3d vascularized human ocular tissue for cell therapy and drug discovery
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Servic
InventorsKapil Bharti, Min Jae Song, Russell Louis Quinn
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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.
Claims34
A method of fabricating a three-dimensional engineered blood retinal barrier (BRB) comprising a choroid and retinal pigmented epithelial cells, the method comprising:
- a. depositing a first bio-ink comprising a hydrogel and a first medium, wherein the hydrogel comprises endothelial cells, onto a biocompatible scaffold comprising first and second opposite surfaces, such that the hydrogel adheres to a first surface of the biocompatible scaffold;
- b. maturing the deposited first bio-ink on the first surface of the biocompatible scaffold in a second medium for at least four days to allow the endothelial cells to form vessels;
- c. depositing retinal pigment epithelial cells in a third medium to form a single cell layer on the second surface of the biocompatible scaffold, such that the biocompatible scaffold is between the endothelial cells and the retinal pigment epithelial cells;
- and
- d. culturing the deposited retinal pigment epithelial cells in the third medium on the biocompatible scaffold so that they proliferate and mature,
- thereby forming the three-dimensional engineered BRB with an artificial choroid on the first surface and an artificial retinal pigment epithelium on the second surface.
A method of fabricating a three-dimensional engineered blood retinal barrier (BRB) comprising a choroid and retinal pigment epithelial cells, the method comprising:
- a. depositing a first bio-ink comprising endothelial cells, fibroblasts and pericytes in a collagen and fibrinogen hydrogel and a first medium comprising thrombin, vascular endothelial grown factor, epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), ascorbic acid, hydrocortisone, heparin sulfate, angiopoietin-1 and aprotinin, onto a biocompatible oxygen plasma treated poly (D, L-lactide co-glycolide) PDGLA scaffold, such that the endothelial cells, fibroblasts and periyctes adhere to a first surface of the biocompatible scaffold;
- b. maturing the deposited first bio-ink in the second medium comprising an effective amount of vascular endothelial grown factor, EGF, FGF, IGF, ascorbic acid, hydrocortisone, heparin sulfate, and angiopoietin-1 and aprotinin, in the absence of thrombin, for at least four days to allow the endothelial cells to form vessels;
- c. depositing retinal pigment epithelial cells in a third medium to form a layer on a second surface of the biocompatible scaffold, wherein the first and the second surface of the biocompatible scaffold are opposite surfaces, such that biocompatible scaffold is between the endothelial cells and the retinal pigment epithelial cells, and wherein the second surface is coated with vitronectin, and wherein the third medium comprises an effective amount of taurine-hydrocortisone-triiodo-thyronin, hydrocortisone, Triiodo-thyronin, fetal bovine serum
- d. culturing the deposited retinal pigment epithelial cells in the third medium comprising the effective amount of taurine-hydrocortisone-triiodo-thyronin, hydrocortisone, triiodo-thyronin, fetal bovine serum so that the deposited retinal pigment epithelial cells proliferate and mature;
- and
- e. culturing the endothelial cells, fibroblasts and the pericytes in a fourth medium comprising an effective amount of vascular endothelial grown factor, wherein the fourth medium does not comprise thrombin and angiopoietin-1;
- f. culturing the endothelial cells, fibroblasts and the pericytes in a fifth medium optionally comprising an effective amount of vascular endothelial grown factor, wherein the fifth medium does not comprise thrombin, angiopoietin-1, and aprotinin;
- and
- g. culturing the retinal pigment epithelial cells in a sixth medium comprising an effective amount of prostaglandin E2 added to the third medium,
- thereby forming the three three-dimensional engineered BRB.
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;
- and
- 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.
-68. (canceled)
A bioprinting insert for bioprinting a scaffold and inserting it against a wall of a culture chamber of a predetermined width, the insert comprising:
- an insert adapted to fit in the culture chamber and form an exposed printing frame for displaying a printing substrate in the printing frame.
70-89. (canceled)
Dependencies are inferred from the claim text.