Scaffold-free 3d bioprinting of porcine cells

Abstract

Provided herein are synthetic, three-dimensional (3D) bioprinted tissue constructs comprising porcine cells and methods of producing and using the same. The synthetic 3D bioprinted tissue constructs are fabricated by bioprinting spheroids comprising porcine cells, including genetically engineered cells, on a microneedle mold and fusing the spheroids to form an engineered tissue construct. Also provided are methods of using scaffold-free 3D bioprinted tissue constructs for applications related to drug screening and toxicity screening.

1 . A method for fabricating a synthetic three-dimensional (3D) porcine tissue construct, wherein the method comprises:
 providing a predetermined arrangement of microneedles;   adding cell spheroids to the microneedles in a computer-controlled manner, wherein the cell spheroids are formed with porcine cells of two or more cell types, wherein at least a portion of the porcine cells are genetically engineered; and   culturing the spheroids on the microneedles for about 5 days whereby the spheroids fuse to form a synthetic 3D porcine tissue construct comprising genetically engineered porcine cells.   
     
     
         2 . The method of  claim 1 , further comprising removing the synthetic 3D porcine tissue construct from the microneedle mold to obtain a scaffold-free synthetic 3D porcine tissue construct comprising genetically engineered porcine cells. 
     
     
         3 . The method of  claim 1 , wherein the cell spheroids comprise porcine cells of two or more cell types selected from the group consisting of porcine hepatocytes, liver sinusoidal endothelial cells (LSECs), stellate cells, Kupffer cells, and fibroblasts, and wherein the synthetic 3D porcine tissue construct is a synthetic 3D porcine liver tissue construct. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the cell spheroids comprise porcine cells of two or more cell types selected from the group consisting of porcine corneal endothelial cells, stromal cells, and corneal epithelial cells, and wherein the synthetic 3D porcine tissue construct is a synthetic 3D porcine cornea tissue construct. 
     
     
         6 . The method of  claim 1 , wherein the cell spheroids comprise porcine fibroblasts and porcine aortic endothelial cells, and wherein the synthetic 3D porcine tissue construct is a synthetic 3D porcine aortic valve tissue construct. 
     
     
         7 . The method of  claim 1 , wherein the cell spheroids comprise porcine cells of two or more cell types selected from the group consisting of porcine alpha cells, beta (islet) cells, fibroblasts, and endothelial cells, and wherein the synthetic 3D porcine tissue construct is a synthetic 3D porcine pancreatic tissue construct. 
     
     
         8 . The method of  claim 1 , wherein the cell spheroids comprise porcine cells of two or more cell types selected from the group consisting of porcine fibroblasts, endothelial cells, and proximal tubule epithelial cells, and wherein the synthetic 3D porcine tissue construct is a synthetic 3D porcine kidney tissue construct. 
     
     
         9 .- 17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the cells of two or more cell types comprise:
 (a) hepatocytes; and   (b) hepatic stellate cells (HSCs).   
     
     
         19 . The method of  claim 18 , wherein the hepatocytes and the hepatic stellate cells are at a ratio of 2-2.5:1. 
     
     
         20 . A cell spheroid comprising one of (a) or (b):
 (a) porcine (1) hepatocytes and (2) hepatic stellate cells at a ratio of 2-2.5:1; or   (b) porcine (1) CD31 +  pulmonary vascular endothelial cells, (2) pulmonary fibroblasts, and (3) pulmonary pneumocytes Type II at a ratio of 1:1:0.5, respectively, wherein the cell spheroid of (b) comprises a total cell number of about 40,000 cells.