# US 11,077,230 B2 · Decellularising tissue

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- Number: US 11,077,230 B2
- Document ID: US11077230B2
- Status: Granted
- Kind: B2
- Application number: 15/769,004
- Filed: 2016-10-18
- Published: 2018-11-01
- Granted: 2021-08-03
- Earliest claimed: 2015-10-21
- Assignees: Videregen Limited
- Applicants: Videregen Limited
- Inventors: Tahera Ansari
- Web page: https://patentgrep.com/patent/US11077230B2

## CPC

- A61L 27/3683: {subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment}
- A61L 27/3633: {Extracellular matrix [ECM]}
- A61L 2430/40: Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)

## Abstract

The invention provides a method for decellularising a tissue the method comprising the steps of: a) adding a tissue to be decellularised to a decellularisation vessel; b) adding a decellularisation medium comprising at least one detergent and/or at least one enzyme to the vessel; c) introducing a supercritical fluid to the vessel; and d) maintaining contact of the tissue with the supercritical fluid for a time sufficient to substantially decellularise the tissue.

## Claims

### Claim 1

A method for decellularising a tissue comprising the steps of:
a) adding a tissue to be decellularised to a decellularisation vessel;
b) adding a decellularisation medium comprising at least one detergent and at least one enzyme selected from the group consisting of a deoxyribonuclease and a ribonuclease to the vessel;
c) introducing supercritical carbon dioxide to the vessel with the tissue to be decellularised and the decellularisation medium present; and
d) maintaining contact of the tissue with the supercritical carbon dioxide for a time sufficient to substantially decellularise the tissue.

### Claim 2 (depends on 1)

The method of claim 1 wherein the detergent comprises sodium dodecyl sulphate, sodium deoxycholate, a detergent comprising hydrophilic polyoxyethylene-oxide and hydrophilic hydrocarbon moieties or any combination thereof.

### Claim 3 (depends on 2)

The method of claim 2 wherein the detergent comprises a mixture of sodium dodecyl sulphate and sodium deoxycholate; a mixture of sodium dodecyl sulphate and a detergent comprising hydrophilic polyoxyethylene-oxide and hydrophilic hydrocarbon moieties; or a mixture of sodium deoxycholate and a detergent comprising hydrophilic polyoxyethylene-oxide and hydrophilic hydrocarbon moieties.

### Claim 4 (depends on 1)

The method of claim 1 wherein the decellularisation medium further comprises an enzyme selected from the group consisting of a proteolytic enzyme, a lipase, a cellulase, and any combination thereof.

### Claim 5 (depends on 4)

The method of claim 4 wherein the proteolytic enzyme comprises trypsin.

### Claim 6 (depends on 4)

The method of claim 4 wherein the detergent of the decellularisation medium is selected from the group consisting of sodium dodecyl sulphate, sodium deoxycholate, a detergent comprising hydrophilic polyoxyethylene-oxide and hydrophilic hydrocarbon moieties, and combinations thereof.

### Claim 7 (depends on 6)

The method of claim 6 wherein the detergent of the decellularisation medium comprises sodium dodecyl sulphate and/or sodium deoxycholate.

### Claim 8 (depends on 1)

The method of claim 1 wherein the vessel is heated to at least 28° C.

### Claim 9 (depends on 1)

The method of claim 1 wherein the supercritical carbon dioxide is maintained at a pressure of above 100 MPa.

### Claim 10 (depends on 1)

The method of claim 1 wherein the tissue is maintained in contact with the supercritical carbon dioxide and decellularisation medium for a period of at least 24 hrs.

### Claim 11 (depends on 10)

The method of claim 10 wherein the tissue is maintained in contact with the supercritical carbon dioxide and decellularisation medium for a period of at least 30 hrs.

### Claim 12 (depends on 1)

The method of claim 1 further comprising a step e) of washing the decellularised tissue with a washing buffer.

### Claim 13 (depends on 12)

The method of claim 12 wherein step e) comprises washing the decellularised tissue with phosphate buffered saline for a period of at least 24 hrs.

### Claim 14

A method of manufacturing a tissue implant or scaffold comprising the steps of:
a) adding a tissue to be decellularised to a decellularisation vessel;
b) adding a decellularisation medium comprising at least one detergent and at least one enzyme selected from the group consisting of a deoxyribonuclease and a ribonuclease to the vessel;
c) introducing supercritical carbon dioxide to the vessel with the tissue to be decellularised and the decellularisation medium present; and
d) maintaining contact of the tissue with the supercritical carbon dioxide and the decellularisation medium for a time sufficient to substantially decellularise the tissue.