Single-cell analysis and sorting using droplet-based microfluidics

Mažutis, Linas; Gilbert, John; Ung, W. Lloyd; Weitz, David A.; Griffiths, Andrew D.; Heyman, John A.

doi:10.1038/nprot.2013.046

Cited by 1,206 publications

(1,115 citation statements)

References 96 publications

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“…Microfluidic chips with flow-focusing geometry ( Fig. S1 B and C) were produced using standard soft lithographic techniques (21,22), i.e., SU-8/Si masters were used to make polydimethylsiloxane (PDMS) slides with 60 × 70 μm and 20 × 22 μm (width × height) orifices. Inlets and outlets were made using a 1.2-mm Harris Uni-Core biopsy punch.…”

Section: Methodsmentioning

confidence: 99%

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Terekhov

Смирнов

Stepanova

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

191

172

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Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

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Section: Methodsmentioning

confidence: 99%

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Terekhov

Смирнов

Stepanova

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

191

172

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“…28 AutoDesk software in AutoCAD was used to design the desired two-dimensional pattern and printed on a photolithography chrome mask. The silicon wafer (University Wafers, Boston, MA) was spin coated with SU-8 3025 photoresist (Michrochem, Newton, MA) to a 30 mm film thickness (Laurell Technologies, North Wales, PA), baked at 65°C for 1 minute and 95°C for 5 minutes, and exposed to UV light (;10 mJ · cm 2 ) through the chrome mask for 30 seconds.…”

Section: Microfluidic Bioreactor Design and Fabricationmentioning

confidence: 99%

Platelet bioreactor-on-a-chip

Thon

Mažutis

et al. 2014

Blood

Self Cite

188

210

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• We have developed a biomimetic microfluidic platelet bioreactor that recapitulates bone marrow and blood vessel microenvironments.• Application of shear stress in this bioreactor triggers physiological proplatelet production, and platelet release.Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. (Blood. 2014;124(12):1857-1867) IntroductionAlthough platelets (PLTs) play critical roles in hemostasis, 1 angiogenesis, 2 and innate immunity, 3 PLT production remains poorly understood. Consequently, PLT units are derived entirely from human donors, despite serious clinical concerns owing to their immunogenicity and associated risk of sepsis. 4 More than 2.17 million apheresisequivalent PLT units are transfused yearly in the United States 5,6 at a cost of .$1 billion per year. Although demand for PLT transfusions has increased markedly in the past decade, a near-static pool of donors and a 5-day PLT unit shelf life resulting from bacterial contamination 7 and storage-related PLT deterioration, 8 have resulted in significant PLT shortages. 9 Furthermore, artificial platelet substitutes have failed to replace physiological platelet products. 10 An efficient, donorindependent PLT bioreactor capable of generating clinically significant numbers of functional human PLTs is necessary to obviate risks associated with PLT procurement and storage, and help meet growing transfusion needs. In vivo, megakaryocytes (MKs) PLT progenitors sit outside blood vessels in the bone marrow (BM) and extend long, branching cellular structures designated proPLTs into the circulation from which PLTs are released. 11-15 Nearly 100% of human adult MKs must produce ;10 3 PLTs each to account for circulating PLT counts. 16 Although functional human PLTs were first grown in vitro in 1995, 17 to date only ;10% of human MKs initiate proPLT production in culture. This results in yields of 10 122 PLTs per CD34 1 cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation c...

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“…To incorporate cells within droplets, a bulk liquid containing cells is typically dispersed into droplets. Here, the probability of finding a certain number of cells inside a droplet follows the Poisson distribution [28]. To avoid droplets that lack cells, one should typically have 10 6 cells/ml at the start for a droplet size of approximately 100 micrometer [29].…”

Section: Cell Biology and Tissue Engineeringmentioning

confidence: 99%

Droplet microfluidics: A tool for biology, chemistry and nanotechnology

Mashaghi

Abbaspourrad

Weitz

et al. 2016

TrAC Trends in Analytical Chemistry

321

188

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The ability to perform laboratory operations on small scales using miniaturized devices provides numerous benefits, including reduced quantities of reagents and waste as well as increased portability and controllability of assays. These operations can involve reaction components in the solution phase and as a result, their miniaturization can be accomplished through microfluidic approaches. One such approach, droplet microfluidics, provides a high-throughput platform for a wide range of assays and approaches in chemistry, biology and nanotechnology. We highlight recent advances in the application of droplet microfluidics in chipbased technologies, such as single-cell analysis tools, small-scale cell cultures, in-droplet chemical synthesis, high-throughput drug screening, and nanodevice fabrication.

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Single-cell analysis and sorting using droplet-based microfluidics

Cited by 1,206 publications

References 96 publications

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Platelet bioreactor-on-a-chip

Droplet microfluidics: A tool for biology, chemistry and nanotechnology

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