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In this study, we report an opto-microfluidic platform integrated with dark-field light scattering imaging and fluorescence microspectroscopy to investigate the temperature-dependent behavior of nanoliposomes. This newly developed experimental platform enabled both in situ measurements of the temperature of the sample solution and observation of individual nanoliposomes. As a proof-of-concept, the temperature-dependent interaction between 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) nanoliposomes and a poly(ethylene glycol) (PEG) grafted surface was investigated using this opto-microfluidic platform. Temperature-dependent detachment behavior of DLPC nanoliposomes, which was because of the phase transition of DLPC, was observed. The opto-microfluidic platform for individual nanoparticle measurements may become a powerful tool for investigating temperature-dependent physicochemical properties of nanoparticles in aqueous solution.
Assuntos
Lipossomos/química , Nanoestruturas/química , Fenômenos Ópticos , Polietilenoglicóis/química , Temperatura , Dispositivos Lab-On-A-Chip , Propriedades de Superfície , Água/químicaRESUMO
In this report, we describe a microfluidic vascular-bed (micro-VB) device providing a platform for 3D tissue engineering with vascular network formation. The micro-VB device allows functional connections between endothelial capillaries of heterogeneous sections (5-100 µm in diameter) and artificial plastic tubes or reservoirs (1-10 mm in diameter). Moreover, the micro-VB device can be installed in a standard 100 mm-diameter Petri dish. Endothelial networks in 3D engineered tissues were obtained by cellular self-assembly on the device, after co-culturing of human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) in fibrin gel. Endothelial capillary connection between vascularized tissues and microfluidic channels, mimicking arteries and veins, was confirmed by perfusion of fluorescent microspheres. The micro-VB devices were compatible with the use of commercially available culture dishes and did not require the involvement of additional equipment. Thus, these micro-VB devices are expected to substantially improve the routine application of 3D tissue engineering to regenerative medicine.
Assuntos
Artérias/citologia , Dispositivos Lab-On-A-Chip , Engenharia Tecidual/instrumentação , Veias/citologia , Desenho de Equipamento , Células Endoteliais da Veia Umbilical Humana/citologia , HumanosRESUMO
We investigated the disposition of ARTCEREB® irrigation and perfusion solution (Artcereb) during intrathecal perfusion in a lateral ventricle-cisternal perfusion model in conscious rats. In this perfusion model, the perfusion rate was set at 0.35 ml/kg/h, taking into consideration the clinical perfusion rate (500 ml/60 kg/d). The influence of Artcereb on electrolytes in cerebrospinal fluid (CSF) and blood were then investigated. After 24 h of ventriculocisternal perfusion with Artcereb using the push-pull method, output of K(+), Na(+) and Cl(-) to the cistern magna was very similar to input of these electrolytes in Artcereb infused intraventricularly. Recovery rates of K(+), Na(+) and Cl(-) after perfusion were 102%, 105% and 100% when calculated using the recovered perfusion solution. In addition, concentrations of K(+), Na(+) and Cl(-) in blood remained almost constant at near baseline levels throughout perfusion. Thus, intrathecally perfused Artcereb did not affect electrolyte balance in the CSF and blood. To confirm the dynamics of Artcereb distribution, a whole body autoradiography study was performed at 1 and 6 h after perfusion with ¹4C-inulin-added Artcereb. Radioactivity was detected in the entire CSF space of the brain, and the cribriform plate in the nasal cavity, and the cerebrospinal cavity. Radioactivity was observed in the bladder, thus suggesting that some ¹4C-inulin was transferred to the bloodstream via a physiological route, and was excreted renally.
Assuntos
Sistema Nervoso Central/cirurgia , Coluna Vertebral/cirurgia , Animais , Masculino , Perfusão , Ratos , Ratos Sprague-Dawley , Irrigação TerapêuticaRESUMO
The micromolding process using biocompatible thermoplastic polymers is highly attractive as a fabrication process of microdevices in biomedical applications. In this study, we investigated the effect of the thermal history in the micromolding process on the crystallinity of semi-crystalline polymers, such as poly (L-lactic acid) (PLLA), during their crystallization from the amorphous and molten states. In particular, the thermal history in the micromolding process using poly(dimethylsiloxane) replica mold embedded with a thermocouple was recorded. The crystallinity of PLLA constructs fabricated using the micromolding process was measured via wide-angle X-ray scattering, and crystallization kinetics was analyzed based on the Kolmogorov-Johnson-Mehl-Avrami equation. A crystallization rate of k = 0.061 min-n was obtained in the micromolding process of PLLA crystallization from the amorphous state, accompanied by the quenching operation, forming a large number of crystal nuclei. Finally, the fabrication of PLLA microneedles was performed using micromolding processes with different thermal histories. The information about the thermal history during the micromolding process is significant in the development of polymer microdevices to achieve better material properties.
RESUMO
CMOS-based opto-electronic neural interface devices are presented. The devices are designed with target application of in vitro and in vivo optogenetics. Two types of the opto-electronic neural interface devices are presented. One is single-chip type device for on-chip optogenetics, and the other is multi-chip type device with flexibility and wide-area coverage for in vivo optogenetics on brain. Design, packaging and functional evaluations are presented.
Assuntos
Interfaces Cérebro-Computador , Metais/química , Dispositivos Ópticos , Optogenética/instrumentação , Óxidos , SemicondutoresRESUMO
Dendritic spine generation and elimination play an important role in learning and memory, the dynamics of which have been examined within the neocortex in vivo. Spine turnover has also been detected in the absence of specific learning tasks, and is frequently exaggerated in animal models of autistic spectrum disorder (ASD). The present study aimed to examine whether the baseline rate of spine turnover was activity-dependent. This was achieved using a microfluidic brain interface and open-dura surgery, with the goal of abolishing neuronal Ca(2+) signaling in the visual cortex of wild-type mice and rodent models of fragile X syndrome (Fmr1 knockout [KO]). In wild-type and Fmr1 KO mice, the majority of baseline turnover was found to be activity-independent. Accordingly, the application of matrix metalloproteinase-9 inhibitors selectively restored the abnormal spine dynamics observed in Fmr1 KO mice, without affecting the intrinsic dynamics of spine turnover in wild-type mice. Such findings indicate that the baseline turnover of dendritic spines is mediated by activity-independent intrinsic dynamics. Furthermore, these results suggest that the targeting of abnormal intrinsic dynamics might pose a novel therapy for ASD.
Assuntos
Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Córtex Visual/metabolismo , Córtex Visual/patologia , Animais , Espinhas Dendríticas/genética , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Camundongos , Camundongos KnockoutRESUMO
A novel flexible sensor was developed for the noninvasive oxygen metabolism measurement of cultivated cells and tissues. This device is composed of a transparent double-layered polymer sheet of ethylene-vinyl alcohol (EVOH) and poly(dimethylsiloxane) (PDMS) having an array of microhole structures of 90 µm diameter and 50 µm depth on its surface. All the microhole structures were equipped with a 1-µm-thick optical chemical sensing layer of platinum porphyrin-fluoropolymer on their bottom. The three-dimensional microstructures of the sensor were fabricated by a newly developed simple and low-cost production method named self-aligned hot embossing. The device was designed to be attached slightly above the cells cultivated on a dish to form a temporarily closed microspace over the target cells during measurement. Since the change in oxygen concentration is relatively fast in the microcompartmentalized culture medium, a rapid evaluation of the oxygen consumption rate is possible by measuring the phosphorescence lifetime of the platinum porphyrin-fluoropolymer. The combined use of the device and an automated optical measurement system enabled the high-throughput sensing of cellular oxygen consumption (100 points/min). We monitored the oxygen metabolism of the human breast cancer cell line MCF7 on a Petri dish and evaluated the oxygen consumption rate to be 0.72 ± 0.12 fmol/min/cell. Furthermore, to demonstrate the utility of the developed sensing system, we demonstrated the mapping of the oxygen consumption rate of rat brain slices and succeeded in visualizing a clear difference among the layer structures of the hippocampus, i.e., the cornu ammonis (CA1 and CA3) and dentate gyrus (DG).
Assuntos
Medições Luminescentes/métodos , Consumo de Oxigênio , Animais , Encéfalo/metabolismo , Dimetilpolisiloxanos/química , Humanos , Células MCF-7 , Platina/química , Porfirinas/química , RatosRESUMO
In this study, we propose an advanced architecture of a smart electrode for neural stimulation of a retinal prosthesis. A feature of the proposed architecture is embedding CMOS microchips into the core of the stimulus electrodes. Microchip integration without dead space on the array is possible. Additionally, higher durability can be expected because the microchips are protected by the stimulus electrodes like a metal casing. Dedicated circular-shaped CMOS microchips were designed and fabricated. The microchip measured 400 µm in diameter. Stimulus electrodes that had a microcavity for embedding the microchip were also fabricated. In the assembly process, the CMOS microchip was mounted on a flexible substrate, and then the stimulus electrode was mounted to cover the microchip. The microchip was completely built into the inside of the electrode. By performing an ex-vivo experiment using the extracted eyeball of a pig, stimulus function of the electrode was demonstrated successfully.
Assuntos
Estimulação Elétrica , Eletrodos Implantados , Retina/cirurgia , Próteses Visuais , Animais , Desenho de Equipamento , Microeletrodos , Microscopia Eletrônica de Varredura , Retina/fisiopatologia , Software , Suínos , Titânio/químicaRESUMO
Cellular self-assembly based on cell-to-cell communication is a well-known tissue organizing process in living bodies. Hence, integrating cellular self-assembly processes into tissue engineering is a promising approach to fabricate well-organized functional tissues. In this research, we investigated the capability of endothelial cells (ECs) to control shape and position of vascular formation using arbitral-assembling techniques in three-dimensional engineered tissues. To quantify the degree of migration of ECs in endothelial network formation, image correlation analysis was conducted. Positive correlation between the original positions of arbitrarily assembled ECs and the positions of formed endothelial networks indicated the potential for controlling shape and position of vascular formations in engineered tissues. To demonstrate the feasibility of controlling vascular formations, engineered tissues with vascular networks in triangle and circle patterns were made. The technique reported here employs cellular self-assembly for tissue engineering and is expected to provide fundamental beneficial methods to supply various functional tissues for drug screening and regenerative medicine.
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Vasos Sanguíneos/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Engenharia Tecidual/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neovascularização FisiológicaRESUMO
The application of the fluorescence imaging method to living animals, together with the use of genetically engineered animals and synthesized photo-responsive compounds, is a powerful method for investigating brain functions. Here, we report a fluorescence imaging method for the brain surface and deep brain tissue that uses compact and mass-producible semiconductor imaging devices based on complementary metal-oxide semiconductor (CMOS) technology. An image sensor chip was designed to be inserted into brain tissue, and its size was 1500 × 450 µm. Sample illumination is also a key issue for intravital fluorescence imaging. Hence, for the uniform illumination of the imaging area, we propose a new method involving the epi-illumination of living biological tissues, and we performed investigations using optical simulations and experimental evaluation.
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The high-resolution imaging of neural cells in vivo has brought about great progress in neuroscience research. Here, we report a novel experimental platform, where the intact brain of a living mouse can be studied with the aid of a surgically implanted micro-optical fluidic device; acting as an interface between neurons and the outer world. The newly developed device provides the functions required for the long-term and high-resolution observation of the fine structures of neurons by two-photon laser scanning microscopy and the microfluidic delivery of chemicals or drugs directly into the brain. A proof-of-concept experiment of single-synapse stimulation by two-photon uncaging of caged glutamate and observation of dendritic spine shrinkage over subsequent days demonstrated a promising use for the present technology.
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Encéfalo/ultraestrutura , Técnicas Analíticas Microfluídicas , Neurônios/ultraestrutura , Animais , Camundongos , Microscopia ConfocalRESUMO
Hydrogels have several excellent characteristics suitable for biomedical use such as softness, biological inertness and solute permeability. Hence, integrating hydrogels into microfluidic devices is a promising approach for providing additional functions such as biocompatibility and porosity, to microfluidic devices. However, the poor mechanical strength of hydrogels has severely limited device design and fabrication. A tetra-poly(ethylene glycol) (tetra-PEG) hydrogel synthesized recently has high mechanical strength and is expected to overcome such a limitation. In this research, we have comprehensively studied the implementation of tetra-PEG gel into microfluidic device technology. First, the fabrication of tetra-PEG gel/PDMS hybrid microchannels was established by developing a simple and robust bonding technique. Second, some fundamental features of tetra-PEG gel/PDMS hybrid microchannels, particularly fluid flow and mass transfer, were studied. Finally, to demonstrate the unique application of tetra-PEG-gel-integrated microfluidic devices, the generation of patterned chemical modulation with the maximum concentration gradient: 10% per 20 µm in a hydrogel was performed. The techniques developed in this study are expected to provide fundamental and beneficial methods of developing various microfluidic devices for life science and biomedical applications.