High Throughput Microfluidics for Analysis of Immune Cell Activation
OwnerRohan Thakur, James Malcolm Heather +1inventors
InventorsRohan Thakur, James Malcolm Heather, Shannon Leigh Stott
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Abstract
Provided herein are microfluidic platforms and methods of use thereof for generating, tracking, monitoring, and analyzing thousands of droplets per second for interactions between two or more particles, such as cells, encapsulated in individual droplets, wherein the individual droplets are uniquely identified by specific ratios of multiple different optical barcodes and at least one sequence barcode per droplet.
Claims35
A method for analyzing an interaction between two or more particles, the method comprising:
- (a) inertially ordering the particles into spaced and ordered streams of particles;
- (b) co-encapsulating in individual droplets two or more of the spaced and ordered particles and an activation reporter to form a plurality of target droplets;
- (c) co-encapsulating in individual droplets a plurality of different optical barcodes to generate a plurality of barcoded droplets, wherein a specific ratio of the different optical barcodes is used to uniquely identify each of the individual barcoded droplets;
- (d) determining interaction between the particles in each target droplet by monitoring each target droplet for a presence or absence of the activation reporter to identify target droplets positive for interacting, activated particles;
- (e) merging each identified target droplet with an adjacent barcoded droplet to generate merged droplets;
- and
- (f) sequencing nucleic acids in the merged droplets to determine the sequence of any nucleic acids in the particles and to determine the sequence of any barcodes in the merged droplets;
- wherein step (c) further comprises co-encapsulating in the individual barcoded droplets at least one sequence barcode to generate a plurality of dual barcoded droplets and step (e) comprises merging each identified target droplet with an adjacent dual barcoded droplet;
- or
- wherein steps (b) and (c) are combined to co-encapsulate in individual target droplets the two or more particles and the activation reporter as well as the plurality of different optical barcodes to generate the target droplets, and step (e) comprises merging each identified target droplet with a second droplet comprising a sequence barcode to form the merged droplet;
- or
- wherein step (c) further comprises merging the target droplets with the barcoded droplets to generate optically barcoded target droplets, and step (e) comprises merging each identified optically barcoded target droplet with a second droplet comprising a sequence barcode.
The method of any one of claims 1-18, wherein the target droplets and the dual barcoded droplets are merged by droplet-stream merger, droplet-jet merger, or both. The method of any one of claims 1-19, wherein the particles are selected from the group consisting of cells, eggs, bacteria, fungi, virus, algae, any prokaryotic or eukaryotic cells, organelles, exosomes, beads, reagents, drugs, small molecules, proteins, antibodies, enzymes, and nucleic acids.
The method of claim 20, wherein the particles comprise a T-cell and an antigen presenting cell (APC) and the activation reporter is a calcium activation reporter.
A microfluidic system comprising:
- (a) a dual barcoded droplet preparation module comprising a plurality of channels for receiving one or more barcodes and a droplet generator comprising a nozzle in fluid communication with the plurality of channels;
- (b) an inertially ordered cell encapsulation module comprising
- (i) a microchannel having an inlet, an outlet, and a minimum cross-sectional dimension D configured to receive a fluid sample containing multiple particles having a maximum individual cross-sectional dimension of at least 0.1 D, and
- (ii) a droplet generator comprising a nozzle in fluid communication with the outlet of the microfluidic channel;
- (c) an incubation and activation profiling module comprising an inlet end, a middle section, and on outlet end, wherein the inlet end comprises a central channel for droplets and a plurality of microchannels arranged in fluid communication with and one or both sides of the central channel to allow excess fluid and other waste materials in the fluid sample to flow out of the central channel while maintaining droplets within the central channel, and wherein the outlet end comprises a narrowing channel to allow the droplets to become arranged in single file when exiting the outlet end;
- (d) an optical barcode detection module comprising one or more optical detection devices;
- and
- (e) a selective droplet merging module comprising a channel and an electrode configured to apply an electric field in the channel sufficient to cause adjacent droplets to merge into one larger droplet.
Dependencies are inferred from the claim text.