Microfluidic devices and methods for use thereof in multicellular assays of secretion

Patent metadata

Field	Value
Publication number	US-10725024-B2
Application number	US-201414773244-A
Country	US
Kind code	B2
Filing date	Mar 28, 2014
Priority date	Mar 28, 2013
Publication date	Jul 28, 2020
Grant date	Jul 28, 2020

Abstract

Methods and devices are provided herein for identifying a cell population comprising an effector cell that exerts an extracellular effect. In one embodiment the method comprises retaining in a microreactor a cell population comprising one or more effector cells, wherein the contents of the microreactor further comprise a readout particle population comprising one or more readout particles, incubating the cell population and the readout particle population within the microreactor, assaying the cell population for the presence of the extracellular effect, wherein the readout particle population or subpopulation thereof provides a direct or indirect readout of the extracellular effect, and determining, based on the results of the assaying step, whether one or more effector cells within the cell population exerts the extracellular effect on the readout particle. If an extracellular effect is measured, the cell population is recovered for further analysis to determine the cell or cells responsible for the effect.

First claim

What is claimed is:

1. A method of identifying a heterologous cell population comprising an antibody secreting cell (ASC) that secretes an antibody that binds to a target epitope, comprising:
retaining, in each of a plurality of microreactors, one of a plurality of heterogeneous cell populations, each population comprising 10 to 500 cells, wherein each heterogeneous cell population comprises one or more ASCs, wherein the contents of each microreactor further comprise a readout particle population comprising one or more readout beads, readout cells, or a combination thereof displaying the target epitope on their surfaces, and wherein individual heterogenous cell populations are retained in individual microreactors,
incubating the heterogeneous cell populations and the readout particle population within the plurality of microreactors to allow for secreted antibody binding to the readout particle population via the target epitope,
introducing a fluorescently labeled secondary antibody into the plurality of microreactors, wherein the fluorescently labeled secondary antibody binds to the secreted antibody,
washing the plurality of microreactors to remove unbound secreted antibody and unbound fluorescently labeled secondary antibody,
assaying the individual microreactors for the presence of a binding interaction between secreted antibody and the target epitope via detection of the fluorescent label, and
determining, based on the results of the assaying step, whether one or more of the heterogenous cell populations includes an ASC that secretes an antibody that binds to the target epitope.

2. The method of claim 1 , wherein the readout particle population is a homogeneous population or a heterogeneous population of readout particles.

3. The method of claim 1 , wherein the readout particle population is immobilized on a surface of the individual microreactors.

4. The method of claim 1 , wherein the binding interaction is an antigen-antibody binding specificity interaction, antigen-antibody binding affinity interaction or antigen-antibody binding kinetic interaction.

5. The method of claims 1 , further comprising maintaining the heterogeneous cell populations in substantially a single plane.

6. The method of claim 1 , further comprising maintaining the readout particle population in substantially a single plane.

7. The method of claim 1 , wherein if a heterogenous cell population includes an ASC that secretes an antibody that binds to the target epitope, the method further comprises recovering the heterogenous cell population or a portion thereof to obtain a recovered cell population.

8. The method of claim 7 , wherein the recovering step comprises positioning the open end of a microcapillary in a microreactor comprising the heterogenous cell population that includes an ASC that secretes an antibody that binds to the target epitope and aspirating the microreactor's contents or a portion thereof to obtain a recovered aspirated cell population.

9. The method of claim 8 , wherein the microcapillary is mounted on a robotic micromanipulation system on a microscope or the microcapillary is controlled robotically.

10. The method of claim 7 , further comprising,
retaining a plurality of cell subpopulations originating from the recovered cell population in a plurality of vessels, wherein each cell subpopulation is present in an individual vessel,
lysing the individual cell subpopulations to provide lysed cell subpopulations, and
amplifying one or more nucleic acids within each of the lysed cell populations.

Assignees

• Univ British Columbia

Inventors

• Ricicova Marketa
• Heyries Kevin Albert
• Zahn Hans
• Petriv Oleh
• Lecault Veronique
• Singhal Anupam
• Da Costa Daniel J
• Hansen Carl L G
• Nelson Brad
• Nielsen Julie
• Lisaingo Kathleen

Classifications

• C07K16/108

  Orthomyxoviridae (F), e.g. influenza virus · CPC title

• C12N5/0635

  B lymphocytes · CPC title

• C12N5/0634

  Cells from the blood or the immune system · CPC title

• G01N33/54313

  the carrier being characterised by its particulate form · CPC title

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title