Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap

Bounab, Yacine; Eyer, Klaus; Dixneuf, Sophie; Rybczyńska, Maria; Chauvel, Cécile; Mistretta, Maxime; Tran, Trang; Aymerich, Nathan; Chenon, Guilhem; Llitjos, Jean-François; Venet, Fabienne; Monneret, Guillaume; Gillespie, Iain A.; Cortez, Pierre; Moucadel, Virginie; Pachot, Alexandre; Troesch, Alain; Leissner, Philippe; Textoris, Julien; Bibette, Jérôme; Guyard, Cyril; Baudry, Jean; Griffiths, Andrew D.; Védrine, Corine

doi:10.1038/s41596-020-0354-0

Cited by 79 publications

(110 citation statements)

References 85 publications

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“…Cell and reagents were prepared and encapsulated in droplet as previously described ( 7 ). Briefly, monocytes from healthy donors or septic shock patients were enriched with an EasySep Direct Human Monocyte Isolation Kit (STEMCELL Technology).…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore enzyme-linked immunospot (ELISPOT), commonly used to detect proteins secreted from individual cells, is limited to endpoint measurements and is poorly quantitative. We have developed an ultrasensitive in-droplet immunoassay, in which an advanced microfluidic assay combined to fluorescence microscopy allows addressing the time course of LPS-elicited TNFα production by single monocytes encapsulated within droplets ( 7 ). Thousands of single in-droplet cells can thereby be monitored at the same time.…”

Section: Introductionmentioning

confidence: 99%

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Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion

et al. 2021

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ObjectiveThe development of advanced single-cell technologies to decipher inter-cellular heterogeneity has enabled the dynamic assessment of individual cells behavior over time, overcoming the limitation of traditional assays. Here, we evaluated the feasibility of an advanced microfluidic assay combined to fluorescence microscopy to address the behavior of circulating monocytes from septic shock patients.MethodsSeven septic shock patients and ten healthy volunteers were enrolled in the study. Using the proposed microfluidic assay we investigated the production over time of LPS-elicited TNFα by single monocytes encapsulated within droplets. Cellular endocytic activity was assessed by internalization of magnetic nanoparticles. Besides, we assessed HLA-DR membrane expression and LPS-induced TNFα production in monocytes through classical flow cytometry assays.ResultsConsistent with the flow cytometry results, the total number of TNFα molecules secreted by encapsulated single monocytes was significantly decreased in septic shock patients compared to healthy donors. TNFα production was dampened as soon as 30 and 60 minutes after LPS stimulation in monocytes from septic patients. Furthermore, the microfluidic assay revealed heterogeneous individual behavior of monocytes from septic shock patients. Of note, monocytes from both healthy donors and patients exhibited similar phagocytic activities over time.ConclusionThe microfluidic assay highlights the functional heterogeneity of monocytes, and provides in-depth resolution in assessing the hallmark monocyte deactivation encountered in post-septic immunosuppression.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion

et al. 2021

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“…In order to characterize directly the secretion rate, specificity, and affinity for GPIIbIIIa of IgG secreted by autoreactive plasma cells and plasmablasts, collectively termed ASC hereunder, without the need to sort, clone, or re-express antibodies, we adapted a single-cell bioassay in microfluidic droplets (termed "DropMap") that we have described previously 22,23 . Mononuclear cells from the spleen, bone marrow, or blood of patients and fluorescent bioassay reagents are co-encapsulated in droplets, immobilized within an observation chamber and imaged over 1 hour by time-lapse fluorescence imaging.…”

Section: Single-cell Bioassay Allows Phenotypic Characterization Of Autoreactive Ascmentioning

confidence: 99%

Single-cell analyses of immune thrombocytopenic patients reveal multiorgan dissemination of high-affinity autoreactive plasma cells

Herrerias

Crickx

Broketa

et al. 2021

Preprint

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The major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin αIIbβ3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

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“…Microfluidics technology developed over the past few decades has greatly impacted biomedical research, therapeutics, and diagnostics. Various applications, such as small compound screening and gene expression studies, greatly benefit from increased throughput and sensitivity provided by microfluidics [1][2][3][4][5][6] . Generally, a large-scale microfluidic assay also requires massive measurement of samples, for example, fluorescenceactivated cell sorting (FACS), through which followed-up analyses, such as counting and phenotyping, are further enabled [7][8][9][10][11] .…”

Section: Introductionmentioning

confidence: 99%

High-speed large-scale 4D activities mapping of moving C. elegans by deep-learning-enabled light-field microscopy on a chip

Peng

Zhu

et al. 2021

Preprint

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We report a novel fusion of microfluidics and light-field microscopy, to achieve high-speed 4D (space + time) imaging of moving C. elegans on a chip. Our approach combines automatic chip-based worm loading / compartmentalization / flushing / reloading with instantaneous deep-learning light-field imaging of moving worm. Taken together, we realized intoto image-based screening of wild-type and uncoordinated-type worms at a volume rate of 33 Hz, with sustained observation of 1 minute per worm, and overall throughput of 42 worms per hour. With quickly yielding over 80000 image volumes that four-dimensionally visualize the dynamics of all the worms, we can quantitatively analyse their behaviours as well as the neural activities, and correlate the phenotypes with the neuron functions. The different types of worms can be readily identified as a result of the high-throughput activity mapping. Our approach shows great potential for various lab-on-a-chip biological studies, such as embryo sorting and cell growth assays.

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Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap

Cited by 79 publications

References 85 publications

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion

Single-cell analyses of immune thrombocytopenic patients reveal multiorgan dissemination of high-affinity autoreactive plasma cells

High-speed large-scale 4D activities mapping of moving C. elegans by deep-learning-enabled light-field microscopy on a chip

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