US 12,345,708 B2
Microfluidic determination of immune and other cells
David A. Weitz, Cambridge, MA (US); Li Sun, Cambridge, MA (US); and John Heyman, Cambridge, MA (US)
Assigned to President and Fellows of Harvard College, Cambridge, MA (US)
Appl. No. 16/087,203
Filed by President and Fellows of Harvard College, Cambridge, MA (US)
PCT Filed Mar. 24, 2017, PCT No. PCT/US2017/024058
§ 371(c)(1), (2) Date Sep. 21, 2018,
PCT Pub. No. WO2017/165791, PCT Pub. Date Sep. 28, 2017.
Claims priority of provisional application 62/313,339, filed on Mar. 25, 2016.
Prior Publication US 2019/0101537 A1, Apr. 4, 2019
Int. Cl. G01N 33/569 (2006.01); B01L 3/00 (2006.01); C12N 5/0781 (2010.01); C12N 5/0783 (2010.01); C12Q 1/6895 (2018.01); G01N 33/573 (2006.01); G01N 33/68 (2006.01)
CPC G01N 33/56972 (2013.01) [B01L 3/502792 (2013.01); C12N 5/0635 (2013.01); C12N 5/0636 (2013.01); C12Q 1/6895 (2013.01); G01N 33/573 (2013.01); G01N 33/6863 (2013.01); C12Q 2600/13 (2013.01)] 20 Claims
 
1. A method, comprising:
flowing immune cells in a first stream within a first microchannel of a microfluidic device, target cells in a second stream within a second microchannel of the microfluidic device, a particle attached to a first antibody with binding specificity for a target cytokine, a second antibody with binding specificity for the target cytokine, and a first label that determines cell viability of the target cell, wherein the particle attached to the first antibody is flowed in either the first stream or the second stream, wherein the second antibody is flowed in either the first stream or the second stream, and wherein the first label is flowed in either the first stream or the second stream;
co-encapsulating, in a droplet in the microfluidic device, only one flowing immune cell from the first stream, only one flowing target cell from the second stream, the flowing particle attached to the first antibody with binding specificity for the target cytokine, the flowing second antibody with binding specificity for the target cytokine, and the flowing first label that determines cell viability of the target cell, wherein the second antibody comprises a second label that is different from the first label, and wherein release of the target cytokine is indicative of activation of the immune cell resulting from an interaction between the target cell and the immune cell, wherein neither the only one immune cell nor the only one target cell are individually encapsulated in a prior droplet before co-encapsulation in the droplet in the microfluidic device;
determining presence or absence of the first label that determines cell viability of the target cell;
determining presence or absence of the target cytokine within the droplet in the microfluidic device by determining presence or absence of the second label concentrated around the particle within the droplet, wherein determination of the presence of the target cytokine occurs after the target cytokine binds to the first antibody and the second antibody; and
sorting the droplet based on the detected presence or absence of the first label and detected presence or absence of the second label, thereby obtaining a population of droplets having a presence of the first label and presence of the second label.