Cells derived from adipose tissue and methods of isolating and using the same

Abstract

Adipose tissue-derived stromal cells and methods of isolating and using the same. In at least one embodiment of isolated adipose tissue-derived stromal cells of the present disclosure, the cells are isolated by performing adipose tissue resection or suction on a mammalian patient, dissecting tissue obtained from said tissue resection or suction and dissociating said tissue into a cell suspension, removing adipocytes from the cell suspension, culturing the adipocyte-depleted cell suspension in EGM-2-MV media, and isolating adipose tissue-derived stromal cells secreting vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and granulocyte-colony stimulating factor (G-CSF).

1 . A method for isolating adipose derived stromal cells, comprising:
 a) dissociating subcutaneous adipose tissue isolated from a mammal into a cell suspension;   b) removing adipocytes from said cell suspension, resulting in an adipocyte-depleted cell suspension; and   c) culturing the adipocyte-depleted cell suspension in a media containing growth factors VEGF, bFGF, EGF, and IGF, such that a mixed population of cells comprising a first population of CD34+/VE-cadherin− cells and a second population of CD34+/VE-cadherin+ endothelial cells are obtained.   
     
     
         2 . The method of  claim 1 , wherein the first population and the second population secrete vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and granulocyte-colony stimulating factor (G-CSF). 
     
     
         3 . The method of  claim 1 , further comprising culturing the cells of step c) in a culture medium of interest. 
     
     
         4 . The method of  claim 1 , further comprising culturing the cells of step c) in DMEM and 10% fetal bovine serum. 
     
     
         5 . The method of  claim 1 , wherein said cells are exposed to a receptor ligand cocktail, said ligands being selected from the group consisting of at least one of VEGF, LIF, bFGF, IGF1, IGF2, HGF, cardiotrophin, myotrophin, nitric oxide synthase 1, nitric oxide synthase 2, nitric oxide synthase 3, tumor necrosis factor alpha, tumor necrosis factor beta, fibroblast growth factor, pleotrophin, endothelin, and angiopoietin. 
     
     
         6 . The method of  claim 1 , further comprising introduction of an exogenous nucleic acid molecule encoding a protein of interest into the cells of step c). 
     
     
         7 . The method of  claim 1 , further comprising exposing the cell suspension to red cell lysis buffer. 
     
     
         8 . The method of  claim 1 , wherein the adipose derived stromal cells do not secrete detectable levels of basic fibroblast growth factor (bFGF). 
     
     
         9 . The method of  claim 1 , further comprising the step of culturing the first population and the second population in a growth-factor free basal medium. 
     
     
         10 . The method of  claim 9 , wherein the growth-factor free basal medium is EBM 2. 
     
     
         11 . The method of  claim 1 , wherein the culturing step is performed on matrigel. 
     
     
         12 . The method of  claim 1 , wherein the media further comprises 5-azacytidine. 
     
     
         13 . The method of  claim 1 , wherein the dissociating step is performed using agitation, sonic energy, or thermal energy. 
     
     
         14 . The method of  claim 1 , wherein the dissociating step is performed using collagenase, dispase, or trypsin.