Microfluidic determination of immune and other cells

Patent metadata

Field	Value
Publication number	US-12345708-B2
Application number	US-201716087203-A
Country	US
Kind code	B2
Filing date	Mar 24, 2017
Priority date	Mar 25, 2016
Publication date	Jul 1, 2025
Grant date	Jul 1, 2025

Abstract

The present invention generally relates to fluidic droplets and systems and methods for determining immune or other cells. Some aspects of the invention are generally directed to assays that combine sensitive detection of secreted products with detection of target cell death in droplets containing an effector cell, systems and methods to isolate droplets in which one or more cell interactions have occurred, or systems and methods to generate nucleic acid information from cell interactions. In addition, some embodiments of the invention are generally directed to containing two (or more) cells in droplets, e.g., an effector cell and one or more target cells, and determining various interactions between the cells within the droplets, such as whether the effector cell kills the target cell, whether the effector cell releases antibodies, cytokines or other substances that are able to interact with the target cell or are released in the presence of the target cell, or the like.

First claim

What is claimed is:

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.

2. The method of claim 1 , wherein the first label comprises a first fluorescent entity.

3. The method of claim 1 , wherein the first label comprises one or more of calcein, a calcein derivative, Alexa Fluor 488, or a fluorescent green dye.

4. The method of claim 1 , wherein the second antibody comprises an anti-interferon-gamma (anti-IFN-gamma) antibody.

5. The method of claim 1 , wherein the particle comprises polystyrene.

6. The method of claim 1 , wherein the particle comprises a streptavidin coating, and the first antibody comprises a biotinylated portion.

7. The method of claim 1 , wherein the target cytokine includes TNF-alpha.

8. The method of claim 1 , wherein the target cytokine includes IFN-gamma.

9. The method of claim 1 , wherein the immune cell is a T-cell.

10. The method of claim 1 , wherein the immune cell is a CD8+ T-cell.

11. The method of claim 1 , wherein the immune cell is a B-cell.

12. The method of claim 1 , wherein the target cell is a cancer cell.

13. The method of claim 1 , wherein the target cell is a virally-infected cell.

14. The method of claim 1 , comprising simultaneously containing the only one immune cell, the only one target cell, the particle attached to the first antibody, and the second antibody in the microfluidic droplet.

15. The method of claim 1 , wherein detection of presence of the target cytokine comprises detecting distribution of a label of the second antibody around the particle attached to the first antibody.

16. The method of claim 1 , wherein determination of the presence or absence of the target cytokine within the droplet does not include a wash step.

17. The method of claim 1 , wherein co-encapsulating, in the droplet in the microfluidic device, comprises:
co-encapsulating the only one immune cell and the only one target cell at a junction downstream from where the first microchannel and the second microchannel meet.

18. The method of claim 1 , further comprising:
collecting the immune cell from the sorted droplet; and
sequencing at least a receptor of the immune cell.

19. The method of claim 1 , wherein the step of co-encapsulating further comprises co-encapsulating one or more additional labels in the droplet that are different from the first label and the second label.

20. The method of claim 19 , further comprising detecting presence or absence of each of the one or more additional labels, wherein the sorting of the droplet is further based on the detected presence or absence of each of the one or more additional labels, and wherein the population of droplets further have a presence of each of the one or more additional labels.

Assignees

• Harvard College

Inventors

• Weitz David A
• Sun Li
• Heyman John

Classifications

• G01N33/6863

  Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors · CPC title

• G01N33/573

  for enzymes or isoenzymes · CPC title

• C12N5/0636

  T lymphocytes · CPC title

• C12N5/0635

  B lymphocytes · CPC title

• B01L3/502792

  for moving individual droplets on a plate, e.g. by locally altering surface tension · CPC title