Droplet microfluidics with reagent micromixing for investigating intrinsic platelet functionality
Droplet microfluidics with reagent micromixing for investigating intrinsic platelet functionality
Introduction: Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets.
Methods: To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets.
Results: The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and
α
IIb
β
3 integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity.
Conclusions: The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology.
Supplementary information: The online version of this article (10.1007/s12195-020-00665-6) contains supplementary material, which is available to authorized users.
Droplet microfluidics, Flow cytometry, Micromixing, Platelets, Single cell analysis
223-230
West, Jonathan
f1c2e060-16c3-44c0-af70-242a1c58b968
Jongen, Maaike Sybilla Anna
47c19196-eb09-421c-ab3a-1330ff9a79b0
Holloway, Paul M
59a865d9-08bc-43c7-aa00-d80509956d86
Lane, Simon I.R.
44286abe-c5d5-49ef-acdd-4c935403c0f6
Englyst, Nicola
f84399af-7265-4224-b556-102c3aa272b0
McCarty, Owen
13e388bc-bd7c-467d-a3b5-0b821577d0f1
June 2021
West, Jonathan
f1c2e060-16c3-44c0-af70-242a1c58b968
Jongen, Maaike Sybilla Anna
47c19196-eb09-421c-ab3a-1330ff9a79b0
Holloway, Paul M
59a865d9-08bc-43c7-aa00-d80509956d86
Lane, Simon I.R.
44286abe-c5d5-49ef-acdd-4c935403c0f6
Englyst, Nicola
f84399af-7265-4224-b556-102c3aa272b0
McCarty, Owen
13e388bc-bd7c-467d-a3b5-0b821577d0f1
West, Jonathan, Jongen, Maaike Sybilla Anna, Holloway, Paul M, Lane, Simon I.R., Englyst, Nicola and McCarty, Owen
(2021)
Droplet microfluidics with reagent micromixing for investigating intrinsic platelet functionality.
Cell and Molecular Bioengineering, 14 (3), .
(doi:10.1007/s12195-020-00665-6).
Abstract
Introduction: Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets.
Methods: To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets.
Results: The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and
α
IIb
β
3 integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity.
Conclusions: The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology.
Supplementary information: The online version of this article (10.1007/s12195-020-00665-6) contains supplementary material, which is available to authorized users.
Text
Droplet Microfluidics
- Accepted Manuscript
More information
Accepted/In Press date: 23 December 2020
e-pub ahead of print date: 21 January 2021
Published date: June 2021
Additional Information:
Funding Information:
The authors are grateful to the blood donors, and also to George Devitt and Dianne M. Lopez for access to a fluorescent plate reader. The research was funded by Marie Curie (333721, J.W.), the British Heart Foundation (FS/13/67/30473, M.S.A.J.), the National Institute of Health (R01 HL101972) and the Medical Research Council (MC_PC_15078, M.S.A.J.). MS.A.J., S.I.R.L., P.M.H. and J.W. undertook the research. M.S.A.J., S.I.R.L. and J.W. prepared the figures and manuscript. P.M.H., N.A.E. and O.J.T.M. revised and edited the manuscript. All authors approved the final version of the manuscript. MS.A.J., S.I.R.L., P.M.H., N.A.E., O.J.T.M. and J.W. declare no conflicts of interest. All human subject research was carried out in accordance with institutional guidelines approved by the University of Southampton Ethics Committee ((ERGO 5538) and South Central ? Hampshire B National Research Ethics Service (REC: 14/SC/0211)). No animal studies were carried out by the authors for this article.
Publisher Copyright:
© 2021, Biomedical Engineering Society.
Keywords:
Droplet microfluidics, Flow cytometry, Micromixing, Platelets, Single cell analysis
Identifiers
Local EPrints ID: 446770
URI: http://eprints.soton.ac.uk/id/eprint/446770
ISSN: 1865-5033
PURE UUID: 822be377-b3ea-412a-af1a-b9fee54023f8
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Date deposited: 22 Feb 2021 17:31
Last modified: 06 Jun 2024 04:18
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Contributors
Author:
Maaike Sybilla Anna Jongen
Author:
Paul M Holloway
Author:
Simon I.R. Lane
Author:
Owen McCarty
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