# US 11,999,934 B2 · Integrated microfluidic system for generation of MicroOrganoSpheres (MOS)

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- Number: US 11,999,934 B2
- Document ID: US11999934B2
- Status: Granted
- Kind: B2
- Application number: 18/378,921
- Filed: 2023-10-11
- Published: 2024-04-18
- Granted: 2024-06-04
- Earliest claimed: 2022-10-11
- Assignees: Xilis, Inc.
- Applicants: Xilis, Inc.
- Inventors: Bradley Scott Thomas; Timothy A. Miller; Daniel Nelson; Raheel Samuel; George Robinson; Tyler Welder
- Web page: https://patentgrep.com/patent/US11999934B2

## CPC

- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- B01L 3/502746: {characterised by the means for controlling flow resistance, e.g. flow controllers, baffles or throttle valves}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 23/40: {Manifolds; Distribution pieces (fluid transfer means B01L3/563)}
- C12M 25/16: {Particles; Beads; Granular material; Encapsulation (chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00)}
- C12M 41/12: {of temperature (controlling the temperature of chemical or physical processes B01J19/0013, heating or cooling apparatus for laboratory use B01L7/00)}
- C12M 41/36: {of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404)}
- C12M 41/40: {of pressure}
- G01N 15/0227: using imaging; using holography
- G01N 15/1425: {using an analyser being characterised by its control arrangement}
- G01N 15/1433: using image recognition
- G01N 15/1434: Optical arrangements
- G01N 15/1459: {the analysis being performed on a sample stream}
- G01N 15/1484: {microstructural devices}
- B01F 23/405: {Methods of mixing liquids with liquids (B01F23/4105 takes precedence)}
- B01F 23/4105: {Methods of emulsifying}
- B01L 2200/061: Counting droplets
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2200/143: Quality control, feedback systems
- B01L 2300/023: Sending and receiving of information, e.g. using Bluetooth®
- B01L 2300/0654: Lenses; Optical fibres
- B01L 2300/0883: Serpentine channels
- B01L 2300/14: Means for pressure control
- B01L 2300/161: Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
- B01L 2300/1805: Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L 2400/082: Active control of flow resistance, e.g. flow controllers
- G01N 2015/1006: {for cytology}
- G01N 2015/1027: {Determining speed or velocity of a particle}
- G01N 2015/144: {Imaging characterised by its optical setup}
- G01N 2015/1486: {Counting the particles}
- G01N 2015/1493: {Particle size}
- 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}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)

## Abstract

A microfluidic apparatus includes a microfluidic chip for MicroOrganoSpheres (MOS) generation. A first channel is defined in a surface of the microfluidic chip and includes: a droplet generation portion including an inlet portion, a junction between the inlet portion and an emulsifying fluid channel, and a chamber downstream of the junction. A cross-sectional area of the chamber is larger than that of the inlet portion. The first channel includes a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration. The apparatus includes a cartridge for MOS demulsification, including: a collection container; a substrate disposed on the collection container, and a membrane disposed between the collection container and the surface of the substrate. A second channel is defined in the surface of the substrate that faces the collection container and is fluidically connected to an output of the polymerization portion of the first channel.

## Claims

### Claim 1

A microfluidic apparatus comprising:
a microfluidic chip for generation of droplets from sample material, in which a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel comprising:
a droplet generation portion comprising:
an inlet portion configured to be connected to a source of the sample material,
a junction between the inlet portion and an emulsifying fluid channel, and
a chamber downstream of the junction, in which a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet portion, and
a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration; and
a cartridge for droplet demulsification, the cartridge comprising:
a collection container defining a cavity;
a substrate disposed on the collection container, in which a second microfluidic channel is defined in a surface of the substrate that faces the cavity of the collection container, and in which the second microfluidic channel is fluidically connected to an output of the polymerization portion of the first microfluidic channel and configured to be connected to an output vial; and
a hydrophobic, oleophilic membrane disposed between the cavity of the collection container and the surface of the substrate,
in which the second microfluidic channel comprises:
an upstream section that has a simple serpentine configuration, and
a downstream section that has a double serpentine configuration.

### Claim 2 (depends on 1)

The microfluidic apparatus of claim 1, in which the surface of the microfluidic chip is a first surface, and in which the polymerization portion of the first microfluidic channel is defined on the first surface of the microfluidic chip and on a second surface of the microfluidic chip opposite the first surface.

### Claim 3 (depends on 1)

The microfluidic apparatus of claim 1, in which the junction comprises a junction between the inlet portion and two emulsifying fluid channels.

### Claim 4 (depends on 1)

The microfluidic apparatus of claim 1, in which the droplet generation portion of the first microfluidic channel comprises an outlet portion downstream of the chamber, in which a cross-sectional area of the chamber is larger than a cross-sectional area of the outlet portion.

### Claim 5

A microfluidic apparatus comprising:
a microfluidic chip for generation of droplets from sample material, in which a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel comprising:
a droplet generation portion comprising:
an inlet portion configured to be connected to a source of the sample material,
a junction between the inlet portion and an emulsifying fluid channel, and
a chamber downstream of the junction, in which a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet portion, and
a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration; and
a cartridge for droplet demulsification, the cartridge comprising:
a collection container defining a cavity;
a substrate disposed on the collection container, in which a second microfluidic channel is defined in a first surface of the substrate that faces the cavity of the collection container, and in which the second microfluidic channel is fluidically connected to an output of the polymerization portion of the first microfluidic channel and configured to be connected to an output vial, and
in which a media inlet channel is defined on a second surface of the substrate opposite the first surface of the substrate, the media inlet channel fluidically connected to an upstream section of the second microfluidic channel and configured to be connected to a media reservoir; and
a hydrophobic, oleophilic membrane disposed between the cavity of the collection container and the first surface of the substrate.

### Claim 6 (depends on 5)

The microfluidic apparatus of claim 5, in which the media inlet channel is fluidically connected to the media reservoir via a tube extending through the substrate and the collection container.

### Claim 7 (depends on 5)

The microfluidic apparatus of claim 5, in which the collection container is disposed in a cavity defined in the media reservoir such that the collection container is positioned between the media reservoir and the substrate.

### Claim 8 (depends on 5)

The microfluidic apparatus of claim 5, in which a bottom surface of the media reservoir is angled relative to a plane of the substrate.

### Claim 9 (depends on 5)

The microfluidic apparatus of claim 5, in which the demulsification cartridge comprises a duckbill valve extending through the substrate and the collection container, the duckbill valve configured to provide fluidic access to the media reservoir.

### Claim 10

A microfluidic apparatus comprising:
a microfluidic chip for generation of droplets from sample material, in which a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel comprising:
a droplet generation portion comprising:
an inlet portion configured to be connected to a source of the sample material,
a junction between the inlet portion and an emulsifying fluid channel, and
a chamber downstream of the junction, in which a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet portion, and
a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration; and
a cartridge for droplet demulsification, the cartridge comprising:
a collection container defining a cavity;
a substrate disposed on the collection container, in which a second microfluidic channel is defined in a surface of the substrate that faces the cavity of the collection container, and in which the second microfluidic channel is fluidically connected to an output of the polymerization portion of the first microfluidic channel and configured to be connected to an output vial;
a hydrophobic material disposed within the collection container; and
a hydrophobic, oleophilic membrane disposed between the cavity of the collection container and the surface of the substrate.

### Claim 11 (depends on 1)

The microfluidic apparatus of claim 1, comprising a reservoir fluidically connected to the first microfluidic channel via an input port defined at an input end of the first microfluidic channel.

### Claim 12 (depends on 11)

The microfluidic apparatus of claim 11, comprising an input port in a cover of the reservoir, the input port comprising a duckbill valve.

### Claim 13 (depends on 11)

The microfluidic apparatus of claim 11, comprising an output port in a cover of the reservoir, the output port connected to a tube extending into a cavity of the reservoir.

### Claim 14 (depends on 11)

The microfluidic apparatus of claim 11, comprising a reservoir holder configured to receive the reservoir, the reservoir holder comprising a cooling system configured to cool the reservoir.

### Claim 15 (depends on 14)

The microfluidic apparatus of claim 14, in which the cooling system comprises a thermoelectric cooling system.

### Claim 16

A microfluidic apparatus comprising:
a microfluidic chip for generation of droplets from sample material, in which a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel comprising:
a droplet generation portion comprising:
an inlet portion configured to be connected to a source of the sample material,
a junction between the inlet portion and an emulsifying fluid channel, and
a chamber downstream of the junction, in which a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet portion, and
a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration,
in which one or more cutouts are defined in the microfluidic chip between the droplet generation portion and the polymerization portion; and
a cartridge for droplet demulsification, the cartridge comprising:
a collection container defining a cavity;
a substrate disposed on the collection container, in which a second microfluidic channel is defined in a surface of the substrate that faces the cavity of the collection container, and in which the second microfluidic channel is fluidically connected to an output of the polymerization portion of the first microfluidic channel and configured to be connected to an output vial; and
a hydrophobic, oleophilic membrane disposed between the cavity of the collection container and the surface of the substrate.

### Claim 17 (depends on 16)

The microfluidic apparatus of claim 16, in which edges of the one or more cutouts are angled relative to the surface of the microfluidic chip.

### Claim 18 (depends on 16)

The microfluidic apparatus of claim 16, in which the one or more cutouts extend through an entire thickness of the microfluidic chip.

### Claim 19 (depends on 1)

The microfluidic apparatus of claim 1, in which multiple first microfluidic channels are defined in the surface of the microfluidic chip, in which the first microfluidic channel is among the multiple first microfluidic channels, and in which the apparatus comprises multiple cartridges, in which the cartridge is among the multiple cartridges, in which the second microfluidic channel of each cartridge is fluidically connected to a corresponding one of the first microfluidic channel of the microfluidic chip.

### Claim 20 (depends on 1)

The microfluidic apparatus of claim 1, in which the apparatus comprises an output vial fluidically connected to the second microfluidic channel via an output port defined at an output end of the second microfluidic channel.

### Claim 21

A system comprising:
a microfluidic apparatus comprising:
a microfluidic chip for generation of droplets from sample material, in which a first microfluidic channel is defined in a surface of the microfluidic chip, the first microfluidic channel comprising:
a droplet generation portion comprising:
an inlet portion configured to be connected to a source of the sample material,
a junction between the inlet portion and an emulsifying fluid channel, and
a chamber downstream of the junction, in which a cross-sectional area of the chamber is larger than a cross-sectional area of the inlet portion, and
a polymerization portion downstream of the droplet generation portion, the polymerization portion having a serpentine configuration;
a cartridge for droplet demulsification, the cartridge comprising:
a collection container defining a cavity;
a substrate disposed on the collection container, in which a second microfluidic channel is defined in a surface of the substrate that faces the cavity of the collection container, and in which the second microfluidic channel is fluidically connected to an output of the polymerization portion of the first microfluidic channel and configured to be connected to an output vial; and
a hydrophobic, oleophilic membrane disposed between the cavity of the collection container and the surface of the substrate;
a housing, in which the microfluidic apparatus is disposed in the housing; and
a polymerization block housed in the housing and positioned to apply a stimulus to the polymerization portion of the first microfluidic channel.

### Claim 22 (depends on 21)

The system of claim 21, in which the polymerization block comprises a thermal polymerization block configured to apply heat to the polymerization portion of the first microfluidic channel.

### Claim 23 (depends on 21)

The system of claim 21, in which the polymerization block comprises a light polymerization block configured to illuminate the polymerization portion of the first microfluidic channel.

### Claim 24 (depends on 21)

The system of claim 21, in which the surface of the microfluidic chip is a first surface, and in which the polymerization block comprises:
a first block disposed adjacent the first surface of the microfluidic chip; and
a second block disposed adjacent a second surface of the microfluidic chip, the second surface opposite the first surface.

### Claim 25 (depends on 21)

The system of claim 21, comprising a reservoir for emulsifying fluid, in which the emulsifying fluid channel of the microfluidic apparatus is fluidically connected to the reservoir.

### Claim 26 (depends on 25)

The system of claim 25, comprising a pump disposed between the reservoir for emulsifying fluid and the emulsifying fluid channel.

### Claim 27 (depends on 26)

The system of claim 26, comprising a controller configured to control operation of the pump to achieve a target fluid velocity in the second microfluidic channel.

### Claim 28 (depends on 21)

The system of claim 21, comprising an imaging system positioned to capture images of at least a portion of the chamber.

### Claim 29 (depends on 28)

The system of claim 28, comprising a controller configured to control a flow rate of fluid through the inlet portion of the microfluidic channel based on the images captured by the imaging system.