Monkey surgeries for this study were conducted by a research group of fully trained ophthalmologists and veterinarians who hold proper licenses.
Six female cynomolgus monkeys (2.0–2.5 kg; Keari Co., Ltd.) were anesthetized intramuscularly with a mixture of ketamine hydrochloride (5 mg/kg; Sankyo, Tokyo, Japan) and xylazine (1 mg/kg; Bayer, Munich, Germany) followed by inhalation anesthesia with isoflurane. Transplantation was performed on the right eye of each animal only and was performed in an animal surgery room at the same levels of cleanliness as for human keratoplasty. During surgical procedures, animals were observed by veterinarians monitoring pulse, blood pressure, and Pao 2. To induce endothelial dysfunction 3-mm limbal–corneal incisions were made in six eyes of six monkeys, and the corneal endothelia were mechanically scraped with a 20-gauge silicone needle (soft tapered needle; Inami, Tokyo, Japan). The scraped area measured at least 9 mm in diameter (the diameter of the cornea is ∼10 mm), and the denuded area was confirmed by 0.04% trypan blue staining during surgery. In preliminary experiments we confirmed that the mechanically scraped area had no cells on Descemet’s membrane, and residual corneal endothelial cells were detected in only a 500- to 600-μm area at the edge of Descemet’s membrane. At surgery, the limbal–corneal incision was spread to 6-mm, and a 6-mm diameter disc of a cultivated MCEC sheet was brought into the anterior chamber in four eyes of four animals on a supportive carrier (Lens Glide; Alcon, Tokyo, Japan) with the corneal endothelial side facing the anterior chamber. In one of the surgeries a DiI-labeled cultivated MCEC sheet was used. In all cases the limbal–corneal incision was closed with 10-0 nylon interrupted sutures, and the cultivated MCEC sheet attached to Descemet’s membrane by air injection. As the control, a collagen sheet without MCECs was transplanted in one eye of one animal with endothelial dysfunction, and a suspension of cultivated MCECs (8 × 104 cells in 50 μL intraocular irrigating solution; OpeGuard Neo Kit; Senju, Osaka, Japan) was injected into the anterior chamber in one eye of another. The number of injected MCECs was the same as the number of MCECs on one 6-mm diameter disc of cultivated MCEC sheet. After surgery, all animals had a subconjunctival injection of dexamethasone (1.0 mg), followed by 0.1% betamethasone ointment once a day for 10 days. No systemic immunosuppression was used.