# US 12,180,446 B2 · Native extracellular matrix-derived membrane inserts for organs-on-chips, multilayer microfluidics microdevices, bioreactors, tissue culture inserts, and two-dimensional and three-dimensional cell culture systems

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- Number: US 12,180,446 B2
- Document ID: US12180446B2
- Status: Granted
- Kind: B2
- Application number: 16/787,275
- Filed: 2020-02-11
- Published: 2020-06-18
- Granted: 2024-12-31
- Earliest claimed: 2017-08-11
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: Dongeun Huh; Mark Mondrinos; Alex Yoon Yi; Jeongyun Seo
- Web page: https://patentgrep.com/patent/US12180446B2

## CPC

- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 23/22: {Transparent or translucent parts (glassware for laboratory use B01L3/00)}
- C12M 23/34: {Internal compartments or partitions}
- C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis}
- C12M 29/10: {Perfusion}
- 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 presently disclosed subject matter provides native extracellular matrix-derived membrane inserts for organs-on-chips, multilayer microfluidics microdevices, bioreactors, tissue culture inserts, and two-dimensional and three-dimensional cell culture systems. A microfluidic cell culture is provided that can include at least one membrane including extracellular matrix (ECM) material. The ECM material can be used to construct a perfusable microfluidic system including a plurality of layers of microfabricated cell culture chambers. The microfluidic cell culture can further include a lower layer including a microchannel on which the at least one membrane is placed and an upper layer including another microchannel. The upper layer can be bonded to the lower layer.

## Claims

### Claim 1

A microfluidic cell culture device, comprising:
at least one membrane that is naturally derived and comprises vitrified extracellular matrix (ECM) material, wherein the at least one membrane is uncoated;
a lower layer comprising a first microchannel on which the at least one membrane is placed;
an upper layer comprising a second microchannel, wherein the upper layer is bonded directly to a surface of the lower layer around the at least one uncoated membrane; and
a layer of cells seeded on at least one surface of the at least one uncoated membrane.

### Claim 2 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the at least one uncoated membrane comprising the vitrified ECM material resembles fibrous architecture of native basement membranes.

### Claim 3 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the vitrified ECM material is integrated into a multilayered microfluidic device mimicking physiological multicellular structures and tissue-tissue interfaces.

### Claim 4 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the at least one uncoated membrane is an application-specific membrane generated by isolating and vitrifying ECM components from an application-specific tissue type of interest.

### Claim 5 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the vitrified ECM material is derived from at least one of animal tissue and human tissue.

### Claim 6 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the vitrified ECM material is formed via evaporation-induced vitrification.

### Claim 7 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the vitrified ECM material is generated by
pooling and processing one or more of engineered tissues and organoids comprising human cells using modified tissue decellularization techniques, the pooling and processing including
removing cells, and
harvesting human cell-derived ECM materials.

### Claim 8 (depends on 7)

The microfluidic cell culture device of claim 7, wherein cells of non-human origin are added to human cell culture in order to increase the rate of ECM production and to control the type of ECM produced.

### Claim 9 (depends on 7)

The microfluidic cell culture device of claim 7, wherein the organoids are treated with biochemical agents to control the process of ECM production and deposition, and wherein the organoids are subjected to mechanical or electrical signals to control ECM production.

### Claim 10 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the vitrified ECM material comprises
(i) collagen type I
(ii) collagen type I and Matrigel, and/or
(iii) collagen type I and alginate.

### Claim 11 (depends on 8)

The microfluidic cell culture device of claim 8, wherein the at least one uncoated membrane is two or more membranes in a stacked arrangement.

### Claim 12 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the at least one surface of the at least one uncoated membrane remains uncoated prior to cell seeding.

### Claim 13 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the layer of cells comprises human adenocarcinoma cells.

### Claim 14 (depends on 1)

The microfluidic cell culture device of claim 1, wherein
the vitrified ECM material comprises collagen type I and Matrigel, and
the layer of cells comprises
a first layer of cells on a first surface of the at least one uncoated membrane facing the first microchannel of the lower layer, and
a second layer of cells on a second surface of the at least one uncoated membrane facing the second microchannel of the upper layer.

### Claim 15 (depends on 14)

The microfluidic cell culture device of claim 14, wherein the first layer of cells comprises human bronchial epithelial cells and the second layer of cells comprises primary human lung fibroblasts.

### Claim 16 (depends on 14)

The microfluidic cell culture device of claim 14, wherein the first layer of cells comprises human bronchial epithelial cells and the second layer of cells comprises human venous endothelial cells.

### Claim 17 (depends on 14)

The microfluidic cell culture device of claim 14, wherein the first layer of cells comprises human venous endothelial cells and the second layer of cells comprises primary human lung fibroblasts.

### Claim 18 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the vitrified ECM material comprises between about 50% and about 80% collagen type I and between about 20% and about 50% Matrigel.

### Claim 19 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the at least one uncoated membrane has a Young's modulus of between about 400 kPa and about 600 kPa.

### Claim 20 (depends on 18)

The microfluidic cell culture device of claim 18, wherein the vitrified ECM material comprises about 80% collagen type 1 and about 20% Matrigel.

### Claim 21 (depends on 10)

The microfluidic cell culture device of claim 10, wherein the vitrified ECM material comprises collagen type 1 and Matrigel.

### Claim 22 (depends on 21)

The microfluidic cell culture device of claim 21, wherein the collagen type I is crosslinked with the Matrigel.

### Claim 23 (depends on 1)

The microfluidic cell culture device of claim 1, wherein the at least one uncoated membrane is optically transparent.