共查询到20条相似文献，搜索用时 15 毫秒
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Kenza Samlali Fatemeh Ahmadi Angela B. V. Quach Guy Soffer Steve C. C. Shih 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(34)
Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co‐planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL‐sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in‐house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI‐H1299 lung squamous cell population resulting from loss‐of‐function eGFP and RAF1 gene knockout transfections. 相似文献
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Xuan Li Mohammad Aghaamoo Shiyue Liu Do‐Hyun Lee Abraham P. Lee 《Small (Weinheim an der Bergstrasse, Germany)》2018,14(40)
While lipoplex (cationic lipid‐nucleic acid complex)‐mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only ≈5% or even lower. Here, efficient and consistent lipoplex‐mediated transfection is demonstrated for hard‐to‐transfect suspension cells via a single‐cell, droplet‐microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co‐confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch‐off junction. The transfection efficiency, examined by the delivery of the pcDNA3‐EGFP plasmid, improves from ≈5% to ≈50% for all three tested suspension cell lines, i.e., K562, THP‐1, Jurkat, and with significantly reduced cell‐to‐cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)–CAS9 (CRISPR‐associated nuclease 9) mechanism is also achieved using this platform. Lipoplex‐mediated single‐cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell‐to‐cell variation for hard‐to‐transfect suspension cells. 相似文献
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Sarah Tuber Eric von Lieres Alexander Grünberger 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(16)
Microfluidic single‐cell cultivation (MSCC) is an emerging field within fundamental as well as applied biology. During the last years, most MSCCs were performed at constant environmental conditions. Recently, MSCC at oscillating and dynamic environmental conditions has started to gain significant interest in the research community for the investigation of cellular behavior. Herein, an overview of this topic is given and microfluidic concepts that enable oscillating and dynamic control of environmental conditions with a focus on medium conditions are discussed, and their application in single‐cell research for the cultivation of both mammalian and microbial cell systems is demonstrated. Furthermore, perspectives for performing MSCC at complex dynamic environmental profiles of single parameters and multiparameters (e.g., pH and O2) in amplitude and time are discussed. The technical progress in this field provides completely new experimental approaches and lays the foundation for systematic analysis of cellular metabolism at fluctuating environments. 相似文献
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Idan Gal Reuven Edri Nadav Noor Matan Rotenberg Michael Namestnikov Itai Cabilly Assaf Shapira Tal Dvir 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(8)
One of the strategies for heart regeneration includes cell delivery to the defected heart. However, most of the injected cells do not form quick cell–cell or cell–matrix interactions, therefore, their ability to engraft at the desired site and improve heart function is poor. Here, the use of a microfluidic system is reported for generating personalized hydrogel‐based cellular microdroplets for cardiac cell delivery. To evaluate the system's limitations, a mathematical model of oxygen diffusion and consumption within the droplet is developed. Following, the microfluidic system's parameters are optimized and cardiac cells from neonatal rats or induced pluripotent stem cells are encapsulated. The morphology and cardiac specific markers are assessed and cell function within the droplets is analyzed. Finally, the cellular droplets are injected to mouse gastrocnemius muscle to validate cell retention, survival, and maturation within the host tissue. These results demonstrate the potential of this approach to generate personalized cellular microtissues, which can be injected to distinct regions in the body for treating damaged tissues. 相似文献
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《Small Methods》2017,1(10)
Single‐cell analyses of secretory proteins are essential to fully understand cellular functional heterogeneity and unravel the underlying mechanisms of intercellular signaling and interactions. Retrieving dynamic information of protein secretion at single‐cell resolution reflects the precise, real‐time functional states of individual cells in physiological processes. Such measurements remain very challenging in single‐cell analysis, which requires highly integrated systems capable of performing on‐chip single‐cell isolation and subsequent real‐time protein detection. Here, recent advances in microfluidics‐based single‐cell manipulation and emerging approaches for label‐free single‐cell biosensing are reviewed. The advantages and limitations of these technologies are summarized and challenges to establish the integrated microfluidic biosensing systems for real‐time single‐cell secretomics are discussed. Recent efforts on integrated platforms for on‐chip single‐cell protein assays are highlighted and some perspectives on future directions in this field are provided. 相似文献
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Hashimoto M Garstecki P Whitesides GM 《Small (Weinheim an der Bergstrasse, Germany)》2007,3(10):1792-1802
A method is described for the formation of stable, composite aqueous emulsions of 1) combinations of distinct families of bubbles of nitrogen, 2) combinations of distinct families of droplets of an organic fluid (either perfluoro(methyl)decalin or hexadecane), and 3) combinations of bubbles and droplets. A system of two or three microfluidic flow-focusing units is coupled to a single outlet channel. The composite emulsions can be precisely tuned, both in their composition and in the number fraction of components--either bubbles or droplets--of different types. The use of microfluidic technology, with closely coupled flow-focusing units, guarantees that the emulsions are mixed locally at a controlled local stoichiometry. The emulsions self-assemble in a nonequilibrium process to form a wide variety of highly organized periodic lattices. 相似文献
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Juan H. Gonzlez‐Estefan Mathieu Gonidec Thi Thiet Vu Nathalie Daro Guillaume Chastanet 《Advanced Materials Technologies》2019,4(8)
Over the last two decades, polydimethylsiloxane (PDMS) has been widely used as the material of choice for fast‐throughput prototyping of microfluidic devices due to the ease of fabrication of PDMS devices by soft‐lithography replica molding methods. Nevertheless, PDMS is known to swell significantly in a variety of organic solvents which has sometimes limited its use in synthetic chemistry and has led the microfluidic community to consider PDMS as being “incompatible” with such solvents. Nevertheless, as shown here, when analyzed deeper and controlled properly, the solvent‐induced swelling of PDMS actually constitutes a potentially useful phenomenon that can become a simple tool to control and adjust finely and dynamically the geometry of microfluidic chips in situ. In this paper, this method is applied to the control of the behavior of a variety of droplet generators. The simplicity and efficiency of this approach make it a great asset for droplet microfluidics synthesis, for which microreactor sizes are a critical parameter that is often overlooked due to its complex implementation. In a more general sense, it is expected that integrating swelling into design should allow to adjust the function of many different types of chips exhibiting geometry‐driven functions. 相似文献
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Lang Nan Man Yuk Alison Lai Matthew Yuk Heng Tang Yau Kei Chan Leo Lit Man Poon Ho Cheung Shum 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(9)
Droplet‐based microfluidic techniques are extensively used in efficient manipulation and genome‐wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single‐droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on‐demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on‐demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell‐to‐cell variations. 相似文献
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Teng Xu Yanhai Gong Xiaolu Su Pengfei Zhu Jing Dai Jian Xu Bo Ma 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(30)
The small size and low DNA amount of bacterial cells have hindered establishing phenome–genome links in a precisely indexed, one‐cell‐per‐reaction manner. Here, Raman‐Activated Gravity‐driven single‐cell Encapsulation and Sequencing (RAGE‐Seq) is presented, where individual cells are phenotypically screened via single‐cell Raman spectra (SCRS) in an aquatic, vitality‐preserving environment, then the cell with targeted SCRS is precisely packaged in a picoliter microdroplet and readily exported in a precisely indexed, “one‐cell‐one‐tube” manner. Such integration of microdroplet encapsulation to Raman‐activated sorting ensures high‐coverage one‐cell genome sequencing or cultivation that is directly linked to metabolic phenotype. For clinical Escherichia coli isolates, genome assemblies derived from precisely one cell via RAGE‐Seq consistently reach >95% coverage. Moreover, directly from a urine sample of urogenital tract infection, metabolic‐activity‐based antimicrobial susceptibility phenotypes and genome sequence of 99.5% coverage are obtained simultaneously from precisely one cell. This single‐cell global mutation map corroborates resistance phenotype and genotype, and unveils epidemiological features with high specificity and sensitivity. The ability to profile and correlate bacterial metabolic phenome and high‐quality genome sequences at one‐cell resolution suggests broad application of RAGE‐Seq. 相似文献
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《Small Methods》2018,2(1)
Considerable advances have been witnessed in the development of biochips that seek to realize various types of immune cell analysis on microscale platforms and enhance both basic and applied immunological research beyond the capability of conventional methods. Here, state‐of‐the‐art designs and examples for biochip‐based analysis and manipulation of immune cells are reviewed, and the potential of this emerging technology to enhance the understanding of immunology and improve disease diagnosis and treatment is discussed. In particular, some of the recent advances in this field, along with the challenges that must be addressed for these technologies, and their potential in precision medicine are highlighted. 相似文献
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Yankai Jia Yukun Ren Likai Hou Weiyu Liu Xiaokang Deng Hongyuan Jiang 《Small (Weinheim an der Bergstrasse, Germany)》2017,13(46)
Advances in microfluidic emulsification have enabled the generation of exquisite multiple‐core droplets, which are promising structures to accommodate microreactions. An essential requirement for conducting reactions is the sequential coalescence of the multiple cores encapsulated within these droplets, therefore, mixing the reagents together in a controlled sequence. Here, a microfluidic approach is reported for the conduction of two‐step microreactions by electrically fusing three cores inside double‐emulsion droplets. Using a microcapillary glass device, monodisperse water‐in‐oil‐in‐water droplets are fabricated with three compartmented reagents encapsulated inside. An AC electric field is then applied through a polydimethylsiloxane chip to trigger the sequential mixing of the reagents, where the precise sequence is guaranteed by the discrepancy of the volume or conductivity of the inner cores. A two‐step reaction in each droplet is ensured by two times of core coalescence, which totally takes 20–40 s depending on varying conditions. The optimal parameters of the AC signal for the sequential fusion of the inner droplets are identified. Moreover, the capability of this technique is demonstrated by conducting an enzyme‐catalyzed reaction used for glucose detection with the double‐emulsion droplets. This technique should benefit a wide range of applications that require multistep reactions in micrometer scale. 相似文献