Methods of making endothelial scaffolds

Patent metadata

Field	Value
Publication number	US-9707256-B2
Application number	US-201314102953-A
Country	US
Kind code	B2
Filing date	Dec 11, 2013
Priority date	Feb 26, 2009
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

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).

First claim

That which is claimed is:

1. A method of making an endothelial scaffold, comprising the steps of:
providing a sectioned corneal stroma, said stroma having a thickness of between 50 and 200 micrometers, wherein said providing comprises sectioning a cornea parallel to the endothelial surface thereof;
decellularizing said stroma to form a scaffold, wherein said decellularizing comprises treating said stroma with a detergent and/or basic solution, or comprises treating said stroma with a hypo-osmotic solution; and
seeding cultured endothelial cells onto said scaffold to form an endothelial scaffold,
wherein said cultured endothelial cells are corneal endothelial cells, and wherein said cultured corneal endothelial cells are positive for the markers: Zona occludin-1 (ZO-1), Na+/K+ ATPase, connexin-43, cytokeratin AE1/AE3, von Willebrand factor (vWF), and VE-Cadherin; and are negative for the marker CD31,
to thereby produce said endothelial scaffold.

2. The method of claim 1 , wherein said cultured endothelial cells form a monolayer of hexagonally-shaped cells on said scaffold.

3. The method of claim 1 , wherein said cells have been passaged between 1 and 20 times.

4. The method of claim 1 , wherein said cornea is a human cornea.

5. The method of claim 1 , wherein said endothelial cells are human endothelial cells.

6. The method of claim 1 , wherein said sectioned corneal stroma has an average thickness of between 75 and 150 micrometers.

7. The method of claim 1 , wherein said sectioned corneal stroma has an average thickness of between 100 and 130 micrometers.

8. The method of claim 1 , wherein said sectioning is carried out with a microtome.

9. The method of claim 1 , wherein said decellularizing comprises treating said stroma with a hypo-osmotic solution.

10. The method of claim 1 , wherein said decellularizing comprises treating said stroma with a detergent and/or basic solution.

Assignees

• Univ Wake Forest Health Sciences
• Wake Forest Inst For Regenerative Medicine

Inventors

• Soker Shay
• Williams J Koudy
• Laber Patrick
• Greven Margaret
• Walter Keith A

Classifications

• A61L2430/16

  for reconstruction of eye parts, e.g. intraocular lens, cornea · CPC title

• A61K35/44 Primary

  Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• A61F2/142 Primary

  Cornea, e.g. artificial corneae, keratoprostheses or corneal implants for repair of defective corneal tissue · CPC title

• A61K9/0051

  Ocular inserts or implants · CPC title