Additive Manufacturing of Functional Myocardial Tissue

Abstract

Methods fabricate an endothelialized myocardium usable for screening of a drug. A microfibrous hydrogel scaffold is manufactured with additive manufacturing that concurrently bioprints endothelial cells directly within the microfibrous hydrogel scaffold. A bioink is bioprinted into an arrangement of one or more microfibers. The bioink includes at least one crosslinking component and suspended endothelial cells. The crosslinking component or components are crosslinked to yield the microfibrous hydrogel scaffold having the endothelial cells embedded directly within. The microfibrous hydrogel scaffold is seeded with cardiomyocytes to yield the endothelialized myocardium with a controlled anisotropy. The endothelialized myocardium can be incubated until the endothelialized myocardium matures into spontaneously beating myocardial tissue having contractions aligned with the controlled anisotropy. The beating myocardial tissue can be used to screen a drug when the beating myocardial tissue is embedded within a microfluidic perfusion bioreactor.

What is claimed is: 
     
         1 . A method for fabricating an endothelialized myocardium, comprising the steps of:
 A) additive manufacturing a microfibrous hydrogel scaffold;   B) bioprinting endothelial cells directly within the microfibrous hydrogel scaffold, said bioprinting step occurring concurrently with said step A); and   C) seeding the result of said step A) and said step B) with cardiomyocytes to yield the endothelialized myocardium, said step C) being accomplished with a controlled anisotropy.   
     
     
         2 . The method of  claim 1 , further comprising the step of:
 D) embedding the endothelialized myocardium from said step C) into a microfluidic perfusion bioreactor.   
     
     
         3 . The method of  claim 1 , wherein the microfibrous hydrogel scaffold from said step A) has a periphery, and said step C) is not accomplished until the endothelial cells from said step B) have migrated to the periphery. 
     
     
         4 . The method of  claim 3 , wherein said step C) is not accomplished until ten to fourteen days after said step B) is accomplished. 
     
     
         5 . The method of  claim 1 , wherein the cardiomyocytes are neonatal rat cardiomyocytes. 
     
     
         6 . The method of  claim 1 , wherein the cardiomyocytes are human-induced Pluripotent Stem Cell (hiPSC) cardiomyocytes. 
     
     
         7 . The method of  claim 1 , wherein said step A) results in the microfibrous hydrogel scaffold having an aspect ratio of the controlled anisotropy selected from the group consisting of 2×3, 2×4, and 2×5. 
     
     
         8 . The method of  claim 1 , wherein the microfibrous hydrogel scaffold is made of a composite alginate and gelatin methacryloyl (GelMA) bioink with the endothelial cells directly embedded within the microfibrous hydrogel scaffold. 
     
     
         9 . The method of  claim 8 , wherein the microfibrous hydrogel scaffold comprises a plurality of serpentine layers each defining a primary axis, and the serpentine layers are successively placed upon one another in a cross-hatch configuration, so that the primary axis of adjacent ones of the serpentine layers are perpendicular. 
     
     
         10 . The method of  claim 9 , wherein the cross-hatch configuration establishes a plurality of rectangular holes, the rectangular holes having an aspect ratio of the controlled anisotropy when viewed in a plan view. 
     
     
         11 . The method of  claim 10 , wherein the aspect ratio of the controlled anisotropy is selected from the group consisting of 2×3, 2×4, and 2×5. 
     
     
         12 . The method of  claim 9 , wherein alternating ones of the serpentine layers having the primary axis in the same direction are placed so that they are offset from each other in a plan view. 
     
     
         13 . A method for providing a device for screening of a drug, comprising the steps of:
 A) bioprinting a bioink into an arrangement of at least one microfiber, the bioink including at least one crosslinking component and suspended endothelial cells;   B) crosslinking the at least one crosslinking component to yield, from the arrangement of the at least one microfiber, a microfibrous hydrogel scaffold having the endothelial cells embedded directly within; and   C) seeding the microfibrous hydrogel scaffold from said step B) with cardiomyocytes to yield an endothelialized myocardium with a controlled anisotropy.   
     
     
         14 . The method of  claim 13 , wherein said step A) includes bioprinting the at least one microfiber, which is a single continuous microfiber, into the arrangement, which is a multilayer serpentine arrangement with the controlled anisotropy. 
     
     
         15 . The method of  claim 13 , wherein the crosslinking of said step B) of the at least one crosslinking component, which is two crosslinking components including an alginate component and a gelatin methacryloyl (GelMA) component, includes rapid ionic crosslinking of the alginate component with a crosslinking solution discharged concurrently with the bioink during said step A), and photo-crosslinking the GelMA component after said step A) is accomplished. 
     
     
         16 . The method of  claim 13 , further comprising the step of:
 D) incubating the endothelialized myocardium until the endothelialized myocardium matures into spontaneously beating myocardial tissue having contractions aligned with the controlled anisotropy.   
     
     
         17 . The method of  claim 13 , further comprising the step of:
 D) embedding the endothelialized myocardium from said step C) into a microfluidic perfusion bioreactor.   
     
     
         18 . The method of  claim 17 , wherein said step C) is not accomplished until ten to fourteen days after said step B) is accomplished, the endothelial cells migrating within the at least one microfiber of the microfibrous hydrogel scaffold during the days. 
     
     
         19 . The method of  claim 17 , wherein the cardiomyocytes are human-induced Pluripotent Stem Cell (hiPSC) cardiomyocytes. 
     
     
         20 . The method of  claim 19 , wherein the hiPSC cardiomyocytes are specific to a patient being proposed for treatment with the drug.