# US 12,600,931 B2 · Microfluidic cell culture plate for air-liquid interface and 3D cultured tissue applications

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- Number: US 12,600,931 B2
- Document ID: US12600931B2
- Status: Granted
- Kind: B2
- Application number: 17/246,028
- Filed: 2021-04-30
- Published: 2021-11-04
- Granted: 2026-04-14
- Earliest claimed: 2020-05-01
- Assignees: The Charles Stark Draper Laboratory, Inc.
- Applicants: The Charles Stark Draper Laboratory Inc.
- Inventors: Jonathan R. Coppeta; Hesham Azizgolshani; Brian P. Cain; Brett C. Isenberg; Joseph L. Charest; Else M. Vedula; Ashley L. Gard; Ryan S. Maloney; Jeffrey T. Borenstein; Rebeccah J. Luu
- Web page: https://patentgrep.com/patent/US12600931B2

## CPC

- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)}
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 23/22: {Transparent or translucent parts (glassware for laboratory use B01L3/00)}
- C12M 25/04: {in combination with well or multiwell plates, i.e. culture inserts}
- C12M 29/00: {Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B)}
- C12M 35/08: {Chemical, biochemical or biological means, e.g. plasma jet, co-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}

## Abstract

The present disclosure describes systems and methods for providing culturing of a number of various tissue types in an air-liquid configuration in a high-throughput format and allowing co-culture of cells as well as application of physiologically relevant flow. A microfluidic cell culturing device is provided that includes a first channel having a first inlet port and a second inlet port, the first channel defined in a first layer. The microfluidic cell culturing device includes a membrane layer having a first surface coupled to the first layer defining the first channel, the membrane layer comprising semipermeable membrane that forms at least a portion of a surface of the first channel. The microfluidic cell culturing device includes a chamber defined in a second layer that exposes a portion the membrane layer to an external environment, wherein the chamber overlaps a portion of the first channel across the membrane layer.

## Claims

### Claim 1

A microfluidic cell culturing device, comprising:
a first channel having a first inlet port and a second inlet port, sidewalls of the first channel defined in a first layer, a bottom surface of the first channel defined by an optically clear layer coupled to the first layer, each of the first inlet port and the second inlet port defined at least in part in a second layer;
a membrane layer having a first surface coupled to the first layer defining sidewalls of the first channel and a second surface coupled to the second layer, the membrane layer comprising a semipermeable membrane that forms a top surface of the first channel, the membrane layer comprising a first opening aligned with the first inlet port and a second opening aligned with the second inlet port; and
a chamber defined as a slot in the second layer coupled to the second surface of the membrane layer, the chamber having a length greater than at least twice a width of the chamber, the chamber comprising a first divot defined in the second layer at a first end of the chamber and a second divot defined in the second layer at a second end of the chamber, the chamber exposing a portion of the second surface of the membrane layer to an external environment via an opening in the second layer, wherein the chamber overlaps a portion of the first channel across the membrane layer.

### Claim 2 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the chamber further comprises one or more conductive traces configured to attach to an electrical measurement device.

### Claim 3 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein at least one of the first inlet port, the second inlet port, or the chamber, is in fluid communication with a respective reservoir of a culture plate.

### Claim 4 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the chamber is configured to receive suspension cells or adherent cells, and the exposed portion of the second surface of the membrane layer is configured to culture one or more cells.

### Claim 5 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the first layer is coupled to the membrane layer using thermal compression.

### Claim 6 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the chamber further comprises a step feature configured to retain cell suspension during seeding of the microfluidic cell culturing device.

### Claim 7 (depends on 6)

The microfluidic cell culturing device of claim 6, wherein the step feature has a height in a range of 250 microns to 2.5 millimeters.

### Claim 8 (depends on 6)

The microfluidic cell culturing device of claim 6, wherein the step feature further comprises a draft angle configured to reduce bulging of a cell suspension droplet positioned in the chamber.

### Claim 9 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the chamber comprises a rectangular footprint or a slotted footprint.

### Claim 10 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the semipermeable membrane comprises pores having a pore size in a range of 0.4 to 8 microns.

### Claim 11 (depends on 1)

The microfluidic cell culturing device of claim 1, wherein the first layer or the second layer comprise one or more of cyclo-olefin polymers, polyetherimide, polystyrene, polycarbonate, or polymethylmethacrylate.

### Claim 12

A culture plate, comprising:
a plurality of well reservoirs; and
a plurality of microfluidic devices, each of the plurality of microfluidic devices comprising:
a first channel having a first inlet port and a second inlet port, sidewalls of the first channel defined in a first layer, a bottom surface of the first channel defined by an optically clear layer coupled to the first layer, each of the first inlet port and the second inlet port defined at least in part in a second layer;
a membrane layer having a first surface coupled to the first layer defining the first channel and a second surface coupled to the second layer, the membrane layer comprising a semipermeable membrane that forms a top surface of the first channel, the membrane layer comprising a first opening aligned with the first inlet port and a second opening aligned with the second inlet port; and
a chamber defined as a slot in the second layer coupled to the second surface of the membrane layer, the chamber having a length greater than at least twice a width of the chamber, the chamber comprising a first divot defined in the second layer at a first end of the chamber and a second divot defined in the second layer at a second end of the chamber, the chamber exposing a portion of the second surface of the membrane layer to an external environment via an opening in the second layer, wherein the chamber overlaps a portion of the first channel across the membrane layer.

### Claim 13 (depends on 12)

The culture plate of claim 12, the chamber of each of the plurality of microfluidic devices further comprises one or more conductive traces configured to attach to an electrical measurement device.

### Claim 14 (depends on 12)

The culture plate of claim 12, wherein at least one of the first inlet port, the second inlet port, or the chamber, of each of the plurality of microfluidic devices, is coupled to a respective well reservoir of the plurality of well reservoirs.

### Claim 15 (depends on 12)

The culture plate of claim 12, wherein the chamber of each of the plurality of microfluidic devices is configured to receive suspension cells or adherent cells, and the exposed portion of the second surface of the membrane layer of each of the plurality of microfluidic devices is configured to culture one or more cells.

### Claim 16 (depends on 12)

The culture plate of claim 12, wherein the chamber of each of the plurality of microfluidic devices further comprises a step feature configured to retain cell suspension during seeding of the plurality of microfluidic devices.

### Claim 17 (depends on 16)

The culture plate of claim 16, wherein the step feature has a height in a range of 250 microns to 2.5 millimeters.

### Claim 18 (depends on 16)

The culture plate of claim 16, wherein the step feature further comprises a draft angle configured to reduce bulging of a cell suspension droplet positioned in the chamber.

### Claim 19 (depends on 16)

The culture plate of claim 16, wherein the first layer or the second layer of each of the plurality of microfluidic devices comprises one or more of cyclo-olefin polymers, polyetherimide, polystyrene, polycarbonate, or polymethylmethacrylate.

### Claim 20 (depends on 16)

The culture plate of claim 16, further comprising one or more pumps each in fluid communication with a respective first inlet port or a respective second inlet port of a respective microfluidic device of the plurality of microfluidic devices.