Search Results (2,363)

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

In vitro cartilage explant culture has been used to assess nutraceuticals on cartilage responses to inflammatory stimuli. However, applying extracts of nutraceuticals directly to cartilage explants does not account for effects of digestion and hepatic biotransformation, or selective exclusion of product metabolites from joint fluid by the synovial membrane. The current study produced a simulated biological extract of a common nutraceutical (glucosamine; G_sim) by exposing it to a simulated upper gastrointestinal tract digestion, hepatic biotransformation by liver microsomes, and purification to a molecular weight cut-off of 50 kDa. This extract was then used to condition cartilage explants cultured for 120 h in the presence or absence of an inflammatory stimulus (lipopolysaccharide). Media samples were analyzed for prostaglandin E₂ (PGE₂), glycosaminoglycan (GAG), and nitric oxide (NO). Tissue was digested and analyzed for GAG content and stained for viability. Conditioning of explants with G_sim significantly reduced media GAG in stimulated and unstimulated explants and reduced nitric oxide production in unstimulated explants. These data provide evidence for the value of glucosamine in protecting cartilage from deterioration following an inflammatory challenge, and the model improves applicability of these in vitro data to the in vivo setting. Full article

Brain organoid technology has revolutionized in vitro modeling of human neurodevelopment and disease, providing unprecedented insights into cortical patterning, neural circuit assembly, and pathogenic mechanisms of neurological disorders. Critically, human brain organoids uniquely recapitulate human-specific developmental processes—such as the expansion of outer radial glia and neuromelanin—that are absent in rodent models, making them indispensable for studying human brain evolution and dysfunction. However, a major bottleneck persists: Extended culture periods (≥6 months) are empirically required to achieve late-stage maturation markers like synaptic refinement, functional network plasticity, and gliogenesis. Yet prolonged conventional 3D culture exacerbates metabolic stress, hypoxia-induced necrosis, and microenvironmental instability, leading to asynchronous tissue maturation—electrophysiologically active superficial layers juxtaposed with degenerating cores. This immaturity/heterogeneity severely limits their utility in modeling adult-onset disorders (e.g., Alzheimer’s disease) and high-fidelity drug screening, as organoids fail to recapitulate postnatal transcriptional signatures or neurovascular interactions without bioengineering interventions. We summarize emerging strategies to decouple maturation milestones from rigid temporal frameworks, emphasizing the synergistic integration of chronological optimization (e.g., vascularized co-cultures) and active bioengineering accelerators (e.g., electrical stimulation and microfluidics). By bridging biological timelines with scalable engineering, this review charts a roadmap to generate translationally relevant, functionally mature brain organoids. Full article

Cacao genotypes propagation through plant tissue culture represents a strategic approach for establishing a core collection of elite plants to be used as a donor material source, necessary for increasing new planting areas of cacao. This study aimed to evaluate somatic embryo regeneration in ten native fine-aroma cacao genotypes (INDES-06, INDES-11, INDES-14, INDES-32, INDES-52, INDES-53, INDES-63, INDES-64, INDES-66, INDES-70) from the INDES-CES germplasm collection, under in vitro conditions using culture medium supplemented with different concentrations of Thidiazuron (0, 10, and 20 nM). Our results showed an average of 20 and 100% of callogenesis in all genotypes evaluated, but the callus development did not appear after early stages of its induction; however, primary somatic embryos were observed after 42 days after TDZ treatment in the INDES-52, INDES-53, INDES-64, INDES-66, INDES-70 genotypes. The INDES-52 genotype was more responsive to under 20 nM of TDZ, generating an average of 17 embryos per explant. This study contributes to the adaptation and establishment of a protocol for somatic embryo regeneration of fine-flavor cacao genotypes. Full article

Aronia melanocarpa (black chokeberry) is a medicinally valuable small fruit species, yet its commercial propagation remains limited by low rooting and genotype-specific responses. This study developed an efficient, callus-free micropropagation and rooting protocol using a Shrub Plant Medium (SPM) supplemented with 5 mg/L BAP in large 660 mL jars, which yielded up to 27 shoots per explant. Optimal rooting (100%) was achieved with 0.5 mg/L NAA + 0.25 mg/L IBA in half-strength SPM. In the second phase, supervised machine learning models, including Random Forest (RF), XGBoost, Gaussian Process (GP), and Multilayer Perceptron (MLP), were employed to predict morphogenic traits based on culture conditions. XGBoost and RF outperformed other models, achieving R² values exceeding 0.95 for key variables such as shoot number and root length. These results demonstrate that data-driven modeling can enhance protocol precision and reduce experimental workload in plant tissue culture. The study also highlights the potential for combining physiological understanding with artificial intelligence to streamline future in vitro applications in woody species. Full article

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Male-factor infertility accounts for approxiamately half of all infertility cases globally, yet therapeutic options remain limited for individuals with no retrievable spermatozoa, such as those with non-obstructive azoospermia (NOA). In recent years, artificial gametogenesis has emerged as a promising avenue for fertility restoration, driven by advances in two complementary strategies: organotypic in vitro spermatogenesis (IVS), which aims to complete spermatogenesis ex vivo using native testicular tissue, and in vitro gametogenesis (IVG), which seeks to generate male gametes de novo from pluripotent or reprogrammed somatic stem cells. To evaluate the current landscape and future potential of these approaches, a narrative, semi-systematic literature search was conducted in PubMed and Scopus for the period January 2010 to February 2025. Additionally, landmark studies published prior to 2010 that contributed foundational knowledge in spermatogenesis and testicular tissue modeling were reviewed to provide historical context. This narrative review synthesizes multidisciplinary evidence from cell biology, tissue engineering, and translational medicine to benchmark IVS and IVG technologies against species-specific developmental milestones, ranging from rodent models to non-human primates and emerging human systems. Key challenges—such as the reconstitution of the blood–testis barrier, stage-specific endocrine signaling, and epigenetic reprogramming—are discussed alongside critical performance metrics of various platforms, including air–liquid interface slice cultures, three-dimensional organoids, microfluidic “testis-on-chip” devices, and stem cell-derived gametogenic protocols. Particular attention is given to clinical applicability in contexts such as NOA, oncofertility preservation in prepubertal patients, genetic syndromes, and reprocutive scenarios involving same-sex or unpartnered individuals. Safety, regulatory, and ethical considerations are critically appraised, and a translational framework is outlined that emphasizes biomimetic scaffold design, multi-omics-guided media optimization, and rigorous genomic and epigenomic quality control. While the generation of functionally mature sperm in vitro remains unachieved, converging progress in animal models and early human systems suggests that clinically revelant IVS and IVG applications are approaching feasibility, offering a paradigm shift in reproductive medicine. Full article

Skin aging is characterized by compromised epidermal homeostasis and dermo-epidermal junction (DEJ) integrity, resulting in reduced stem cell potential and impaired tissue regeneration. This study investigated the effects of Andrographis paniculata extract (APE) on keratinocyte stem cells (KSCs) and DEJ composition in human skin. Using human skin explants and cell culture models, we demonstrated that APE treatment enhances DEJ composition by increasing Collagen IV and Laminin production while decreasing MMP-9 expression, without altering epidermal structure or differentiation. In the same model, APE preserved stemness potential by upregulating markers related to niche components (collagen XVII and β1-integrin), proliferation (Ki-67 and KRT15), and stem cell capacity (Survivin and LRIG1). In vitro studies revealed that APE selectively stimulated KSC proliferation without affecting transit amplifying cells and promoted Collagen IV and Laminin secretion, particularly in KSCs. Furthermore, in a co-culture model simulating a compromised DEJ (UVB-induced), APE increased Laminin production in KSCs, suggesting a protective effect against photo-damage. These findings indicate that APE enhances DEJ composition and preserves stem cell potential, highlighting its promise as a candidate for skin anti-aging strategies targeting stem cell maintenance and extracellular matrix stability to promote skin regeneration and repair. Full article

Bioactive glass materials have been used for decades in orthopedic surgery, traumatology, and oral and maxillofacial surgery to repair bone defects. This study aimed to evaluate in vitro the survival and proliferation of MG63 human osteosarcoma-derived cells on S53P4 bioactive glass (BonAlive^® granules). Microscopic visualization was performed to directly observe the interactions between the cells and the material. Osteoblast-like cells were examined on non-adherent test plates, on tissue culture (TC)-treated plates and on the surface of the bioglass to assess the differences. Cell survival and proliferation were monitored using a CCK-8 optical density assay. Comparing the mean OD of MG63 cells in MEM on TC-treated plates with cells on BG, we detected a significant difference (p < 0.05), over each time of observation. The sustained cell proliferation confirmed the non-cytotoxic property of the bioglass, as the cell number increased continuously at 48, 72, 96, and 168 h and even did not plateau after 168 h. Since the properties of bioglasses can vary significantly depending on their composition and environment, a thorough characterization of their biocompatibility is crucial to ensure their effective and appropriate application—for example, during hip and knee prosthesis insertion. Full article

Moringa peregrina (Forssk.) Fiori is a tropical tree in southern Iran known as the most important natural coagulant in the world. Today, plant tissue culture is a new method that has a very high potential to produce valuable medicinal compounds on a commercial level. Advances in in vitro cultivation methods have increased the usefulness of plants as renewable resources. In this study, in addition to the phytochemical analysis of the extract of M. peregrina using HPLC, the interaction effect of different concentrations of aqueous extract of M. peregrina (0, 1, 1.5, and 3 mg/L) in two types of MS and ½ MS basal culture media over three weeks on the in vitro growth of Staurogyne repens (Nees) Kuntze was studied. The amounts of quercetin, gallic acid, caffeic acid, and myricetin in the aqueous extract of M. peregrina were 64.9, 374.8, 42, and 4.6 mg/g, respectively. The results showed that using M. peregrina leaf aqueous extract had a positive effect on the length of the branches, the percentage of green leaves, rooting, and the fresh and dry weight of S. repens samples. The highest increase in growth indices was observed in the MS culture medium supplemented with 3 mg/L of M. peregrina leaf aqueous extract after three weeks of cultivation. Of course, this effect was significantly greater in the MS medium and at higher concentrations compared to the ½ MS medium. Three weeks after cultivation at a concentration of 3 mg/L of the extract, the length of the S. repens branches was 5.3 and 1.8 cm in the two basic MS and ½ MS culture media, and the percentage of green leaves was 14 and 4 percent, respectively. Also, rooting was measured at 9.6 and 3.6 percent, fresh weight at 6 and 1.4 g, and dry weight at 1.1 and 0.03 g, respectively. Therefore, adding M. peregrina leaf aqueous extract as a stimulant significantly increased the in vitro growth of S. repens. Full article

Roses are one of the most essential ornamental flowers in the world. At present, traditional techniques such as cross breeding are mainly used in rose breeding. The inefficiency of the in vitro regeneration system has become the limiting step for the innovation and genetic improvement of rose germplasm resources. A tissue culture rapid propagation system of Rosa ‘Pompon Veranda’ was established using the stem segments with shoots as the initial experimental material. The results showed that the best disinfection method was to soak the explants in 75% ethanol for 1 min, and then soak them in 15% sodium hypochlorite solution for 15 min. The contamination rate was only about 6%. The best rooting medium for tissue culture seedlings was 1/2MS with 0.1 mg∙L⁻¹ NAA, and the rooting rate can reach around 95%. On this basis, calluses were induced by using leaflets of tissue-cultured seedlings as explants. The results showed that the optimal medium for inducing callus tissue was MS + 5.0 mg∙L⁻¹ 2,4-D, with an induction rate of 100%. The calluses were cultured in the medium of MS with 0.01 mg∙L⁻¹ NAA, 1.5 mg∙L⁻¹ TDZ and 0.1 mg∙L⁻¹ GA₃ for 12 days in the dark and then transferred to light conditions. The differentiation rate of callus was 10.87%. On the medium of MS with 0.5 mg∙L⁻¹ 6-BA, 0.004 mg∙L⁻¹ NAA and 0.1 mg∙L⁻¹ GA₃, the shoots could regenerate into whole plants. This study has established an in vitro regeneration system of R. ‘Pompon Veranda’, which is a key perquisite for the subsequent establishment of its genetic transformation system. Moreover, this method will also be an important reference for studies on quality traits such as floral scent and prickles of Rosa plants. 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

Osteoporosis is a multifactorial, polygenic disease characterized by reduced bone mineral density (BMD) and increased fracture risk. Genome-wide association studies (GWASs) have identified numerous loci associated with BMD and/or bone fractures, but functional characterization of these target genes is essential to understand the biological mechanisms underlying osteoporosis. This review focuses on current methodologies and key examples of successful functional studies aimed at evaluating gene function in osteoporosis research. Functional evaluation typically follows a multi-step approach. In silico analyses using omics datasets expression quantitative trait loci (eQTLs), protein quantitative trait loci (pQTLs), and DNA methylation quantitative trait loci (mQTLs) help prioritize candidate genes and predict relevant biological pathways. In vitro models, including immortalized bone-derived cell lines and primary mesenchymal stem cells (MSCs), are used to explore gene function in osteogenesis. Advanced three-dimensional culture systems provide additional physiological relevance for studying bone-related cellular processes. In situ analyses of patient-derived bone and muscle tissues offer validation in a disease-relevant context, while in vivo studies using mouse and zebrafish models enable comprehensive assessment of gene function in skeletal development and maintenance. Integration of these complementary methodologies helps translate GWAS findings into biological insights and supports the identification of novel therapeutic targets for osteoporosis. 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