Cornea is a transparent tissue comprising five cellular layers, i.e. corneal epithelium, Bowman membrane, corneal stromal layer, Descemet's membrane, and corneal endothelium from outside to inside in the order, contains no blood vessel but nerves. Transparency of cornea is maintained by corneal stromal layer and corneal epithelium.
Corneal endothelial dysfunction is one of the main causes of blindness. The loss of function of corneal endothelium for maintaining transparency of cornea results in the impairment of eyesight. Corneal endothelial dysfunction is a disease caused by the degeneration, necrosis or physical loss of corneal endothelium or the like. Bullous keratopathy is a disease caused by severe corneal endothelial dysfunction, in which a pomp function, one of the functions of corneal endothelial cell, to regulate the amount of water in cornea is impaired, and as a result, the water in the cornea in not discharged and the cornea becomes edematous and opacified. In addition, there are cases where corneal epithelium stripped off by edema.
The density of corneal endothelial cells on normal cornea is about 2500 to 3000 cell/mm2, but
the density decreases in a condition of corneal endothelial dysfunction. When the cell density becomes about 500 cells/mm2 or less, the pumping function and the barrier function of corneal endothelial cells decline resulting in edema.
Corneal endothelial cells cannot regenerate, once injured. As the therapy for the corneal endothelial dysfunction, penetrating keratoplasty has been performed to recover the function of corneal endothelium and the transparency of cornea as well, where healthy cornea which possesses the three-layer structure comprising epithelium, stroma, and endothelium is transplanted. However penetrating keratoplasty has a problem such as vulnerability of eyeball caused by dissection of all the layer of cornea. Therefore, DSAEK (Descemet's Stripping Automated Endothelial Keratoplasty) in which only corneal endothelium has been transplanted, and DMEK (Descemet's Membrane Endothelial Keratoplasty) in which corneal endothelium has been transplanted with Descemet's membrane have been recently prevailing (NPL 1). The examples of diseases caused by the dysfunction of corneal endothelium and requiring corneal transplantation, include corneal edema, corneal leukoma, conical cornea, and the like, in addition to bullous keratopathy. In Japan, the number of patients waiting for corneal transplantation is about 5500, but the annual number of corneal transplantation is about 2700. The current situation is that sufficient actions to the patients cannot be taken due to donor shortage.
As the method to solve donor shortage as above, studies have been made to culture corneal endothelial cells and transplant them to the patients with corneal endothelial dysfunction to recover the function of corneal endothelium. For example, a method for production of a corneal endothelium-like sheet for transplantation by culturing corneal endothelial cells on amnion has been reported (PTL 1 and 2). In these reports, DMEM medium containing 10% FCS, 2 ng/mL of b-FGF, and antibiotics (PTL 1), or DMEM medium containing 10% FCS (PTL 2) was used as the medium for corneal endothelial cell culture. Furthermore a method for production of a corneal endothelium-like sheet for transplantation by culturing corneal endothelial cells on a collagen sheet coated with fibronectin has been reported (PTL 3), and a method for production of a corneal endothelium-like sheet for transplantation by culturing corneal endothelial cells on a cellulose substrate has been reported (PTL 4). In these reports, low glucose DMEM medium supplemented with 15% FCS, 2 ng/mL of b-FGF, and antibiotics was used as a medium for corneal endothelial cell culture. And it has been reported that hyaluronic acid, EGF, and the like can be added to the medium. Several media have been reported as the media for corneal endothelial cell culture (NPL 2).
There is a problem with the culture of corneal endothelial cells, in which corneal endothelial cells morphologically change to fibroblast-like cell during culture period (PTL 4 and NPT 3).
A method to produce a corneal endothelial cell layer with high cell density for transplantation has been reported, in which the corneal endothelial cells are cultured in the presence of a Rho kinase inhibitor to prevent the morphological change of the cells and to promote the cell attachment (PTL 5). In this report, a low glucose DMEM medium supplemented with 15% FCS, 2 ng/mL of b-FGF, and antibiotics has been used as a basal medium for corneal endothelial cell culture (NPT 4). It has been known that Rho kinase is activated when cell death of human ES cells occurs during the culture, and ROCK inhibitors, such as (+)trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl) cyclohexane and 1-(5-Isoquinolinesulfonyl)homopiperazine, prevent this cell death (NPT 5).
Furthermore, a method for culturing corneal endothelial cells has been known, in which Opti-MEM-1 (mfd. by Gibco) supplemented with 20 ng/mL of NGF, 5 ng/mL of EGF, 20 μg/mL of ascorbic acid, 200 μg/mL of CaCl2, 100 μg/mL of pituitary extract, 0.08% (w/v) of chondroitin sulfate and antibiotics is used (NPT 6). However, transplant therapy has not yet been in practical use for improving the function of corneal endothelium of the patient with corneal endothelial dysfunction using the cells obtained by culturing corneal endothelial cells.