Method for producing human corneal epithelium sheet

Patent metadata

Field	Value
Publication number	US-2016008408-A1
Application number	US-201414772854-A
Country	US
Kind code	A1
Filing date	Mar 9, 2014
Priority date	Mar 11, 2013
Publication date	Jan 14, 2016
Grant date	—

Abstract

[Problem] To provide a method for producing human corneal epithelial sheet, wherein the human corneal epithelial-derived cells obtained by culturing human corneal epithelial cells are cultured on an amnion substrate.
[Solution] A method for culturing human corneal epithelial cells using mesenchymal stem cells as the feeder cells; and a method for culturing human corneal epithelial cells using a medium containing a ROCK inhibitor, a phosphodiesterase inhibitor, a MAP kinase inhibitor and a TGF-β receptor inhibitor in various combinations.

First claim

1 . A method for producing cells derived from human corneal epithelial cells, which comprises a process wherein, in a culture vessel separated into first and second chambers by a membrane having micropores that disallow cells to pass through, feeder cells are added to the first chamber and human corneal epithelial cells are added to the second chamber, said human corneal epithelial cells are cultured by using a first medium containing a ROCK inhibitor and a phosphodiesterase inhibitor, and then said human corneal epithelial cells are cultured by using a second medium containing a phosphodiesterase inhibitor, a MAP kinase inhibitor and a TGF-β receptor inhibitor.

2 . The method according to claim 1 , wherein said culture vessel is separated by the membrane having micropores into two chambers up and down, so as to give an upper chamber and a lower chamber.

3 . The method according to claim 2 , wherein said lower chamber is the first chamber and said upper chamber is the second chamber, and said human corneal epithelial cells are cultured on the membrane having micropores.

4 . The method according to claim 3 , wherein said human corneal epithelial cells are cultured on the membrane having micropores that disallow said cells to pass through and retained in said culture vessel without direct contact with the feeder cells.

5 . The method according to claim 1 , wherein the ROCK inhibitor contained in said first medium is Y27632, the phosphodiesterase inhibitor contained in said first and second media is 3-isobutyl-1-methylxanthine, the MAP kinase inhibitor contained in said second medium is selected from SB203580, SB239063 and a combination thereof, and the TGF-β receptor inhibitor contained in said second medium is selected from SB431542, LY364947, A83-01 and a combination thereof.

6 . The method according to claim 1 , wherein the ROCK inhibitor contained in said first medium is Y27632, the phosphodiesterase inhibitor contained in said first and second media is 3-isobutyl-1-methylxanthine, the MAP kinase inhibitor contained in said second medium is SB203580, and the TGF-β receptor inhibitor contained in said second medium is SB431542.

7 . The method according to claim 6 , wherein the concentrations of Y27632 contained in said first medium, 3-isobutyl-1-methylxanthine contained in said first and second media, SB203580 contained in said second medium, and SB431542 contained in said second medium are 1 to 20 μM, 50 to 150 μM, 0.2 to 2 μM, and 0.2 to 2 μM, respectively.

8 . The method according to claim 6 , wherein the concentrations of Y27632 contained in said first medium, 3-isobutyl-1-methylxanthine contained in said first and second media, SB203580 contained in said second medium, and SB431542 contained in said second medium are about 10 μM, about 100 μM, about 1 μM, and about 1 μM, respectively.

9 . The method according to claim 1 , wherein said feeder cells are human mesenchymal stem cells.

10 . The method of claim 1 , wherein the method further comprises a step wherein the cells obtained by the method according to claim 1 are frozen by suspending in a cell-freezing liquid, so as to obtain frozen cells.

11 . Cells derived from the human corneal epithelial cells which are obtained by the method according to claim 1 .

12 . Cells derived from the frozen human corneal epithelial cells which are obtained by the method according to claim 10 .

13 . A method for producing human corneal epithelial sheet, which comprises a step wherein, after thawing the cells derived from the human corneal epithelial cells according to claim 11 , the cells derived from the human corneal epithelial cells according to claim 11 are cultured in a culture vessel containing feeder cells on an amnion substrate which is placed on the membrane having micropores that disallow said feeder cells to pass through and retained in said culture vessel with a third medium containing a ROCK inhibitor, and then cultured by using a fourth medium containing a MAP kinase inhibitor and a TGF-β receptor inhibitor, so as to form cell layer comprising the cells derived from said human corneal epithelial cells on said amnion substrate, and a subsequent step wherein said cell layer is cultured by using said fourth medium in a condition where all or part of the surface of said cell layer is not covered with the medium.

14 . The method according to claim 13 , wherein said amnion substrate is placed in said culture vessel with the side stripping off the amnion epithelium facing upward.

15 . The method according to claim 13 , wherein said ROCK inhibitor contained in said third medium is Y27632, said MAP kinase inhibitor contained in said fourth medium is SB203580, and said TGF-β receptor inhibitor contained in said fourth medium is SB431542.

16 . The method according to claim 15 , wherein the concentrations of Y27632 contained in said third medium, SB203580 contained in said fourth medium, and SB431542 contained in said fourth medium are 1 to 20 μM, 0.2 to 2 μM, and 0.2 to 2 μM, respectively.

17 . The method according to claim 15 , wherein the concentrations of Y27632 contained in said third medium, SB203580 contained in said fourth medium, and SB431542 contained in said fourth medium are about 10 μM, 1 μM, and about 1 μM, respectively.

18 . The method according to claim 13 , wherein said feeder cells are human mesenchymal stem cells.

19 . A human corneal epithelial sheet obtained by the method according to claim 13 , which comprises said amnion substrate and the cells derived from said human corneal epithelial cells.

20 . The human corneal epithelial sheet according to claim 19 , wherein 95% or more of an area of the side stripping off the amnion epithelium of said amnion substrate is covered with the cell layer formed by the cells derived from said human corneal epithelial cells.

21 . The human corneal epithelial sheet according to claim 20 , wherein 90% or more of said cell layer has a layered structure comprising two or more cell layers.

22 . The human corneal epithelial sheet according to claim 19 , wherein 98% or more of the cells derived from said human corneal epithelial cells is cytokeratin AE1/AE3-positive, and 80% or more is cytokeratin 12-positive.

23 . The human corneal epithelial sheet according to claim 19 , wherein 99% or more of the cells derived from said human corneal epithelial cells is cytokeratin AE1/AE3-positive, and 85% or more is cytokeratin 12-positive.

24 . The human corneal epithelial sheet according to claim 19 for treatment of a corneal disease.

25 . The human corneal epithelial sheet according to claim 24 , wherein said corneal disease is the disease caused by the irreversible impairment of the barrier functions of the corneal epithelium.

26 . The human corneal epithelial sheet according to claim 24 , wherein said corneal disease is selected from the group consisting of corneal dystrophy, Stevens-Johnson syndrome, chemical injury and corneal epithelial stem cell deficiency.

27 . A human corneal epithelial sheet wherein 99.5% or more of the surface area of an amnion substrate is covered with the cell layer formed by the cells derived from human corneal epithelial cells, 98% or more of the cells is cytokeratin AE1/AE3-positive, 80% or more of the cells is cytokeratin 12-positive, 95% or more of the cell layer has a layered structure comprising four or more cell layers, 90% or more of the sur

Assignees

• Japan Chem Res
• Kyoto Prefectural Public Univ Corp

Inventors

• Imagawa Kiwamu
• Maeda Kenichi
• HOSODA Yuki
• Yokoyama Shuichi
• Okumura Naoki
• Koizumi Noriko
• Kinoshita Shigeru

Classifications

• A61P27/02

  Ophthalmic agents · CPC title

• C12N5/0602

  Vertebrate cells · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title