Search Results (713)

Pulmonary fibrosis (PF) is a progressive and fatal lung disease characterized by irreversible alveolar destruction and pathological extracellular matrix (ECM) deposition. Currently approved agents (pirfenidone and nintedanib) slow functional decline but do not reverse established fibrosis or restore functional alveoli. Multifunctional bioscaffolds present a promising therapeutic strategy through targeted modulation of critical cellular processes, including proliferation, migration, and differentiation. This review synthesizes recent advances in scaffold-based interventions for PF, with a focus on their dual mechano-epigenetic regulatory functions. We delineate how scaffold properties (elastic modulus, stiffness gradients, dynamic mechanical cues) direct cell fate decisions via mechanotransduction pathways, exemplified by focal adhesion–cytoskeleton coupling. Critically, we highlight how pathological mechanical inputs establish and perpetuate self-reinforcing epigenetic barriers to regeneration through aberrant chromatin states. Furthermore, we examine scaffolds as platforms for precision epigenetic drug delivery, particularly controlled release of inhibitors targeting DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) to disrupt this mechano-reinforced barrier. Evidence from PF murine models and ex vivo lung slice cultures demonstrate scaffold-mediated remodeling of the fibrotic niche, with key studies reporting substantial reductions in collagen deposition and significant increases in alveolar epithelial cell markers following intervention. These quantitative outcomes highlight enhanced alveolar epithelial plasticity and upregulating antifibrotic gene networks. Emerging integration of stimuli-responsive biomaterials, CRISPR/dCas9-based epigenetic editors, and AI-driven design to enhance scaffold functionality is discussed. Collectively, multifunctional bioscaffolds hold significant potential for clinical translation by uniquely co-targeting mechanotransduction and epigenetic reprogramming. Future work will need to resolve persistent challenges, including the erasure of pathological mechanical memory and precise spatiotemporal control of epigenetic modifiers in vivo, to unlock their full therapeutic potential. Full article

Background/Objectives: The interaction between brain-metastasizing melanoma cells and surrounding microglia shapes the immune tumor microenvironment and influences tumor progression. Gene expression analysis revealed that sphingosine-1-phosphate receptor 1 (S1PR1), encoding the S1P1 receptor, is upregulated in microglia upon interaction with melanoma cells. Here, we investigated the functions of S1P1 in microglia and its contribution to melanoma–microglia crosstalk. Methods: We examined the effects of S1P1 inhibition on microglia and four brain-metastasizing human melanoma cell lines in monocultures and co-cultures using the selective S1P1 antagonist NIBR0213 and S1PR1 gene knockdown. Results: We found that melanoma-secreted IL-6 upregulated S1PR1 expression in microglia. S1P1 inhibition increased expression of CD32, CD150, and CD163 in microglia; however, CD150 and CD163 upregulation was abolished in the presence of melanoma cells. S1P1 inhibition downregulated immunosuppressive and anti-inflammatory factors in microglia, including CD274, SOCS3, TGFBR1, TGFBR2, and JunB, promoting a pro-inflammatory phenotype. It also reduced viability of both melanoma and microglia cells, inducing apoptosis in melanoma-associated microglia, possibly via downregulation of CH25H, an upstream regulator of SREBPs. In co-cultures, melanoma cells were more sensitive than microglia to NIBR0213-induced growth arrest. In 3D spheroid cultures, NIBR0213 delayed melanoma–microglia aggregation. Combined treatment with the BRAF inhibitor Vemurafenib and NIBR0213 enhanced Vemurafenib efficacy in three of four melanoma lines. Conclusions: S1P1 contributes to the immunosuppressive phenotype of microglia. Inhibiting the S1P/S1P1 axis impairs viability and crosstalk between melanoma cells and tumor-activated microglia, offering a potential therapeutic strategy for melanoma brain metastases. Full article

Mesenchymal stem cells (MSCs) spontaneously assemble into three-dimensional (3D) spheroids under matrix-deficient conditions such as the synovial cavity, although their functional significance has yet to be fully elucidated. In this study, we used concave microwell cultures to promote the spontaneous aggregation of adipose-derived MSCs (ASCs) from OA patients, thereby mimicking the intra-articular microenvironment. We analyzed the paracrine factors of ASC aggregates and compared it with that of conventional 2D monolayer cultures. Notably, 3D aggregation significantly increased the secretion of HGF and VEGF, whereas FGF2 levels remained relatively unchanged. These results indicate that the structural characteristics of ASC aggregates enhance the secretion of key paracrine factors involved in angiogenesis and tissue repair. To functionally evaluate the biological relevance of the secreted factors, conditioned media (CM) from ASC aggregates were applied to human articular chondrocytes. The CM significantly promoted chondrocyte proliferation, an effect that was abolished by the addition of HGF-neutralizing antibodies, thereby highlighting HGF as a central mediator of the regenerative response. Additionally, we further explored whether extracellular factors could modulate growth factor expression such as HGF. In this context, we investigated the impact of low-concentration hyaluronic acid (HA), a key synovial component widely used in OA treatment. Co-treatment with HA not only amplified the expression and secretion of HGF, VEGF, and FGF2, but also promoted ASC proliferation. ASCs forming functional aggregates may exert regenerative effects as active paracrine modulators, and the addition of low-dose hyaluronic acid is expected to further enhance this function, offering a promising strategy for MSC-based osteoarthritis therapy. Full article

Organoids, self-organizing, three-dimensional (3D) multicellular structures derived from tissues or stem cells, offer physiologically relevant models for studying human development and disease. Compared to conventional two-dimensional (2D) cell cultures and animal models, organoids more accurately recapitulate the architecture and function of human organs. Among the critical microenvironmental cues influencing organoid behavior, hypoxia and multilineage communication are particularly important for guiding cell fate, tissue organization, and pathological modeling. Hypoxia, primarily regulated by hypoxia-inducible factors (HIFs), modulates cellular proliferation, differentiation, metabolism, and gene expression, making it a key component in disease modeling. Similarly, multilineage communication, facilitated by intercellular interactions and extracellular matrix (ECM) remodeling, enhances organoid complexity and immunological relevance. This review explores the dynamic interplay between hypoxia and multilineage signaling in 3D organoid-based disease models, emphasizing recent advances in engineering hypoxic niches and co-culture systems to improve preclinical research fidelity. We also discuss their translational implications for drug screening, regenerative medicine, and precision therapies, while highlighting current challenges and future opportunities. By integrating biophysical, biochemical, and computational approaches, next-generation organoid models may be further optimized for translational research and therapeutic innovation. Full article

Articular cartilage regeneration remains a major challenge due to its limited self-repair capacity. Bone marrow-derived skeletal stem cells (SSCs) and mesenchymal stem cells (MSCs) are promising candidates for cartilage engineering, although they differ in their chondrogenic potential. This study explored whether co-culturing SSCs and MSCs in three-dimensional (3D) organoid systems under cartilage physioxia (5% O₂) and chondrogenic induction could improve cartilage tissue formation. SSCs, MSCs, and SSC–MSC co-cultures were characterized for morphology, phenotype, and differentiation capacity. Organoids were generated and cultured for 10 days, followed by analysis of morphology, viability, gene expression (SOX9, RUNX2, ACAN, COL2A1, COL10A1, PRG4, and PDPN), chondrocyte-associated antigens (CD44, CD105, CD146, and PDPN), and cartilage ECM proteins (aggrecan, collagen types I, II, and X, and PRG4). SSCs showed robust chondrogenic and osteogenic potential, while MSCs exhibited a balanced multipotency. Co-culture-derived organoids enhanced chondrogenesis and reduced adipogenesis, with higher expression of cartilage-specific ECM and lower hypertrophic marker levels. These findings highlight the functional synergy between SSCs and MSCs in co-culture, promoting the formation of stable, cartilage-like structures under physioxia. The approach offers a promising strategy for generating preclinical models and advancing regenerative therapies for hyaline cartilage repair. Full article

The basal forebrain (BF)-hippocampus (HPC) circuit is indispensable for learning and memory, and in vitro models are essential for dissecting its age-related decline. Nonetheless, current culture methods endure brief survival or confine cells to two dimensions, leaving the circuit’s progressive degeneration refractory to long-term investigation. Here, we developed a simple, three-dimensional (3D) compartmentalized co-culture model that mimics the anatomical organization of BF and HPC neurons. Results demonstrate that basal forebrain cholinergic neurons (BFCNs) co-cultured with primary HPC neurons remain viable for more than two months without exogenous growth factors, significantly promoting BFCNs growth, polarity development, and functional maturation. In this system, BFCNs somata were confined within the hydrogel, whereas cholinergic axons extended toward adjacent hippocampal area, reaching 1681.9 ± 351.8 μm by week 5—significantly longer than in BFCNs monocultures. This model can successfully recapitulate age-dependent progressive neuronal degeneration during long-term culture, validating this long-term co-culture as a platform for studying circuit aging and degeneration. Therefore, this low-cost and highly physiological platform provides a new avenue for in-depth investigations into the mechanisms of neurodegenerative diseases. Full article

Syngas fermentation in combination with chain elongation offers great promise for sustainable medium-chain fatty acid production. While immobilization has proven effective for stabilizing monocultures of C. kluyveri for chain elongation, its applicability to co-cultures involving C. carboxidivorans for simultaneous syngas fermentation remains unexplored. This study investigates the physiological compatibility of C. carboxidivorans with agar-based hydrogel immobilization and its co-cultivation potential with C. kluyveri in a synthetic dual-layer biofilm reactor. First, we conducted autotrophic batch fermentations using suspended and immobilized cells, proving metabolic activity similar for both. Applying different sulfur feeding rates, experiments showed best ethanol formation with C. carboxidivorans at increased sulfur feeding, enabling better conditions for chain elongation with C. kluyveri. In the synthetic dual-layer biofilm reactor, with the C. carboxidivorans biofilm in contact with the CO-containing gas phase above the C. kluyveri biofilm, the formation of 1-butyrate and 1-hexanoate was observed with product formation rates of 0.46 g L⁻¹ d⁻¹ 1-butyrate, and 0.91 g L⁻¹ d⁻¹ 1-hexanoate, respectively. The formation rate of 1-hexanoate in the dual-layer biofilm reactor was approximately 7.6 times higher than that reported with suspended cells in a stirred tank bioreactor. Spatial analysis revealed species-specific migration behavior and confirmed that C. carboxidivorans reduced local CO concentrations, improving the environment for C. kluyveri. Full article

Intestinal disorders such as inflammatory bowel diseases (IBDs), Crohn’s disease, malabsorption syndromes, and gastrointestinal fistulae (GIFs) are often characterized by chronic inflammation, epithelial barrier disruption, impaired stromal remodeling, and defective angiogenesis. These multifactorial alterations hinder tissue repair and contribute to poor clinical outcomes, with limited efficacy from current therapeutic options. Despite recent advances in surgical and endoscopic techniques, current treatment options remain limited and are frequently accompanied by high morbidity and costs. In this context, regenerative medicine offers a promising avenue to support tissue repair and improve patient care Regenerative medicine offers a promising avenue to restore intestinal homeostasis using advanced biomaterials and cell-based therapies. In this study, we developed a 3D-bioprinted model based on patient-derived stromal vascular fraction (SVF) embedded in a GelMA hydrogel, designed to promote intestinal tissue regeneration. To identify the most suitable hydrogel for bioprinting, we initially evaluated the mechanical properties and biocompatibility of four distinct matrices using bone marrow-derived mesenchymal stromal cells (BM-MSCs). Among the tested formulations, GelMA demonstrated optimal support for cell viability, low oxidative stress, and structural stability in physiologically relevant conditions. Based on these results, GelMA was selected for subsequent bioprinting of freshly isolated SVF. The resulting bioprinted constructs enhanced key regenerative processes across multiple compartments. The SVF-laden constructs significantly enhanced intestinal epithelial cell viability and tight junction formation, as shown by increased trans-epithelial electrical resistance (TEER). Co-culture with fibroblasts accelerated wound closure, while endothelial cells exhibited increased tube formation in the presence of SVF. Together with VEGF secretion, indicating strong paracrine and angiogenic effects. By supporting epithelial, stromal, and vascular regeneration, this approach provides a versatile and translational platform for treating a broad spectrum of intestinal pathologies. Full article

Bone remodeling relies on the coordinated activity of osteoblasts (OBs) and osteoclasts (OCs). Disruptions in OB-OC balance can lead to diseases such as periodontitis, a chronic microbial-induced inflammatory disease. To investigate how inflammation affects OB-OC interactions, we standardized an in vitro 2D indirect co-culture system using primary human OB and OC precursors from peripheral blood mononuclear cells in a transwell setup, which allows paracrine signaling and separate analysis of each cell type. When exposed to bacterial lipopolysaccharides (Aa LPS and E. coli LPS) and proinflammatory cytokines (IL-6 and TNF-α), we observed that inflammatory stimuli significantly increased OC differentiation, particularly TNF-α, while E. coli LPS specifically suppressed OB activity as observed by the expression of key markers and cellular staining. These results demonstrate that microbial and host-derived inflammatory factors can differentially modulate bone cell behavior. This approach offers a physiologically relevant and ethically advantageous alternative to animal models to screen dual-targeted bone therapies to restore perturbed metabolism. Full article

The Helicobacter pylori vapD gene is transcribed and expressed when the bacteria are within the gastric cell. In this current study, we investigated how vapD knockout affects the survival of H. pylori inside human gastric adenocarcinoma cells. We constructed an H. pylori 26695 vapD (Hp ΔvapD) mutant strain. H. pylori 26695 wt and Hp ΔvapD strains were grown in synthetic media and were co-cultured with AGS cells. From the start, the growth curve, total protein concentration and colony-forming units (CFUs) of each strain were measured. From each co-culture, CFUs and total RNA were obtained, and transcript levels of GAPDH, vapD, vacA, ureA, and 16s Hp were measured by qRT-PCR. Hp ΔvapD did not affect the growth rate of the strain in synthetic media, showing that the vapD gene is not necessary when the bacteria grow outside eukaryote cells. However, in the intracellular environment, the number of CFUs recovered from the Hp ΔvapD strain from AGS cells decreased after 36 h. Transcription levels of the vacA gene from the Hp ΔvapD strain were 10,000-fold lower than those of H. pylori wt, to the point of being undetectable. The results suggest that the vapD gene contributed to maintaining H. pylori inside gastric cells. Full article

Vitiligo is a chronic autoimmune dermatosis defined by selective melanocyte depletion and patchy depigmentation. IFN–γ-driven recruitment of autoreactive CD8⁺ T cells and induction of melanocyte apoptosis are central to its pathogenesis. Current therapies—including UVB phototherapy, tacrolimus, vitamin D3 analogs, and surgical methods—show limited and inconsistent efficacy. Emerging treatments like JAK inhibitors and WNT activators offer potential but require further validation. Translational progress is hindered by a lack of scalable human models. Here, we describe a tunable in vitro vitiligo platform in which human iPSC-derived melanocytes (iMc) are co-cultured with keratinocytes on Matrigel and exposed to precise graded IFN-γ concentrations. Our data revealed dose-dependent decreases in iMc survival and dendritic structure, faithfully mirroring derived melanocyte pathology. Leveraging this platform, we first evaluated the short-term efficacy of the ROCK inhibitor Y27632 under early-stage patient IFN-γ concentrations representative of patient lesional thresholds. At three days, Y27632 significantly upregulated adhesion molecules E-cadherin and DDR1, and two central factors—ET1 and bFGF. Importantly, ROCK inhibition reversed dendritic retraction and improved overall viability of iMc-keratinocytes. These findings position ROCK blockade as a promising adjunctive strategy and establish a pre-clinical platform for evaluating combination therapies for durable pigment restoration. Full article

Plexiform neurofibromas (hereafter called pNF1) are often diagnosed in early childhood and occur in about 30% of neurofibromatosis type 1 (NF1) patients. pNF1 exhibits aggressive growth along a nerve in the body and has substantial potential for progression to malignant peripheral nerve sheath tumors that are rarely curable. There are two recently FDA-approved drugs, selumetinib and mirdametinib, for pNF1 patients who have symptomatic and inoperable plexiform neurofibromas; however, these treatments achieve only approximately 30% tumor shrinkage. Fibroblasts, the most abundant cell types within the pNF1 tumor microenvironment, are implicated in pNF1 growth and invasion; however, how fibroblasts affect a drug response of pNF1 remains poorly understood. In the present study, we focused on contributions of fibroblasts to the drug resistance in pNF1 via their secretome. We employed our established three-dimensional (3D) culture system incorporating human pNF1 tumor cells (Nf1^−/−) and primary fibroblasts (Nf1^+/−) grown in our patented microfluidic culture chips for monocultures and parallel cocultures in which 3D pNF1 structures and fibroblasts share their secretome without direct cell-to-cell contact. Three-dimensional pNF1 structures in 3D parallel cocultures with fibroblasts exhibited greater drug resistance than ones in monocultures. We found that pNF1 tumor cells showed increased P-glycoprotein expression when incubated with fibroblast-derived conditioned media or parallel cocultured with fibroblasts, compared to control conditions. Pharmacological inhibition of P-glycoprotein partially restored drug sensitivity. Additionally, fibroblasts showed higher resistance to selumetinib and mirdametinib than pNF1 tumor structures, likely due to elevated P-glycoprotein levels. This study is the first to define precise roles of fibroblasts in pNF1 drug resistance, emphasizing the potential of fibroblast-targeted therapies as a promising approach to improve pNF1 treatment outcomes. Full article

Adipose tissue enlargement in obesity leads to hypoxia, which may promote premature aging. This study aimed to understand the hypoxic response in 3D cultures of SGBS cells, a model for brown-like adipose tissue expressing uncoupling protein 1 (UCP1). Single-nucleus RNA sequencing of SGBS organoids revealed a heterogeneous composition and sub-population-specific responses to hypoxia. The analysis identified a cluster of transcriptional repression, indicating dying cells, and implied a role of ferroptosis in this model. Further experiments with SGBS cells and white adipose tissue-derived stem/progenitor cells showed that Acyl-CoA synthetase long-chain family member 4 (ACSL4), a key enzyme in ferroptosis, is expressed only in the presence of browning factors. Hypoxia downregulated ACSL4 protein in SGBS organoids but induced an inflammaging phenotype. Analysis of brown-like epicardial adipose tissue from cardiac surgery patients revealed a significant positive correlation of ACSL4 mRNA with UCP1 and hypoxia-inducible pro-inflammatory markers, while ACSL4 protein appeared to be inversely correlated. In conclusion, this study demonstrates that adipocytes’ capability to undergo ACSL4-mediated ferroptosis is linked to brown-like adipogenesis, suggesting an opportunity to modulate ferroptotic signaling in adipose tissue. The dual role of hypoxia by inhibiting ACSL4 but promoting inflammaging indicates a relationship between ferroptosis and aging that warrants further investigation. Full article

Background/Objectives: Paclitaxel (PTX) faces clinical limitations in melanoma treatment due to poor solubility, P-glycoprotein (P-gp)-mediated efflux, and systemic toxicity. This study aimed to develop PTX-loaded mesoporous silica nanoparticles (PS), which would be co-administered with curcumin (CUR) and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) to enhance intracellular accumulation and improve anti-tumor activity. CUR and TPGS were integrated with PS to inhibit P-gp-mediated PTX-efflux, to enhance the intracellular accumulation of PTX, and to improve anti-tumor activity in B16F10 cells. Methods: The physicochemical properties of PS were analyzed using standard characterization methods. The antitumor activity of PS co-administered with CUR and TPGS was evaluated using two-dimensional (2D) culture and three-dimensional (3D) spheroid assays, and also assessed in B16F10 tumor-bearing mice. The therapeutic mechanism of the PS combination was compared using apoptosis and microtubule disruption through flow cytometry and confocal microscopy. The pharmacokinetics and biodistribution of the PS combination were compared in B16F10 tumor-bearing mice. Results: PS formulations exhibited amorphous transformation with an approximate particle size of 200 nm. PS co-administered with CUR and TPGS reduced the IC₅₀ to 178.7 nM compared with 283.3 nM for free PTX in B16F10 melanoma cells and achieved significant tumor growth inhibition in B16F10 melanoma spheroid culture. The intracellular accumulation of PTX correlated with its therapeutic efficacy. Flow cytometry revealed a significant induction of both early and late apoptosis in cells treated with the PS + CUR + TPGS combination, while confocal imaging confirmed enhanced microtubule disruption. In B16F10 tumor-bearing mice, PS co-administered with CUR and TPGS demonstrated higher and selective distribution of PTX into tumor tissue without affecting systemic exposure of PTX in B16F10-xenografted mice. Conclusions: PS + CUR + TPGS combination enhanced PTX delivery by improving solubility and enhancing distribution to tumor tissue through P-gp inhibition, thereby increasing its therapeutic potential. The combination of CUR and TPGS offers synergistic apoptosis induction and microtubule disruption. Thus, the PS + CUR + TPGS combination represents a promising approach for treating drug-resistant melanomas. Full article

Two-dimensional cell culture systems lack the ability to replicate the complex, three-dimensional (3D) architecture and cellular microenvironments found in vivo. Multicellular spheroids (MCSs) present a promising alternative, with the ability to mimic native cell–cell and cell–matrix interactions and provide 3D architectures similar to in vivo conditions. These factors are critical for various biomedical applications, including cancer research, tissue engineering, and drug discovery and development. Polymeric materials such as hydrogels, solid scaffolds, and ultra-low attachment surfaces serve as versatile platforms for 3D cell culture, offering tailored biochemical and mechanical cues to support cellular organization. This review article focuses on the structure–property relationships of polymeric biomaterials that influence MCS formation, growth, and functionality. Specifically, we highlight their physicochemical properties and their influence on spheroid formation using key natural polymers, including collagen, hyaluronic acid, chitosan, and synthetic polymers like poly(lactic-co-glycolic acid) and poly(N-isopropylacrylamide) as examples. Despite recent advances, several challenges persist, including spheroid loss during media changes, limited viability or function in long-term cultures, and difficulties in scaling for high-throughput applications. Importantly, the development of MCS platforms also supports the 3R principle (Replacement, Reduction, and Refinement) by offering ethical and physiologically relevant alternatives to animal testing. This review emphasizes the need for innovative biomaterials and methodologies to overcome these limitations, ultimately advancing the utility of MCSs in biomedical research. Full article