DS03 - Stimuler le renouveau industriel

A generic Microfluidic Approach for Deciphering Nanoscale biovesiclES propertieS – MADNESS

MADNESS

A generic Microfluidic Approach for Deciphering Nanoscale biovesiclES propertieS

Project main objective

The overall objective of the project is to develop a miniaturised platform for the isolation, fractionation and qualification of extracellular microvesicles. These nanobioobjects, produced by all cell types, circulate in biological fluids, and include exosomes, ectosomes and apoptotic bodies, of a size ranging from 30 to 1000 nm . We propose a microfluidic approach combining a hydrodynamic separation module of these particles with analysis and reaction chambers. The microfluidic module will perform size separation in the range 0-500nm and will be interconnected to a multiplex immunocapture module to perform specific subpopulation capture. The trapped EVs will then be characterized from a nanometrological point of view by AFM. The sorted sample will be collected at the output of the sorting device in order to be qualified and quantified by a multi omic approach by coupling to the mass spectrometry techniques available in our technological environment. Proof of concept will be performed on the representative case of platelet derived microvesicles (PMVs). This multi-parameter, multi-functional approach will pave the way for generic instrumentation for the manipulation and qualification of biological nanovesicles and allow for new diagnostic or prognostic tests.

A first generation of separation devices have been designed. It first targets the collection of vesicles with a diameter of less than 700nm, which corresponds to the majority population of platelet vesicles.
In parallel, the development of the nanobioanalysis module was initiated. It was designed as an antibody micro-arrays chip, integrated into a microfluidic module compatible with the sorting module. It reproduces the parameters of use of an SPR chip, that is to say a low pressure operation (10mbar) so low flow and a low injection volume (3µL). The chemical functionalization of the capture chip uses a technology based on single self-assembled coatings grafted onto a gold surface developed specifically for FEMTO

The sorting devices were made in the clean room on the basis of polymer dry film (epoxy) technology . We validated the separation and agreement with specifications using synthetic calibrated beads of diameters 200, 500 and 1000 nm. The separation is operational at low pressure (2bar) even in highly concentrated media (2 e09 to 2 e11 particles/mL) and we demonstrated that it is not sensitive to pressure or flow fluctuations, making it a robust principle. In order to increase the filtered volume ( 35µL in 15 min), we designed a more complex device (100 lateral channels for collecting the filtered sample, 4x2cm chip) involving the patterning of channels on two different levels.This structure required adaptation of the manufacturing process. The chips are being tested. A device with a smaller cut-off diameter (200 nm corresponding to the upper limit of exosomes) was also designed. The possibility of using a thermal gradient to pre-concentrate vesicles before filtration is under theoretical study. Biocapture proof of concept was performed using microbeads (480 and 920 nm calibrators) biofonctionalized to mimic the capture of biological vesicles on the surface of the biopucent have been successfully performed demonstrating the feasibility of the approach.

The work continues with a study of biological samples. Similarly, the device with a smaller cut-off diameter (200 nm corresponding to the upper limit of exosomes) will be validated in the same way in the coming months. Depending on the theoretical results on thermophoretic pre-concentration, this solution will be implemented in microfluidic chips.
As regards the trapping module, the work is now focussing on optimizing the operating conditions on the one hand and on the natural sample processing on the other.

L. Pillemont, D. Guneysu, C. Elie-Caille, W. Boireau and A.-M. Gué. Towards on-chip EVs separation: a lab-on-chip approach. J Extracell Vesicles. 2019; 8(Suppl 1): 1593587, p 310. doi: 10.1080/20013078.2019.1593587
L. Pillemont, D. Guneysu, C. Elie-Caille, W. Boireau and A.-M. Gué A generic Microfluidic Approach for Deciphering Nanoscale biovesiclES propertieS, 3rd Caparica Conference on Sample Pretreatment, Caparica, Portugal, 3-6 December 2018
L. Pillemont, D. Guneysu, C. Elie-Caille, W. Boireau and A.-M. Gué Towards on-chip EVs separation: a lab on chip approach, Annual Meeting of the International Society of Extracellular Vesicles (ISEV), 24-28 april 2019, Kyoto, Japan
Obeid S., Sung, P-S., Le Roy B., Chou M-L., Shieh S-L., Elie-Caille C., Burnouf T., Boireau W. NanoBioAnalytical characterization of extracellular vesicles in 75-nm nanofiltered human plasma for transfusion: a tool to improve transfusion safety. NANOMEDICINE: NANOTECHNOLOGY, BIOLOGY, AND MEDICINE 2019
B. Namasivayam, Yu-Wen Wu, L. Delila, A. Frelet-Barrand, T. Burnouf, C. Elie-Caille and W. Boireau. The nanobioanalytical platform, a tuneable tool for a sensitive detection & characterization of extracellular vesicles subsets from biological samples. J Extracell Vesicles. 2019; 8(Suppl 1): 1593587, p 82. doi: 10.1080/20013078.2019.1593587
Thery C, C Elie-Caille, A Frelet-Barrand, W Boireau et al, Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines, J Extracell Vesicles 2018, VOL. 8, 1535750
B. Namasivayam, Wu Yu-Wen, L. Delila, A. Frelet-Barrand, T. Burnouf, C. Elie-Caille, W. Boireau. The Nanobioanalytical Platform, a tuneable tool for a sensitive detection/characterization of extracellular vesicles subsets from biological sample. Annual Meeting of the International Society of Extracellular Vesicles (ISEV2019), Kyoto, Japan, 24-28 april 2019

Cell-derived microvesicles (EVs) are nanometer size vesicles released under physiological conditions by essentially all cells present in the body. They are circulating in body fluids and are increasingly regarded as promising indicators of pathologies (i.e., biomarker). Therefore identifying and characterizing nanoscale vesicles in biofluids could lead to unprecedented diagnosis/prognosis assays for a wide set of pathologies. Owe to their complexity in size, origin, membrane markers, there is currently no ideal technology available yet to quantify and characterize cell-derived microvesicles, and fully relate their structure and functions. Indeed, all currently available methods (including flow-cytometry, dynamic light scattering, tunable resistive pulse sensing, nanoparticle tracking analysis, etc.) have limits in their ability to capture the whole diversity of cell microvesicles populations in terms of size, membrane markers expression, and structure-function properties. Indeed, only a combination of technologies can provide the required set of information needed for assessing EVs. In addition, current methodologies are not amenable to automation and large-scale analysis of numerous samples. These issues represent serious bottlenecks, which impede fundamental progress as well as practical diagnostic breakthroughs. In that context, the overall objective of this proposal is to develop a miniaturized conceptual platform allowing the isolation, fractionation and classification of EVs. We propose a microfluidic approach coupling a hydrodynamic separation module with analysis and reaction chambers. The microfluidic module will perform size fractionation within the 100-500 nm range where flow cytometers cannot operate. In front of each collecting chambers, we envision to interconnect miniaturized immuno (and ligands)-µarray in microchannels in order to perform specific captures in a multiplex format followed by nanometrological investigation of trapped species with an AFM instrumentation.
Then, each fractionated samples, thus screened, will be collected in output in order to be quantified and qualified with multi-omics approaches by coupling mass spectrometers available in our instrumental park. Although this project focusses on instrumental issues, we propose to make the proof of concept on the representative case of platelet microvesicles (PMVs). Depending on the stimulus responsible for their production, PMVs exert different biological functions (i.e., capacity to stimulate or not endothelial cells, dendritic cells). This will be used as a functional read-out to analysed fractioned PMV samples. This multiparametric and multifunctional approach will pave the way to a generic instrumentation for bio-nanoparticles qualification enabling new diagnostic/prognostic assays.
The “MADNESS” project involves the scientific and complementary expertise of 3 laboratories from Toulouse and Besançon: LAAS-CNRS (Microfabrication and Microfluidics), FEMTO-ST (Microfabrication and bioanalysis) and EFS-UFC (bioanalysis and biology).

Project coordinator

Madame Anne-Marie Gué (Laboratoire d'Analyse et d'Architecture des Systèmes)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

FEMTO-ST
U001098 INTERACTION HOTE-GREFFON-TUMEUR/INGENIERIE CELLULAIRE ET GENIQUE
LAAS-CNRS Laboratoire d'Analyse et d'Architecture des Systèmes

Help of the ANR 358,020 euros
Beginning and duration of the scientific project: October 2017 - 36 Months

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