# US 9,566,369 B2 · Methods for tissue decellularization

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- Number: US 9,566,369 B2
- Document ID: US9566369B2
- Status: Granted
- Kind: B2
- Application number: 13/580,367
- Filed: 2011-02-25
- Published: 2013-01-31
- Granted: 2017-02-14
- Earliest claimed: 2010-02-26
- Assignees: Decell Technologies Inc.
- Applicants: Paul F. Gratzer
- Inventors: Paul F. Gratzer
- Web page: https://patentgrep.com/patent/US9566369B2

## 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 method for producing a human bioprosthetic tissue comprising:
contacting a human tissue with a hypotonic solution to produce a lysed tissue, the human tissue having an epidermal cellular layer with an underlying dermis and an underlying basement membrane matrix;
contacting the lysed tissue with a first surfactant solution to produce a surfactant-treated tissue;
contacting the surfactant-treated tissue with a nuclease enzyme solution to produce an enzyme-treated tissue;
contacting the enzyme-treated tissue with a cleaning solution comprising a second surfactant, a chaotropic agent, or a mixture thereof, to produce a decellularized tissue;
and
contacting the decellularized tissue with a bioburden reducing agent solution to produce the human bioprosthetic tissue,
wherein the bioburden reducing agent solution comprises 1% v/v Peracetic Acid;
wherein the human tissue's epidermal cellular layer has been removed, leaving the underlying basement membrane matrix intact in the human bioprosthetic tissue.

### Claim 2 (depends on 1)

The method of claim 1, wherein the human tissue is human skin tissue, wherein the hypotonic solution comprises 10 mM Tris buffer, wherein the first surfactant solution comprises 1% v/v Triton X-100®, wherein the nuclease enzyme solution comprises RNase and DNase, wherein the cleaning solution comprises 1% v/v tri-n-butyl phosphate, wherein the bioburden reducing agent solution comprises 1% v/v Peracetic Acid, wherein each step of the method is performed separately from the other steps of the method, and wherein each step of the method is immediately followed by a rinsing step prior to the start of the next step of the method.

### Claim 3 (depends on 1)

The method of claim 1, further comprising contacting one or more of the tissue with a physiologically isotonic solution.

### Claim 4 (depends on 1)

The method of claim 1, wherein one or more of the solutions further comprises a protease inhibitor, and wherein the protease inhibitor is a serine protease inhibitor, a metalloprotease inhibitor, ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride or a combination thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein one or more of the solutions further comprises a bioburden reducing agent, and wherein the bioburden reducing agent comprises penicillin, streptomycin, peracetic acid, ethanol, or a combination thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the hypotonic solution comprises one or more organic or inorganic buffers, one or more antibiotics or antimycotics, an alkaline pH, and wherein the osmolarity of the hypotonic solution is maintained as hyptotonic to cells, and wherein the first surfactant solution comprises a salt selected from the group consisting of KCl and NaCl, one or more organic or inorganic buffers, one or more antibiotics or antimycotics, an alkaline pH, one or more protease inhibitors, and 0.2-3% v/v of a anionic, non-ionic, zwitterionic or cationic detergent selected from the group consisting of Triton X-100, Triton X-200, Tween 20, Tween 80, sodium deoxycholate, CHAPS, N-lauroyl-sarcosinate, Igepal CA630, and Sulfobetain-10 and -16, and wherein the nuclease enzyme solution comprises an endonuclease selected from the group consisting of DNAse, RNAse, and Benzonase, wherein the nuclease enzyme solution is prepared with a physiological buffer selected from the group consisting of Hanks' Balanced Salt Solution, HEPES, Phosphate Buffered Saline, Tris-Buffered Saline, and wherein the nuclease enzyme solution is maintained at a pH of 6-8, and wherein the cleaning solution comprises 0.2-3% v/v of an anionic, non-ionic, zwitterionic or cationic detergent selected from the group consisting of Triton X-100, Triton X-200, Tween 20, Tween 80, sodium deoxycholate, CHAPS, N-lauroyl-sarcosinate, Igepal CA630, and Sulfobetain-10 and -16 or Tri-n-butyl phosphate, one or more organic or inorganic buffers, one or more antibiotics or antimycotics, an alkaline pH, and wherein the cleaning solution is prepared in either an aqueous or a 70% ethanol solvent.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first surfactant solution comprises an anionic surfactant, and wherein the anionic surfactant is Triton X-100.

### Claim 8 (depends on 1)

The method of claim 1, further comprising carrying out one or more steps at a temperature of between about 22° C. and 40° C.

### Claim 9 (depends on 1)

The method of claim 1, wherein the human tissue is a human skin tissue, and wherein the human skin tissue is human allogeneic skin.

### Claim 10 (depends on 1)

The method of claim 1, wherein the human tissue is a soft human tissue, and wherein the soft human tissue is a heart valve, tendon, ligament, artery, vein, diaphragm, pericardium, fascia, dura mater, tympanic membrane, aortic conduit, or cartilage.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

The method of claim 1, wherein the cleaning solution further comprises tri-n-butyl phosphate.

### Claim 13 (depends on 1)

A bioprosthetic tissue according to the method of claim 1.

### Claim 14 (depends on 1)

The method of claim 1, wherein the bioburden reducing agent solution comprises 1% v/v Peracetic Acid in ethanol.

### Claim 15 (depends on 1)

The method of claim 1, wherein the cleaning solution comprises 1% v/v TnBP in 70% ethanol solvent.

### Claim 16 (depends on 1)

The method of claim 1, wherein in the hypotonic solution the epidermal layer of the human tissue separates from the underlying dermis while leaving the basement membrane intact.