# US 12,179,191 B2 · Methods for encapsulating and assaying cells

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- Number: US 12,179,191 B2
- Document ID: US12179191B2
- Status: Granted
- Kind: B2
- Application number: 18/167,638
- Filed: 2023-02-10
- Published: 2023-06-29
- Granted: 2024-12-31
- Earliest claimed: 2019-04-30
- Assignees: Bruker Cellular Analysis, Inc.
- Applicants: Bruker Cellular Analysis, Inc.
- Inventors: Volker L. S. Kurz; Jason M. McEwen; Kellen C. Mobilia; Alexander J. Mastroianni; Joshua J. Cardiel Rivera
- Web page: https://patentgrep.com/patent/US12179191B2

## CPC

- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/502761: {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}
- B01L 3/502792: {for moving individual droplets on a plate, e.g. by locally altering surface tension}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 25/16: {Particles; Beads; Granular material; Encapsulation (chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00)}
- B01L 2200/0642: Filling fluids into wells by specific techniques
- B01L 2200/0668: Trapping microscopic beads
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2200/0689: Sealing
- B01L 2200/16: Reagents, handling or storing thereof
- B01L 2300/0636: Integrated biosensor, microarrays
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/087: Multiple sequential chambers
- B01L 2300/0877: Flow chambers
- B01L 2300/0893: having a very large number of wells, microfabricated wells
- B01L 2400/0424: Dielectrophoretic forces
- B01L 2400/0427: Electrowetting
- B01L 2400/0677: phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

In biosciences and related fields, it can be useful to study cells in isolation so that cells having unique and desirable properties can be identified within a heterogenous mixture of cells. Processes and methods disclosed herein provide for encapsulating cells within a microfluidic device and assaying the encapsulated cells. Encapsulation can, among other benefits, facilitate analyses of cells that generate secretions of interest which would otherwise rapidly diffuse away or mix with the secretions of other cells.

## Claims

### Claim 1

A process for encapsulating micro-objects in a microfluidic device, wherein the microfluidic device comprises an enclosure comprising a channel and a first chamber having an opening fluidically connecting the first chamber to the channel, wherein one or both of (i) at least a portion of surfaces forming the channel proximal to the opening of the first chamber and (ii) at least a portion of surfaces forming the first chamber proximal to the channel comprises a first hydrophobic coating, the process comprising:
filling the channel and the first chamber in the enclosure of the microfluidic device with a first aqueous medium;
disposing a first micro-object in the first chamber; and
flowing a water immiscible fluidic medium into the channel, displacing substantially all of the first aqueous medium in the channel without substantially displacing the first aqueous medium in the first chamber, thereby reversibly encapsulating the first micro-object in the first chamber.

### Claim 2 (depends on 1)

The process of claim 1, wherein all channel surfaces proximal to and surrounding the opening to the first chamber comprise the first hydrophobic coating.

### Claim 3 (depends on 2)

The process of claim 2, wherein all channel surfaces within 10 microns of the opening to the first chamber comprises the first hydrophobic coating.

### Claim 4 (depends on 3)

The process of claim 3, wherein the first hydrophobic coating has a contact angle from about 45 degrees to about 100 degrees.

### Claim 5 (depends on 1)

The process of claim 1, wherein the first micro-object is configured to express a molecule of interest or a reporter molecule.

### Claim 6 (depends on 5)

The process of claim 5, wherein the molecule of interest is a small molecule, a carbohydrate, a peptide, a protein, or a nucleic acid.

### Claim 7 (depends on 1)

The process of claim 1, wherein the first micro-object is a biological cell or a bead.

### Claim 8 (depends on 1)

The process of claim 1, further comprising disposing a second micro-object into the first chamber.

### Claim 9 (depends on 8)

The process of claim 8, further comprising monitoring a biological activity of the second micro-object.

### Claim 10 (depends on 8)

The process of claim 8, wherein the second micro-object is a biological cell or a bead.

### Claim 11 (depends on 10)

The process of claim 10, wherein the second micro-object is a capture bead, wherein the capture bead is configured to bind a biological material.

### Claim 12 (depends on 1)

The process of claim 1, further comprising adding one or both of (i) an assay media and (ii) an assay reagent to the first chamber.

### Claim 13 (depends on 1)

The process of claim 1, further comprising aspirating the water immiscible fluidic medium out of the channel.

### Claim 14 (depends on 13)

The process of claim 13, wherein aspirating the water immiscible fluidic medium further comprises subsequently aspirating a second aqueous medium into the channel.

### Claim 15 (depends on 13)

The process of claim 13, wherein all chamber surfaces proximal to and surrounding the opening of the first chamber comprise the first hydrophobic coating, the process further comprises:
generating an encapsulation layer of water immiscible fluidic media in the first chamber, wherein the encapsulation layer of water immiscible fluidic media in the first chamber is located immediately adjacent to the channel and shares an interface with the first aqueous medium in the first chamber so as to separate the first aqueous medium in the first chamber from a medium present in the channel.

### Claim 16 (depends on 15)

The process of claim 15, wherein all chamber surfaces within 10 microns of the opening of the first chamber comprises the first hydrophobic coating.

### Claim 17 (depends on 15)

The process of claim 15, wherein the encapsulation layer of water immiscible fluidic media in the first chamber has an average thickness of about 5 μm to about 50 μm.

### Claim 18 (depends on 15)

The process of claim 15, further comprising: removing the encapsulation layer of water immiscible fluidic media in the first chamber at the opening to the channel thereby generating a de-encapsulated chamber.

### Claim 19 (depends on 18)

The process of claim 18 further comprising: flowing a third aqueous medium into the channel, wherein the third aqueous medium comprises an assay reagent, a lytic reagent, or an export buffer.

### Claim 20 (depends on 1)

The process of claim 1, wherein the first chamber comprises a plurality of surfaces forming the first chamber, wherein at least one chamber surface of the plurality surfaces comprises a hydrophilic coating.

### Claim 21 (depends on 20)

The process of claim 20, wherein the hydrophilic coating comprises a plurality of second covalently bound surface modifications, each comprising a second linking group, and a second moiety, wherein the second moiety is polar.

### Claim 22 (depends on 1)

The process of claim 1, wherein the first hydrophobic coating comprises a first covalently bound surface modification comprising: a first linking group, and a first moiety, wherein the first moiety is nonpolar.

### Claim 23 (depends on 1)

The process of claim 1, wherein the water immiscible fluidic medium is an alkane, a fluoroalkane, an oil, a hydrophobic polymer, or any combination thereof.

### Claim 24 (depends on 1)

The process of claim 1, wherein the microfluidic device further comprises a second chamber having an opening fluidically connecting the second chamber to the channel, wherein one or both of (i) at least a portion of surfaces forming the channel proximal to the opening of the second chamber and (ii) at least a portion of surfaces forming the second chamber proximal to the channel comprises a second hydrophobic coating,
wherein the process further comprises: filling the second chamber in the enclosure of the microfluidic device with the first aqueous medium; and disposing a second cell in the second chamber;
wherein flowing a water immiscible fluidic medium into the channel and displacing substantially all of the first aqueous medium in the channel does not substantially displacing the first aqueous medium in the second chamber.