Biomatrix scaffolds
The present invention provides biomatrix scaffolds, a tissue extract enriched for extracellular matrix components and bound growth factors, cytokines and hormones, and methods of making and using same.
1. A method of producing a biomatrix scaffold from biological tissue, comprising:
a) perfusing or homogenizing biological tissue with a first basal medium, 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, and wherein the osmolality of said first medium is from about 250 mOsm/kg to about 350 mOsm/kg and said first medium is serum free and at neutral pH; then
b) perfusing the biological tissue or extracting the homogenate of step (a) with a delipidating buffer comprising lipases and detergents in said first medium, wherein the detergents do not comprise sodium dodecyl sulfate or Triton X-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 2.0M NaCl to about 5.0M, 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 osomolality 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.
2. 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; cetylpyridinium; 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 α-D-maltopyranoside; n-dodecyl β-D-maltoside; n-octyl β-D-glucopyranoside; sorbitan esters; n-tetradecyl β-D-maltoside; tritons; Nonidet-P-40; Poloxamer 188; sodium lauryl sulfate; and sodium deoxycholate.
3. The method of claim 1 , wherein the lipases are phospholipases.
4. The method of claim 1 , wherein the basal medium is selected from the group consisting of RPMI 1640, DME/F12, DME, F12, Waymouth's, and William's medium.
5. The method of claim 1 , wherein the second medium comprises at least one of the constituents present in interstitial fluid.
6. The method of claim 1 , wherein the delipidating buffer of step (b) comprises from about 20 units/L to about 50 units/L, phospholipase A2 and about 1% sodium deoxycholate in the first medium.
7. The method of claim 1 , wherein the salt concentration of the buffer of step (c) is from about 3.4M NaCl to about 3.5M NaCl when used for scaffold preparation from an adult liver and is from about 4.0M NaCl to about 4.5M NaCl when used for scaffold preparation from a fetal liver.
8. The method of claim 1 , wherein the buffer of step (c) further comprises a protease inhibitor.
9. The method of claim 8 , wherein the protease inhibitor is soybean trypsin inhibitor.
10. The method of claim 1 , wherein the buffer of step (d) fur comprises a protease inhibitor.
11. The method of claim 10 , wherein the protease inhibitor is soybean trypsin inhibitor.
12. The method 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.
13. The method of claim 1 further comprising sterilizing the biomatrix scaffold.
14. The method of claim 13 , wherein the sterilizing step comprises gamma irradiation of the biomatrix scaffold.
15. 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 issue.
16. The method of claim 1 , wherein the biological tissue is from a mammal.