Self-assembling multicellular bodies and methods of producing a three-dimensional biological structure using the same

Abstract

Structures and methods for tissue engineering include a multicellular body including a plurality of living cells. A plurality of multicellular bodies can be arranged in a pattern and allowed to fuse to form an engineered tissue. The arrangement can include filler bodies including a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies and that is resistant to adherence of cells to it. Three-dimensional constructs can be assembled by printing or otherwise stacking the multicellular bodies and filler bodies such that there is direct contact between adjoining multicellular bodies, suitably along a contact area that has a substantial length. The direct contact between the multicellular bodies promotes efficient and reliable fusion. The increased contact area between adjoining multicellular bodies also promotes efficient and reliable fusion. Methods of producing multicellular bodies having characteristics that facilitate assembly of the three-dimensional constructs are also provided.

1 - 174 . (canceled) 
     
     
         175 . A method of producing a multicellular body comprising a plurality of living cells, the method comprising:
 shaping a cell paste comprising a plurality of living cells in a device that holds the cell paste in a three-dimensional shape; and   incubating the shaped cell paste in a controlled environment while it is held in said three-dimensional shape to produce a cohesive body that is capable of supporting itself on a flat surface.   
     
     
         176 . The method of  claim 175 , further comprising producing the cell paste by mixing a plurality of living cells with a tissue culture medium. 
     
     
         177 . The method of  claim 176 , wherein the producing comprises compacting the living cells. 
     
     
         178 . The method of  claim 177 , wherein the compacting comprises centrifuging a cell suspension comprising the plurality of living cells. 
     
     
         179 . The method of  claim 175 , wherein the shaping comprises retaining the cell paste in a first shaping device to allow the cells to partially cohere to one another in the first shaping device, transferring the partially cohered cell paste to a second shaping device, and retaining the partially cohered cell paste in the second shaping device to form the multicellular body. 
     
     
         180 . The method of  claim 179 , wherein the first shaping device comprises a capillary pipette and the second shaping device comprises a device that allows nutrients and oxygen to be supplied to the cells while they are retained in the second shaping device. 
     
     
         181 . The method of  claim 179 , wherein the second shaping device comprises a mold made of a biocompatible material which resists migration and ingrowth of cells into it and resists adherence of cells to it. 
     
     
         182 . The method of  claim 181 , wherein the biocompatible material is selected from the group consisting of polytetrafluoroethylene (PTFE), stainless steel, hyaluronic acid, agarose, agar, and polyethylene glycol. 
     
     
         183 . The method of  claim 175 , further comprising producing the cell paste by mixing a plurality of living cells with a solution comprising about 10 to about 30 percent gelatin to form a cell suspension and compacting the cell suspension to form the cell paste. 
     
     
         184 . The method of  claim 175 , further comprising producing the cell paste by mixing a plurality of living cells with a solution comprising about 10-80 mg/ml fibrinogen to form a cell suspension and compacting the cell suspension to form the cell paste. 
     
     
         185 . The method of  claim 175 , wherein the cell paste consists essentially of a plurality of living cells of a single cell type. 
     
     
         186 . The method of  claim 175 , wherein the cell paste includes living cells of a first cell type and at least about 90 percent of the cells are cells of the first cell type. 
     
     
         187 . The method of  claim 175 , wherein the cell paste comprises a plurality of living cells of a first cell type and a plurality of living cells of a second cell type, the second cell type being different from the first cell type. 
     
     
         188 . The method of  claim 187 , wherein the cell paste comprises a plurality of endothelial cells and a plurality of smooth muscle cells. 
     
     
         189 . The method of  claim 175 , wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length of at least about 1000 microns. 
     
     
         190 . The method of  claim 175 , wherein shaping the cell paste comprises shaping the cell paste into an elongate shape having a length of less than about 30 centimeters. 
     
     
         191 . The method of  claim 175 , wherein shaping the cell paste comprises shaping the cell paste into a substantially cylindrical shape having a substantially circular cross-section. 
     
     
         192 . The method of  claim 175 , wherein shaping the cell paste comprises shaping a cell paste comprising a plurality of living cells and one or more extracellular matrix components or one or more derivatives of one or more extracellular matrix components. 
     
     
         193 . The method of  claim 192 , wherein shaping the cell paste comprises shaping a cell paste comprising a plurality of living cells and gelatin. 
     
     
         194 . The method of  192 , wherein shaping the cell paste comprises shaping a cell paste comprising a plurality of living cells and fibrinogen.