Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

Prakadan, Sanjay M.; Shalek, Alex K.; Weitz, David A.

doi:10.1038/nrg.2017.15

Cited by 293 publications

(246 citation statements)

References 155 publications

(259 reference statements)

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“…In addition, neutrophil studies using genomics and proteomic approaches are limited by laborious isolation processes and the need for milliliter volumes of blood (≈4–8 mL) to purify sufficient protein and nuclei acid content. While these processes have been successfully miniaturized using microfluidics technologies for sample preparation and high‐throughput “‐omics” analyses, they are still labor intensive and require technical expertise, and thus do not translate easily for bedside testing.…”

Section: Introductionmentioning

confidence: 99%

A Novel Microdevice for Rapid Neutrophil Purification and Phenotyping in Type 2 Diabetes Mellitus

Tay

Dalan

et al. 2017

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Neutrophil dysfunction is strongly linked to type 2 diabetes mellitus (T2DM) pathophysiology, but the prognostic potential of neutrophil biomarkers remains largely unexplored due to arduous leukocyte isolation methods. Herein, a novel integrated microdevice is reported for single-step neutrophil sorting and phenotyping (chemotaxis and formation of neutrophil extracellular traps (NETosis)) using small blood volumes (fingerprick). Untouched neutrophils are purified on-chip from whole blood directly using biomimetic cell margination and affinity-based capture, and are exposed to preloaded chemoattractant or NETosis stimulant to initiate chemotaxis or NETosis, respectively. Device performance is first characterized using healthy and in vitro inflamed blood samples (tumor necrosis factor alpha, high glucose), followed by clinical risk stratification in a cohort of subjects with T2DM. Interestingly, "high-risk" T2DM patients characterized by severe chemotaxis impairment reveal significantly higher C-reactive protein levels and poor lipid metabolism characteristics as compared to "low-risk" subjects, and their neutrophil chemotaxis responses can be mitigated after in vitro metformin treatment. Overall, this unique and user-friendly microfluidics immune health profiling strategy can significantly aid the quantification of chemotaxis and NETosis in clinical settings, and be further translated into a tool for risk stratification and precision medicine methods in subjects with metabolic diseases such as T2DM.

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Section: Introductionmentioning

confidence: 99%

A Novel Microdevice for Rapid Neutrophil Purification and Phenotyping in Type 2 Diabetes Mellitus

Tay

Dalan

et al. 2017

Small

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“…Experimental omics approaches 39,40 to separately process multiple single cells are increasingly important. Four types of approaches are available for partitioning the molecular contents of one cell from another: 1) dilution and separately processing; 2) statistical dilution, tagging and pooling, 41 2) droplet, 42,43 3) micro/nanowell, 21,44 4) microfluidic trapping.…”

Section: Discussionmentioning

confidence: 99%

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

et al. 2018

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Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

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“…Single‐cell preparations for MS have also provided interesting results, although these techniques can be technically more challenging than, for instance, single‐cell RNA sequencing (Peterson et al, ). Recent advances in single‐cell separation techniques and molecular barcoding (Prakadan, Shalek, & Weitz, ) have unraveled complex interactions and heterogeneous responses among cell types upon stimulation (Satija & Shalek, ; Shalek et al, ; Tirosh et al, ). A detailed description of these novel techniques is outside the scope of the current review, but can be found elsewhere (Svensson, Vento‐Tormo, & Teichmann, ).…”

Section: Tissue Classification and Collectionmentioning

confidence: 99%

Proteome analysis of tissues by mass spectrometry

Đapić

Baljeu‐Neuman

Uwugiaren

et al. 2019

Mass Spectrometry Reviews

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Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh‐frozen and formalin‐fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue‐sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom‐up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys‐C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue‐specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.

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Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

Cited by 293 publications

References 155 publications

A Novel Microdevice for Rapid Neutrophil Purification and Phenotyping in Type 2 Diabetes Mellitus

A Novel Microdevice for Rapid Neutrophil Purification and Phenotyping in Type 2 Diabetes Mellitus

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Proteome analysis of tissues by mass spectrometry

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