Integrated organ and tissue printing methods, system and apparatus

Abstract

A method of making an organ or tissue comprises: (a) providing a first dispenser containing a structural support polymer and a second dispenser containing a live cell-containing composition; (b) depositing a layer on said support from said first and second dispenser, said layer comprising a structural support polymer and said cell-containing composition; and then (c) iteratively repeating said depositing step a plurality of times to form a plurality of layers one on another, with separate and discrete regions in each of said layers comprising one or the other of said support polymer or said cell-containing composition, to thereby produce provide a composite three dimensional structure containing both structural support regions and cell-containing regions. Apparatus for carrying out the method and composite products produced by the method are also described.

1 . A method of printing an organ or tissue in a plurality of layers, comprising:
 (a) providing a first dispenser containing a structural support polymer and a second dispenser containing a live cell-containing composition;   (b) depositing a layer on said support from said first and second dispenser, said layer comprising a structural support polymer and said cell-containing composition; and then   (c) iteratively repeating said depositing step a plurality of times to form a plurality of layers one on another, with separate and discrete regions in each of said layers separately deposited by one or the other of said syringes, to thereby produce provide a composite three dimensional structure containing both structural support regions and cell-containing regions.   
     
     
         2 . The method of  claim 1 , wherein said structural support polymer is deposited in a plurality of regions in a plurality of adjacent layers at least partially overlapping one another to form a supporting matrix interconnecting said plurality of adjacent layers. 
     
     
         3 . The method of  claim 1 , wherein said cell containing composition comprises a hydrogel. 
     
     
         4 . The method of  claim 3 , wherein said hydrogel is crosslinked after said depositing step. 
     
     
         5 . The method of  claim 1 , further comprising:
 chilling said cell-containing composition during said depositing step; and   heating said structural support polymer during said depositing step.   
     
     
         6 . The method of  claim 1 , further comprising:
 providing a third dispenser containing a sacrificial polymer different from said structural support polymer,   said depositing step further comprising depositing said sacrificial polymer in at least some of said layers immediately adjacent and in contact with said cell-containing composition.   
     
     
         7 . The method of  claim 6 , wherein said depositing step comprises depositing said sacrificial polymer in a plurality of adjacent layers at least partially overlapping one another, to form a sacrificial network interconnecting a plurality of adjacent layers. 
     
     
         8 . The method of  claim 6 , wherein said sacrificial polymer comprises a hydrogel that is a liquid at room temperature. 
     
     
         9 . The method of  claim 8 , further comprising chilling said hydrogel during said dispensing step to a temperature at which said hydrogel forms a stable gel and retains shape imparted during said depositing step until the subsequent removal thereof. 
     
     
         10 . The method of  claim 6 , wherein a plurality of deposited layers has an axis of orientation; and
 at least some of said layers are deposited at an axis of orientation sufficient different from the axis of orientation of others of said layers to enhance the structural rigidity of said composite.   
     
     
         11 . The method of  claim 6 , further comprising the step of removing said sacrificial polymer. 
     
     
         12 . The method of  claim 10 , wherein said removing step is carried out by washing said composite with an aqueous solution. 
     
     
         13 . The method of  claim 1 , wherein said iteratively repeating is carried out from 5 to 5000 times, and each of said layers is from 5 to 1000 um thick. 
     
     
         14 . The method of  claim 1 , wherein said structural support polymer is dispensed at a temperature of at least about 25 degrees Celsius. 
     
     
         15 . The method of  claim 1 , wherein said cell-containing composition is dispensed at a temperature of not more than about 20 degrees Celsius. 
     
     
         16 . The method of  claims 1 , wherein said cell-containing composition is dispensed at a temperature at least 3 degrees Celsius less than the temperature at which said structural support polymer is dispensed. 
     
     
         17 . An organ and tissue printing system, comprising:
 (a) a support stage for supporting an organ to be printed thereon;   (b) a positioning unit operatively associated with said support stage;   (c) at least a pair of syringes connected to said positioning unit and positioned above said support stage, each of said syringes comprising a nozzle,   (d) a syringe activator operatively associated with each of said pair of syringes for independently and selectively dispensing the contents thereof;   (d) a temperature regulator operatively associated with at least one of said syringes for independently regulating the temperature of at least one of said pair of syringes; and   (e) a positioning controller operatively associated with said positioning unit, said support stage, or both said positioning unit and said support stage, for adjusting in three dimensions the position of said support stage in relation to said at least a pair of syringes during printing of an organ or tissue on said support stage.   
     
     
         18 . The apparatus of  claim 17 , wherein said temperature regulator comprises a thermal element operatively associated with said syringe. 
     
     
         19 . The apparatus of  claim 18 , wherein said temperature regulator further comprises:
 a temperature sensor operatively associated with said syringe; and   a temperature controller operatively associated with said temperature sensor and said thermal element for independently controlling the temperature of at least one of said pair of syringes.   
     
     
         20 . The apparatus of  claim 17 , wherein a first member of said pair of syringes has a heater operatively associated with said syringe. 
     
     
         21 . The apparatus of  claim 17 , wherein a second member of said pair of syringes has a cooler or heat exchanger operatively associated with said syringe reservoir. 
     
     
         22 . The apparatus of  claim 17 , further comprising a third syringe, wherein said third syringe has a cooler or heat exchanger operatively therewith (e.g., for cooling sacrificial hydrogel dispensed by said syringe). 
     
     
         23 . The apparatus of  claims 17 , wherein said positioning unit comprises a 3-axis stage system. 
     
     
         24 . The apparatus of  claim 17 , further comprising a CAD/CAM system operatively associated with said positioning controller and configured for adjusting in three dimensions in a predetermined pattern the position of said support stage in relation to said at least a pair of syringes during printing of an organ or tissue in a plurality of layers on said support stage. 
     
     
         25 . The apparatus of  claim 24 , said CAD/CAM system operatively associated with said syringe activators and configured to selectively deposit the contents of each of said syringes in independent locations in a predetermined pattern in each of said layers. 
     
     
         26 . The apparatus of  claims 17 , wherein each of said syringes is activated by pneumatic pressure, hydraulic pressure, mechanical pressure or pumping;
 and optionally, wherein said syringe for dispensing a cell support composition is an inkjet printer head or a solid-freeform fabrication syringe.   
     
     
         27 . The apparatus of  claims 17 , wherein each of said nozzles has an inner diameter of from 5 um to 1000 um 
     
     
         28 . The apparatus of  claims 17 , wherein each of said syringes further comprises a reservoir, with said reservoir optionally but preferably mounted on said positioning unit. 
     
     
         29 . A three dimensional tissue or organ construct comprising:
 a solid support in the form of a three dimensional scaffold of a structural support polymer in combination with   a live cell-containing composition in separate and discrete regions therein.   
     
     
         30 . A construct according to  claim 29 , said construct further containing openings formed therein. 
     
     
         31 . The construct of  claim 30 , wherein said openings comprise pores, cavities, chambers, vessel lumens or channels formed therein. 
     
     
         32 . The construct of  claim 30 , wherein said scaffold comprises a plurality of layers, and said openings are formed within said layers, between said layers, or both within and between said layers. 
     
     
         33 . A construct according to  claim 29 , wherein said live cell containing composition comprises a hydrogel. 
     
     
         34 . The construct of  claim 29 , wherein said composite comprises 2 or more different tissues, said two or more different tissues sharing a common, integrally formed and connected, solid support scaffold. 
     
     
         35 . The construct of  claim 29 , wherein said composite comprises a plurality of layers, each of said layers having an axis of orientation, with at least some of said layers having an axis of orientation sufficient different from the axis of orientation of other of said layers to enhance the structural rigidity of said scaffold.