# US 11,027,048 B2 · Methods for tissue decellularization

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- Number: US 11,027,048 B2
- Document ID: US11027048B2
- Status: Granted
- Kind: B2
- Application number: 15/254,177
- Filed: 2016-09-01
- Published: 2016-12-22
- Granted: 2021-06-08
- Earliest claimed: 2010-02-26
- Assignees: Decell Technologies Inc.
- Applicants: DeCell Technologies Inc.
- Inventors: Paul Frank Gratzer
- Web page: https://patentgrep.com/patent/US11027048B2

## CPC

- A61L 27/60: Materials for use in artificial skin
- A61L 27/24: Collagen
- A61L 27/362: {Skin, e.g. dermal papillae}
- A61L 27/3641: {characterised by the site of application in the body (materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60)}
- 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 27/3687: {characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents}
- 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

A method for producing sterile, decellurized bioprosthetic tissue comprising: (a) contacting a human tissue with a hypotonic solution to produce a lysed tissue, (b) contacting the lysed tissue with a first surfactant to produce a surfactant-treated tissue, (c) contacting the surfactant-treated tissue with a nuclease enzyme solution to produce an enzyme-treated tissue, (d) contacting the enzyme-treated tissue with a cleaning solution comprising a second surfactant, a chaotropic agent or a mixture thereof to produce a decellurized tissue and (e) contacting the decellurized tissue with a bioburden reducing agent to produce the final bioprosthetic tissue. Kits to be used in conjunction with said method, as well as, the bioprosthetic tissue produced via said method are also provided.

## Claims

### Claim 1

A bioprosthetic tissue, wherein the tissue is 95±5% free of nucleic acids, wherein the tissue is 95±5% free of major histocompatibility molecules, wherein the tissue is 95±5% free of Staphylococcus bacteria, Streptococcus bacteria, Enterococcus bacteria, and Bacillus bacteria, or complies with American Association of Tissue Banks Standards for Tissue Banking under K2.200, wherein the collagen structure of the tissue is not altered compared to a fresh control tissue as measured by a Hydrothermal Isometric Tension test, and wherein the bioprosthetic tissue has been prepared from a human tissue having an epidermal cellular layer with an underlying basement membrane matrix such that the human tissue's epidermal cellular layer has been removed, leaving the underlying basement membrane matrix intact in the bioprosthetic tissue.

### Claim 2 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the tissue is 95±5% free of Staphylococcus aureus bacteria, Streptococcus pyogenes bacteria, Enterococcus bacteria, Bacillus subtilis, and fungus, or complies with American Association of Tissue Banks Standards for Tissue Banking under K2.200.

### Claim 3 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the tissue is human skin.

### Claim 4 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the nucleic acid is DNA or RNA, and wherein the tissue has less than 0.5 ng/mg dry weight of tissue of DNA.

### Claim 5 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the major histocompatibility complex (MHC) is HLA-DR, and wherein the tissue is 95±5% free of HLA-DR as measured by immunohistochemistry, or wherein the MHC is HLA-A, B, C, and wherein the tissue is 95±5% free of HLA-A, B, C as measured by immunohistochemistry.

### Claim 6 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the tissue is 95±5% free of Vimentin as measured by immunohistochemistry, and wherein the tissue is 95±5% free of Beta-Actin as measured by immunohistochemistry.

### Claim 7 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the tissue comprises elastin as measured by histology using Van Gieson stain, and wherein the tissue comprises one or more proteoglycans as measured by histology using Masson's Trichrome stain.

### Claim 8 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the collagen structure of the tissue is not altered compared to a fresh control tissue as assessed by thermal stability of collagen.

### Claim 9 (depends on 1)

The bioprosthetic tissue of claim 1, wherein the denaturation temperature of the tissue is not altered compared to a fresh control tissue, and wherein a denaturation temperature of the tissue is about 64, 65, 66, 67, 68, or 69° C. as measured by a Hydrothermal Isometric Tension test.