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Gene therapy using adeno-associated virus (AAV) has potential as a radical treatment modality for genetic diseases such as sensorineural deafness. To establish clinical applications, it is necessary to avoid immune response to AAV by controlled release system of AAV. Here, a near-infrared (NIR)-triggered on-demand AAV release system using alginate hydrogel microbeads with a heat transducer is proposed. By using a centrifuge-based microdroplet shooting device, the microbeads encapsulating AAV with Fe3 O4 microparticles (Fe3 O4 -MPs) as a heat transducer are fabricated. Fe3 O4 -MPs generated heat by NIR enhanced the diffusion speed of the AAV, resulting in the AAV being released from the microbeads. By irradiating the microbeads encapsulating fluorescent polystyrene nanoparticles (FP-NPs) (viral model) with NIR, the fluorescence intensity decreased only for FP-NPs with a diameter of 20 nm and not for 100 or 200 nm, confirming that this system can release virus with a diameter of several tens of nanometers. By irradiating NIR to the AAV-encapsulating microbeads with Fe3 O4 -MPs, the AAV is released on demand, and gene transfection to cells by AAV is confirmed without loss of viral activity. The NIR-triggered AAV release system proposed in this study increases the number of alternatives for the method of drug release in gene therapy.
Assuntos
Dependovirus , Hidrogéis , Dependovirus/genética , Temperatura Alta , Alginatos , Microesferas , Preparações de Ação Retardada , Terapia GenéticaRESUMO
Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l-lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.
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Dependovirus , Hidrogéis , Dependovirus/genética , Alginatos , Microesferas , Preparações de Ação Retardada , Vetores GenéticosRESUMO
This paper describes up-scalable microfiber-shaped tissues for macroscale tendon tissue reconstruction in vitro. C3H10T1/2 cells were encapsulated in a calcium alginate hydrogel microfiber that was fabricated via a double coaxial microfluidic device. The C3H10T1/2 cells gradually merged to construct the microfiber-shaped tendon-like tissue. Our microfiber-shaped tendon-like tissues were alive and maintained their microfiber-shaped morphology over 600 days. Immunostaining and real-time quantitative polymerase chain reaction analyses showed that our fabricated microfiber-shaped tendon-like tissue properly expressed tenomodulin and the orientation of the filaments of actin, which are one of the characteristics of tendon tissue in vivo. Furthermore, a macroscale tendon tissue assembly with â¼1 cm in length and â¼200 µm in thickness was successfully constructed by bundling the microfiber-shaped tendon-like tissues together. This feature enabled us to fabricate a macroscale tendon tissue with uniform cell distribution. We believe that our fabricated microfiber-shaped tendon-like tissue would be a suitable strategy to reconstruct tendon tissue in vitro for the treatments of tendon-related injuries.
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Alginatos , Hidrogéis , Contagem de Células , Dispositivos Lab-On-A-Chip , Tendões , Engenharia TecidualRESUMO
To generate three-dimensional tissue in vitro, promoting vasculogenesis in cell aggregates is an important factor. Here, we found that ultrasound promoted vasculogenesis of human umbilical vein endothelial cells (HUVECs). Promotion of HUVEC network formation and lumen formation were observed using our method. In addition to morphological evaluations, protein expression was quantified by western blot assays. As a result, expression of proteins related to vasculogenesis and the response to mechanical stress on cells was enhanced by exposure to ultrasound. Although several previous studies have shown that ultrasound may promote vasculogenesis, the effect of ultrasound was unclear because of unregulated ultrasound, the complex culture environment, or two-dimensional-cultured HUVECs that cannot form a lumen structure. In this study, regulated ultrasound was propagated on three-dimensional-monocultured HUVECs, which clarified the effect of ultrasound on vasculogenesis. We believe this finding may be an innovation in the tissue engineering field.
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Técnicas de Cultura de Células , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Ondas Ultrassônicas , Células Endoteliais da Veia Umbilical Humana/citologia , HumanosRESUMO
We present fluorescent Janus hydrogel microbeads for continuous glucose sensing with pH calibration. The Janus hydrogel microbeads, that consist of fluorescent glucose and pH sensors, were fabricated with a UV-assisted centrifugal microfluidic device. The microbead can calibrate the pH values of its surroundings and enables accurate measurements of glucose within various pH conditions. As a proof of concept, we succeeded in obtaining the accurate value of glucose concentration in a body-fluid-like sample solution. We believe that our fluorescent microbeads, with pH calibration capability, could be applied to fully implantable sensors for continuous glucose monitoring.
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Automonitorização da Glicemia , Hidrogéis , Glicemia , Calibragem , Glucose , Concentração de Íons de Hidrogênio , MicroesferasRESUMO
Nerve guidance conduits (NGCs) composed of biocompatible polymers have been attracting attention as an alternative for autograft surgery in peripheral nerve regeneration. However, the nerve tissues repaired by NGCs often tend to be inadequate and lead to functional failure because of the lack of cellular supports. This paper presents a chitosan-collagen hydrogel conduit containing cells to induce peripheral nerve regeneration with cellular support. The conduit composed of two coaxial hydrogel layers of chitosan and collagen is simply made by molding and mechanical anchoring attachment with holes made on the hydrogel tube. A chitosan layer strengthens the conduit mechanically, and a collagen layer provides a scaffold for cells supporting the axonal extension. The conduits of different diameters (outer diameter approximately 2-4 mm) are fabricated. The conduit is bioabsorbable with lysozyme, and biocompatible even under bio absorption. In the neuron culture demonstration, the conduit containing Schwann cells induced the extension of the axon of neurons directed to the conduit. Our easily fabricated conduit could help the high-quality regeneration of peripheral nerves and contribute to the nerve repair surgery.
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Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Quitosana/química , Colágeno/química , Hidrogéis/química , Regeneração Nervosa/efeitos dos fármacos , Nervos Periféricos/fisiologia , Cápsulas , Nervos Periféricos/citologia , Células de Schwann/citologia , Engenharia TecidualRESUMO
The spatially-targeted fabrication of bimetallic microstructures coexisting in the supporting hydrogel is demonstrated by multi-photon photoreduction. Microstructures composed of gold and silver were fabricated along a predefined trajectory by taking advantages of the hydrogel's ionic permeability. Different resonant wavelengths of optical absorption were obtained for gold, silver, and their bimetallic structures. Transmission electron microscopy and energy dispersive X-ray analysis revealed that the optical properties are attributable to the formation of bimetallic structure consisted of core-shell nanoparticles. The fabrication of dissimilar metal structures within hydrogel is a promising technique for optically driven actuators in soft robotics and sensing applications by allowing for site-selective optical properties.
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This paper describes three-dimensional (3D) tissue shape control of mouse neural stem cell (mNSC) micro tissues by using closed agarose microchambers for effective differentiation induction of neurons in vitro. Our agarose microchambers, made by micromolding, can be sealed with an agarose sheet to form the mNSC tissues along the shape of microchambers. We constructed lane-shaped mNSC tissues with different width (â¼60-210 µm) and thickness (â¼25-95 µm) dimensions and induced differentiation to neurons with differentiation medium. We found that in thick tissues (thickness: >60 µm), distribution of differentiated neurons was not uniform, whereas in thin tissues (thickness: â¼30 µm), differentiated neurons were uniformly distributed with high differentiation efficiency. Our system to construct in vitro 3D neural tissues having uniformly distributed neurons at high differentiation ratio, could become an effective tool for drug screening using 3D neural tissues and 3D mNSC tissues under differentiation induction.
Assuntos
Diferenciação Celular , Técnicas Citológicas/métodos , Tecido Nervoso/citologia , Células-Tronco Neurais/fisiologia , Engenharia Tecidual/métodos , Animais , Técnicas Citológicas/instrumentação , Camundongos , Sefarose , Engenharia Tecidual/instrumentaçãoRESUMO
We herein report the micropatterning of flexible multiple photonic colloidal crystal gels (PCCGs) using single-layered microchannels. These patterned PCCGs exhibit structural colors that can be tuned by adjustment of the diameter and concentration of the colloidal particles in precursor solutions of N-isopropylacrylamide (NIPAM) or polyethylene glycol diacrylate (PEGDA). The precursor solutions containing dispersed colloidal particles were selectively injected into single-layered microchannels where they polymerized rapidly. The shape, density, and height of the patterned PCCG pixels were determined by the microchannels, which in turn determined the optical properties of the PCCG arrays. Furthermore, the preparation of three different types of PCCGs exhibiting three different structural colors at a high pixel density was demonstrated successfully using the single-layered polydimethylsiloxane (PDMS) microchannels. Finally, the optical reflective properties and the mechanical flexibility of the patterned multiple PCCG arrays were evaluated. We expect that our method for the preparation of such patterned PCCG arrays will contribute to the development of flexible optical devices.
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Stimuli-responsive microfibers are fabricated by extruding mixed solutions of poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-AAc) and sodium alginate (Na-alginate) using a microfluidic spinning system. The fabricated microfibers shrink and swell with temperature and/or pH. By controlling the extruded laminar flow, microfibers capable of anisotropic shrinkage are fabricated. Cross-sectional microscale geometries of microfibers, including double layering and hollowness, are successfully controlled by patterning the laminar flow during microfiber formation, resulting in hydrogels capable of folding/unfolding motions and fluid pumping. In addition, macroscopic 3D-bundle structures are assembled with these microfibers. We believe that our microfibers can be applied to various applications such as soft actuators, soft robots, and micropumps.
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This paper reports a twisting microfluidic method utilising a centrifuge-based fluid extruding system in a planetary centrifuge which simultaneously generates an orbital rotation and an axial spin. In this method, fluid extrusion from a micro-scale capillary to an 'open-space' solution or air enables release of the fluid from the capillary-based microchannel, which physically means that there is a release of fluids from a confined low-Reynolds-number environment to an open non-low-Reynolds-number environment. As a result, the extruded fluids are separated from the axial spin of the capillary, and the difference in the angular rates of the axial spin between the capillary and the extruded fluids produces the 'twisting' of the fluid. In this study, we achieve control of the twist of highly viscous fluids, and we construct a simple physical model for the fluid twist. In addition, we demonstrate the formation of twisted hydrogel microstructures (stripe-patterned microbeads and multi-helical microfibres) with control over the stripe pattern and the helical pitch length. We believe that this method will enable the generation of more sophisticated microstructures which cannot easily be formed by usual channel-based microfluidic devices. This method can also provide advanced control of microfluids, as in the case of rapid mixing of highly viscous fluids. This method can contribute to a wide range of applications in materials science, biophysics, biomedical science, and microengineering in the future.
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A vertical confocal observation system capable of high-resolution observation of intracellular structure is demonstrated. The system consists of magnet-active microplates to rotate, incline, and translate single adherent cells in the applied magnetic field. Appended to conventional confocal microscopes, this system enables high-resolution cross-sectional imaging with single-molecule sensitivity in single scanning.
Assuntos
Desenho de Equipamento , Microscopia Confocal/instrumentação , Humanos , MagnetismoRESUMO
Previous studies have demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) into the lesioned spinal cord can promote functional recovery following incomplete spinal cord injury (SCI) in animal models. However, this strategy is insufficient following complete SCI because of the gap at the lesion epicenter. To obtain functional recovery in a mouse model of complete SCI, this study uses a novel collagen-based microfiber as a scaffold for engrafted NS/PCs. We hypothesized that the NS/PC-microfiber combination would facilitate lesion closure as well as transplant survival in the transected spinal cord. NS/PCs were seeded inside the novel microfibers, where they maintained their capacity to differentiate and proliferate. After transplantation, the stumps of the transected spinal cord were successfully bridged by the NS/PC-laden microfibers. Moreover, the transplanted cells migrated into the host spinal cord and differentiated into three neural lineages (astrocytes, neurons, and oligodendrocytes). However, the NS/PC-laden scaffold could not achieve a neural connection between the rostral end of the injury and the intact caudal area of the spinal cord, nor could it achieve recovery of motor function. To obtain optimal functional recovery, a microfiber design with a modified composition may be useful. Furthermore, combinatorial therapy with rehabilitation and/or medications should also be considered for practical success of biomaterial/cell transplantation-based approaches to regenerative medicine.
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Fibras Nervosas/fisiologia , Células-Tronco Neurais/fisiologia , Doenças da Medula Espinal/mortalidade , Doenças da Medula Espinal/cirurgia , Transplante de Células-Tronco/métodos , Análise de Variância , Animais , Materiais Biocompatíveis/uso terapêutico , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/transplante , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2â µL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems.
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Centrifugação/instrumentação , Filtração/instrumentação , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Lipossomos/química , Desenho de Equipamento , Bicamadas Lipídicas/síntese química , Lipossomos/síntese química , Lipossomos/ultraestrutura , Tamanho da PartículaRESUMO
An approach for manipulating single adherent cells is developed that is integrated with an enzymatic batch release. This strategy uses an array of releasable microfabricated mobile substrates, termed microplates, formed from a biocompatible polymer, parylene. A parylene microplate array of 10-70 µm in diameter can be formed on an alginate hydrogel sacrificial layer by using a standard photolithographic process. The parylene surfaces are modified with fibronectin to enhance cell attachment, growth, and stretching. To load single cells onto these microplates, cells are initially placed in suspension at an optimized seeding density and are allowed to settle, stretch, and grow on individual microplates. The sacrificial layer underneath the microplate array can be dissolved on a time-scale of several seconds without cytotoxicity. This system allows the inspection of selected single adherent cells. The ability to assess single cells while maintaining their adhesive properties will broaden the examination of a variety of attributes, such as cell shape and cytoskeletal properties.
Assuntos
Enzimas/metabolismo , Fibroblastos/citologia , Polímeros/química , Análise de Célula Única/instrumentação , Xilenos/química , Animais , Adesão Celular , Linhagem Celular , Fibroblastos/parasitologia , Humanos , Masculino , Camundongos , Ratos , Toxoplasma/fisiologiaRESUMO
Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.
Assuntos
Materiais Biocompatíveis , Diabetes Mellitus Experimental/terapia , Matriz Extracelular , Transplante das Ilhotas Pancreáticas/métodos , Técnicas Analíticas Microfluídicas , Alginatos , Animais , Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Técnicas Analíticas Microfluídicas/instrumentação , Células Musculares/citologia , Miócitos Cardíacos , Células NIH 3T3 , Ratos , Engenharia Tecidual/métodosRESUMO
Detection of chemical substances is essential for living a healthy and cultural life in the modern world. One type of chemical sensing technology, biosensing, uses biological components with molecular recognition abilities, enabling a broad spectrum of sensing targets. Short single-stranded nucleic acids called aptamers are one of the biological molecules used in biosensing, and sensing methods combining aptamers and hydrogels have been researched for simple sensing applications. In this research, we propose a hydrogel-based biosensor that uses aptamer recognition and DNA-driven swelling hydrogels for the rapid detection of histamine. Aptamer recognition and DNA-driven swelling hydrogels are directly linked via DNA molecular reactions, enabling rapid sensing. We selected histamine, a major food poisoning toxin, as our sensing target and detected the existence of histamine within 10 min with significance. Because this sensing foundation uses aptamers, which have a vast library of targets, we believe this system can be expanded to various targets, broadening the application of hydrogel-based biosensors.
Assuntos
Aptâmeros de Nucleotídeos , Materiais Biocompatíveis , Técnicas Biossensoriais , Histamina , Hidrogéis , Teste de Materiais , Aptâmeros de Nucleotídeos/química , Hidrogéis/química , Histamina/análise , Histamina/química , Materiais Biocompatíveis/química , Tamanho da Partícula , DNA/químicaRESUMO
Here an electrical stimulation system is described for maturing microfiber-shaped cardiac tissue (cardiac microfibers, CMFs). The system enables stable culturing of CMFs with electrical stimulation by placing the tissue between electrodes. The electrical stimulation device provides an electric field covering whole CMFs within the stimulation area and can control the beating of the cardiac microfibers. In addition, CMFs under electrical stimulation with different frequencies are examined to evaluate the maturation levels by their sarcomere lengths, electrophysiological characteristics, and gene expression. Sarcomere elongation (14% increase compared to control) is observed at day 10, and a significant upregulation of electrodynamic properties such as gap junction protein alpha 1 (GJA1) and potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) (maximum fourfold increase compared to control) is observed at day 30. These results suggest that electrically stimulated cultures can accelerate the maturation of microfiber-shaped cardiac tissues compared to those without electrical stimulation. This model will contribute to the pathological research of unexplained cardiac diseases and pharmacologic testing by stably constructing matured CMFs.
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This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.
Assuntos
Alginatos , Colágeno , Dependovirus , Microesferas , Dependovirus/genética , Alginatos/química , Animais , Colágeno/química , Camundongos , Técnicas de Transferência de Genes , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Hidrogéis/química , Géis/químicaRESUMO
Cellular response to dynamic chemical stimulation encodes rich information about the underlying reaction pathways and their kinetics. Microfluidic chemical stimulators play a key role in generating dynamic concentration waveforms by mixing several aqueous solutions. In this article, we propose a multi-layer microfluidic chemical stimulator capable of modulating chemical concentrations by a simple binary logic based on the electronic-hydraulic analogy of electronic R-2R ladder circuits. The proposed device, which we call L-2L ladder digital-to-analogue converter (DAC), allows us to systematically modulate 2 n levels of concentrations from single sources of solution and solvent by a single operation of 2n membrane valves, which contrasts with existing devices that require complex channel geometry with multiple input sources and valve operations. We fabricated the L-2L ladder DAC with n = 3 bit resolution and verified the concept by comparing the generated waveforms with computational simulations. The response time of the proposed DAC was within the order of seconds because of its simple operation logic of membrane valves. Furthermore, detailed analysis of the waveforms revealed that the transient concentration can be systematically predicted by a simple addition of the transient waveforms of 2n = 6 base patterns, enabling facile optimization of the channel geometry to fine-tune the output waveforms.