Decelluarlized Tissue Engineered Constructs and Tissues

• US 20080248080A1
• Filed: 05/21/2008
• Published: 10/09/2008
• Est. Priority Date: 08/16/2000
• Status: Abandoned Application

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Prabhakar, Vikas, Niklason, Laura, Mitchell, Shannon, Koh, Jennifer
• Application Number:
  US12/124,521
• Publication Number:
  US 20080248080A1
• Time in Patent Office: Days
• US Class Current: 424/423
• CPC Class Codes: A61L 27/3691 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

New methods for producing tissue engineered constructs and engineered native tissues are disclosed. The methods include producing a tissue engineered construct by growing cells in vitro on a substrate and then decellularizing the construct to produce a decellularized construct consisting largely of extracellular matrix components. The construct can be used immediately or stored until needed. The decellularized construct can be used for further tissue engineering, which may include seeding the construct with cells obtained from the intended recipient of the construct. During any of the growth phases required for production of the construct, the developing construct may be subjected to various tissue engineering steps such as application of mechanical stimuli including pulsatile forces. The methods also include producing an engineered native tissue by harvesting tissue from an animal or human, performing one or more tissue engineering steps on the tissue, and subjecting the tissue to decellularization. The decellularized, engineered native tissue may then be subjected to further tissue engineering steps.

Claims

1. A method for preparing a construct for use as a tissue engineering scaffold or for implanting into a human comprising:

culturing and maintaining human smooth muscle cells, endothelial cells, or fibroblast cells under conditions suitable for growth of the cells, whereby a proteinaceous extracellular matrix surrounding said cells is formed; and

,

decellularizing the proteinaceous extracellular matrix,

thereby forming a tissue engineered construct comprising a 3-dimensional proteinaceous extracellular matrix.

2. The method of claim 1 further comprising:

implanting the tissue engineered construct into a human.

3. The method of claim 2 further comprising:

testing serum concentrations in the human of IgG and IgM antibodies against antigens present in extracts made from the proteinaceous extracellular matrix surrounding said cells which has not been decellularized.

4. The method of claim 1 further comprising:

testing the tissue engineered construct comprising a 3-dimensional proteinaceous extracellular matrix for reduced immunogenicity by histopathologic analysis of tissue samples obtained from rabbits implanted with said tissue engineered construct compared to the proteinaceous extracellular matrix surrounding said cells that have not been decellularized.

5. A method for preparing a construct for use as a tissue engineering scaffold or for implanting into a human comprising:

maintaining human smooth muscle, endothelial or fibroblast cells under conditions suitable for growth of the cells, whereby a proteinaceous extracellular matrix surrounding said cells is formed, and

decellularizing said proteinaceous extracellular matrix.

6. A method of treating a tissue-engineered construct comprising a 3-dimensional proteinaceous extracellular matrix, comprising:

decellularizing a tissue-engineered construct comprising a 3-dimensional proteinaceous extracellular matrix, thereby providing a matrix with reduced immunogenicity.

7. The method of claim 6 wherein the matrix is at least 50 microns in its smallest dimension.

8. The method of claim 6 further comprising:

testing the matrix for reduced immunogenicity by assaying IgG or IgM antibodies in serum of a human in whom the matrix has been implanted, wherein the antibodies are against antigens present in extracts made from non-decellularized constructs.

9. The method of claim 6 wherein the step of decellularizing employs a nuclease.

10. The method of claim 6 wherein the step of decellularizing employs a protease inhibitor.

11. The method of claim 6 wherein the step of decellularizing employs freezing and thawing.

12. The method of claim 6 wherein the step of decellularizing employs a phospholipase.

13. The method of claim 6 wherein the step of decellularizing employs a detergent.