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Transplantation of a sheet of human corneal endothelial cell in a rabbit model.

Hitani K, Yokoo S, Honda N, Usui T, Yamagami S, Amano S - Mol. Vis. (2008)

Bottom Line: After HCEC sheet transplantation, corneal edema decreased much earlier in the HCEC group than in the control group.The average endothelial cell density in the HCEC sheet group seven days postoperatively was 2,244 cells/mm(2).This technique for producing an HCEC sheet might be useful in regenerative medicine for the cornea and reconstruction of the corneal endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

ABSTRACT

Purpose: To develop a novel method for constructing a sheet of human corneal endothelial cells (HCECs) and examine the properties of the HCEC sheet.

Methods: HCECs were cultured on a cell culture insert for a week; ethylenediamine tetraacetic acid was applied from the bottom of the cell culture insert to attenuate the attachment of HCECs. The sheet of HCECs was constructed by bluntly detaching the cell sheet with a spatula. HCEC cell sheets were placed on the posterior surface of excised rabbit corneal buttons and transplanted onto the corneal beds of donor rabbits. In two eyes from the HCEC sheet group, cultured HCECs were labeled with PKH26 to observe the localization of HCECs after transplantation.

Results: Cultured HCECs could be bluntly detached en bloc from the bottom of a culture insert. Immunostaining for ZO-1, Na+, K+-ATPase, laminin, fibronectin, and type IV collagen was positive in the cell sheet. The average cell density in a HCEC sheet was 2,425 cells/mm(2). After HCEC sheet transplantation, corneal edema decreased much earlier in the HCEC group than in the control group. In the HCEC sheet group, the monolayer of continuous cells attached to the posterior surface of the transplanted rabbit cornea and the posterior surface of transplanted cornea was covered with PKH26-labeled cells. The average endothelial cell density in the HCEC sheet group seven days postoperatively was 2,244 cells/mm(2).

Conclusions: This technique for producing an HCEC sheet might be useful in regenerative medicine for the cornea and reconstruction of the corneal endothelium.

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Related in: MedlinePlus

Immunohistologic findings of the human corneal endothelial cell sheet. A and B: In the flat mount immunohistochemical observation, immunostaining of both ZO-1 (A) and Na+, K+-ATPase (B) were positive at most cellular boundaries. The cellular shape demarcated with immunostaining was quasi-regular with well defined cell boundaries. C: In the transverse observation of extracellular matrix, immunostaining for laminin was positive at the basal side, superficial side, and intercellular space. D and E: Immunostaining of fibronectin (D) and type IV collagen (E) was weak at the surface of the sheet, but positive at the intercellular space. The scale bars are equal to 50 μm.
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f4: Immunohistologic findings of the human corneal endothelial cell sheet. A and B: In the flat mount immunohistochemical observation, immunostaining of both ZO-1 (A) and Na+, K+-ATPase (B) were positive at most cellular boundaries. The cellular shape demarcated with immunostaining was quasi-regular with well defined cell boundaries. C: In the transverse observation of extracellular matrix, immunostaining for laminin was positive at the basal side, superficial side, and intercellular space. D and E: Immunostaining of fibronectin (D) and type IV collagen (E) was weak at the surface of the sheet, but positive at the intercellular space. The scale bars are equal to 50 μm.

Mentions: By flat mount immunohistochemical observation, immunostaining of both ZO-1, a tight junction-associated protein, and Na+, K+-ATPase were positive at most cell boundaries (Figure 4), suggesting proper function of cellular junctions and Na+, K+-ATPase pumps. The cellular shape demarcated with immunostaining was quasi-regular with well defined cell boundaries. The average ±standard deviation of cell density in a ZO-1 stained HCEC sheet was 2,425 \pom 83 cells/mm2.


Transplantation of a sheet of human corneal endothelial cell in a rabbit model.

Hitani K, Yokoo S, Honda N, Usui T, Yamagami S, Amano S - Mol. Vis. (2008)

Immunohistologic findings of the human corneal endothelial cell sheet. A and B: In the flat mount immunohistochemical observation, immunostaining of both ZO-1 (A) and Na+, K+-ATPase (B) were positive at most cellular boundaries. The cellular shape demarcated with immunostaining was quasi-regular with well defined cell boundaries. C: In the transverse observation of extracellular matrix, immunostaining for laminin was positive at the basal side, superficial side, and intercellular space. D and E: Immunostaining of fibronectin (D) and type IV collagen (E) was weak at the surface of the sheet, but positive at the intercellular space. The scale bars are equal to 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267690&req=5

f4: Immunohistologic findings of the human corneal endothelial cell sheet. A and B: In the flat mount immunohistochemical observation, immunostaining of both ZO-1 (A) and Na+, K+-ATPase (B) were positive at most cellular boundaries. The cellular shape demarcated with immunostaining was quasi-regular with well defined cell boundaries. C: In the transverse observation of extracellular matrix, immunostaining for laminin was positive at the basal side, superficial side, and intercellular space. D and E: Immunostaining of fibronectin (D) and type IV collagen (E) was weak at the surface of the sheet, but positive at the intercellular space. The scale bars are equal to 50 μm.
Mentions: By flat mount immunohistochemical observation, immunostaining of both ZO-1, a tight junction-associated protein, and Na+, K+-ATPase were positive at most cell boundaries (Figure 4), suggesting proper function of cellular junctions and Na+, K+-ATPase pumps. The cellular shape demarcated with immunostaining was quasi-regular with well defined cell boundaries. The average ±standard deviation of cell density in a ZO-1 stained HCEC sheet was 2,425 \pom 83 cells/mm2.

Bottom Line: After HCEC sheet transplantation, corneal edema decreased much earlier in the HCEC group than in the control group.The average endothelial cell density in the HCEC sheet group seven days postoperatively was 2,244 cells/mm(2).This technique for producing an HCEC sheet might be useful in regenerative medicine for the cornea and reconstruction of the corneal endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

ABSTRACT

Purpose: To develop a novel method for constructing a sheet of human corneal endothelial cells (HCECs) and examine the properties of the HCEC sheet.

Methods: HCECs were cultured on a cell culture insert for a week; ethylenediamine tetraacetic acid was applied from the bottom of the cell culture insert to attenuate the attachment of HCECs. The sheet of HCECs was constructed by bluntly detaching the cell sheet with a spatula. HCEC cell sheets were placed on the posterior surface of excised rabbit corneal buttons and transplanted onto the corneal beds of donor rabbits. In two eyes from the HCEC sheet group, cultured HCECs were labeled with PKH26 to observe the localization of HCECs after transplantation.

Results: Cultured HCECs could be bluntly detached en bloc from the bottom of a culture insert. Immunostaining for ZO-1, Na+, K+-ATPase, laminin, fibronectin, and type IV collagen was positive in the cell sheet. The average cell density in a HCEC sheet was 2,425 cells/mm(2). After HCEC sheet transplantation, corneal edema decreased much earlier in the HCEC group than in the control group. In the HCEC sheet group, the monolayer of continuous cells attached to the posterior surface of the transplanted rabbit cornea and the posterior surface of transplanted cornea was covered with PKH26-labeled cells. The average endothelial cell density in the HCEC sheet group seven days postoperatively was 2,244 cells/mm(2).

Conclusions: This technique for producing an HCEC sheet might be useful in regenerative medicine for the cornea and reconstruction of the corneal endothelium.

Show MeSH
Related in: MedlinePlus