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Mapping cellular activities over large areas is crucial for understanding the collective behaviors of multicellular systems. Biomechanical properties, such as cellular traction force, serve as critical regulators of physiological states and molecular configurations. However, existing technologies for mapping large-area biomechanical dynamics are limited by the small field of view and scanning nature. To address this, we propose a novel platform that utilizes a vast number of optical diffractive elements for mapping large-area biomechanical dynamics. This platform achieves a field-of-view of 10.6 mm X 10.6 mm, a three-orders-of-magnitude improvement over traditional traction force microscopy. Transient mechanical waves generated by monolayer neonatal rat ventricular myocytes were captured with high spatiotemporal resolution (130 fps and 20 µm for temporal and spatial resolution, respectively). Furthermore, its label-free nature allows for long-term observations extended to a week, with minimal disruption of cellular functions. Finally, simultaneous measurements of calcium ions concentrations and biomechanical dynamics are demonstrated.
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We report a massive field-of-view and high-speed videography platform for measuring the sub-cellular traction forces of more than 10,000 biological cells over 13 mm2 at 83 frames per second. Our Single-Pixel Optical Tracers (SPOT) tool uses 2-dimensional diffraction gratings embedded into a soft substrate to convert cells' mechanical traction force into optical colors detectable by a video camera. The platform measures the sub-cellular traction forces of diverse cell types, including tightly connected tissue sheets and near isolated cells. We used this platform to explore the mechanical wave propagation in a tightly connected sheet of Neonatal Rat Ventricular Myocytes (NRVMs) and discovered that the activation time of some tissue regions are heterogeneous from the overall spiral wave behavior of the cardiac wave.
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Miocitos Cardíacos , Animales , Ratas , Miocitos Cardíacos/citología , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Grabación en Video , Células CultivadasRESUMEN
Chemodynamic therapy (CDT) uses the Fenton or Fenton-like reaction to yield toxic â§OH following H2O2 â â§OH for tumoral therapy. Unfortunately, H2O2 is often taken from the limited endogenous supply of H2O2 in cancer cells. A water oxidation CoFe Prussian blue (CFPB) nanoframes is presented to provide sustained, external energy-free self-supply of â§OH from H2O to process CDT and/or photothermal therapy (PTT). Unexpectedly, the as-prepared CFPB nanocubes with no near-infrared (NIR) absorption is transformed into CFPB nanoframes with NIR absorption due to the increased Fe3+-N ≡ C-Fe2+ composition through the proposed proton-induced metal replacement reactions. Surprisingly, both the CFPB nanocubes and nanoframes provide for the self-supply of O2, H2O2, and â§OH from H2O, with the nanoframe outperforming in the production of â§OH. Simulation analysis indicates separated active sites in catalyzation of water oxidation, oxygen reduction, and Fenton-like reactions from CFPB. The liposome-covered CFPB nanoframes prepared for controllable water-driven CDT for male tumoral mice treatments.
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Nanopartículas , Neoplasias , Masculino , Animales , Ratones , Dominio Catalítico , Peróxido de Hidrógeno , Catálisis , Agua , Línea Celular TumoralRESUMEN
We report a large field-of-view and high-speed videography platform for measuring the sub-cellular traction forces of more than 10,000 biological cells over 13mm 2 at 83 frames per second. Our Single-Pixel Optical Tracers (SPOT) tool uses 2-dimensional diffraction gratings embedded into a soft substrate to convert cells' mechanical traction stress into optical colors detectable by a video camera. The platform measures the sub-cellular traction forces of diverse cell types, including tightly connected tissue sheets and near isolated cells. We used this platform to explore the mechanical wave propagation in a tightly connected sheet of Neonatal Rat Ventricular Myocytes (NRVMs) and discovered that the activation time of some tissue regions are heterogeneous from the overall spiral wave behavior of the cardiac wave. One-Sentence Summary: An optical platform for fast, concurrent measurements of cell mechanics at 83 frames per second, over a large area of 13mm 2 .
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Acoustic patterning of micro-particles has many important biomedical applications. However, fabrication of such microdevices is costly and labor-intensive. Among conventional fabrication methods, photo-lithography provides high resolution but is expensive and time consuming, and not ideal for rapid prototyping and testing for academic applications. In this work, we demonstrate a highly efficient method for rapid prototyping of acoustic patterning devices using laser manufacturing. With this method we can fabricate a newly designed functional acoustic device in 4 hours. The acoustic devices fabricated using this method can achieve sub-wavelength, complex and non-periodic patterning of microparticles and biological objects with a spatial resolution of 60 µm across a large active manipulation area of 10 × 10 mm2.
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Acústica , Rayos LáserRESUMEN
Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.
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Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Pirimidinas/biosíntesis , Pirofosfatasas/metabolismo , Regeneración , Transducción de Señal , Adenosina Monofosfato/genética , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Lesiones Cardíacas/genética , Lesiones Cardíacas/metabolismo , Ratones , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genéticaRESUMEN
This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types. For complete details on the use and execution of this protocol, please refer to Sercel et al. (2021) and Patananan et al. (2020).
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ADN Mitocondrial , Mitocondrias , Animales , Células Cultivadas , Células Clonales/metabolismo , ADN Mitocondrial/genética , Mamíferos/genética , Mitocondrias/genéticaRESUMEN
Generating mammalian cells with specific mitochondrial DNA (mtDNA)-nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.
Mitochondria are specialized structures within cells that generate vital energy and biological building blocks. Mitochondria have a double membrane and contain many copies of their own circular DNA (mitochondrial DNA), which include the blueprints to create just thirteen essential mitochondrial proteins. Like all genetic material, mitochondrial DNA can become damaged or mutated, and these changes can be passed on to offspring. Some of these alterations are linked to severe and debilitating diseases. Both the double membrane of the mitochondria and their high number of DNA copies make treating such diseases difficult. A successful therapy must be capable of correcting almost every copy of mitochondrial DNA. However, the multiple copies of mitochondrial DNA create a problem for genetic research as current techniques are unable to reliably introduce particular mitochondrial mutations to all types of human cells to investigate how they may alter cell function. Sercel, Patananan et al. have developed a method to deliver new mitochondria into thousands of cells at the same time. This technique, called MitoPunch, uses a pressure-driven device to propel mitochondria taken from donor cells into recipient cells without mitochondrial DNA to reestablish their function. Using human cancer cells and healthy skin cells that lack mitochondrial DNA, Sercel, Patananan et al. showed that cells that received mitochondria retained the new mitochondrial DNA. The technique uses readily accessible parts, meaning it can be performed quickly and inexpensively in any laboratory. It further only requires a small amount of donor starting material, meaning that even precious samples with limited material could be used as mitochondrial donors. This new technique has several important potential applications for mitochondrial DNA research. It could be used in the lab to create large numbers of cell lineswith known mutations in the mitochondrial DNA to establish new systems that test drugs or probe the interaction between mitochondrial and nuclear DNA. It could be used to study a broad spectrum of biological questions since mitochondrial function is essential for several processes required for life. Critically, it could also be used as a starting point to develop next-generation therapies capable of treating inherited mitochondrial genetic diseases in severely affected patients.
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Diferenciación Celular , Núcleo Celular/metabolismo , ADN Mitocondrial/genética , Mitocondrias/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , RatonesRESUMEN
We demonstrate a novel platform for mapping the pressure distribution of complex microfluidics networks with high spatial resolution. Our approach utilizes colorimetric interferometers enabled by lossy optical resonant cavities embedded in a silicon substrate. Detection of local pressures in real-time within a fluid network occurs by monitoring a reflected color emanating from each optical cavity. Pressure distribution measurements spanning a 1 cm2 area with a spatial resolution of 50 µm have been achieved. We applied a machine-learning-assisted sensor calibration method to generate a dynamic measurement range from 0 to 5.0 psi, with 0.2 psi accuracy. Adjustments to this dynamic measurement range are possible to meet different application needs for monitoring flow conditions in complex microfluidics networks, for the timely detection of anomalies such as clogging or leakage at their occurring locations.
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Colorimetría , Microfluídica , Calibración , SilicioRESUMEN
In molecular and cellular biological research, cell isolation and sorting are required for accurate investigation of cell populations of specific physical or biological characteristics. By employing unique cell properties to distinguish between heterogeneous cell populations, rapid and accurate sorting with high efficiency is possible. Dielectrophoresis-based cell manipulation has significant promise for separation of cells based on their physical properties and is used in diverse areas ranging from cellular diagnostics to therapeutic applications. In this study, we present a microfluidic device that can achieve label-free and size-based cell separation with high size differential resolution from a mono-cellular population or complex sample matrices. It was realized by using the tunnel dielectrophoresis (TDEP) technique to manipulate the spatial position of individual cells three dimensionally with high resolution. Cells were processed in high speed flows in high ionic strength buffers. A mixture of different sizes of polystyrene micro-particles with a size difference as small as 1 µm can be separated with high purity (>90%). For the first time, high-pass, low-pass, and band-pass filtering within a mono-cellular mammalian cell population were demonstrated with a tunable bandwidth as small as 3 µm. In addition, leukocyte subtype separation was demonstrated by sorting monocytes out of peripheral blood mononuclear cells (PBMCs) from whole blood with high purity (>85%). Its ability to deliver real-time adjustable cut-off threshold size-based cell sorting and its capability to provide an arbitrary cell size pick-up band could potentially enable many research and clinical applications.
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Dispositivos Laboratorio en un Chip , Leucocitos Mononucleares , Animales , Separación Celular , Monocitos , PoliestirenosRESUMEN
Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.
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Reprogramación Celular , Fibroblastos/metabolismo , Técnicas de Transferencia de Gen , Ensayos Analíticos de Alto Rendimiento/métodos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/trasplante , Animales , Diferenciación Celular , Línea Celular , ADN Mitocondrial/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Metaboloma , Ratones , Ratones Endogámicos C57BL , TranscriptomaRESUMEN
Mammalian STE20-like kinase 1 (MST1/STK4/KRS2) encodes a serine/threonine kinase that is the mammalian homolog of Drosophila Hippo. STK4 plays an important role in controlling cell growth, apoptosis, and organ size. STK4 has been studied in many cancers with previous studies indicating an involvement in colon cancer lymph node metastasis and highlighting its potential as a diagnostic marker for colon cancer. However, the role of STK4 defect in promoting colon cancer progression is still understudied. Here, we found that STK4 was significantly downregulated in colon cancer and was associated with distal metastasis and poor survival. Furthermore, STK4 knockdown enhanced sphere formation and metastasis in vitro and promoted tumor development in vivo. We found that STK4 colocalized with ß-catenin and directly phosphorylated ß-catenin resulting in its degradation via the ubiquitin-mediated pathway. This may suggest that STK4 knockdown causes ß-catenin phosphorylation failure and subsequently ß-catenin accumulation, consequently leading to anchorage-independent growth and metastasis in colon cancer. Our results support that STK4 may act as a potential candidate for the assessment of ß-catenin-mediated colon cancer prognosis.
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Movimiento Celular/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , beta Catenina/metabolismo , Anciano , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones Endogámicos NOD , Ratones SCID , Modelos Biológicos , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Supervivencia , Transcripción GenéticaRESUMEN
Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.
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Cicatriz/metabolismo , Colágeno Tipo V/deficiencia , Colágeno Tipo V/metabolismo , Lesiones Cardíacas/metabolismo , Contracción Miocárdica/genética , Miofibroblastos/metabolismo , Animales , Cicatriz/genética , Cicatriz/fisiopatología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Fibrosis/genética , Fibrosis/metabolismo , Regulación de la Expresión Génica/genética , Integrinas/antagonistas & inhibidores , Integrinas/genética , Integrinas/metabolismo , Isoproterenol/farmacología , Masculino , Mecanotransducción Celular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía de Fuerza Atómica/instrumentación , Microscopía Electrónica de Transmisión , Contracción Miocárdica/efectos de los fármacos , Miofibroblastos/citología , Miofibroblastos/patología , Miofibroblastos/ultraestructura , Análisis de Componente Principal , Proteómica , RNA-Seq , Análisis de la Célula IndividualRESUMEN
A multifunctional chemical neural probe fabrication process exploiting PDMS thin-film transfer to incorporate a microfluidic channel onto a silicon-based microelectrode array (MEA) platform, and enzyme microstamping to provide multi-analyte detection is described. The Si/PDMS hybrid chemtrode, modified with a nano-based on-probe IrOx reference electrode, was validated in brain phantoms and in rat brain.
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Microfluídica , Prótesis e Implantes , Animales , Microelectrodos , RatasRESUMEN
INTRODUCTION: Metastasis is a fundamentally physical process in which cells deform through narrow gaps and generate forces to invade surrounding tissues. While it is commonly thought that increased cell deformability is an advantage for invading cells, we previously found that more invasive pancreatic ductal adenocarcinoma (PDAC) cells are stiffer than less invasive PDAC cells. Here we investigate potential mechanisms of the simultaneous increase in PDAC cell stiffness and invasion, focusing on the contributions of myosin II, Arp2/3, and formins. METHOD: We measure cell invasion using a 3D scratch wound invasion assay and cell stiffness using atomic force microscopy (AFM). To determine the effects of actin- and myosin-mediated force generation on cell stiffness and invasion, we treat cells with pharmacologic inhibitors of myosin II (blebbistatin), Arp2/3 (CK-666), and formins (SMIFH2). RESULTS: We find that the activity of myosin II, Arp2/3, and formins all contribute to the stiffness of PDAC cells. Interestingly, we find that the invasion of PDAC cell lines is differentially affected when the activity of myosin II, Arp2/3, or formins is inhibited, suggesting that despite having similar tissue origins, different PDAC cell lines may rely on different mechanisms for invasion. CONCLUSIONS: These findings deepen our knowledge of the factors that regulate cancer cell mechanotype and invasion, and incite further studies to develop therapeutics that target multiple mechanisms of invasion for improved clinical benefit.
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Local heating using pulsed laser-induced photothermal effects on plasmonic nanostructured substrates can be used for intracellular delivery applications. However, the fabrication of plasmonic nanostructured interfaces is hampered by complex nanomanufacturing schemes. Here, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350 nm in diameter) were fabricated using chemical lift-off lithography (CLL). Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. Delivery efficiencies of >98% were achieved using the cell impermeable dye calcein (0.6 kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations.
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Arbitrary patterning of micro-objects in liquid is crucial to many biomedical applications. Among conventional methodologies, acoustic approaches provide superior biocompatibility but are intrinsically limited to producing periodic patterns at low resolution due to the nature of standing waves and the coupling between fluid and structure vibrations. This work demonstrates a near-field acoustic platform capable of synthesizing high resolution, complex and non-periodic energy potential wells. A thin and viscoelastic membrane is utilized to modulate the acoustic wavefront on a deep, sub-wavelength scale by suppressing the structural vibration selectively on the platform. Using 3 MHz excitation (λâ¼ 500 µm in water), we have experimentally validated such a concept by realizing patterning of microparticles and cells with a line resolution of 50 µm (one tenth of the wavelength). Furthermore, massively parallel patterning across a 3 × 3 mm2 area has been achieved. This new acoustic wavefront modulation mechanism is powerful for manufacturing complex biologic products.
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Aire , Membranas Artificiales , Sonido , Propiedades de SuperficieRESUMEN
Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100â¯000 cells delivered per minute. The bacterial enzyme ß-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.
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Hipertermia Inducida , Espacio Intracelular/química , Nanopartículas/química , Fototerapia , Línea Celular Tumoral , Supervivencia Celular , Análisis de Elementos Finitos , Gravitación , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Rayos Láser , Suspensiones , beta-Lactamasas/metabolismoRESUMEN
Flexible neural probes have been pursued previously to minimize the mechanical mismatch between soft neural tissues and implants and thereby improve long-term performance. However, difficulties with insertion of such probes deep into the brain severely restricts their utility. We describe a solution to this problem using gallium (Ga) in probe construction, taking advantage of the solid-to-liquid phase change of the metal at body temperature and probe shape deformation to provide temperature-dependent control of stiffness over 5 orders of magnitude. Probes in the stiff state were successfully inserted 2â¯cm-deep into agarose gel "brain phantoms" and into rat brains under cooled conditions where, upon Ga melting, they became ultra soft, flexible, and stretchable in all directions. The current 30⯵m-thick probes incorporated multilayer, deformable microfluidic channels for chemical agent delivery, electrical interconnects through Ga wires, and high-performance electrochemical glutamate sensing. These PDMS-based microprobes of ultra-large tunable stiffness (ULTS) should serve as an attractive platform for multifunctional chronic neural implants.
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Técnicas Biosensibles , Encéfalo/efectos de los fármacos , Galio/administración & dosificación , Animales , Encéfalo/patología , Electrodos Implantados , Galio/química , Humanos , Polímeros/química , Ratas , TemperaturaRESUMEN
We developed a highly efficient method for patterning cells by a novel and simple technique called lift-off cell lithography (LCL). Our approach borrows the key concept of lift-off lithography from microfabrication and utilizes a fully biocompatible process to achieve high-throughput, high-efficiency cell patterning with nearly zero background defects across a large surface area. Using LCL, we reproducibly achieved >70% patterning efficiency for both adherent and non-adherent cells with <1% defects in undesired areas.