Methods of making endothelial scaffolds
Inventors
Soker, Shay • Williams, J. Koudy • Laber, Patrick • Greven, Margaret • Walter, Keith A.
Assignees
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Publication Number
US-9707256-B2
Publication Date
2017-07-18
Expiration Date
Abstract
Provided herein is an endothelial scaffold comprising, consisting of, or consisting essentially of decellularized corneal stroma. In some embodiments, the scaffold has cultured endothelial cells seeded thereon. Methods of treating a patient in need of corneal endothelial transplant are also provided, including implanting the scaffold as described herein onto a cornea of the patient (e.g., by deep keratectomy).
Core Innovation
The invention provides an endothelial scaffold made from decellularized corneal stroma. The corneal stroma is sectioned into a thickness between 50 and 200 micrometers, and the decellularized stroma forms a scaffold for endothelial cell seeding. The scaffold is used to produce an endothelial scaffold by combining the decellularized corneal stroma with cultured corneal endothelial cells.
The scaffold is seeded with cultured corneal endothelial cells that are positive for ZO-1, Na+/K+ ATPase, connexin-43, cytokeratin AE1/AE3, von Willebrand factor (vWF), and VE-Cadherin, and are negative for CD31. Cultured corneal endothelial cells form an endothelial layer on the scaffold, including maintenance of marker expression and confluence as described.
The decellularized scaffold is produced by treating the sectioned corneal stroma with a detergent and/or basic solution or by treating with a hypo-osmotic solution. The document reports that decellularization preserves mechanical properties and that the endothelialized constructs support sustained endothelial marker expression in an in vivo rabbit transplantation context.
Claims Coverage
The partial content includes one independent claim, which covers a method of making an endothelial scaffold. The claim includes three main inventive elements: sectioning corneal stroma of a defined thickness, decellularizing with specified solution classes to form a scaffold, and seeding cultured corneal endothelial cells with a defined marker-positive and CD31-negative profile.
Endothelial scaffold made from sectioned corneal stroma and seeded marker-defined corneal endothelial cells
Section a cornea parallel to the endothelial surface to provide sectioned corneal stroma with a thickness between 50 and 200 micrometers; decellularize the stroma by treating with a detergent and/or basic solution or treating with a hypo-osmotic solution to form a scaffold; seed cultured endothelial cells onto the scaffold to form an endothelial scaffold, wherein the cultured corneal endothelial cells are positive for ZO-1, Na+/K+ ATPase, connexin-43, cytokeratin AE1/AE3, vWF, and VE-Cadherin and are negative for CD31.
Overall, the independent claim ties together sectioned corneal stroma within a defined thickness range, decellularization using detergent/basic and/or hypo-osmotic treatment classes, and seeding of cultured corneal endothelial cells defined by endothelial marker positivity and CD31-negativity to produce the endothelial scaffold.
Stated Advantages
Preserves mechanical properties after decellularization, with mechanical properties similar to native (tensile strength, elongation at break, and Young’s modulus reported as similar).
Supports endothelial marker expression and confluence/coverage after seeding, including sustained endothelial marker expression after in vivo rabbit transplantation for a described period.
Produces endothelialized constructs with cultured corneal endothelial cells that maintain defined marker expression on decellularized corneal stroma.
Documented Applications
Corneal endothelial transplant, including implanting an endothelial scaffold into a cornea via deep keratectomy, as described in the treatment context.
In vivo rabbit transplantation to epithelialized/acellular corneas, reporting sustained endothelial marker expression after the described interval.
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