# US 2013/0028981 A1 · Methods for tissue decellularization

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- Number: US 2013/0028981 A1
- Document ID: US20130028981A1
- Status: Published application
- Kind: A1
- Application number: 13/580,367
- Filed: 2011-02-25
- Published: 2013-01-31
- Earliest claimed: 2010-02-26
- Assignees: Decell Technologies Inc.
- Inventors: Paul F. Gratzer
- Web page: https://patentgrep.com/patent/US20130028981A1

## 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;
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.

### 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® (octyl phenoxy polyethoxyethanol), wherein the nuclease enzyme solution comprises RNase and DNase, wherein the cleaning solution comprises 1% (v/v) tri-n-butyl phosphate (TnBP), 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 tissues 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

(canceled)

### Claim 6

(canceled)

### Claim 7

(canceled)

### Claim 8 (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 9

(canceled)

### Claim 10 (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 solution is maintained as hypotonic 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, sodium dodecyl sulfate (SDS), 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 solution is prepared with a physiological buffer selected from the group consisting of Hanks' Balanced Salt Solution (HBSS), HEPES, Phosphate Buffered Saline (PBS), Tris-Buffered Saline (TBS), and wherein the 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, sodium dodecyl sulfate (SDS), N-lauroyl-sarcosinate, Igepal CA630, and Sulfobetain-10 and -16 or Tri-n-butyl phosphate (TnBP), one or more organic or inorganic buffers, one or more antibiotics or antimycotics, an alkaline pH, and wherein the solution is prepared in either an aqueos or a 70% ethanol solvent.

### Claim 11

(canceled)

### Claim 12

(canceled)

### Claim 13 (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 14

(canceled)

### Claim 15

(canceled)

### Claim 16

(canceled)

### Claim 17

(canceled)

### Claim 18

(canceled)

### Claim 19 (depends on 1)

The method of claim 1, further comprising sonicating one or more of the tissues.

### Claim 20

(canceled)

### Claim 21 (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 22

(canceled)

### Claim 23 (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 24 (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 25

(canceled)

### Claim 26 (depends on 1)

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

### Claim 27

(canceled)

### Claim 28

(canceled)

### Claim 29

(canceled)

### Claim 30

(canceled)

### Claim 31

(canceled)

### Claim 32

(canceled)

### Claim 33

(canceled)

### Claim 34

(canceled)

### Claim 35

(canceled)

### Claim 36

(canceled)

### Claim 37 (depends on 1)

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

### Claim 38

(canceled)

### Claim 39 (depends on 1)

A bioprosthetic tissue produced according to the method of claim 1.

### Claim 40

A bioprosthetic tissue, wherein the tissue is substantially free of nucleic acids, wherein the tissue is substantially free of major histocompatibility molecules (MHC), wherein the tissue is substantially free of staphylococcus bacteria, wherein the tissue is substantially free of streptococcus bacteria, wherein the tissue is substantially free of enterococcus bacteria, wherein the tissue is substantially free of bacillus bacteria, and wherein the collagen structure of the tissue is not substantially altered compared to a fresh control tissue.

### Claim 41 (depends on 40)

The bioprosthetic tissue of claim 40, wherein the tissue is substantially free of Staphylococcus aureus bacteria, wherein the tissue is substantially free of Streptococcus pyogenes bacteria, wherein the tissue is substantially free of Enterococcus bacteria, and wherein the tissue is substantially free of Bacillus subtilis, and wherein the tissue is substantially free of fungus.

### Claim 42 (depends on 40)

The bioprosthetic tissue of claim 40, wherein the tissue is human skin.

### Claim 43 (depends on 40)

The bioprosthetic tissue of claim 40, 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 44

(canceled)

### Claim 45

(canceled)

### Claim 46

(canceled)

### Claim 47 (depends on 40)

The bioprosthetic tissue of claim 40, wherein the MHC is HLA-DR, and wherein the tissue is substantially free of HLA-DR as measured by immunohistochemistry, and wherein the MHC is HLA-A,B,C, and wherein the tissue is substantially free of HLA-A,B,C as measured by immunohistochemistry.

### Claim 48

(canceled)

### Claim 49 (depends on 40)

The bioprosthetic tissue of claim 40, wherein the tissue is substantially free of Vimentin as measured by immunohistochemistry, and wherein the tissue is substantially free of Beta-Actin as measured by immunohistochemistry.

### Claim 50

(canceled)

### Claim 51 (depends on 40)

The bioprosthetic tissue of claim 40, 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 52

(canceled)

### Claim 53

(canceled)

### Claim 54 (depends on 40)

The bioprosthetic tissue of claim 40, wherein the collagen structure of the tissue is not substantially altered compared to a fresh control tissue as assessed by the thermal stability of collagen.

### Claim 55 (depends on 40)

The bioprosthetic tissue of claim 40, wherein the denaturation temperature of the tissue is not substantially altered compared to a fresh control tissue, and wherein the denaturation temperature of the tissue is about 64, 65, 66, 67, 68, or 69° C. as measured by a Hydrothermal Isometric Tension (HIT) test.

### Claim 56

(canceled)

### Claim 57

A kit, comprising a hyptotonic solution, a first surfactant solution, a nuclease enzyme solution, a cleaning solution, a bioburden reducing agent solution, and instructions for contacting a human tissue with each of the solutions.

### Claim 58

(canceled)

### Claim 59

(canceled)

### Claim 60

(canceled)

### Claim 61

(canceled)

### Claim 62

(canceled)