Search Results (3,620)

Since the last century, it has been shown that dedifferentiated cells of Camellia sinensis can produce catechins and other secondary metabolites under in vitro conditions, with potential applications in the cosmetic, pharmaceutical and food industries. In this work, cell suspension cultures of a C. sinensis cell line (LSC-5Y) were established in a liquid medium in order to optimize the biomass productivity, catechin monomer (GC, EGC, C, EC, CG, and ECG) and alkaloid (TB and CAF) productivity. The following factors were evaluated: concentration of growth regulators (BA and IBA), inoculum size, age of the cell line, light exposure, and effect of biotic elicitors (MeJA and extracts of Ciborinia camelliae). GC, EGC, and ECG increased approximately 1.80-fold when the auxin IBA concentration was increased from 0.1 to 2.0 mg/L. In addition, better productivity of EGC, C, EC, and CAF was achieved by using inoculum densities between 50 and 100 g/L. Although lower inoculum densities (25 g/L) showed a higher growth rate (0.20 d⁻¹), the use of inoculum densities higher than 25 g/L favors a 2–4-fold increase in total catechin (TC) productivity, with maximum productivity being reached after 21 days of culture. However, the cell line showed instability in TC productivity: in the short term (in three successive subcultures), the coefficient of variation was 32.80%, and catechin production capacity was 2.5 years with maximum productivity at 0.5 years. Finally, it was observed that ethanol, used as an elicitor solvent, has a strong elicitor effect capable of increasing the accumulation of catechins up to 5.24 times compared to the treatment without an elicitor. Full article

Korean mistletoe (Viscum album L. var. coloratum) is renowned for its medicinal properties, including anti-cancer and immunoadjuvant effects. This study aimed to elucidate the mechanisms by which Korean mistletoe lectin (V. album L. var. coloratum agglutinin; VCA) modulates breast cancer cell apoptosis and macrophage polarization. The specific objectives were to (1) investigate the direct effects of VCA on MCF-7 breast cancer cells and THP-1-derived M1/M2 macrophages; (2) analyze the impact of VCA on the paracrine interactions between these cell types; and (3) compare the efficacy of VCA in 2D vs. 3D co-culture models to bridge the gap between in vitro and in vivo studies. We employed both 2D and 3D models, co-culturing human M1/M2 macrophages with human MCF-7 breast cancer cells in a Transwell system. Our research demonstrated that M1 and M2 macrophages significantly influenced the immune and apoptotic responses of breast cancer cells when exposed to VCA. M1 macrophages exhibited cytotoxic characteristics and enhanced VCA-induced apoptosis in both 2D and 3D co-culture models. Conversely, M2 macrophages initially displayed a protective effect by reducing apoptosis in breast cancer cells, but this protective effect was reversed upon exposure to VCA. Furthermore, our findings illustrate VCA’s ability to modulate M1 and M2 polarization in breast cancer cells. Finally, the use of magnetic 3D cell cultures suggests their potential to yield results comparable to conventional 2D cultures, bridging the gap between in vitro and in vivo studies. Full article

Diabetic retinopathy (DR) is one of the most prevalent secondary complications associated with diabetes. Specifically, Type 1 Diabetes Mellitus (T1D) has an immune component that may determine the evolution of DR by compromising the immune response of the retina, which is mediated by microglia. In the early stages of DR, the permeabilization of the blood–retinal barrier allows immune cells from the peripheral system to interact with the retinal immune system. The use of new bioactive molecules, such as 3-(2,4-dihydroxyphenyl)phthalide (M9), with powerful anti-inflammatory activity, might represent an advance in the treatment of diseases like DR by targeting the immune systems responsible for its onset and progression. Our research aimed to investigate the molecular mechanisms involved in the interaction of specific cells of the innate immune system during the progression of DR and the reduction in inflammatory processes contributing to the pathology. In vitro studies were conducted exposing Bv.2 microglial and Raw264.7 macrophage cells to proinflammatory stimuli for 24 h, in the presence or absence of M9. Ex vivo and in vivo approaches were performed in BB rats, an animal model for T1D. Retinal explants from BB rats were cultured with M9. Retinas from BB rats treated for 15 days with M9 via intraperitoneal injection were analyzed to determine survival, cellular signaling, and inflammatory markers using qPCR, Western blot, or immunofluorescence approaches. Retinal structure images were acquired via Spectral-Domain–Optical Coherence Tomography (SD-OCT). Our results show that the treatment with M9 significantly reduces inflammatory processes in in vitro, ex vivo, and in vivo models of DR. M9 works by inhibiting the proinflammatory responses during DR progression mainly affecting immune cell responses. It also induces an anti-inflammatory response, primarily mediated by microglial cells, leading to the synthesis of Arginase-1 and Hemeoxygenase-1(HO-1). Ultimately, in vivo administration of M9 preserves the retinal integrity from the degeneration associated with DR progression. Our findings demonstrate a specific interaction between both retinal and systemic immune cells in the progression of DR, with a differential response to treatment, mainly driven by microglia in the anti-inflammatory action. In vivo treatment with M9 induces a switch in immune cell phenotypes and functions that contributes to delaying the DR progression, positioning microglial cells as a new and specific therapeutic target in DR. Full article

Stem cell-based therapy holds promise for cartilage regeneration in treating knee osteoarthritis (KOA). Injectable hydrogels have been developed to mimic the extracellular matrix (ECM) and facilitate stem cell growth, proliferation, and differentiation. However, these hydrogels face limitations such as poor mechanical strength, inadequate biocompatibility, and suboptimal biodegradability, collectively hindering their effectiveness in cartilage regeneration. This study introduces an injectable, biodegradable, and self-healing hydrogel composed of chitosan–PEG and PEG–dialdehyde for stem cell delivery. This hydrogel can form in situ by blending two polymer solutions through injection at physiological temperature, encapsulating human adipose-derived stem cells (hADSCs) during the gelation process. Featuring a 3D porous structure with large pore size, optimal mechanical properties, biodegradability, easy injectability, and rapid self-healing capability, the hydrogel supports the growth, proliferation, and differentiation of hADSCs. Notably, encapsulated hADSCs form 3D spheroids during proliferation, with their sizes increasing over time alongside hydrogel degradation while maintaining high viability for at least 10 days. Additionally, hADSCs encapsulated in this hydrogel exhibit upregulated expression of chondrogenic differentiation genes and proteins compared to those cultured on 2D surfaces. These characteristics make the chitosan–PEG/PEG–dialdehyde hydrogel–stem cell construct suitable for direct implantation through minimally invasive injection, enhancing stem cell-based therapy for KOA and other cell-based treatments. Full article

Globally, a number of diseases impact us and while treatment options exist, it is often found that similar treatments have variable effects on different patients with the same disease. Particularly in the case of conditions that are closely associated with genetics (like cancer), the intensity and results of a treatment vary between patients. Even for diseases like arthritis it is not uncommon for only a fraction of patients to achieve remission with the same therapeutic approach. With millions suffering from diseases like cancer and arthritis, precision medicine (PM) has been at the forefront of biomedical and pharmaceutical research since 2015. PM focusses on understanding the genetic and environmental factors affecting the patients and has several platforms. One of the platforms is the use of three-dimensional (3D) in vitro models, especially those derived from the patient themselves. These models, like organ-on-chip (OOC), organoid and spheroid models, 3D biomaterial scaffolds and others, have several advantages over traditional two-dimensional (2D) cell culture approaches. In this opinion paper, the author briefly discusses the different platforms used for PM. Then, the advantages that 3D in vitro models have over traditional 2D models and in vivo models are considered and an overview of their applications is provided. Finally, the author outlines the challenges and future directions and shares their opinion about using 3D in vitro models as a tool for PM towards enhanced patient outcomes. Full article

The close interaction between neurons and astrocytes has been extensively studied. However, the specific behavior of these cells after ischemia-reperfusion injury and hypothermia remains poorly characterized. A growing body of evidence suggests that mitochondria function and putative transference between neurons and astrocytes may play a fundamental role in adaptive and homeostatic responses after systemic insults such as cardiac arrest, which highlights the importance of a better understanding of how neurons and astrocytes behave individually in these settings. Brain injury is one of the most important challenges in post-cardiac arrest syndrome, and therapeutic hypothermia remains the single, gold standard treatment for neuroprotection after cardiac arrest. In our study, we modeled ischemia-reperfusion injury by using in vitro enhanced oxygen-glucose deprivation and reperfusion (eOGD-R) and subsequent hypothermia (HPT) (31.5 °C) to cell lines of neurons (HT-22) and astrocytes (C8-D1A) with/without hypothermia. Using cell lysis (LDH; lactate dehydrogenase) as a measure of membrane integrity and cell viability, we found that neurons were more susceptible to eOGD-R when compared with astrocytes. However, they benefited significantly from HPT, while the HPT effect after eOGD-R on astrocytes was negligible. Similarly, eOGD-R caused a more significant reduction in adenosine triphosphate (ATP) in neurons than astrocytes, and the ATP-enhancing effects from HPT were more prominent in neurons than astrocytes. In both neurons and astrocytes, measurement of reactive oxygen species (ROS) revealed higher ROS output following eOGD-R, with a non-significant trend of differential reduction observed in neurons. HPT after eOGD-R effectively downregulated ROS in both cells; however, the effect was significantly more effective in neurons. Lipid peroxidation was higher after eOGD-R in neurons, while in astrocytes, the increase was not statistically significant. Interestingly, HPT had similar effects on the reduction in lipoperoxidation after eOGD-R with both types of cells. While glutathione (GSH) levels were downregulated after eOGD-R in both cells, HPT enhanced GSH in astrocytes, but worsened GSH in neurons. In conclusion, neuron and astrocyte cultures respond differently to eOGD-R and eOGD-R + HTP treatments. Neurons showed higher sensitivity to ischemia-reperfusion insults than astrocytes; however, they benefited more from HPT therapy. These data suggest that given the differential effects from HPT in neurons and astrocytes, future therapeutic developments could potentially enhance HPT outcomes by means of neuronal and astrocytic targeted therapies. Full article

Human mesenchymal stromal cells (hMSCs), whether used alone or together with three-dimensional scaffolds, are the best-studied postnatal stem cells in regenerative medicine. In this study, innovative composite scaffolds consisting of a core–shell architecture were seeded with bone-marrow-derived hMSCs (BM-hMSCs) and tested for their biocompatibility and remarkable capacity to promote and support bone regeneration and mineralization. The scaffolds were prepared by grafting three different amounts of gelatin–chitosan (CH) hydrogel into a 3D-printed polylactic acid (PLA) core (PLA-CH), and the mechanical and degradation properties were analyzed. The BM-hMSCs were cultured in the scaffolds with the presence of growth medium (GM) or osteogenic medium (OM) with differentiation stimuli in combination with fetal bovine serum (FBS) or human platelet lysate (hPL). The primary objective was to determine the viability, proliferation, morphology, and spreading capacity of BM-hMSCs within the scaffolds, thereby confirming their biocompatibility. Secondly, the BM-hMSCs were shown to differentiate into osteoblasts and to facilitate scaffold mineralization. This was evinced by a positive Von Kossa result, the modulation of differentiation markers (osteocalcin and osteopontin), an expression of a marker of extracellular matrix remodeling (bone morphogenetic protein-2), and collagen I. The results of the energy-dispersive X-ray analysis (EDS) clearly demonstrate the presence of calcium and phosphorus in the samples that were incubated in OM, in the presence of FBS and hPL, but not in GM. The chemical distribution maps of calcium and phosphorus indicate that these elements are co-localized in the same areas of the sections, demonstrating the formation of hydroxyapatite. In conclusion, our findings show that the combination of BM-hMSCs and PLA-CH, regardless of the amount of hydrogel content, in the presence of differentiation stimuli, can provide a construct with enhanced osteogenicity for clinically relevant bone regeneration. Full article

Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks. Full article

The regeneration of plant somatic cells is a prerequisite for their biological breeding. Identification of key genes controlling embryogenic callus (EC) differentiation and investigation of the genetic mechanism of cell fate determination are important for improving plant variety. In this study, we used the maize inbred line KN5585 and its gene-edited mutants Zmprx19-1, Zmprx19-2 and Zmprx19-3 as plant materials. Three somatic regeneration-related traits, the embryogenic callus induction rate (EIR), green callus rate (GCR) and plantlet regeneration rate (PRR), were identified by tissue culture of immature embryos. Additionally, the ECs at different differentiation stages (0 d, 5 d, 10 d and 15 d) were subjected to RNA-seq, and comparative transcriptome analyses were performed. The results showed that the somatic regeneration traits of the mutants were all highly significantly lower than those of the wild type (p < 0.01). The PRR value of KN5585 was 75.25%, while the highest PRR of the mutants was only 15.08%, indicating that knockdown of ZmPRX19 inhibited EC regeneration. Transcriptome sequencing yielded a total of 200.30 Gb of clean data from 24 libraries, with an average of 6.53 Gb of clean data per library. Mutant and wild-type gene expression data were compared separately at four differentiation stages, and 689 common differentially expressed genes (DEGs) were screened. WGCNA was used to classify these genes into nine modules, which were subsequently subjected to GO and KEGG enrichment analyses. In total, 40, 23, 17 and 5 genes were significantly (q < 0.05) enriched in plant hormone signal transduction, the MAPK signaling pathway-plant, phenylpropanoid biosynthesis and photosynthesis, respectively. Moreover, protein–protein interaction (PPI) network analysis revealed five MAPKKK17_18 hub nodes involved in the MAPK pathway-plant, which may be the key genes controlling plantlet differentiation from ECs. The above results provide a basis for the final elucidation of the molecular mechanism of plant somatic regeneration. Full article

The microvasculature, which makes up the majority of the cardiovascular system, plays a crucial role in the process of thrombosis, with the pathological formation of blood clots inside blood vessels. Since blood microflow conditions significantly influence platelet activation and thrombosis, accurately mimicking the structure of bifurcating microvascular networks and emulating local physiological blood flow conditions are valuable for understanding blood clot formation. In this work, we present an in vitro model for blood clotting in microvessels, focusing on 3D bifurcations that align with Murray’s law, which guides vascular networks by maintaining a constant wall shear rate throughout. Using these models, we demonstrate that microvascular bifurcations act as sites facilitating thrombus formation compared to straight models. Additionally, by culturing endothelial cells on the luminal surfaces of the models, we show the potential of using our in vitro platforms to recapitulate the initial clotting in diseases involving endothelial dysfunction, such as Thrombotic Thrombocytopenic Purpura. Full article

Nano-hydroxyapatite (nHA) demonstrates favorable biological activity, cell adhesion, cell proliferation, and osteoconductivity, making it highly valuable in biomedicine. It is extensively used as a bone substitute and in bone transplantation within the dental and orthopedic fields. This study employed oyster shells as a calcium source to synthesize nHA at 150 °C with various hydrothermal reaction durations (10 min, 1 h, 6 h, and 12 h). As a control, HA synthesized via a wet precipitation method for 1 h at room temperature was utilized. Subsequent material analyses, including XRD, FE-SEM, FTIR, and ICP-MS, were conducted, followed by comprehensive evaluations of the bioactivity, cell attachment, cell proliferation, and sintering properties of the synthesized nHA. The results indicated that nHA synthesized through the hydrothermal reaction produced nanoscale crystals, with the aspect ratio of nHA particles increasing with the duration of hydrothermal treatment. Notably, rod-like nHA particles became prominent with hydrothermal durations exceeding 6 h. nHA particles derived from oyster shells contained carbonate and trace elements (Na, Mg, K, and Sr), similar to constituents found in human hard tissue such as bone and teeth. The immersion of nHA synthesized at 150 °C for 1 h (HT2) in simulated body fluid (SBF) for 28 d led to the formation of a bone-like apatite layer on the surface, indicating the excellent bioactivity of the synthesized nHA. The cell culture results revealed superior cell attachment and proliferation for nHA (HT2). Following the sequential formation and sintering at 1200 °C for 4 h, HT2 ceramics exhibited enhanced microhardness (5.65 GPa) and fracture toughness (1.23 MPa·m^0.5), surpassing those of human tooth enamel. Full article

Osteoporotic vertebral compression fractures (OVCFs) are the most prevalent fractures among patients with osteoporosis, leading to severe pain, deformities, and even death. This study explored the use of ectopic embryonic calvaria derived mesenchymal stem cells (EE-cMSCs), which are known for their superior differentiation and proliferation capabilities, as a potential treatment for bone regeneration in OVCFs. We evaluated the impact of EE-cMSCs on osteoclastogenesis in a RAW264.7 cell environment, which was induced by the receptor activator of nuclear factor kappa-beta ligand (RANKL), using cytochemical staining and quantitative real-time PCR. The osteogenic potential of EE-cMSCs was evaluated under various hydrogel conditions. An osteoporotic vertebral body bone defect model was established by inducing osteoporosis in rats through bilateral ovariectomy and creating defects in their coccygeal vertebral bodies. The effects of EE-cMSCs were examined using micro-computed tomography (μCT) and histology, including immunohistochemical analyses. In vitro, EE-cMSCs inhibited osteoclast differentiation and promoted osteogenesis in a 3D cell culture environment using fibrin hydrogel. Moreover, μCT and histological staining demonstrated increased new bone formation in the group treated with EE-cMSCs and fibrin. Immunostaining showed reduced osteoclast activity and bone resorption, alongside increased angiogenesis. Thus, EE-cMSCs can effectively promote bone regeneration and may represent a promising therapeutic approach for treating OVCFs. Full article

There is great interest in developing effective therapies for the treatment of skin wounds accompanied by deep tissue losses and severe infections. We have attempted to prepare biohybrids formed of agglomerates of mesenchymal stem cells (MSCs) with gelatin hydrogel beads (GEL beads) delivering bacteriophages (phages) as antibacterial agents and/or basic fibroblast growth factor (bFGF) for faster and better healing, providing combined therapies for these types of skin wounds. The gelatin beads were produced through a two-step process using basic and/or acidic gelatins with different isoelectric points. Escherichia coli (E. coli) and its specific T4 phages were propagated. Phages and/or bFGF were loaded within the GELs and their release rates and modes were obtained. The phage release from the basic GEL beads was quite fast; in contrast, the bFGF release from the acidic GEL beads was sustained, as anticipated. MSCs were isolated from mouse adipose tissues and 2D-cultured. Agglomerates of these MSCs with GEL beads were formed and maturated in 3D cultures, and their time-dependent changes were followed. In these 3D culture experiments, it was observed that the agglomerates with GEL beads were very healthy and the MSCs formed tissue-like structures in 7 days, while the MSC agglomerates were not healthy and shrunk considerably as a result of cell death. Full article

Mesoporous bioactive glass nanoparticles (MBGNs) doped with therapeutical ions present multifunctional systems that enable a synergistic outcome through the dual delivery of drugs and ions. The aim of this study was to evaluate influence of co-doping with strontium and magnesium ions (SrMg-MBGNs) on the properties of MBGNs. A modified microemulsion-assisted sol–gel synthesis was used to obtain particles, and their physicochemical properties, bioactivity, and drug-loading/release ability were evaluated. Indirect biological assays using 2D and 3D cell culture models on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and endothelial EA.hy926 cells, respectively, were used to determine biocompatibility of MBGNs, their influence on alkaline phosphatase (ALP) production, calcium deposition, and cytoskeletal organization. Results showed that Sr,Mg-doping increased pore volume and solubility, and changed the mesoporous structure from worm-like to radial–dendritic, which led to a slightly accelerated drug release compared to pristine MBGNs. Biological assays confirmed that particles are biocompatible, and have ability to slightly induce ALP production and calcium deposition of hBM-MSCs, as well as to significantly improve the proliferation of EA.hy926 compared to biochemical stimulation via vascular endothelial growth factor (VEGF) administration or regular media. Fluorescence staining revealed that SrMg-MBGNs had a similar effect on EA.hy926 cytoskeletal organization to the VEGF group. In conclusion, Sr,Mg-MBGNs might be considered promising biomaterial for biomedical applications. Full article

Bone marrow has raised a great deal of scientific interest, since it is responsible for the vital process of hematopoiesis and is affiliated with many normal and pathological conditions of the human body. In recent years, organs-on-chips (OoCs) have emerged as the epitome of biomimetic systems, combining the advantages of microfluidic technology with cellular biology to surpass conventional 2D/3D cell culture techniques and animal testing. Bone-marrow-on-a-chip (BMoC) devices are usually focused only on the maintenance of the hematopoietic niche; otherwise, they incorporate at least three types of cells for on-chip generation. We, thereby, introduce a BMoC device that aspires to the purely in vitro generation and maintenance of the hematopoietic niche, using solely mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs), and relying on the spontaneous formation of the niche without the inclusion of gels or scaffolds. The fabrication process of this poly(dimethylsiloxane) (PDMS)-based device, based on replica molding, is presented, and two membranes, a perforated, in-house-fabricated PDMS membrane and a commercial poly(ethylene terephthalate) (PET) one, were tested and their performances were compared. The device was submerged in a culture dish filled with medium for passive perfusion via diffusion in order to prevent on-chip bubble accumulation. The passively perfused BMoC device, having incorporated a commercial poly(ethylene terephthalate) (PET) membrane, allows for a sustainable MSC and HSPC co-culture and proliferation for three days, a promising indication for the future creation of a hematopoietic bone marrow organoid. Full article