Biomatrix scaffolds for industrial scale dispersal

• US 9,102,913 B2
• Filed: 07/01/2011
• Issued: 08/11/2015
• Est. Priority Date: 07/02/2010
• Status: Active Grant

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: Phoenixsongs Biologicals, University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Roach, Marsha Lynn, Malavarca, Richard Harold, Wang, Yunfang, Reid, Lola Cynthia McAdams
• Primary Examiner(s): Fox, Allison
• Assistant Examiner(s): Paguio Frising, Michelle F
• Application Number:
  US13/807,260
• Publication Number:
  US 20130137176A1
• Time in Patent Office: 1,502 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61L 2430/28 for liver reconstruction
• CPC Class Codes: A61L 27/24 Collagen
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: 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
• 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: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2501/20 Cytokines; Chemokines
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/14 from hepatocytes
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• 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
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0693 Tumour cells; Cancer cells
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention provides biomatrix scaffolds for industrial scale dispersal.

Claims

1. A method of producing a biomatrix scaffold from biological tissue for industrial scale dispersal onto culture apparatus, comprising:

(a) perfusing or homogenizing biological tissue with a buffer comprising a salt concentration from about 3.5 M NaCl to about 4.5 M NaCl, wherein the tissue is selected from the group consisting of liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart and intestines;

then

(b) perfusing the biological tissue or extracting the homogenate of step (a) with a delipidating buffer comprising lipases and detergents in a serum free first medium, having a neutral pH and an osmolality from about 250 mOsm/kg to about 350 mOsm/kg, wherein the detergents do not comprise sodium dodecyl sulfate or TritonX-100;

then

(c) perfusing the tissue or extracting the homogenate of step (b) with a buffer at a neutral pH and comprising a salt concentration from about 2.0 M NaCl to about 5.0 M NaCl, the concentration chosen to keep insoluble collagens identified in the biological tissue;

then

(d) perfusing the tissue or extracting the homogenate of step (c) with RNase and DNase in a buffer; and

then

(e) rinsing the tissue or homogenate of step (d) with a second medium that is at neutral pH, is serum-free and has an osmolality from about 250 mOsm/kg to about 350 mOsm/kg, thereby producing an intact or homogenized biomatrix scaffold from the biological tissue, said biomatrix scaffold comprising at least 95% of the collagens and most collagen-associated matrix components and matrix bound growth factors, hormones and cytokines of the biological tissue;

(f) diluting the biomatrix scaffold in basal medium;

(g) freezing the biomatrix scaffold of (f);

(h) pulverizing the biomatrix scaffold of (g) by cryogenic grinding into biomatrix particles ranging in size from about 1 nm to about 100 μ

m;

(i) thawing the biomatrix particles of (h) in suspension in basal medium; and

(j) dispersing the biomatrix particles of step (i) onto a culture apparatus, thereby producing a biomatrix scaffold from biological tissue for industrial scale dispersal onto culture apparatus.

2. The method of claim 1, further comprising the step of sterilizing the biomatrix scaffold.

3. The method of claim 2, wherein the sterilizing of step is carried out by gamma irradiation.

4. The method of claim 1, wherein the biomatrix scaffold of (f) is diluted at about a 1:

6 ratio in the basal medium.

5. The method of claim 1, wherein the biomatrix particles of (i) are diluted at about a 1:

24 ratio in the basal medium.

6. The method of claim 1, wherein the detergents are selected from the group consisting of salts of deoxycholic acid, 1-heptanesulfonic acid, N-laurylsarcosine, lauryl sulfate, 1-octane sulfonic acid and taurocholic acid;

benzalkonium chloride;

cetylpyridimum;

methylbenzethonium chloride;

decamethonium bromide;

alkyl betaines;

alkyl amidoalkyl betaines;

N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;

phosphatidylcholine;

n-decyl α

-D-glucopyranoside;

n-decyl β

-D-maltoside;

tritons;

Nonidet-P-40;

Poloxamer 188;

sodium lauryl sulfate; and

sodium deoxycholate.

7. The method of claim 1, wherein the lipases are phospholipases.

8. The method of claim 1, wherein the first medium is a basal medium selected from the group consisting of RPMI 1640, DME/F12, DME, F12, Waymouth'"'"'s, and William'"'"'s medium.

9. The method of claim 1, wherein the second medium comprises at least one of the constituents present in interstitial fluid.

10. The method of claim 1, wherein the delipidating buffer of step (b) comprises front about 20 units/L to about 50 units/L phospholipase A2 and about 1% sodium deoxycholate in the first medium.

11. The method of claim 1, wherein the salt concentration of the buffer of step (c) is from about 3.4 M NaCl to about 3.5 M NaCl when used for scaffold preparation from an adult liver and is from about 4.0 M NaCl to about 4.5 M NaCl when used for scaffold preparation from a fetal liver.

12. The method of claim 1, wherein the buffer of step (c) further comprises a protease inhibitor.

13. The method of claim 12, wherein the protease inhibitor is soybean trypsin inhibitor.

14. The method of claim 1, wherein the buffer of step (d) further comprises a protease inhibitor.

15. The method of claim 13, wherein the protease inhibitor is soybean trypsin inhibitor.

16. The method of claim 1, wherein all media and buffers of steps (a) through (e) are free of a detectable amount of an enzyme that degrades extracellular matrix components.

17. The method of claim 1, wherein the biological tissue is selected from the group consisting of liver tissue, lung tissue, pancreatic tissue, thyroid tissue, intestinal tissue, skin tissue, blood vessel tissue, bladder tissue, heart tissue and kidney tissue.

18. The method of claim 1, wherein the biological tissue is from a mammal.