# US 2023/0264191 A1 · Systems and methods for fabricating physiologically relevant in vitro vessels

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- Number: US 2023/0264191 A1
- Document ID: US20230264191A1
- Status: Published application
- Kind: A1
- Application number: 18/112,279
- Filed: 2023-02-21
- Published: 2023-08-24
- Earliest claimed: 2022-02-21
- Assignees: The Texas A&M University System
- Applicants: The Texas A&M University System
- Inventors: Abhishek Jain; Amirali Selahi
- Web page: https://patentgrep.com/patent/US20230264191A1

## CPC

- B01L 3/502707: {characterised by the manufacture of the container or its components}
- C12N 5/069: {Vascular Endothelial cells}
- 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}
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12N 5/0602: {Vertebrate cells}
- C12N 5/0691: {Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels}
- C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)}
- B01L 2200/025: Align devices or objects to ensure defined positions relative to each other
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- 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 2400/0487: fluid pressure, pneumatics
- C12N 2501/25: Tumour necrosing factors [TNF]
- C12N 2521/00: Culture process characterised by the use of hydrostatic pressure, flow or shear forces
- C12N 2533/54: Collagen; Gelatin
- C12N 2535/10: Patterned coating
- 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)
- 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

A method for fabricating an in vitro vessel includes forming a substrate that defines a microfluidic passage therein extending along a longitudinal axis and defined by an inner surface, positioning the substrate in a vertical orientation whereby an acute angle is formed between the longitudinal axis of the microfluidic passage and the direction of gravity, and culturing a plurality of first cells in the microfluidic passage while the substrate is disposed in the vertical orientation whereby an annular layer of the plurality of first cells is formed in the microfluidic channel, wherein the layer of the plurality of first cells defines a lumen extending longitudinally through the microfluidic channel.

## Claims

### Claim 1

A method for fabricating an in vitro vessel, the method comprising:
(a) forming a substrate that defines a microfluidic passage therein extending along a longitudinal axis and defined by an inner surface;
(b) positioning the substrate in a vertical orientation whereby an acute angle is formed between the longitudinal axis of the microfluidic passage and the direction of gravity; and
(c) culturing a plurality of first cells in the microfluidic passage while the substrate is disposed in the vertical orientation whereby an annular layer of the plurality of first cells is formed in the microfluidic channel, wherein the layer of the plurality of first cells defines a lumen extending longitudinally through the microfluidic channel.

### Claim 2 (depends on 1)

The method of claim 1, wherein the plurality of first cells comprise at least one of lymphatic endothelial cells (LECs), vascular endothelial cells (VECs), lymphatic muscle cells (LMCs), and vascular muscle cells (VMCs).

### Claim 3 (depends on 1)

The method of claim 1, further comprising:
(d) culturing a plurality of second cells that are different from plurality of first cells in the microfluidic passage whereby an annular layer of the plurality of second cells is formed in the microfluidic channel.

### Claim 4 (depends on 3)

The method of claim 3, wherein the layer of the plurality of first cells forms an annular inner layer of the first cells in the microfluidic channel and the layer of the plurality of second cells forms an outer layer of the plurality of second cells in the microfluidic channel that is radially positioned between the inner layer and the inner surface of the microfluidic channel.

### Claim 5 (depends on 3)

The method of claim 3, wherein (d) is performed prior to (b).

### Claim 6 (depends on 1)

The method of claim 1, wherein the lumen has an elliptical cross-section.

### Claim 7

An in vitro vessel comprising:
a substrate forming a microfluidic channel extending along a longitudinal axis and defined by an inner surface, wherein the microfluidic channel extends between a fluid inlet at a first end of the microfluidic channel and a fluid outlet located at a longitudinally opposed second end of the microfluidic channel;
an annular outer layer of muscle cells positioned in the microfluidic channel and extending entirely around the longitudinal axis of the microfluidic channel; and
an annular inner layer of endothelial cells positioned in the outer layer of muscle cells within the microfluidic channel and extending entirely around the longitudinal axis of the microfluidic channel, wherein the inner layer of endothelial cells defines a lumen extending longitudinally through the microfluidic channel and in fluid communication with both the fluid inlet and the fluid outlet formed in the substrate.

### Claim 8 (depends on 7)

The in vitro vessel of claim 7, wherein the muscle cells of the outer layer of muscle cells comprise lymphatic muscle cells (LMCs) and the endothelial cells of the inner layer of endothelial cells comprise lymphatic endothelial cells (LECs).

### Claim 9 (depends on 7)

The in vitro vessel of claim 7, wherein the muscle cells of the outer layer of muscle cells comprise vascular muscle cells (VMCs) and the endothelial cells of the inner layer of endothelial cells comprise vascular endothelial cells (VECs).

### Claim 10 (depends on 7)

The in vitro vessel of claim 7, wherein the outer layer of muscle cells is embedded in an annular extracellular matrix (ECM) positioned in the microfluidic channel and containing collagen.

### Claim 11 (depends on 7)

The in vitro vessel of claim 7, wherein a majority of the muscle cells comprising the outer layer of muscle cells are aligned substantially perpendicular to the longitudinal axis of the microfluidic channel.

### Claim 12 (depends on 7)

The in vitro vessel of claim 7, wherein a majority of the endothelial cells comprising the inner layer of endothelial cells are aligned substantially parallel to the longitudinal axis of the microfluidic channel.

### Claim 13 (depends on 7)

The in vitro vessel of claim 7, wherein a majority of the endothelial cells comprising the inner layer of endothelial cells are aligned substantially perpendicular to a majority of the muscle cells comprising the outer layer of muscle cells.

### Claim 14 (depends on 7)

The in vitro vessel of claim 7, wherein the lumen has an elliptical cross-section.

### Claim 15

An in vitro vessel comprising:
a substrate forming a microfluidic channel extending along a longitudinal axis and defined by an inner surface, wherein the microfluidic channel extends between a fluid inlet at a first end of the microfluidic channel and a fluid outlet located at a longitudinally opposed second end of the microfluidic channel; and
an annular layer of endothelial cells positioned within the microfluidic channel and extending entirely around the longitudinal axis of the microfluidic channel, wherein the layer of endothelial cells defines a lumen having an elliptical cross-section and extending longitudinally through the microfluidic channel and in fluid communication with both the fluid inlet and the fluid outlet formed in the substrate.

### Claim 16 (depends on 15)

The in vitro vessel of claim 15, wherein the endothelial cells of the layer of endothelial cells comprise lymphatic endothelial cells (LECs).

### Claim 17 (depends on 15)

The in vitro vessel of claim 15, wherein the endothelial cells of the layer of endothelial cells comprise vascular endothelial cells (VECs).

### Claim 18 (depends on 15)

The in vitro vessel of claim 15, wherein the elliptical cross-section of the lumen is defined by a major axis and a minor axis extending orthogonal to the major axis of the elliptical cross-section of the lumen, and wherein a ratio of the major axis to the minor axis is between 1.1:1 and 5:1.

### Claim 19 (depends on 18)

The in vitro vessel of claim 18, wherein the elliptical cross-section of the lumen is defined by a major axis and a minor axis extending orthogonal to the major axis of the elliptical cross-section of the lumen, and wherein the ratio of the major axis to the minor axis is between 1:1 and 3:1.

### Claim 20 (depends on 18)

The in vitro vessel of claim 18, wherein a ratio of the minimum radial thickness of the layer of endothelial cells to the maximum radial thickness of the layer of endothelial cells is between 0.5:1 and 2:1.

### Claim 21 (depends on 18)

The in vitro vessel of claim 18, wherein:
the layer of endothelial cells positioned within the microfluidic channel defines an annular inner layer of endothelial cells; and
an annular outer layer of muscle cells positioned in the microfluidic channel radially between the inner layer of endothelial cells and the inner surface of the microfluidic channel, and wherein the outer layer of muscle cells extends entirely around the longitudinal axis of the microfluidic channel.

### Claim 22 (depends on 21)

The in vitro vessel of claim 21, wherein a ratio of the minimum radial thickness of the layer of muscle cells to the maximum radial thickness of the layer of muscle cells is between 0.5:1 and 2:1.