Biomedical Microdevices最新文献

Extracellular vesicles (EVs) are nanosized particles secreted by most cells for information transmission, which affects the microenvironment. EVs are known to follow the characteristics and conditions of their mother cells and have attracted considerable attention for disease diagnosis and therapeutic effects. In particular, mesenchymal stem cell (MSC)-derived EVs have shown potential for facilitating regenerative wound healing, modulating immune responses, and inhibiting inflammatory diseases. However, previous isolation methods demonstrated limited EV yield, purity, and filter capacity. Here, we report a two-step tangential flow filtration (TFF) system using track-etched membranes with uniform cylindrical nanopores for effectively isolating EVs with high purity and yield. Using two different uniform nanoporous track-etched membranes (50 and 200 nm), only the particles in the small EV (sEV) size range were separated through a size-exclusion mechanism. Comparative analysis with the existing ultrafiltration membrane-based TFF system revealed that the nanoporous membrane-based TFF (Nano-TFF) system exhibited a separation efficiency (yield) exceeding twofold, achieving sEVs purity surpassing 90%. The efficacy of the highly purified sEVs was validated by incorporating them into wound dressing material and applying them to a wound animal model. Notably, the sEVs-loaded wound dressing group demonstrated enhanced wound recovery compared to control groups. The Nano-TFF system, which provides precise separation and high efficiency, can be applied to separate various bioactive agents, including sEVs, that require high-purity isolation.

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Mononuclear cells (MNCs), a type of leukocyte, require enrichment owing to their rarity for research and clinical applications. The enrichment of MNCs is generally performed via conventional methods (e.g., density gradient centrifugation). However, these methods have downsides, such as being labor intensive, energy and time consuming, and requiring advanced equipment. Therefore, inertial microfluidics has recently drawn widespread attention as a way to overcome these limitations. This work aims to investigate MNC separation using a novel spiral inertial microfluidic system design. After MNCs were enriched by Ficoll stratification, the cells were separated according to their size and deformability properties by passing through the microfluidic system. In the final step, various cell markers were examined for characterization in these cells collected at outlets. In this paper, we determined that MNCs obtained from three different hematological products could be sorted with a recovery rate of 97.5% and a purity level of 84%, whereas red blood cells (RBCs) had a depletion ratio of 80% using Sunflower-designed microfluidic system. The loss of MNCs in this system was much lower than that in density gradient centrifugation. The separation technique studied here has several advantages, such as continuous processing, a high operation flow rate (e.g., 0.7 ml/min), simplifying the operative procedures for automation, and creating no clogging problems. Additionally, this technique can be easily integrated with downstream applications, such as direct analysis of MNCs via a flow cytometer, and can reduce the number of man-hand manipulation processes.

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Conventional in vitro and preclinical animal models often fail to accurately replicate the complexity of human diseases, limiting the success of translational studies and contributing to the low success rate of clinical trials (Ingber 2016). In response, research has increasingly focused on organ-on-chip technology, which better mimics human tissue interfaces and organ functionality. In this study, we describe the fabrication of a novel biomembrane made of porous silicon (PSi) for use in organ-on-chip systems. This biomembrane more accurately simulates the complex tissue interfaces observed in vivo compared to conventional organ-on-chip interfaces. By leveraging established semiconductor techniques, such as anisotropic chemical etching and electrochemical anodization, we developed a reproducible method to create ultra-thin freestanding PSi biomembranes. These membranes were thinned to approximately 10 μm and anodized to contain nanoporous structures (~ 15 nm diameter) that permeate the entire membrane. The incorporation of these membranes into organ-on-chip-like devices demonstrated their functionality in a lung-on-a-chip (LOAC) model system. The results indicate that the PSi biomembranes support cellular viability and adhesion, and are consistent with the expected diffusion of nutrients and signaling molecules between distinct cell types. This novel approach provides a reliable method for generating PSi biomembranes tailored to mimic tissue interfaces. The study underscores the potential of PSi-based membranes to enhance the accuracy and functionality of organ-on-chip devices in translational research.

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Membrane-bound extracellular vesicles (EVs) are more than mere messengers; they are the carriers of intercellular communication, carrying biomolecules for regulatory processes. They have potential in biomarker discovery and disease diagnosis for clinical applications. However, the exploration and utilization of EVs are currently constrained by the existing processing methodologies. Microfluidic technology is a versatile platform, achieving the efficient, consistent, and precise separation and aggregation of particles from the nanoscale to the microscale. It has great potential for EVs, enabling precise manipulation, separation, and aggregation in microchannels. This review explores active and passive microfluidic techniques, presenting a cost-effective and scalable solution for label-free separation. Their development is important for EV research, unlocking value in the in-depth study. Their innovative biomedical applications can revolutionize laboratory medicine, drug delivery, and regenerative medicine by fully realizing and harnessing the potential of EVs.

Intracellular delivery of therapeutic materials remains challenging, with conventional micropattern-assisted optoporation methods making it difficult to analyze the spatial effects of individual laser pulses. Here, we show that pigmented SU-8 microdisks enable precise analysis of distance-dependent shockwave effects on cell membrane permeabilization, achieving delivery yields up to 60% in optimized conditions. Using 20 μm and 50 μm microdisks irradiated by nanosecond laser pulses, we discovered that larger patterns generate more extensive shockwaves leading to increased cell damage over broader ranges, while smaller patterns maintain high delivery efficiency with minimal cellular disruption. Furthermore, cellular adhesion strength critically influences treatment outcomes: strongly adherent SAOS-2 cells showed remarkable resilience while weakly adherent HEK-293 cells experienced extensive damage at greater distances. Our results demonstrate how micropattern size and cell-specific properties determine the spatial extent and efficiency of shockwave-mediated delivery, providing a framework for optimizing intracellular delivery strategies while preserving cell viability.

Glioblastoma is a highly malignant brain tumor with limited survival rates due to challenges in complete surgical excision, high recurrence (> 90%), and the inefficacy of systemic drug delivery. Significant efforts have been made to develop drug-loaded brain implants, catheters, and wafers aimed at enhancing survival rates by suppressing tumor recurrence. However, these devices often fail due to clogging, reflux, and the inability to be fully implanted intracranially. Furthermore, a lack of tissue penetration, diffusion distance, and duration of therapy have limited effectiveness of these implants. To address existing challenges, this study reports an osmosis-driven, 3D-printed brain implant with the potential for precise device customization to meet therapeutic needs, while negating systemic toxicity. It is capable of being loaded with two distinct therapeutic agents and implanted directly into the tumor resection cavity during surgery. The device features dual reservoirs, osmotic membranes, and precision-engineered needles for anchoring the device in the resection cavity and perfusing. Further, the device was characterized in vitro using 0.2% agarose gel as a brain tissue analog, with food dye as a drug analog and sodium chloride serving as an osmogen. A design of experiment approach was implemented to investigate various parameters, including membrane pore size, osmogen concentration, needle length, and their effects on release rates. The results demonstrated that the optimized implant achieves flow rates of 2.5 ± 0.1 µl/Hr and diffusion distance of up to 15.5 ± 0.4 mm, using 25 nm pore osmotic membranes with 25.3% osmogen concentration, aligning with model predictions.

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Chlorhexidine (Chx) is a commonly used antimicrobial agent in dentistry, but its effectiveness can be limited due to rapid clearance, potential cytotoxicity, and insufficient tissue penetration. Nanomaterials have been developed as carriers for Chx, can offer a solution by adapting to environmental changes during disease states and enabling targeted drug delivery. This study explores Chx-loaded nanomaterials, which show enhanced antibacterial properties, promote tissue regeneration, and facilitate drug diffusion. Results show sustained drug release profiles and significantly enhanced antimicrobial activity compared to free Chx. In vitro studies confirm their effectiveness against key dental pathogens while maintaining excellent biocompatibility with human gingival fibroblasts and periodontal ligament cells. Future research should focus on optimizing the formulation and delivery methods of these nanomaterials to ensure safe, effective treatment of dental infections.

The clinical need for minimally invasive inner ear diagnostics and therapeutics has grown rapidly in recent years, particularly with the development of gene therapies for treating hearing and balance disorders. These therapies often require delivery of large injectate volumes that can cause hearing damage. In response to this challenge, dual-lumen microneedles, with two separate fluidic pathways controlled independently by micropumps, were designed for simultaneous aspiration and delivery to the inner ear across the round window membrane (RWM) and were fabricated using 2-photon polymerization (2PP). To assess the proof of concept of the dual-lumen microneedle device, simultaneous injection of 5 µL of adeno-associated virus (AAV) expressing green fluorescent protein (GFP) and aspiration of 5 µL of perilymph was performed in guinea pigs in vivo. Hearing thresholds were measured using auditory brainstem response (ABR) at time points before and 1 week after the procedure. Confocal imaging of the cochlea, the utricle, and the contralateral inner ear was employed to quantify and characterize the spatial distribution of hair cells with AAV transduction. Dual-lumen microneedle devices were found to be functional in the surgical setting. There was hearing loss limited to higher frequencies of 24 kHz and 28 kHz with ABR mean threshold shifts of 13 dB sound pressure level (SPL) (p = 0.03) and 23 dB SPL (p < 0.01), respectively. Furthermore, cochlear AAV transduction with a stereotypical basoapical gradient was observed in all animals (n = 5). Thus, dual-lumen microneedles can facilitate delivery of large volumes of therapeutic material into the inner ear, overcoming the limitations of single-lumen microneedles.

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Cancer early detection is one of the most challenging purposes of preventive medicine. Liquid biopsy represents a revolutionary approach, fostering access to early screening and increasing patients’ compliance, two crucial issues in reaching the largest possible audience in prevention campaigns. To facilitate this approach, the deployment of innovative methods for easy manipulation of biological fluids and the availability of devices for the rapid and low-cost detection of biomarkers is essential. The aim of this study was the optimization of multifunctional Lab-On-Chips with the final aim of realizing a platform for oral carcinoma cells trapping from a complex biological fluid as saliva and for specific subcellular components like extracellular vesicles (EVs) from the neuroblastoma cell model. A set of different microfluidic building blocks was realized through poly-methyl methacrylate (PMMA) micromilling, microfabricated and functionalized to optimize surface chemistry for capturing tumor cells or EVs in multiple channels, assess working concentration for biological fluids and combine sample preparation with detection modules all in the same chip. After optimization, a proof-of-concept device was realized mimicking liquid biopsy analysis from saliva, a biological fluid readily available and with a high compliance from patients, useful for the early diagnosis of cancer.

Conventional in vitro cancer models often fail to replicate the complexity of the tumor microenvironment. We have developed a 3D micro-engineered vascularized organoid chip (VOC) platform to enhance the physiological relevance of in vivo tumor models. This platform incorporates patient-derived tumor spheroids from head and neck cancer patients, providing a more accurate simulation of the native tumor microenvironment. We evaluated the efficacy of 5-fluorouracil (5-FU) and sunitinib on angiogenic sprouting and cell viability of red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs) and head and neck cancer patient-derived tumor spheroids cultured in the VOC platform. A 3D micro-engineered VOC platform was developed to provide a physiologically relevant environment for RFP-HUVECs and head and neck cancer patient-derived tumor spheroids. Cellular responses to 5-FU and sunitinib were examined over 14 days, focusing on interactions and behavior in the VOC setup. 5-FU and sunitinib significantly inhibited angiogenic sprouting and reduced cell viability. Notably, these drugs induced changes in cellular network formation and disrupted the structural integrity of patient-derived spheroids, emphasizing the effectiveness of these drugs in a model that closely simulates the tumor microenvironment of head and neck cancer. Our study demonstrates the potential of the 3D vascularized tumor spheroid microfluidic chip as a valuable tool for personalized treatment and investigation of head and neck squamous cell carcinoma. This platform simulates the tumor microenvironment and offers exceptional precision in evaluating drug efficacy.