3d vascularized human ocular tissue for cell therapy and drug discovery

Abstract

Methods are disclosed for fabricating a three-dimensional engineered blood retinal barrier (BRB) comprising a choroid and retinal pigment epithelial cells. The methods include the use of bioprinting. Also disclosed is a three-dimensional engineered BRB, and its use. Methods are also disclosed for using the three-dimensional engineered BRB, such as for the treatment of retinal degeneration in a subject or screening. A three-dimensional printing insert that is adapted for bioprinting on a culture substrate sheet that is securely retained within and exposed through a printing frame is also disclosed.

We claim: 
     
         1 . A three-dimensional, engineered ocular tissue model comprising:
 a three-dimensional, engineered outer blood retinal barrier comprising
 a first layer comprising a choroid, the choroid comprising a first bio-ink, wherein the first bio-ink comprises a plurality of endothelial cells; 
 a second layer comprising a plurality of retinal pigment epithelial cells, the first layer and the second layer forming the three-dimensional, engineered outer blood retinal barrier; and 
   a biocompatible scaffold, wherein the biocompatible scaffold is between the first layer and the second layer.   
     
     
         2 . The ocular tissue model of  claim 1 , the biocompatible scaffold comprises poly (D, L-lactide co-glycolide) (PDGLA), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(-L-lactic acid) (PLLA), poly (glycolic) acid (PGA), poly caprolactone (PCL), poly ethylene glycol (PEG), silk, fibroin, collagen (vitrified or recombinant), or a combination thereof. 
     
     
         3 . The ocular tissue model of  claim 1 , wherein the biocompatible scaffold comprises PDGLA. 
     
     
         4 . The ocular tissue model of  claim 3 , wherein the PDGLA is cross-linked PDGLA. 
     
     
         5 . The ocular tissue model of  claim 3 , wherein the PDGLA is oxygen-plasma treated PDGLA. 
     
     
         6 . The ocular tissue model of  claim 1 , wherein the first bio-ink further comprises a plurality of fibroblasts. 
     
     
         7 . The ocular tissue model of  claim 1 , wherein the first bio-ink further comprises a plurality of pericytes. 
     
     
         8 . The ocular tissue model of  claim 1 , wherein the first bio-ink comprises about 5 to about million endothelial cells per milliliter. 
     
     
         9 . The ocular tissue model of  claim 6 , wherein the first bio-ink comprises about 10 to about 50 million fibroblasts per milliliter. 
     
     
         10 . The ocular tissue model of  claim 7 , wherein the first bio-ink comprises about 0.5 to about 3 million pericytes per milliliter. 
     
     
         11 . The ocular tissue model of  claim 1 , wherein the first bio-ink further comprises fibroblasts and pericytes, and wherein the endothelial cells, the fibroblasts and the pericytes are present in the first bio-ink at a ratio of 1:0.3:0.1 to 1:10:1, respectively. 
     
     
         12 . The ocular tissue model of  claim 1 , wherein the first bio-ink further comprises fibroblasts and pericytes, and wherein the endothelial cells, the fibroblasts and the pericytes are present in the first bio-ink at a ratio of 1:2:0.5, respectively. 
     
     
         13 . The ocular tissue model of  claim 1 , wherein the first bio-ink further comprises a hydrogel. 
     
     
         14 . The ocular tissue model of  claim 13 , wherein the hydrogel comprises a collagen-based hydrogel. 
     
     
         15 . The ocular tissue model of  claim 14 , wherein the hydrogel comprises a gelatin hydrogel, a collagen hydrogel, a fibrin hydrogel, a polysaccharide hydrogel, an alginate hydrogel, a laminin hydrogel, a fibronectin hydrogel, a laminin hydrogel, a vitronectin hydrogel, a polyethylene glycol hydrogel, a gelatin methacryloyl hydrogel, or a combination thereof. 
     
     
         16 . The ocular tissue model of  claim 1 , wherein the biocompatible scaffold further comprises an extracellular matrix that is coated on a surface of the biocompatible scaffold. 
     
     
         17 . The ocular tissue model of  claim 16 , wherein the extracellular matrix comprises a collagen, a laminin, a gelatin, a chondroitin sulfate, a proteoglycans, an elastin, a hyaluronic acid, avitronectin, a fibronectin or a combination thereof. 
     
     
         18 . The ocular tissue model of  claim 11 , wherein one or more of the retinal pigment epithelial cells, endothelial cells, fibroblasts and pericytes are produced from induced pluripotent stem cells. 
     
     
         19 . The ocular tissue model of  claim 11 , wherein one or more of the retinal pigment epithelial cells, endothelial cells, fibroblasts and pericytes are diseased cells. 
     
     
         20 . The ocular tissue model of  claim 1 , comprising about 100,000 to 400,000 retinal pigment epithelial cells per 1 centimeter of a surface of the biocompatible scaffold. 
     
     
         21 . The ocular tissue model of  claim 1 , wherein the endothelial cells and the retinal pigment epithelial cells are human cells. 
     
     
         22 . The ocular tissue model of  claim 11 , wherein the endothelial cells, fibroblasts, pericytes, and/or retinal pigment epithelial cells are human cells. 
     
     
         23 . The ocular tissue model of  claim 11 , wherein one or more of the retinal pigment epithelial cells, endothelial cells, fibroblasts and pericytes are diseased cells from diseased human donors. 
     
     
         24 . The ocular tissue model of  claim 1 , wherein the model is non-innervated.