Biomatrix scaffolds

Abstract

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 - 46 . (canceled) 
     
     
         47 . A method of producing a biomatrix scaffold from biological tissue, comprising the steps of:
 a) perfusing the biological tissue or homogenizing the biological tissue with a first medium, 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/or detergents in said first medium; 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.   
     
     
         48 . The method of  claim 47 , wherein the second medium comprises at least one of the constituents present in interstitial fluid. 
     
     
         49 . The method of  claim 47 , wherein the delipidating buffer comprises from about 20 units/L to about 50 units/L phospholipase A2 and about 1% sodium deoxycholate in the first medium. 
     
     
         50 . The method of  claim 47 , 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. 
     
     
         51 . The method of  claim 47 , wherein the buffer of step (c) further comprises a protease inhibitor. 
     
     
         52 . The method of  claim 51 , wherein the protease inhibitor is soybean trypsin inhibitor. 
     
     
         53 . The method of  claim 47 , wherein the buffer of step (d) further comprises a protease inhibitor. 
     
     
         54 . The method of  claim 53 , wherein the protease inhibitor is soybean trypsin inhibitor. 
     
     
         55 . The method of  claim 47 , 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. 
     
     
         56 . A biomatrix scaffold produced by the method of  claim 47 . 
     
     
         57 . A method of producing a cell culture, comprising:
 (a) contacting the biomatrix scaffold of  claim 56  with cell culture medium in a culture apparatus; and   (b) seeding the biomatrix scaffold of step (b) with cells, thereby producing a cell culture.   
     
     
         58 . The method of  claim 57 , wherein the cell culture medium comprises at least one of the constituents present in interstitial fluid, wherein the osmolality of said medium is from about 250 mOsm/kg to about 350 mOsm/kg and wherein said medium is serum free. 
     
     
         59 . The method of  claim 57 , wherein the cells are selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, determined stem cells, perinatal stem cells, amniotic fluid-derived stem cells (AFSCs), mesenchymal stem cells (MSCs) from any source, committed progenitors or adult cells of any tissue type, mature cells, normal cells, diseased cells, tumor cells and any combination thereof. 
     
     
         60 . The method of  claim 57 , wherein the cells are selected from the group consisting of liver cells, parenchymal cells, stellate cells, endothelial cells, hepatocytes, cholangiocytes, biliary tree cells that are not cholangiocytes and pancreatic cells. 
     
     
         61 . A method of producing a graft for transplantation into a host animal, comprising:
 (a) contacting the biomatrix scaffold of  claim 56  with cell culture medium in a culture apparatus;   (b) seeding the biomatrix scaffold with cells;   (c) maintaining the biomatrix scaffold under culture conditions; and   (d) establishing a population of the cells on the biomatrix scaffold,   
       thereby producing a tumor graft for transplantation into the host animal. 
     
     
         62 . The method of  claim 61 , further comprising the step of transplanting the graft into the host animal. 
     
     
         63 . The method of  claim 61 , wherein the graft is syngeneic, allogeneic or xenogenic to the host animal. 
     
     
         64 . A method of producing a protein of interest in cells cultured on a biomatrix scaffold, comprising:
 (a) contacting the biomatrix scaffold of  claim 56  with cell culture medium in a culture apparatus;   (b) seeding the biomatrix scaffold with cells that produce the protein of interest;   (c) maintaining the cells on the biomatrix scaffold under culture conditions; and   (d) collecting the protein of interest produced by the cells,   
       thereby producing a protein of interest in cells cultured on a biomatrix scaffold. 
     
     
         65 . The method of  claim 56 , further comprising the step of purifying the protein of interest.