# US 12,110,479 B2 · Microfluidic devices, systems, and methods for investigating three-dimensional cellular structures

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- Number: US 12,110,479 B2
- Document ID: US12110479B2
- Status: Granted
- Kind: B2
- Application number: 16/438,356
- Filed: 2019-06-11
- Published: 2019-12-12
- Granted: 2024-10-08
- Earliest claimed: 2018-06-11
- Assignees: The University of Chicago
- Applicants: The University of Chicago
- Inventors: Savas Tay; Michael Junkin; Brooke Schuster
- Web page: https://patentgrep.com/patent/US12110479B2

## CPC

- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- 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 3/50853: {with covers or lids (closures for test tubes B01L3/50825)}
- C12M 23/22: {Transparent or translucent parts (glassware for laboratory use B01L3/00)}
- C12M 29/00: {Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B)}
- C12N 5/0693: {Tumour cells; Cancer cells}
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/5088: {of vertebrates}
- B01L 3/502707: {characterised by the manufacture of the container or its components}
- B01L 2300/047: Additional chamber, reservoir
- B01L 2300/0874: Three dimensional network
- C12N 2513/00: 3D culture
- 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}
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

This disclosure includes microfluidic devices, systems, and methods for investigating three-dimensional (3D) cell structures (e.g., organoids, spheroids, scaffolds, hydrogels, and the like). Some examples of the present microfluidic devices comprise: a body having a first end, a second end, to lateral edges, an upper side, a lower side, a first layer region between a medial plane of the body and the lower side, and a second layer region between the medial plane and the upper side. In some such examples, the first layer region defines one or more rows of wells, with each well open at the medial plane and having a bottom between the medial plane and the lower side. In some such examples, the second layer region can defines one or more elongated channels open at the medial plane and having a top between the medial plane and the upper side, with each of the elongated channels aligned with one of the rows of wells such that the respective elongated channel is in fluid communication with each of the wells in the respective row of wells.

## Claims

### Claim 1

A microfluidic device for investigating three-dimensional (3D) cell structures, the microfluidic device comprising:
a body having a first end, a second end, two lateral edges, an upper side, a lower side, a first layer region between a medial plane of the body and the lower side, and a second layer region between the medial plane and the upper side;
where the first layer region defines one or more rows of wells, each well including an open top portion at the medial plane and a bottom between the medial plane and the lower side, and where each well has a minimum transverse dimension of 1300 microns;
where the second layer region defines one or more elongated channels that each include an open bottom portion at the medial plane and a top between the medial plane and the upper side, each elongated channel of the one or more elongated channels aligned with a respective one of the rows of wells such that the open bottom portion of each elongated channel opposes the open top portion of each well in the respective one of the rows of wells such that each elongated channel is in fluid communication with each well in the respective one of the rows of wells; and
where the second layer region further defines a first port, a second port, and a third port that each extend from an exterior surface of the body and are each in fluid communication with an elongated channel of the one or more elongated channels such that a first flow path from the first port to the second port corresponds to a first set of wells of a row of wells aligned with the elongated channel and a second flow path from the second port to the third port corresponds to a second set of wells of the row of wells that is different than the first set of wells.

### Claim 2 (depends on 1)

The microfluidic device of claim 1, where:
the first layer region is defined by a first material layer having a first surface and a second surface;
the second layer region is defined by a second material layer having a first surface and a second surface; and
the second surface of the first material layer defines the lower side of the body;
the first surface of the second material layer defines the upper side of the body; and
the first surface of the first material layer contacts the second surface of the second material layer along the medial plane.

### Claim 3 (depends on 1)

The microfluidic device of claim 1, where the first layer region is coupled to a first glass slide.

### Claim 4 (depends on 1)

The microfluidic device of claim 1, further comprising:
a clamp having a first frame and a second frame, the body positioned between the first and second frames, where the first frame comprises a transparent portion disposed adjacent the upper side of the second layer region; where the second frame is closer to the lower side of the body frame than the upper side of the body; and where the second frame is configured to permit imaging of at least one well through the lower side of the body; and
one or more fluid conduits extending through the transparent portion and the second layer region into the one or more elongated channels such that the one or more fluid conduits are in fluid communication with the one or more elongated channels.

### Claim 5 (depends on 1)

The microfluidic device of claim 1, where the first port is positioned at a first end of the elongated channel, and the third port is positioned at a second end of the elongated channel.

### Claim 6 (depends on 1)

The microfluidic device of claim 1, where the upper side of the body includes the exterior surface of the body.

### Claim 7 (depends on 1)

The microfluidic device of claim 1, wherein each well has a depth within a range of 400 to 800 microns, inclusive.

### Claim 8 (depends on 1)

The microfluidic device of claim 1, wherein a third flow path from the first port to the third port corresponds to both the first set of wells and the second set of wells.

### Claim 9 (depends on 1)

The microfluidic device of claim 1, wherein the wells of the first set of wells are distinct from the wells of the second set of wells.

### Claim 10

A microfluidic device for investigating three-dimensional (3D) cell structures, the microfluidic device comprising:
a body having a first end, a second end, two lateral edges, an upper side, a lower side, a first layer region between a medial plane of the body and the lower side, and a second layer region between the medial plane and the upper side;
where the first layer region defines one or more rows of wells, each well open at the medial plane and having a bottom between the medial plane and the lower side, and where each well has a minimum transverse dimension of 1300 microns;
where the second layer region defines one or more elongated channels open at the medial plane and having a top between the medial plane and the upper side, each elongated channel of the one or more elongated channels aligned along a length of the elongated channel with a respective one of the rows of wells such that the elongated channel is in fluid communication with each well in the respective one of the row of wells, and where the elongated channel has a width greater than the minimum transverse dimension of each well in the respective one of the row of wells; and
where the second layer region further defines a first port, a second port, and a third port that each extend from an exterior surface of the body and are each in fluid communication with an elongated channel of the one or more elongated channels such that a first flow path from the first port to the second port corresponds to a first set of wells of a row of wells aligned with the elongated channel and a second flow path from the second port to the third port corresponds to a second set of wells of the row of wells that is different than the first set of wells.

### Claim 11 (depends on 10)

The microfluidic device of claim 10, where each well includes an open top portion, each elongated channel includes an open bottom portion, and the open bottom portion of each elongated channel opposes the open top portion of each well in the respective one of the rows of wells.

### Claim 12 (depends on 10)

The microfluidic device of claim 10, wherein a plane perpendicular to both the medial plane and an elongated channel of the one or more elongated channels extends through a well of the one or more rows of wells and the elongated channel.