Primary culture method

Abstract

A primary culture method in which cells contained in a tissue collected from a living body are primary cultured in vitro, in which the cells in the tissue collected from the living body are seeded and cultured on a top surface of a cell structure containing cells constituting a stroma and composed of a single layer or two or more cell layers laminated in the thickness direction.

What is claimed is: 
     
         1 . A primary culture method comprising:
 forming a cell structure being composed of a single layer of first cells constituting the stroma or two or more cell layers of the first cells laminated in the thickness direction, which comprises:
 obtaining a mixture by mixing the first cells, a strong polyelectrolyte, and an extracellular matrix component in a cationic buffer solution; 
 seeding the mixture in a cell culture vessel; and 
 obtaining the cell structure in which first cells are laminated in multiple layers in the cell culture vessel after seeding the mixture, and 
   seeding second cells on and in direct contact with a top surface of the cell structure, the second cells recovered a tissue collected from a living body.   
     
     
         2 . A primary culture method according to  claim 1 , wherein
 the second cells are cells recovered from a fragmented material of the tissue collected from the aforementioned living body or cells recovered from an enzyme-treated product of the tissue collected from the living body.   
     
     
         3 . A primary culture method according to  claim 1 , wherein
 the cell structure include one or more members selected from the group consisting of fibroblasts, pericytes, endothelial cells, and immune cells as the second cells.   
     
     
         4 . A primary culture method according to  claim 3 , wherein
 the endothelial cells are one or more members selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells.   
     
     
         5 . A primary culture method according to  claim 1 , wherein
 the cell structure include a vascular network structure.   
     
     
         6 . A primary culture method according to  claim 1 , wherein
 the thickness of the cell structure is 5 μm or more.   
     
     
         7 . A primary culture method according to  claim 1 , wherein
 the thickness of the cell structure is 150 μm or more.   
     
     
         8 . A primary culture method according to  claim 1 , wherein
 the tissue collected from the living body contains a tumor tissue.   
     
     
         9 . A primary culture method according to  claim 8 , further comprising:
 fragmenting the tissue collected from the living body; and   sorting the second cells which are cancer cells from an obtained fragmented material and culturing the second cells on the top surface of the cell structure.   
     
     
         10 . A primary culture method according to  claim 9 , wherein
 sorting the second cells from the fragmented material by one or more techniques selected from the group consisting of flow cytometry, magnetic separation, dielectrophoresis, size fractionation, and density gradient fractionation.   
     
     
         11 . A primary cultured cell-containing cell structure comprising:
 a stromal cell layer comprising first cells constituting a stroma, a strong polyelectrolyte and an extracellular matrix component, the stromal cell layer comprising a single layer or two or more cell layers laminated in the thickness direction; and   a cell layer formed from second cells recovered from a tissue collected from a living body on a top surface of the stromal cell layer, wherein   a thickness of the stromal cell layer is 150 μm or more.   
     
     
         12 . A primary cultured cell-containing cell structure according to  claim 11 , wherein
 the second cells are cells recovered from a fragmented material of the tissue collected from the aforementioned living body or cells recovered from an enzyme-treated product of the tissue collected from the living body.   
     
     
         13 . A primary cultured cell-containing cell structure according to  claim 11 , wherein
 the cell structure include one or more members selected from the group consisting of fibroblasts, pericytes, endothelial cells, and immune cells as the second cells.   
     
     
         14 . A primary culture method according to  claim 3 , wherein
 the endothelial cells are one or more members selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells.   
     
     
         15 . A primary culture method according to  claim 1 , wherein
 the cell structure include a vascular network structure.   
     
     
         16 . A primary culture method according to  claim 1 , wherein
 the tissue collected from the living body contains a tumor tissue.