Search Results (19)

Stem-cell-based therapies rely on the transplantation of stem cells or stem-cell-derived organotypic cells into injured tissues in order to improve or restore tissue function that has been impaired by various diseases. The potential of induced pluripotent stem cells has created many applications in the field of cell therapy, for example. Some applications, for example, those in cardiac cell therapy, suffer from low or very low efficiencies of cell engraftment. Therefore, magnetic cell targeting can be discussed as a method for capturing superparamagnetic nanoparticle-labelled cells in the tissue. Here, we employ superparamagnetic iron oxide nanoparticles (SPIONs) for the intracellular magnetic loading of mesenchymal stem cells (MSCs). In addition, we test a novel strategy of labelling MSCs with ferromagnetic particles. The adhesion assays demonstrate a faster adhesion kinetic of SPIONs-loaded MSC spheroids when a magnetic field was applied, resulting in >50% spheroid adhesion after 30 min. Clustering of cells inside the magnetic field is a second potential mechanism of magnetic cell retention and >80% of cells were found to be aggregated in clusters when placed in a magnetic field for 10 min. SPIONs-loaded and ferromagnetic-particle-loaded cells performed equally in the cell clustering assay. In conclusion, the clustering of SPION-labelled cells explains the observation that magnetic targeting reaches maximal efficiency in vivo after only 10 min of magnetic field application. This has significant implications for magnetic-targeting-assisted stem cell and cell replacement therapies. Full article

Microgravity, defined by minimal gravitational forces, represents a unique environment that profoundly influences biological systems, including human cells. This review examines the effects of microgravity on biological processes and their implications for human health. Microgravity significantly impacts the immune system by disrupting key mechanisms, such as T cell activation, cytokine production, and macrophage differentiation, leading to increased susceptibility to infections. In cancer biology, it promotes the formation of spheroids in cancer stem cells and thyroid cancer cells, which closely mimic in vivo tumor dynamics, providing novel insights for oncology research. Additionally, microgravity enhances tissue regeneration by modulating critical pathways, including Hippo and PI3K-Akt, thereby improving stem cell differentiation into hematopoietic and cardiomyocyte lineages. At the organ level, microgravity induces notable changes in hepatic metabolism, endothelial function, and bone mechanotransduction, contributing to lipid dysregulation, vascular remodeling, and accelerated bone loss. Notably, cardiomyocytes derived from human pluripotent stem cells and cultured under microgravity exhibit enhanced mitochondrial biogenesis, improved calcium handling, and advanced structural maturation, including increased sarcomere length and nuclear eccentricity. These advancements enable the development of functional cardiomyocytes, presenting promising therapeutic opportunities for treating cardiac diseases, such as myocardial infarctions. These findings underscore the dual implications of microgravity for space medicine and terrestrial health. They highlight its potential to drive advances in regenerative therapies, oncology, and immunological interventions. Continued research into the biological effects of microgravity is essential for protecting astronaut health during prolonged space missions and fostering biomedical innovations with transformative applications on Earth. Full article

Spheroids serve as the building blocks for three-dimensional (3D) bioprinted tissue patches. When larger than 500 μm, the desired size for 3D bioprinting, they tend to have a hypoxic core with necrotic cells. Therefore, it is critical to assess the viability of spheroids in order to ensure the successful fabrication of high-viability patches. However, current viability assays are time-consuming, labor-intensive, require specialized training, or are subject to human bias. In this study, we build a convolutional neural network (CNN) model to efficiently and accurately predict spheroid viability, using a phase-contrast image of a spheroid as its input. A comprehensive dataset of mouse mesenchymal stem cell (mMSC) spheroids of varying sizes with corresponding viability percentages, which was obtained through CCK-8 assays, was established and used to train and validate the model. The model was trained to automatically classify spheroids into one of four distinct categories based on their predicted viability: 0–20%, 20–40%, 40–70%, and 70–100%. The model achieved an average accuracy of 92%, with a consistent loss below 0.2. This deep-learning model offers a non-invasive, efficient, and accurate method to streamline the assessment of spheroid quality, thereby accelerating the development of bioengineered cardiac tissue patches for cardiovascular disease therapies. Full article

Three-dimensional (3D) tissue culture models provide in vivo-like conditions for studying cell physiology. This study aimed to examine the efficiency of pyramidal microwell geometries in microfluidic devices on spheroid formation, cell growth, viability, and differentiation in mouse embryonic stem cells (mESCs). The static culture using the hanging drop (HD) method served as a control. The microfluidic chips were fabricated to have varying pyramidal tip angles, including 66°, 90°, and 106°. From flow simulations, when the tip angle increased, streamline distortion decreased, resulting in more uniform flow and a lower velocity gradient near the spheroids. These findings demonstrate the significant influence of microwell geometry on fluid dynamics. The 90° microwells provide optimal conditions, including uniform flow and reduced shear stress, while maintaining the ability for waste removal, resulting in superior spheroid growth compared to the HD method and other microwell designs. From the experiments, by Day 3, spheroids in the 90° microwells reached approximately 400 µm in diameter which was significantly larger than those in the 66° microwells, 106° microwells, and HD cultures. Brachyury gene expression in the 90° microwells was four times higher than the HD method, indicating enhanced mesodermal differentiation essential for cardiac differentiation. Immunofluorescence staining confirmed cardiomyocyte differentiation. In conclusion, microwell geometry significantly influences 3D cell culture outcomes. The pyramidal microwells with a 90° tip angle proved most effective in promoting spheroid growth and cardiac differentiation of mESC differentiation, providing insights for optimizing microfluidic systems in tissue engineering and regenerative medicine. Full article

We present a case of fatal disseminated neosporosis in a 3-month-old Cane Corso puppy. The puppy presented to the Purdue Veterinary Hospital emergency service with a 1-week history of progressive left hindlimb lameness. On presentation, the puppy was nonambulatory paraparetic. Signs progressed over two days to nonambulatory tetraparesis, and decreased to absent limb reflexes, cranial nerve deficits, and dull mentation. Blood work showed eosinophilia, elevated ALT, and extremely elevated creatine kinase at 36,000 IU/L (reference interval 22–491 IU/L). Two days after diagnostics were performed, the puppy experienced acute cardiac arrest, and the body was submitted for necropsy. At necropsy, skeletal muscle in all four limbs was diffusely pale tan. Tan streaks were disseminated through the diaphragm, abdominal wall, and myocardium. Histologically, Skeletal myocytes and cardiomyocytes were frequently degenerative, with abundant lymphohistiocytic inflammation and fibrosis. White matter within the brain and spinal cord was inflamed and contained frequent dilated myelin sheaths and spheroids. A few protozoal cysts were within the brain and skeletal myocytes. Antemortem antibody titers were positive for IgG against Neospora caninum (dilution titer of 1:4096, baseline 1:32), confirming protozoal cysts as Neospora caninum. Disseminated neosporosis is an uncommon but important clinical differential for ascending paresis in young dogs. Full article

A three-dimensional (3D) cell culture can more precisely mimic tissues architecture and functionality, being a promising alternative model to study disease pathophysiology and drug screening. Chagas disease (CD) is a neglected parasitosis that affects 7 million people worldwide. Trypanosoma cruzi’s (T. cruzi) mechanisms of invasion/persistence continue to be elucidated. Benznidazole (BZ) and Nifurtimox (NF) are trypanocidal drugs with few effects on the clinical manifestations of the chronic disease. Chronic Chagas cardiomyopathy (CCC) is the main manifestation of CD due to its frequency and severity. The development of fibrosis and hypertrophy in cardiac tissue can lead to heart failure and sudden death. Thus, there is an urgent need for novel therapeutic options. Our group has more than fifteen years of expertise using 3D primary cardiac cell cultures, being the first to reproduce fibrosis and hypertrophy induced by T. cruzi infection in vitro. These primary cardiac spheroids exhibit morphological and functional characteristics that are similar to heart tissue, making them an interesting model for studying CD cardiac fibrosis. Here, we aim to demonstrate that our primary cardiac spheroids are great preclinical models which can be used to develop new insights into CD cardiac fibrosis, presenting advances already achieved in the field, including disease modeling and drug screening. Full article

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes toward perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labeled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A₁₆₅ on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP-MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial–mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analyzed the pericyte-like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis. Full article

Chagas disease therapy still relies on two nitroderivatives, nifurtimox and benznidazole (Bz), which have important limitations and serious adverse effects. New therapeutic alternatives for this silent disease, which has become a worldwide public health problem, are essential for its control and elimination. In this study, 1,2,3-triazole analogues were evaluated for efficacy against T. cruzi. Three triazole derivatives, 1d (0.21 µM), 1f (1.23 µM), and 1g (2.28 µM), showed potent activity against trypomastigotes, reaching IC₅₀ values 10 to 100 times greater than Bz (22.79 µM). Promising candidates are active against intracellular amastigotes (IC₅₀ ≤ 6.20 µM). Treatment of 3D cardiac spheroids, a translational in vitro model, significantly reduced parasite load, indicating good drug diffusion and efficacy. Oral bioavailability was predicted for triazole derivatives. Although infection was significantly reduced without drug pressure in a washout assay, the triazole derivatives did not inhibit parasite resurgence. An isobologram analysis revealed an additive interaction when 1,2,3-triazole analogs and Bz were combined in vitro. These data indicate a strengthened potential of the triazole scaffold and encourage optimization based on an analysis of the structure–activity relationship aimed at identifying new compounds potentially active against T. cruzi. Full article

Background: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. Methods: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. Results: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. Conclusions: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology. Full article

Balloon pumps are employed to assist cardiac function in cases of acute myocardial infarction, ventricular arrhythmias, cardiogenic shock, unstable angina, refractory ventricular failure, or cardiac surgery. Counterpulsation, through increasing the diastolic pressure and reducing the systolic pressure, increases coronary perfusion and assists the heart to pump more blood at each contraction. An expanding-contracting balloon, modifying the Poiseuille flow in a straight circular duct, is examined in this study. The balloon is spheroid-shaped, with the length of its minor axis, which is perpendicular to the flow direction, changing in time following a sinusoidal law. The inlet flow volume rate is steady while the rate that the fluid volume leaves the duct varies in time due to the presence of the balloon. For a pulsation frequency of 60 pulses/min, the pressure difference across the pulsating balloon exhibits significant phase lagging behind the outflow volume waveform. The outlet pressure depends on the balloon radius oscillation amplitude and is computed for a range of such. The flow field around the spheroid, periodically expanding-contracting balloon in the steady flow stream is presented, in which the exact pattern of the gradual downstream intensification of the flow pulsation alongside the spheroid body is also identified. Full article

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant global health issue. This novel virus’s high morbidity and mortality rates have prompted the scientific community to quickly find the best COVID-19 model to investigate all pathological processes underlining its activity and, more importantly, search for optimal drug therapy with minimal toxicity risk. The gold standard in disease modeling involves animal and monolayer culture models; however, these models do not fully reflect the response to human tissues affected by the virus. However, more physiological 3D in vitro culture models, such as spheroids and organoids derived from induced pluripotent stem cells (iPSCs), could serve as promising alternatives. Different iPSC-derived organoids, such as lung, cardiac, brain, intestinal, kidney, liver, nasal, retinal, skin, and pancreatic organoids, have already shown immense potential in COVID-19 modeling. In the present comprehensive review article, we summarize the current knowledge on COVID-19 modeling and drug screening using selected iPSC-derived 3D culture models, including lung, brain, intestinal, cardiac, blood vessels, liver, kidney, and inner ear organoids. Undoubtedly, according to reviewed studies, organoids are the state-of-the-art approach to COVID-19 modeling. Full article

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na⁺ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na⁺ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K⁺ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K⁺ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K⁺ uptake and severe K⁺ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed. Full article

Natural biopolymer scaffolds and conductive nanomaterials have been widely used in cardiac tissue engineering; however, there are still challenges in the scaffold fabrication, which include enhancing nutrient delivery, biocompatibility and properties that favor the growth, maturation and functionality of the generated tissue for therapeutic application. In the present work, different scaffolds prepared with sodium alginate and chitosan (alginate/chitosan) were fabricated with and without the addition of metal nanoparticles and how their fabrication affects cardiomyocyte growth was evaluated. The scaffolds (hydrogels) were dried by freeze drying using calcium gluconate as a crosslinking agent, and two types of metal nanoparticles were incorporated, gold (AuNp) and gold plus sodium alginate (AuNp+Alg). A physicochemical characterization of the scaffolds was carried out by swelling, degradation, permeability and infrared spectroscopy studies. The results show that the scaffolds obtained were highly porous (>90%) and hydrophilic, with swelling percentages of around 3000% and permeability of the order of 1 × 10⁻⁸ m². In addition, the scaffolds proposed favored adhesion and spheroid formation, with cardiac markers expression such as tropomyosin, troponin I and cardiac myosin. The incorporation of AuNp+Alg increased cardiac protein expression and cell proliferation, thus demonstrating their potential use in cardiac tissue engineering. Full article

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research. Full article

The Notch intercellular signaling pathways play significant roles in cardiovascular development, disease, and regeneration through modulating cardiovascular cell specification, proliferation, differentiation, and morphogenesis. The dysregulation of Notch signaling leads to malfunction and maldevelopment of the cardiovascular system. Currently, most findings on Notch signaling rely on animal models and a few clinical studies, which significantly bottleneck the understanding of Notch signaling-associated human cardiovascular development and disease. Recent advances in the bioengineering systems and human pluripotent stem cell-derived cardiovascular cells pave the way to decipher the role of Notch signaling in cardiovascular-related cells (endothelial cells, cardiomyocytes, smooth muscle cells, fibroblasts, and immune cells), and intercellular crosstalk in the physiological, pathological, and regenerative context of the complex human cardiovascular system. In this review, we first summarize the significant roles of Notch signaling in individual cardiac cell types. We then cover the bioengineering systems of microfluidics, hydrogel, spheroid, and 3D bioprinting, which are currently being used for modeling and studying Notch signaling in the cardiovascular system. At last, we provide insights into ancillary supports of bioengineering systems, varied types of cardiovascular cells, and advanced characterization approaches in further refining Notch signaling in cardiovascular development, disease, and regeneration. Full article