Cell culture of corneal endothelial cells

Patent metadata

Field	Value
Publication number	US-10017735-B2
Application number	US-201314415972-A
Country	US
Kind code	B2
Filing date	Jul 18, 2013
Priority date	Jul 18, 2012
Publication date	Jul 10, 2018
Grant date	Jul 10, 2018

Abstract

The invention relates to a dual-media approach for culturing isolated corneal endothelial cells. Isolated corneal endothelial cells are first contacted with a proliferative medium to propagate and/or expand the endothelial cells followed by a maintenance medium to preserve the morphology and/or characteristics of the corneal endothelial cells. The invention includes the proliferative medium and the maintenance medium and also a combination of the two medium.

First claim

The invention claimed is:

1. A method for culturing isolated corneal endothelial cells comprising the steps of:
(i) contacting isolated primary corneal endothelial cells with a maintenance medium comprising Human Endothelial-Serum Free Medium (Human Endothelial-SFM) to stabilize the corneal endothelial cells
(ii) contacting the isolated stabilized corneal endothelial cells with a proliferative medium to propagate and/or expand the corneal endothelial cells; and
(iii) contacting the propagated and/or expanded corneal endothelial cells with the maintenance medium comprising Human Endothelial-SFM to substantially preserve the morphology and characteristics of the corneal endothelial cells.

2. The method according to claim 1 , wherein step (ii) comprises culturing the isolated corneal endothelial cells in the proliferative medium.

3. The method according to claim 1 , wherein step (iii) is performed before the isolated corneal endothelial cells differentiate or change morphology and/or characteristics.

4. The method according to claim 1 wherein step (iii) comprises replacing the proliferative medium with the maintenance medium.

5. The method according to claim 1 , wherein step (iii) comprises passaging the propagated and/or expanded corneal endothelial cells in the maintenance medium.

6. The method according to claim 1 , wherein the proliferative medium comprises a supplemented basal medium.

7. The method according to claim 6 , wherein the proliferative medium comprises a basal medium supplemented with at least one supplement selected from the group consisting of amino acids, antimicrobials, cholera toxin, chondroitin, Dimethyl sulphoxide (DMSO), Endothelial Cell Growth Supplement (ECGS), growth factors, insulin, steroids, minerals, pituitary extract, transferrin and vitamins.

8. The method according to claim 1 , wherein the proliferative medium comprises a basal medium supplemented with serum.

9. The method according to claim 1 , wherein the proliferative medium comprises a basal medium supplemented with at least 2% serum.

10. The method according claim 1 , wherein the proliferative medium comprises a basal medium supplemented with 5% serum.

11. The method according to claim 1 , wherein the maintenance medium comprises Human Endothelial-SFM supplemented with serum.

12. The method according to claim 11 , wherein the maintenance medium comprises Human Endothelial-SFM supplemented with at least 2% serum.

13. The method according to claim 11 or 12 , wherein the maintenance medium comprises Human Endothelial-SFM supplemented with 2.5% or 5% serum.

14. The method according to claim 1 , wherein the maintenance medium comprises Human Endothelial-SFM supplemented with at least one antimicrobial.

15. The method according to claim 1 , wherein the proliferative medium comprises M2 or M4 medium.

Assignees

• Singapore Health Serv Pte Ltd

Inventors

• Tan Donald
• Mehta Jodhbir
• Peh Swee Lim Gary

Classifications

• C12N2500/38

  Vitamins · CPC title

• C12N2501/31

  Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins · CPC title

• C12N2500/24

  Iron; Fe chelators; Transferrin · CPC title

• C12N2500/34

  Sugars · CPC title

• C12N5/0621 Primary

  Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062) · CPC title