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1.
Biffi E Piraino F Pedrocchi A Fiore GB Ferrigno G Redaelli A Menegon A Rasponi M 《Biomicrofluidics》2012,6(2):24106-2410610
Spatially and temporally resolved delivery of soluble factors is a key feature for pharmacological applications. In this framework, microfluidics coupled to multisite electrophysiology offers great advantages in neuropharmacology and toxicology. In this work, a microfluidic device for biochemical stimulation of neuronal networks was developed. A micro-chamber for cell culturing, previously developed and tested for long term neuronal growth by our group, was provided with a thin wall, which partially divided the cell culture region in two sub-compartments. The device was reversibly coupled to a flat micro electrode array and used to culture primary neurons in the same microenvironment. We demonstrated that the two fluidically connected compartments were able to originate two parallel neuronal networks with similar electrophysiological activity but functionally independent. Furthermore, the device allowed to connect the outlet port to a syringe pump and to transform the static culture chamber in a perfused one. At 14 days invitro, sub-networks were independently stimulated with a test molecule, tetrodotoxin, a neurotoxin known to block action potentials, by means of continuous delivery. Electrical activity recordings proved the ability of the device configuration to selectively stimulate each neuronal network individually. The proposed microfluidic approach represents an innovative methodology to perform biological, pharmacological, and electrophysiological experiments on neuronal networks. Indeed, it allows for controlled delivery of substances to cells, and it overcomes the limitations due to standard drug stimulation techniques. Finally, the twin network configuration reduces biological variability, which has important outcomes on pharmacological and drug screening. 相似文献
2.
Axon path-finding plays an important role in normal and pathogenic brain development as well as in neurological regenerative medicine. In both scenarios, axonal growth is influenced by the microenvironment including the soluble molecules and contact-mediated signaling from guiding cells and cellular matrix. Microfluidic devices are a powerful tool for creating a microenvironment at the single cell level. In this paper, an asymmetrical-channel-based biochip, which can be later incorporated into microfluidic devices for neuronal network study, was developed to investigate geometric as well as supporting cell control of polarized axonal growth in forming a defined neuronal circuitry. A laser cell deposition system was used to place single cells, including neuron-glia pairs, into specific microwells of the device, enabling axonal growth without the influence of cytophilic∕phobic surface patterns. Phase microscopy showed that a novel "snag" channel structure influenced axonal growth in the intended direction 4:1 over the opposite direction. In heterotypic experiments, glial cell influence over the axonal growth path was observed with time-lapse microscopy. Thus, it is shown that single cell and heterotypic neuronal path-finding models can be developed in laser patterned biochips. 相似文献
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In this work a disposable, parallel microbioreactor (MBR) suitable for screening in batch or continuous mode is presented. The reactor consists of five parallel microchambers made of poly(dimethylsiloxane) bonded to a glass substrate. A grid structure is engraved on each chamber, allowing subsequent morphology imaging. Measurements are recorded over the entire cultivation period with constant parameters, namely, position and focus in the z-axis. The microdevice may be used for either parallel, uni- or multiparametric screening, and overcomes the drawback of gridless microwell plates which require expensive equipment such as an inverted microscope with an automatic stage. To validate the scalability from laboratory scale to microscale, and thus the cultivation protocol in the MBR, the germination of fungal spores (A. ochraceus) is evaluated for two different key magnitudes (pH and temperature) and compared to the results obtained from conventional laboratory scale systems (flasks and agar plates). Information on germination capacity with regard to interspecies' variability allows for optimization of industrial processes as optimal pH and temperature matched to the mesoscopic cultivation systems. The germination conditions therefore remain unaffected inside the MBR, while providing the following advantages: (i) dramatic reduction of medium consumption, (ii) submerged cultivation with constant oxygen supply, (iii) assured low cost and disposability, and (iv) possibility of a continuous cultivation mode. 相似文献
5.
Shenqiang Wang Letao Yang Bolei Cai Fuwei Liu Yannan Hou Hua Zheng Fang Cheng Hepeng Zhang Le Wang Xiaoyi Wang Qianxin Lv Liang Kong Ki-Bum Lee Qiuyu Zhang 《国家科学评论(英文版)》2022,9(4)
Cartilage injuries are often devastating and most cannot be cured because of the intrinsically low regenerative capacity of cartilage tissues. Although stem-cell therapy has shown enormous potential for cartilage repair, the therapeutic outcome has been restricted by low survival rates and poor chondrocyte differentiation in vivo. Here, we report an injectable hybrid inorganic (IHI) nanoscaffold that facilitates fast assembly, enhances survival and regulates chondrogenic differentiation of stem cells. IHI nanoscaffolds that strongly bind to extracellular matrix (ECM) proteins assemble stem cells through synergistic 3D cell–cell and cell–matrix interactions, creating a favorable physical microenvironment for stem-cell survival and differentiation in vitro and in vivo. Additionally, chondrogenic factors can be loaded into nanoscaffolds with a high capacity, which allows deep, homogenous drug delivery into assembled 3D stem-cell-derived tissues for effective control over the soluble microenvironment of stem cells. The developed IHI nanoscaffolds that assemble with stem cells are injectable. They also scavenge reactive oxygen species and timely biodegrade for proper integration into injured cartilage tissues. Implantation of stem-cell-assembled IHI nanoscaffolds into injured cartilage results in accelerated tissue regeneration and functional recovery. By establishing our IHI nanoscaffold-templated 3D stem-cell assembly method, we provide a promising approach to better overcoming the inhibitory microenvironment associated with cartilage injuries and to advance current stem-cell-based tissue engineering. 相似文献
6.
神经干细胞（NSCs）是具有高度自我更新能力并能分化为神经元、星形胶质细胞和少突胶质细胞的神经前体细胞,具有广阔的临床应用前景。神经营养素家族对神经干细胞的分化有一定影响。其包括：神经生长因子（NGF）、脑源性神经生长因子（BDNF）、神经营养素-3（NT-3）、神经营养素-4/5（NT-4/5）以及神经营养素-6（NT-6）。本文就目前神经营养素家族对NSCs分化的影响的研究现状进行综述。 相似文献
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Control of the 3D microenvironment for cultured cells is essential for understanding the complex relationships that biomolecular concentration gradients have on cellular growth, regeneration, and differentiation. This paper reports a microfluidic device for delivering gradients of soluble molecules to cells in an open reservoir without exposing the cells to flow. The cells are cultured on a polyester membrane that shields them from the flow that delivers the gradient. A novel "lid" design is implemented which prevents leakage from around the membrane without requiring sealing agents or adhesives. Once layers are molded, device fabrication can be performed within minutes while at room temperature. Surface gradients were characterized with epifluorescence microscopy; image analysis verified that sharp gradients (～33 μm wide) can be reproducibly generated. We show that heterogeneous laminar flow patterns of Orange and Green Cell Tracker (CT) applied beneath the membrane can be localized to cells cultured on the other side; concentration profile scans show the extent of CT diffusion parallel to the membrane's surface to be 10-20 μm. Our device is ideal for conventional cell culture because the cell culture surface is readily accessible to physical manipulation (e.g., micropipette access), the cell culture medium is in direct contact with the incubator atmosphere (i.e., no special protocols for ensuring proper equilibration of gas concentrations are required), and the cells are not subjected to flow-induced shear forces, which are advantageous attributes not commonly found in closed-channel microfluidic designs. 相似文献
8.
Angela R. Dixon Shrinidhi Rajan Chuan-Hsien Kuo Tom Bersano Rachel Wold Nobuyuki Futai Shuichi Takayama Geeta Mehta 《Biomicrofluidics》2014,8(1)
We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells. 相似文献
9.
Continuous cell tracking by time-lapse microscopy has led to detailed study of cell differentiation pathways using single cell fate maps. There are a multitude of cell fate outcomes, so hundreds of clonal division histories are required to measure these stochastic branching processes. This study examines the principle of condensing cell imaging information into a relatively small region to maximize live cell imaging throughput. High throughput clonal analysis of non-adherent cells by continuous live cell tracking was possible using a microwell perfusion array with an internal volume of 16 μl and 600 microwells at the base. This study includes examination of biocompatibility of buffer systems, connecting tubing, cell culture substrates, and media degradation. An intermittent perfusion protocol was selected for long-term time-lapse imaging of KG1a cells in the microwell array; 1500 clones were simultaneously cultured and scanned every 3 min at 100 × magnifications for 6 days. The advantages of perfusion microwell culture are continuous long-term cell tracking, higher cell imaging throughput, and greater control over cell microenvironment. Microwell devices facilitate high throughput analysis of cell lineage development and measurement of the probability distribution for cell life events such as mitosis. 相似文献
10.
研究不同浓度钙离子对人成骨样细胞MG-63分化的影响.结果表明, 5.0mmol/L Ca2+促进 CaSR、ALP、OC、Runx 2、IGF-1和BMP2 mRNA表达,而加入CaSR和ERK抑制剂后,则下调 ALP、OC、Runx 2、IGF-1和BMP2 mRNA表达.不同浓度Ca2+处理后,磷酸化-ERK(p-ERK)蛋白表达明显增加.因此,5.0mmol/L Ca2+通过上调CaSR基因的表达,激活ERK通路,促进MG-63成骨样细胞的分化. 相似文献
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Embryoid body (EB) formation forms an important step in embryonic stem cell differentiation invivo. In murine embryonic stem cell (mESC) cultures EB formation is inhibited by the inclusion of leukaemic inhibitory factor (LIF) in the medium. Assembly of mESCs into aggregates by positive dielectrophoresis (DEP) in high field regions between interdigitated oppositely castellated electrodes was found to initiate EB formation. Embryoid body formation in aggregates formed with DEP occurred at a more rapid rate-in fact faster compared to conventional methods-in medium without LIF. However, EB formation also occurred in medium in which LIF was present when the cells were aggregated with DEP. The optimum characteristic size for the electrodes for EB formation with DEP was found to be 75-100 microns; aggregates smaller than this tended to merge, whilst aggregates larger than this tended to split to form multiple EBs. Experiments with ESCs in which green fluorescent protein (GFP) production was targeted to the mesodermal gene brachyury indicated that differentiation within embryoid bodies of this size may preferentially occur along the mesoderm lineage. As hematopoietic lineages during normal development derive from mesoderm, the finding points to a possible application of DEP formed EBs in the production of blood-based products from ESCs. 相似文献
12.
企业价格及定位竞争模型研究 总被引：8，自引：0，他引：8
本文主要分析了在产品多方面差异条件下企业的价格和定位竞争。本文的模型可以说明（1）在多方面差异条件下，企业可以索取超过边际成本的价格，从而打破伯川德悖论；（2）如果企业的价格竞争受到限制，那么企业将追求最小的差异化；（3）当企业可以对价格和定位进行选择时，价格和定位与差异化程度相关。本文的模型从不同的方面深化了古典差异化模型的结果。 相似文献
13.
Microorganisms have a significant influence on the environmental fate of radionuclides in aquatic and terrestrial ecosystems with a multiplicity of physico-chemical and biological mechanisms effecting changes in mobility and speciation. Physico-chemical mechanisms of removal include association with extracellular materials, metabolites and cell walls which are features of living and dead organisms. In living cells, some physico-chemical processes are reversible, influenced by metabolism and changing environmental conditions. Metabolism-dependent mechanisms of radionuclide immobilization include sulphide precipitation, transport and intracellular compartmentation and/or sequestration by proteins and peptides. In addition, chemical reduction to less soluble forms can result in immobilization. Microbial processes involved in radionuclide solubilization include autotrophic and heterotrophic leaching, and complexation by siderophores and other metabolites. Such mechanisms are important components of biogeochemical cycles for radionuclides and should be considered in any analyses of environmental radionuclide contamination. In addition, several microorganism-based biotechnologies are receiving interest as potential treatment methods. 相似文献
14.
Patric Wallin Carl Zandén Bj?rn Carlberg Nina Hellstr?m Erkenstam Johan Liu Julie Gold 《Biomicrofluidics》2012,6(2)
The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble microenvironment on cellular fate processes. 相似文献
15.
The research presented in this paper is the first wave of a longitudinal study of a Cambodian information and communication
for development (ICT4D) project, iREACH, aimed at testing a framework for evaluating whether and how such initiatives can
contribute to capabilities, empowerment and sustainability. The framework is informed by Amartya Sen’s capability approach
(CA), uses a participatory methodology, considers the micro-, meso-, and macro- levels in understanding the role ICT can play
in the development process, and adopts a forward-looking longitudinal perspective. Key findings of this research are that
the project had contributed to livelihoods and other aspects of well-being in diverse ways, primarily in education, health,
and farming. Participants also valued the project because of its contribution to empowerment, particularly gender empowerment.
Another way in which participants valued iREACH was of a more intrinsic nature, manifested in a general appreciation of just
being part of the world and knowing about events in other parts of Cambodia and beyond. These findings are consistent with
the CA’s emphasis on development being about more than economic growth and support the importance of considering external
factors, conceptualised here as the meso- and macro- levels, in the conversion of commodities in the form of services provided
at iREACH, to capabilities. 相似文献
16.
本文首次记载了对分布于峨眉山的凤仙花属Impatiens 12种植物的种子表面显微结构的观察，
并分析了种子表面形态在该属种水平上的分类价值及可能的系统学意义。种皮表层细胞特化、排列方
式、隆起状态、种脊上近合点端的突起、种子末端附属物的有无及其形态等性状被视为凤仙花属种子表
面的主要特征。依据这些性状12种凤仙花种子形态分为两种类型：1．种子表面光滑，无明显大、小细胞
分化。如白花凤仙I．wilsoni可能具3沟花粉凤仙花的种子形态的特点。2．表面粗糙，有明显大、小细
胞的分化及不同程度的细胞隆起，并呈现出多种特异形态．这代表了以一年生草本，4沟花粉为特点的
凤仙花种子形态特征。种子特征与植物体习性、花形态及花粉形态相关，在一定程度上反映了属内类群的分化，因而在凤仙花科、属的分类和系统研究中是不可忽视的性状。 相似文献
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High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment 总被引：1，自引：0，他引：1
Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis. 相似文献
19.
Cancer cell migration through tissue pores and tracks into the bloodstream is a critical biological step for cancer metastasis. Although in vivo studies have shown that expression of vimentin can induce invasive cell lines, its role in cell cytoskeleton reorganization and cell motility under in vitro physical confinement remains unknown. Here, a microfluidic device with cell culture chamber and collagen-coated microchannels was developed as an in vitro model for physiological confinement environments. Using this microchannel assay, we demonstrated that the knockdown of vimentin decreases 3T3 fibroblast cell directional migration speed in confined microchannels. Additionally, as cells form dynamic membranes that define the leading edge of motile cells, different leading edge morphologies of 3T3 fibroblast and 3T3 vimentin knockdown cells were observed. The leading edge morphology change under confinement can be explained by the effect of vimentin on cytoskeletal organization and focal adhesion. The microfluidic device integrated with a time-lapse microscope provided a new approach to study the effect of vimentin on cell adhesion, migration, and invasiveness. 相似文献
20.
Seidi A Kaji H Annabi N Ostrovidov S Ramalingam M Khademhosseini A 《Biomicrofluidics》2011,5(2):22214
In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinson's disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications. 相似文献