3D vascularized human ocular tissue for cell therapy and drug discovery
Inventors
Bharti, Kapil • SONG, Min Jae • Quinn, Russell Louis
Assignees
US Department of Health and Human Services
Publication Number
US-11458225-B2
Publication Date
2022-10-04
Expiration Date
2037-11-08
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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.
Core Innovation
The invention discloses methods for fabricating a three-dimensional engineered blood retinal barrier (BRB) comprising a choroid and retinal pigment epithelial cells using bioprinting. The method involves depositing a first bio-ink with endothelial cells onto a biocompatible scaffold, maturing the bio-ink to form vessels, depositing retinal pigment epithelial cells on the opposite surface of the scaffold to form a monolayer, and culturing these cells so they proliferate and mature. This results in a three-dimensional engineered BRB having an artificial choroid and an artificial retinal pigment epithelium on opposite sides of the scaffold.
The problem addressed is the need for an engineered vascularized outer BRB for transplantation, evaluation of therapeutic agents, and treatment of retinal degenerative diseases and injuries. Existing therapies are limited because transplantation of retinal pigment epithelial cells as single cell suspensions leads to cell death and loss of essential monolayer structure, which is crucial for RPE function. Therefore, there is a need for a three dimensional engineered tissue that recapitulates the native architecture of the BRB for use in cell therapy and drug discovery.
The engineered BRB mimics physiological retinal architecture by including layers of endothelial cells forming vessels, optionally fibroblasts and pericytes, on one side of a biocompatible scaffold and a monolayer of retinal pigment epithelial cells on the opposite side. The methods also include culture media formulations and scaffold treatments to optimize cell adherence, proliferation, and maturation. The engineered tissue can model natural or diseased states, be used for drug screening, and serve as a transplantable cell-based therapy to treat retinal degeneration.
Claims Coverage
The patent discloses two independent claims focused on the method of fabricating a three-dimensional engineered blood retinal barrier (BRB) comprising a choroid and retinal pigment epithelial cells, and a method for determining the effect of a test agent using the engineered BRB. The main inventive features involve bioprinting of bio-inks on a biocompatible scaffold and culturing protocols.
Method for fabricating a three-dimensional engineered BRB using bioprinting
A sequential process of depositing a first bio-ink comprising endothelial cells in a hydrogel on a first surface of a biocompatible scaffold, maturing the bio-ink to allow vessel formation, depositing retinal pigment epithelial cells as a single cell layer on the opposite surface such that the scaffold lies between the two cell types, and culturing the RPE cells to proliferate and mature, resulting in a three-dimensional engineered BRB with an artificial choroid and retinal pigment epithelium.
Use of specific biocompatible scaffolds and scaffold treatments
Use of biocompatible scaffolds comprising or consisting of poly (D, L-lactide co-glycolide) (PDGLA) or similar polymers, optionally cross-linked and treated with oxygen plasma to enhance cell adherence prior to bio-ink deposition.
Bio-ink composition including multiple cell types and hydrogels
First bio-ink compositions comprising endothelial cells, fibroblasts, and pericytes in specified cellular ratios suspended in collagen, fibrinogen, or gelatin-based hydrogels designed for bioprinting. The endothelial cells, fibroblasts, and pericytes can be human cells, preferably derived from induced pluripotent stem cells.
Culture media formulations and timing for maturation
Use of defined culture media containing vascular endothelial growth factor (VEGF), angiopoietin-1, epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), ascorbic acid, hydrocortisone, heparin sulfate, aprotinin, and thrombin during specific stages to promote vessel formation and RPE maturation, including culturing for at least four days after deposition of bio-ink and addition of prostaglandin E2 for RPE maturation.
Deposition methods and scaffold coating
Bioprinting by extrusion or spraying of bio-ink onto the scaffold surfaces with optional coating of the scaffold's second surface with extracellular matrix components such as vitronectin to promote RPE adhesion and growth.
Method for screening test agents using engineered BRB
A method of determining the effect of a test agent by contacting the engineered three-dimensional BRB with the agent and evaluating phenotypic or structural changes in the choroid and retinal pigment epithelial cells as an indicator of biological effect.
Transplantation of the engineered BRB for retinal degeneration treatment
Methods of treating retinal degenerative diseases by transplanting the engineered BRB comprising choroid and retinal pigment epithelial cells into the eye of a subject with conditions such as acute macular degeneration.
The claims collectively cover methods for fabricating a three-dimensional engineered BRB by bioprinting specific bio-inks on biocompatible scaffolds with detailed compositions and culture conditions, methods of using the engineered BRB for screening agents, and transplantation methods for treatment of retinal degeneration.
Stated Advantages
Provides an engineered vascularized outer blood retinal barrier that recapitulates native retinal structure including both a choroidal vascular layer and polarized retinal pigment epithelium, enhancing the efficacy of transplantation therapies.
Enables production of a three-dimensional tissue model usable for the evaluation of therapeutic agents, drug screening, and testing of gene therapies, thereby facilitating discovery and development of treatments for retinal diseases.
Allows use of patient-specific or genetically modified induced pluripotent stem cells to create disease models and personalized tissue therapies, potentially reducing immune rejection.
Supports complex tissue maturation in vitro with simultaneous development of multiple cell types, improving the likelihood that engineered tissues will perform like native tissues following transplantation.
Documented Applications
Use as a transplantable cell-based therapy for treating retinal degenerative diseases and injuries, including age-related macular degeneration, macular dystrophies such as Stargardt's disease and Best disease, retinitis pigmentosa, and acute macular degeneration.
In vitro models for evaluating the effects of test agents on the blood retinal barrier to identify and test therapeutic agents, including drug discovery, gene therapy testing, and biomarker discovery.
Production of three-dimensional engineered ocular tissue models to study normal and diseased eye conditions, to investigate molecular pathways in diseases of the blood retinal barrier, and to assess drug and gene therapy effects.
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