Fabrication of vascularized tissue using microfabricated two-dimensional molds

Abstract

A method and materials to create complex vascularized living tissue in three dimensions from a two-dimension microfabricated mold has been developed. The method involved creating a two dimensional surface having a branching structure etched into the surface. The pattern begins with one or more large channels which serially branch into a large array of channels as small as individual capillaries, then converge to one or more large channels. The etched surface serves a template within a mold formed with the etched surface for the circulation of an individual tissue or organ. Living vascular cells are then seeded onto the mold, where they form living vascular channels based on the pattern etched in the mold. Once formed and sustained by their own matrix, the top of the mold is removed. The organ or tissue specific cells are then added to the etched surface, where they attach and proliferate to form a thin, vascularized sheet of tissue. The tissue can then be gently lifted from the mold using techniques such as fluid flow and other supporting material, as necessary. The tissue can then be systematically folded and compacted into a three-dimensional vascularized structure. This structure can then be implanted into animals or patients by directly connecting the blood vessels to flow into and out of the device. Immediate perfusion of oxygenated blood occurs, which allows survival and function of the entire living mass.

1 . A system for creating thin tissue layers comprising: 
 (a) a mold comprising a support surface having a pattern of channels thereon, suitable for attachment and culturing of cells to form lumens within the channels, and suitable for attachment and culturing of a different type of cells in the areas on the surface surrounding the channels.    
   
   
       2 . The mold of  claim 1  wherein the system further comprises 
 (b) connections and means for circulation of culture fluid through the mold for culturing of the cells attached thereto.    
   
   
       3 . The system of  claim 1  wherein the mold has branched channels therein, beginning from one or more inlets, expanding into more channels, and then converging back into one or more outlets.  
   
   
       4 . The system of  claim 1  further comprising pumping means for circulating fluid through the mold.  
   
   
       5 . The system of  claim 1  wherein the mold is formed from a material selected from the group consisting of silicon, glass, natural cell substrates like hydroxyapatite, and polymer.  
   
   
       6 . The system of  claim 1  further comprising a coating on the channels which promotes adhesion and lifting of cells as intact sheets from the mold.  
   
   
       7 . A method for making a mold comprising a support surface having a pattern of channels thereon, suitable for attachment and culturing of cells to form lumens within the channels, and suitable for attachment and culturing of a different type of cells in the areas on the surface surrounding the channels, comprising: 
 (a) selecting a material for forming the support surface from the group consisting of silicon, metals, polymers, and natural cell substrates like hydroxyapatite,    (b) patterning channels in the surface of the material to create lumens which can be seeded with cells to form tubular structures for fluid flow, and    (c) forming the mold so that cells can be seeded within the mold to form a thin layer of tissue surrounding the tubular structures.    
   
   
       8 . The method of  claim 7  wherein the patterning is done by a process selected from the group consisting of micromachining, lithography, etching, or molding.  
   
   
       9 . A method for forming complex tissues comprising 
 (a) selecting a mold having a complex pattern of channels formed into at least one surface into which cells can be seeded;    (b) seeding cells into the channels to form blood vessels or other lumens;    (c) culturing the cells under conditions until they form vessels or lumens;    (d) seeding other type(s) of cells onto or into the mold so that a tissue is formed incorporating the vessels or lumens; and    (e) removing the tissue layer.    
   
   
       10 . The method of  claim 9  further comprising 
 (f) assembling multiple layers of the tissue layers until the desired complex structure is formed.    
   
   
       11 . The method of  claim 10  further comprising 
 (g) implanting into a body the complex structure and anastomizing the vessles with the blood supply or lumens in other organs or tissues within the body.    
   
   
       12 . The method of  claim 11  further comprising applying to the channel a coating on the channels which promotes adhesion and lifting of cells as intact sheets from the mold.  
   
   
       13 . A complex structure comprising multiple layers of tissue and vasculature formed by a method comprising 
 (a) selecting a mold having a complex pattern of channels formed into at least one surface into which cells can be seeded;    (b) seeding cells into the channels to form blood vessels or other lumens;    (c) culturing the cells under conditions until they form vessels or lumens;    (d) seeding other type(s) of cells onto or into the mold so that a tissue is formed incorporating the vessels or lumens; and    (e) removing the tissue layer.    
   
   
       14 . The structure of  claim 13  wherein the vasculature comprises endothelial cells.  
   
   
       15 . The structure of  claim 14  wherein the other cells are selected from the group consisting of parenchymal cells, cells forming cartilage or bone, muscle cells, and nerve cells.  
   
   
       16 . The structure of  claim 15  wherein the parenchymal cells are derived from organs selected from the group consisting of heart, liver, pancreas, intestine, brain, kidney, reproductive tissues and lung.