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Connie B. Chang James N. Wilking Shin‐Hyun Kim Ho Cheung Shum David A. Weitz 《Small (Weinheim an der Bergstrasse, Germany)》2015,11(32):3954-3961
In this work, microfluidic technology is used to rapidly create hundreds of thousands of monodisperse double and triple emulsion drops that serve as 3D microenvironments for the containment and growth of bacterial biofilms. The size of these drops, with diameters from tens to hundreds of micrometers, makes them amenable to rapid manipulation and analysis. This is demonstrated by using microscopy to visualize cellular differentiation of Bacillus subtilis biofilm communities within each drop and the bacterial biofilm microstructure. Biofilm growth is explored upon specific interfaces in double and triple emulsions and upon negative and positive radii of curvature. Biofilm attachment of matrix and flagella mutants is studied as well as biofilms of Pseudomonas aeruginosa. This is the first demonstration of biofilms grown in microscale emulsion drops, which serve as both templates and containers for biofilm growth and attachment. These microenvironments have the potential to transform existing high‐throughput screening methods for bacterial biofilms. 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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Nondestructive Real‐Time Monitoring of Enhanced Stem Cell Differentiation Using a Graphene‐Au Hybrid Nanoelectrode Array
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Jin‐Ho Lee Hye Kyu Choi Letao Yang Sy‐Tsong Dean Chueng Jeong‐Woo Choi Ki‐Bum Lee 《Advanced materials (Deerfield Beach, Fla.)》2018,30(39)
Stem cells have attracted increasing research interest in the field of regenerative medicine because of their unique ability to differentiate into multiple cell lineages. However, controlling stem cell differentiation efficiently and improving the current destructive characterization methods for monitoring stem cell differentiation are the critical issues. To this end, multifunctional graphene–gold (Au) hybrid nanoelectrode arrays (NEAs) to: (i) investigate the effects of combinatorial physicochemical cues on stem cell differentiation, (ii) enhance stem cell differentiation efficiency through biophysical cues, and (iii) characterize stem cell differentiation in a nondestructive real‐time manner are developed. Through the synergistic effects of physiochemical properties of graphene and biophysical cues from nanoarrays, the graphene‐Au hybrid NEAs facilitate highly enhanced cell adhesion and spreading behaviors. In addition, by varying the dimensions of the graphene‐Au hybrid NEAs, improved stem cell differentiation efficiency, resulting from the increased focal adhesion signal, is shown. Furthermore, graphene‐Au hybrid NEAs are utilized to monitor osteogenic differentiation of stem cells electrochemically in a nondestructive real‐time manner. Collectively, it is believed the unique multifunctional graphene‐Au hybrid NEAs can significantly advance stem‐cell‐based biomedical applications. 相似文献
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Rohollah Nasiri Amir Shamloo Samad Ahadian Leyla Amirifar Javad Akbari Marcus J. Goudie KangJu Lee Nureddin Ashammakhi Mehmet R. Dokmeci Dino Di Carlo Ali Khademhosseini 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(29)
Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high‐efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label‐free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor‐intensive steps of labeling molecular signatures of cells. In general, microfluidic‐based cell sorting approaches can separate cells using “intrinsic” (e.g., fluid dynamic forces) versus “extrinsic” external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label‐free microfluidic‐based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic‐based cell separation methods are discussed. 相似文献
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Centering Single Cells in Microgels via Delayed Crosslinking Supports Long‐Term 3D Culture by Preventing Cell Escape
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Tom Kamperman Sieger Henke Claas Willem Visser Marcel Karperien Jeroen Leijten 《Small (Weinheim an der Bergstrasse, Germany)》2017,13(22)
Single‐cell‐laden microgels support physiological 3D culture conditions while enabling straightforward handling and high‐resolution readouts of individual cells. However, their widespread adoption for long‐term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off‐center encapsulated cells. High‐speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off‐center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long‐term culture of individual cells within <5‐µm‐thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell‐centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures. 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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Effective Gene Delivery into Human Stem Cells with a Cell‐Targeting Peptide‐Modified Bioreducible Polymer
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Jagadish Beloor Suresh Ramakrishna Kihoon Nam Chang Seon Choi Jongkil Kim Sung Hwa Kim Hyong Jin Cho HeungSoo Shin Hyongbum Kim Sung Wan Kim Sang‐Kyung Lee Priti Kumar 《Small (Weinheim an der Bergstrasse, Germany)》2015,11(17):2069-2079
Stem cells are poorly permissive to non‐viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell‐binding ligand with a polymer that releases nucleic acids in a cytoplasm‐responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9‐derived hESC. Conjugating RVG to a redox‐sensitive biodegradable dendrimer‐type arginine‐grafted polymer (PAM‐ABP) enabled nanoparticle formation with plasmid DNA without altering the environment‐sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG‐PAM‐ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. ~60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG‐PAM‐ABP is thus a novel bioreducible, biocompatible, non‐toxic, synthetic gene delivery system for nAchR‐expressing stem cells. Our data also demonstrates that a cell‐binding ligand like RVG can cooperate with a gene delivery system like PAM‐ABP to enable transfection of poorly‐permissive cells. 相似文献
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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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Xing Xu Junxia Wang Lingling Wu Jingjing Guo Yanling Song Tian Tian Wei Wang Zhi Zhu Chaoyong Yang 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(9)
The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed. 相似文献
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Xiaobao Cao Ying Du Andreas Küffner Jordan Van Wyk Paolo Arosio Jing Wang Peter Fischer Stavros Stavrakis Andrew deMello 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(20)
Fluorescence‐based detection schemes provide for multiparameter analysis in a broad range of applications in the chemical and biological sciences. Toward the realization of fully portable analysis systems, microfluidic devices integrating diverse functional components have been implemented in a range of out‐of‐lab environments. That said, there still exits an unmet and recognized need for miniaturized, low‐cost, and sensitive optical detection systems, which provide not only for efficient molecular excitation, but also enhanced photon collection capabilities. To this end, an optofluidic platform that is adept at enhancing fluorescence light collection from microfluidic channels is presented. The central component of the detection module is a monolithic parabolic mirror located directly above the microfluidic channel, which acts to enhance the number of emitted photons reflected toward the detector. In addition, two‐photon polymerization is used to print a microscale‐lens below the microfluidic flow channel and directly opposite the mirror, to enhance the delivery of excitation radiation into the channel. Using such an approach, it is demonstrated that fluorescence signals can be enhanced by over two orders of magnitude, with component parallelization enabling the detection of pL‐volume droplets at rates up to 40 000 droplets per second. 相似文献
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Stem Cell Differentiation: Nondestructive Real‐Time Monitoring of Enhanced Stem Cell Differentiation Using a Graphene‐Au Hybrid Nanoelectrode Array (Adv. Mater. 39/2018)
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Jin‐Ho Lee Hye Kyu Choi Letao Yang Sy‐Tsong Dean Chueng Jeong‐Woo Choi Ki‐Bum Lee 《Advanced materials (Deerfield Beach, Fla.)》2018,30(39)