# US 11,898,167 B2 · Reversible stencils for fabricating micro-tissues

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- Number: US 11,898,167 B2
- Document ID: US11898167B2
- Status: Granted
- Kind: B2
- Application number: 17/079,720
- Filed: 2020-10-26
- Published: 2021-04-29
- Granted: 2024-02-13
- Earliest claimed: 2014-08-07
- Assignees: The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Gladstone; The Regents of the University of California
- Applicants: The Regents of the University of California; The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Gladstone
- Inventors: Nathaniel Huebsch; Bruce Conklin; Kevin E. Healy; Peter Loskill
- Web page: https://patentgrep.com/patent/US11898167B2

## CPC

- C12N 5/0657: {Cardiomyocytes; Heart cells}
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12N 2533/30: Synthetic polymers
- C12N 2535/10: Patterned coating
- 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

The invention relates to devices, methods, kits, and compositions for in vitro generation of three-dimensional micro-tissues that are accurate models of heart, skeletal muscle, neuronal, and other tissues.

## Claims

### Claim 1

A device for confining mammalian cells, forcing cellular/tissue alignment and tissue self-assembly, comprising:
a cell adhesion substrate; and
a removable elastomeric stencil overlay;
wherein the elastomeric stencil has one or more cut-out patterned microwells comprising two or more circular, oval, rectangular, square, V-shaped, or triangular holes, each hole joined to an adjacent hole by a canal (shaft); and
wherein the cell adhesion substrate binds cells within the holes and the canals of the cut-out pattern; and
wherein at least one of the microwells has at least two square holes with a side length “L” of 250 μm to 1000 μm, and the canal joining the two square holes has a longitudinal length “X” of 250-1000 μm and a traverse width “Y” of 50-200 μm.

### Claim 2 (depends on 1)

The device of claim 1, wherein the removable elastomeric stencil comprises polydimethylsiloxane (PDMS), surface functionalized PDMS, polyimide, polyurethane, SU8, thermoplastics, poly(methylmethacrylate) (PMMA), polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), poly(vinyl chloride) (PVC), fibrin, glass, quartz, silicon, hydrogel forming polymers including polyacrylamide, polyethylene glycol, alginate, agarose, gelatin or collagen, or any combination thereof.

### Claim 3 (depends on 1)

The device of claim 1, wherein each of the microwells has a depth of at least 250 μm; wherein each of the canals of each of the microwells is separately about 50 μm to about 150 μm wide; wherein the width of each of the canals compared to the width of the holes is about 1:3 to about 1:10; or a combination thereof.

### Claim 4 (depends on 1)

The device of claim 1, wherein the stencil, including the walls of the holes, is coated with a blocking agent to inhibit cell adhesion to the stencil.

### Claim 5 (depends on 4)

The device of claim 4, wherein the blocking agent comprises Pluronics, polyethylene oxide, alginate, poly-N-isopropylacrylamide, bovine serum albumin, bisacrylamide, alginate, agarose, polyethylene glycol diacrylate, or any combination thereof.

### Claim 6 (depends on 1)

The device of claim 1, wherein the substrate comprises glass, silicon, polyolefin, polystyrene, poly(alkyl)methacrylate, poly(alkyl)acrylate, poly(acrylamide), poly(carbonate), poly(ethylene glycol), poly(N-isopropyl acrylamide), polyacrylonitrile, poly(vinylacetate), poly(vinyl alcohol), a chlorine-containing polymer, poly(vinyl)chloride, polyoxymethylene, polycarbonate, polyamide, polyimide, polyurethane, polyvinylidene difluoride (PVDF), phenolic, amino-epoxy resin, polyester, polyether, polyethylene terephthalate (PET), polyglycolic acid (PGA), poly-(p-phenyleneterephthalamide), polyphosphazene, polypropylene, or a combination thereof.

### Claim 7 (depends on 1)

The device of claim 1, wherein the stencil is permanently or reversibly covalently bonded to the substrate.

### Claim 8 (depends on 1)

The device of claim 1, in which the substrate comprises a porous membrane that prevents cellular flux but allows fluid flow.

### Claim 9 (depends on 1)

The device of claim 1, wherein the substrate comprises a network of microfluidic channels beneath a membrane.

### Claim 10 (depends on 9)

The device of claim 9, wherein the network of microfluidic channels is operably connected to one or more reservoirs, each reservoir thereby operably connected to a microwell.

### Claim 11 (depends on 10)

The device of claim 10, wherein one or more reservoirs each separately comprise one or more test compounds, drugs, proteins, oligonucleotides, nucleic acids, or a combination thereof.

### Claim 12 (depends on 10)

The device of claim 10, wherein the network of microfluidic channels further comprises one or more micropump to remove and deliver fluids to one or more to microwells.

### Claim 13 (depends on 1)

The device of claim 1, wherein the substrate comprises a network of microfluidic channels beneath a membrane, with one or more microchannels operably connected to one or more microwells.

### Claim 14 (depends on 1)

The device of claim 1, wherein the substrate comprises a coating of cell adhesion molecules.

### Claim 15 (depends on 14)

The device of claim 14, wherein the cell adhesion molecules comprise fibronectin, alginate, E-selectin, gelatin, laminin, matrigel, collagen, fibrinogen, bisacrylamide, RGD peptides, PHSRN peptides, and DGEA peptides, and combinations thereof.

### Claim 16 (depends on 1)

The device of claim 1, wherein the substrate comprises a culture dish or a multi-well culture dish with at least one device in each well.

### Claim 17 (depends on 1)

The device of claim 1, further comprising mammalian cells in the microwells.

### Claim 18 (depends on 17)

The device of claim 17, further comprising cells that comprise a heterologous marker gene, a heterologous reporter gene, a mutant gene, or a combination thereof.

### Claim 19 (depends on 18)

The device of claim 18, wherein the mutant gene contributes to a disease or condition.

### Claim 20 (depends on 1)

The device of claim 1, further comprising a mixture of mammalian cell types in the microwells.

### Claim 21 (depends on 1)

The device of claim 1, further comprising about 1000 to about 10,000 cells in at least one microwell.

### Claim 22 (depends on 1)

The device of claim 1, further comprising mammalian cells in at least two adjacent holes of a microwell, wherein the at least two holes are joined by a canal.