Self-assembling tissue modules

Abstract

The disclosure relates to a new approach to constructing cellular aggregates in vitro and their use in methods for producing 3D-tissue constructs in a modular way. In particular, the disclosure is directed to a method for in vitro producing a tissue construct comprising: a) combining living cells to form supracellular aggregates using spatial confinement; b) combining two or more of the supracellular aggregates in a mold or on a biomaterial; c) applying conditions that induce self-assembly within the combined supracellular aggregates to obtain the tissue construct; and d) applying conditions that induce tissue morphogenesis in the tissue construct.

What is claimed is: 
     
         1 . A method for in vitro producing a tissue construct comprising:
 a) combining living cells to form supracellular aggregates using spatial confinement;   b) combining two or more of the supracellular aggregates in a mold or on a biomaterial;   c) applying conditions that induce self-assembly within the combined supracellular aggregates to obtain the tissue construct; and   d) applying conditions that induce tissue morphogenesis in the tissue construct.   
     
     
         2 . A method according to  claim 1 , wherein said tissue morphogenesis comprises migration and/or differentiation of cells. 
     
     
         3 . A method according to  claim 1 , wherein said spatial confinement comprises one selected from an array of microwells, Hanging drop method, or microfluidic channels. 
     
     
         4 . A method according to  claim 3 , wherein said spatial confinement comprises an array of microwells, said microwells having an enveloping diameter in the range of 50-500 μm and a depth in the range of 100-1,000 μm. 
     
     
         5 . A method according to  claim 3 , wherein the microwells have a shape that is different from a cylinder. 
     
     
         6 . A method according to  claim 5 , wherein the shape of at least some of the microwells is such that the resulting aggregates can self-assemble according to the lock-and-key principle. 
     
     
         7 . A method according to  claim 1 , wherein 2-500,000 cells per microwell are combined to form a supracellular aggregate, preferably 10-100,000 cells per microwell, more preferably 10-10,000 cells per microwell. 
     
     
         8 . A method according to  claim 1 , wherein the living cells of the same or different cell type are combined to form the supracellular aggregates. 
     
     
         9 . A method according to  claim 1 , wherein the cells are selected from the group consisting of endothelial cells, smooth muscle cells, striated muscle cells, neural cells, connective tissue cells, osteoblasts, osteoclasts, chondrocytes, hepatocytes, cardiomyocytes, myocytes, Schwann cells, urothelial cells, parenchymal cells, epithelial cells, exocrine secretory epithelial cells, epithelial absorptive cells, keratinizing epithelial cells, extracellular matrix secretion cells, or undifferentiated cells, such as embryonic cells, progenitor cells, (mesenchymal) stem cells, bone marrow cells, satellite cells, fibroblasts, and other precursor cells. 
     
     
         10 . A method according to  claim 1 , wherein the supracellular aggregates have a mean particle size of 20-400 μm as measured by light microscopy. 
     
     
         11 . A method according to  claim 1 , wherein the biomaterial is selected from the group consisting of ceramics, (bio)glasses, polymeric materials (biodegradable or non-biodegradable), and metals. 
     
     
         12 . A method according to  claim 3 , wherein the array of microwells is prepared by microchip technology, hot embossing, selective laser sintering, solid free-form fabrication, and phase separation micro-molding. 
     
     
         13 . A method according to  claim 3 , wherein the array of microwells comprises at least two microwells having a substantially different size and/or shape. 
     
     
         14 . A method according to  claim 3 , wherein the microwells are made of agarose, PEG (polyethyleneglycol) or PDMS. 
     
     
         15 . A method according to  claim 3 , wherein the microwell surface is coated with one or more compounds capable of reducing and/or preventing cellular adhesion, such as PEG, BSA, collagen and/or fibronectin. 
     
     
         16 . A method according to  claim 1 , wherein the living cells are combined in the presence of fibronectin and/or collagen. 
     
     
         17 . A method according to  claim 1 , wherein the surface properties, the magnetic charge, and/or the electrical charge of the supracellular aggregates are modified before combining two or more of the supracellular aggregates. 
     
     
         18 . A method according to  claim 1 , wherein the supracellular aggregates are combined in a moving liquid, for instance, a microfluidic chamber and channel. 
     
     
         19 . A method according to  claim 1 , wherein the supracellular aggregates are combined in a well having an enveloping diameter of at least 500 μm. 
     
     
         20 . A method according to  claim 1 , wherein the conditions in step c) comprise one or more selected from mechanical constraints, compression, shaking, electrical fields, magnetic fields, and gradients of morphogens or growth factors. 
     
     
         21 . A method according to  claim 1 , wherein step d) comprises compaction of the cellular aggregates, preferably by applying geometrical constraints to the tissue construct. 
     
     
         22 . A method according to  claim 1 , wherein in step a) or b) the living cells or the supracellular aggregate is combined with an object and/or wherein in step c) or d) the tissue construct is combined with an object, preferably a biodegradable object and/or a metallic object, to induce a local response.