristics marked by downregulated E-cadherin and cytoskeleton structural changes. Whereas heightened JAK2/STAT3 signaling correlated with elevated leptin dosages, STAT3 inhibition using AG490 reversed leptin-induced migration while reinstating E-cadherin levels to baseline. Furthermore, leptin upregulated NCOA1, an essential STAT3 coactivator, facilitating increased expression of Cyclin D1 and VEGF target genes. Clinical positive relationships were seen between LEP/LEPR expressions and NCOA1 levels and between NCOA1 and various gene signatures related to STAT3/P-STAT3 within BC specimens. Conclusions: Obesity-associated hyperleptinemia enhances aggressiveness in BC through a mechanism involving LEPR-mediated activation pathways encompassing NCOA1/STAT3, which drive proliferation, migration, and EMT. This assigns a potential therapeutic utility for obesity-related advancements found within BC pathology. Full article

Background/Objectives: Three-dimensional (3D) cell cultures are increasingly used because 3D cell aggregates better mimic tissue-level biological mechanisms and support studies of tissue physiology and drug screening. However, existing laboratory methods and commercial microwell platforms often yield inconsistent results and can be error-prone, time-consuming, or costly. The objective of this work was to develop a reproducible, high-yield, and cost-effective approach for generating homogeneous cell aggregates using custom 3D-printed microwell stamps. Methods: Custom conical and semi-spherical microwell stamps were fabricated using 3D printing. Stamp resolution was characterized by scanning electron microscopy (SEM). Negative imprints were cast in polydimethylsiloxane (PDMS), a biocompatible and hydrophobic polymer conducive to cell aggregation. These PDMS microwells were then used to generate pluripotent stem cell aggregates (embryoid bodies, EBs) and tumor spheroids from adherent cancer cell lines. Results: The 3D-printed stamps produced high-resolution conical and semi-spherical microwells in PDMS. Semi-spherical microwells enabled rapid, simple, and cost-effective formation of pluripotent stem cell aggregates that were homogeneous in size and shape. These aggregates outperformed those produced using commercial microwell plates and ultra-low attachment plates. The fabricated microwells also generated uniform tumor spheroids from adherent cancer cells, demonstrating their versatility. Conclusions: The in-house 3D-printed microwell stamps offer a reproducible, efficient, and economical platform for producing homogeneous cell aggregates. This system improves upon commercial alternatives and supports a broad range of applications, including pluripotent stem cell embryoid body formation and tumor spheroid generation. Full article

Microfluidic cell culture systems and organ-on-a-chip platforms provide powerful tools for modeling physiological processes, disease progression, and drug responses under controlled microenvironmental conditions. These technologies rely on diverse cell culture methodologies, including 2D and 3D culture formats, spheroids, scaffold-based systems, hydrogels, and organoid models, to recapitulate tissue-level functions and generate rich, multiparametric datasets through high-resolution imaging, integrated sensors, and biochemical assays. The heterogeneity and volume of these data introduce substantial challenges in pre-processing, feature extraction, multimodal integration, and biological interpretation. Artificial intelligence (AI), particularly machine learning and deep learning, offers solutions to these analytical bottlenecks by enabling automated phenotyping, predictive modeling, and real-time control of microfluidic environments. Recent advances also highlight the importance of technical frameworks such as dimensionality reduction, explainable feature selection, spectral pre-processing, lightweight on-chip inference models, and privacy-preserving approaches that support robust and deployable AI–microfluidic workflows. AI-enabled microfluidic and organ-on-a-chip systems now span a broad application spectrum, including cancer biology, drug screening, toxicity testing, microbial and environmental monitoring, pathogen detection, angiogenesis studies, nerve-on-a-chip models, and exosome-based diagnostics. These platforms also hold increasing potential for precision medicine, where AI can support individualized therapeutic prediction using patient-derived cells and organoids. As the field moves toward more interpretable and autonomous systems, explainable AI will be essential for ensuring transparency, regulatory acceptance, and biological insight. Recent AI-enabled applications in cancer modeling, drug screening, etc., highlight how deep learning can enable precise detection of phenotypic shifts, classify therapeutic responses with high accuracy, and support closed-loop regulation of microfluidic environments. These studies demonstrate that AI can transform microfluidic systems from static culture platforms into adaptive, data-driven experimental tools capable of enhancing assay reproducibility, accelerating drug discovery, and supporting personalized therapeutic decision-making. This narrative review synthesizes current progress, technical challenges, and future opportunities at the intersection of AI, microfluidic cell culture platforms, and advanced organ-on-a-chip systems, highlighting their emerging role in precision health and next-generation biomedical research. Full article

Introduction: A 3D endothelial spheroid model expressing mosaic gain-of-function KRAS mutations was established to further understand the molecular changes associated with sporadic brain arteriovenous malformations (AVMs). Methods: Repellent 96-well U-bottom plates were seeded with human cerebral microvascular endothelial cells and resultant spheroids transduced with recombinant adeno-associated virus expressing KRAS^G12V. Spheroids were monitored using live-cell imaging for extended culture periods. Results: In the early growth period, KRAS^G12V expression increased spheroid growth rates and enhanced spheroid sprouting on gel matrices consistent with known AVM characteristics. With extended culture, novel endothelial characteristics were observed. KRAS^G12V-expressing spheroids displayed dynamic blebbing associated with the formation of rounded, hypertrophic cells disposed to engage in spheroid escape. These cells displayed reduced cell–cell adherence with rapid plasma membrane blebbing characteristic of amoeboid-like migration and mesenchymal-to-amoeboid transition. Spheroid growth and blebbing were reversed with MEK and mTOR inhibitors; Rho/ROCK inhibition specifically targeted the blebbing phenotype. Conclusions: Endothelial spheroids expressing KRAS^G12V exhibit characteristic features associated with abnormal vessel development in brain AVMs as well as novel phenotypes not previously observed in 2D monolayers. The ability to extend culture periods in this simple 3D model may allow further phenotypic exploration of important AVM driver mutations. Full article

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a high rate of recurrence and a dismal prognosis. Studies have shown that pancreatic cancer stem cells (PCSCs) are a subpopulation that contributes to tumor progression, resistance to therapeutics, and metastasis, making them a key subpopulation to target for treatment. Gedunin (GD), a natural compound derived from Azadirachta indica (neem), has shown anticancer properties in pancreatic cancer cells, but its effects on PCSCs remains unclear. This study evaluated the effects of GD in pancreatic cancer stem cells, highlighting its impacts on tumor growth and progression and focusing on its impact on the sonic hedgehog (Shh) signaling pathway. Methods: Functional assays were performed to assess the effect of GD on the sphere-forming ability, colony formation, and self-renewal of PCSCs. Athymic mice xenograft models were utilized to evaluate the tumor suppression effect of GD in vivo. Furthermore, the anticancer effect of GD on PCSCs was assessed using both in vitro and in vivo limiting dilution assay. GD-induced changes in Shh signaling and key stem cell marker expressions in PCSCs were evaluated. Results: GD effectively inhibited tumor growth in xenograft models and reduced the percentage of PCSCs. GD was effective in decreasing PCSCs’ proliferative, self-renewal, and colony-forming capacity. GD decreased the protein expression levels of key Shh signaling markers Gli1 and Shh, stem cell markers SOX2, Nanog, and Oct4, metastasis-related proteins MMP-2, MMP-3, and MMP-9, and EMT markers Tgf1, Slug, Snail, and Twist in both PDAC cells and PCSCs. We demonstrated a significant decrease in the spheroid formation and self-renewal capacity of the (ALDH+) PCSC population following GD treatment in HPAC cells, indicating its potential antagonistic effects on PCSCs. GD was highly effective in reducing tumor volume, stemness, and metastasis in both early and late chemotherapy. In vivo limiting dilution assay using CD133+/LGR5+ PCSC xenografts demonstrated that GD reduces tumor growth, metastasis, and stemness associated with PCSCs by downregulating the expression of Shh and Gli1. GD treatment also reduced micrometastatic lesions in the lung, liver, and brain, as identified using H&E staining. Conclusions: The findings highlight GD’s potential as a promising therapeutic candidate for PDAC, with the ability to target both bulk tumor cells and PCSCs. By simultaneously suppressing tumor growth, stemness, and metastatic spread, GD may contribute to more effective treatment strategies and improved patient outcomes. Full article

Myopathy encompasses a group of diseases characterized by abnormalities in both muscle function and structure. However, the underlying regulatory mechanisms of newly formed myofiber development remain poorly defined. No promising therapeutic approach has been developed, but numerous medication options are available to alleviate symptoms. Our previous studies demonstrated that adenosine kinase (ADK) is critical in regulating adenosine metabolism, pathological angiogenesis, pathological vascular remodeling, and vascular inflammatory diseases. Adenosine dynamically distributes between extracellular and intracellular, and adenosine concentration regulates ADK expression. However, the mechanism by which adenosine triggers an ADK-dependent intracellular signaling pathway to regulate skeletal muscle regeneration is not well defined. This study aimed to evaluate whether the adenosine-induced intracellular signaling pathway is involved in regulating myopathy, and how it regulates the development of newly formed myofibers. In this study, an intramuscular injection of cardiotoxin was used to induce a skeletal muscle injury model; satellite cells and C2C12 cells were employed. Whether adenosine regulates satellite cell activity, new myofiber formation and differentiation, as well as fusion of myofibers, were determined by H&E staining, BrdU incorporation assay, and spheroid sprouting assay. Interaction between ADK and PFKFB3 was evaluated by IF staining, PPI network analysis, molecular docking simulation, and CO-immunoprecipitation assay. The results demonstrated that adenosine dynamically distributes between extracellular and intracellular through concentrative nucleoside transports or equilibrative nucleoside transporters, and it rapidly induces an ADK-dependent intracellular signaling pathway, which interacts with PFKFB3-mediated glycolytic metabolism to promote satellite cell activity, new myofiber formation, differentiation, and fusion, and eventually enhances skeletal muscle regeneration after injury stress. The remarkable endogenous regeneration capacity of skeletal muscle, which is regulated by adenosine-triggered intracellular signaling, presents a promising therapeutic strategy for treating muscle trauma and muscular dystrophies. Full article

Cardiospheres (CSs) are widely used to boost the pro-reparative potential of adult cardiac cells, mediated through their unique secretome profile. The original CS generation method relies on self-assembly of cardiac explant-derived cells (EDCs) on poly-D-lysine (PDL)-coated plates, but yields inconsistently sized spheroids, restricting broader applications. To address this, we employed ultra-low attachment (ULA) U-well plates to promote uniform spheroid assembly. We systematically compared CSs generated from mouse EDCs using the standard method, based on PDL-coated plates, and the alternative approach, based on ULA U-well plates. Both methods produced viable CSs mimicking the cardiac microenvironment, including mesenchymal cells/fibroblasts, smooth muscle, endothelial, and progenitor cells. PDL-formed CSs were characterized by size heterogeneity, increased stiffness, and reduced endothelial cell content. Despite that, they demonstrated elevated secretion of angiogenesis-related factors and robust proangiogenic potential in vivo. In contrast, generation of mCSs on ULA U-well plates resulted in the formation of soft spheroids with uniform size, enhanced vascularization (CD31+ cells), and increased MCP-1 secretion. In summary, the alternative U-well-based approach enables the generation of uniform spheroids with high spontaneous vascularization, while traditionally formed CSs using PDL-coated plates maintain their superior proangiogenic potential. Full article