Method for obtaining mesenchymal stem cells

Abstract

The invention relates to the field of cell biology. In detail it relates to the obtainment of mesenchymal stem cells from human tissue. This invention might be applicable in healing within the frame of the treatment of several diseases. Due to the invention it will be possible to obtain mesenchymal stem cells from human tissue with high homogeneity of the cell suspension, since the used method for obtaining mesenchymal stem cells from human tissue comprises the crushing and enzymatical treatment of the tissue with collagenase solution in Eagle medium in the Dulbecco modification, removal of erythrocytes by the aid of the lysis solution and subsequent filtration of the prepared suspension; as human tissue fat tissue or decidual or amniotic placenta membrane or chorion placenta stroma is used, whereas the filtration is performed sequentially by the use of filters comprising a pore size of 100 μm and 10 μm. In the enzymatical treatment of the fat tissue, of the decidual or amniotic placenta membrane collagenase of the type I is used, and in the enzymatical treatment of the chorion placenta stroma collagenase of the type IV is used.

1 . Method for obtaining and/or isolating mesenchymal stem cells from human tissue, comprising the following steps: 
 providing human tissue,    enzymatical and mechanical treatment of said human tissue for obtaining a cell suspension,    removal of erythrocytes from said suspension, and    filtration of said suspension for obtaining mesenchymal stem cells,    wherein in step (a) fat tissue or/and placenta tissue is used as human tissue.    
   
   
       2 . The method according to  claim 1 , wherein in step (a) the decidual membrane is used as human tissue.  
   
   
       3 . The method according to  claim 1 , wherein in step (a) the amniotic membrane is used as human tissue.  
   
   
       4 . The method according to  claim 1 , wherein in step (a) chorion stroma is used as human tissue.  
   
   
       5 . The method according to  claim 1 , wherein in step (d) at least one filter is used comprising filter pores which are of a size suitable for removing cell residues and/or debris.  
   
   
       6 . The method according to  claim 1 , wherein in step (d) said suspension is sequentially filtered by the use of filters comprising a pore size of about 50 μm to 150 μm, and of about 5 μm to 15 μm.  
   
   
       7 . The method according to  claim 1 , wherein in step (d) said suspension is sequentially filtered by the use of filters comprising a pore size of about 100 μm, and of about 110 μm.  
   
   
       8 . The method according to  claim 1 , wherein in step (d) said suspension is firstly filtered through a first filter comprising a pore size of about 50 μm to 150 μm, and secondly filtered through a second filter comprising a pore size of about 5 μm to 15 μm.  
   
   
       9 . The method according to  claim 1 , wherein in step (d) said suspension is firstly filtered through a first filter comprising a pore size of about 100 μm, and secondly filtered through a second filter comprising a pore size of about 10 μm.  
   
   
       10 . The method according to  claim 1 , wherein in step (b) for said enzymatic treatment collagenase is used.  
   
   
       11 . The method according to  claim 1 , wherein in step (b) collagenase of type I is used in case where decidual membrane, amniotic membrane, and/or fat tissue is used as human tissue, and collagenase of type IV is used in case where chorion stroma is used as human tissue.  
   
   
       12 . The method according to  claim 1 , wherein in step (b) for said enzymatic treatment collagenase is provided in Eagle medium in the Dulbecco modification.  
   
   
       13 . The method according to  claim 1 , wherein in step (b) for said enzymatic treatment collagenase is provided in a final concentration of about 0.075%.  
   
   
       14 . The method according to  claim 1 , wherein in step (c) said removal of erythrocytes is performed by the aid of buffer capable of lysing said erythrocytes.  
   
   
       15 . Method for obtaining mesenchymal stem cells from human tissue, comprising the crushing and enzymatical treatment of said tissue with collagenase solution in Eagle medium in the Dulbecco modification, removal of erythrocytes by the aid of the lysis solution and subsequent filtration of the prepared suspension, wherein as human tissue fat tissue or decidual membrane or amniotic membrane or chorion placenta stroma is used, whereas the filtration is performed sequentially by the use of filters comprising a pore size of 100 μm and 10 μm.  
   
   
       16 . Method according to  claim 1 , wherein in the enzymatic treatment of the fat tissue, the decidual or amniotic placenta membrane collagenase of type I is used, and in the enzymatic treatment of chorion placenta stroma collagenase of type IV is used.