Bss plus

BBG 250® (BBG; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in balanced saline solution (BSS® or BSS-plus®; Alcon, Osaka, Japan) to 0.1% (w/v). All grafts were peeled as described previously. BBG (0.1%, w/v) was used to stain the graft edges during peeling. A punch was gently placed on the endothelial surface to indent a circle 7.75, 8.0, or 8.25 mm in diameter. Subsequently, 1.0 and 1.5 mm- diameter dermatological biopsy punches (Kai Industries, Seki, Japan) were used to place asymmetric marks on the edges of the identified circles [19 (link)]. Donor grafts thus marked were stained with 0.1% (w/v) BBG (1.0 mg/mL) for 1 min and stored in BSS prior to insertion, 30 min later [20 (link)]. Because unfolding time depends on donor age, we usually selected donors over 60 years old.

Silicone oil removal was performed via a pars plana entry. First, an infusion cannula was inserted into the vitreous cavity from an incision at the inferotemporal quadrant 3.0 mm away from the limbus in aphakic and pseudophakic eyes or 3.5 mm in phakic eyes. Then, another two pars plana incisions were made at the superotemporal and superonasal quadrants. One incision was used for illuminating the fundus to detect any redetachment at the time of the SO removal. SO was aspirated with a syringe by active aspiration. Then, anterior chamber was washed with a balanced salt solution (BSS Plus, Alcon Laboratories, Fort Worth, Texas, USA) to remove all SO droplets. Incision sites were closed with 7-0 vicryl suture.

The oil droplet was removed from the anterior chamber at 3wpi. Mice were anesthetized by intraperitoneal injection of Avertin (0.3 mg/g) and placed in a lateral position on a surgery platform. Prior to injection, one drop of 0.5% proparacaine hydrochloride (Akorn, Somerset, New Jersey) was applied to the cornea to reduce its sensitivity during the procedure. Then two corneal tunnel incisions were made using a 32G needle: one tunnel incision superior and one tunnel incision inferior to the center of the cornea, each at the edge of the oil droplet. A 33G needle attached to an elevated balanced salt solution plus (BSS Plus, Alcon Laboratories, Ft. Worth, Texas) drip (110 cm H₂O height, equal to 81 mmHg) was inserted through the superior corneal incision to flow BSS into anterior chamber to maintain its volume. At the same time, another 33G needle attached to a 1 mL syringe with the plunger removed, was inserted through the inferior tunnel incision to allow SO outflow. After removing the oil, a small air bubble was injected by a glass micropipette into anterior chamber to maintain the volume of anterior chamber and temporarily seal the corneal incision. Veterinary antibiotic ointment (BNP ophthalmic ointment) was applied to the surface of the eye.

The completely random series was created by the commercial software (SPSS, ver. 13.0; SPSS, Chicago, USA). To dilate pupil for surgery, one eye of each patient was randomly selected as the intracameral group to intracamerally receive 0.1 mL epinephrine hydrochloride 0.01% (adrenaline hydrochloride, Tianjin Jinyao Amino Acid, China) in 0.9 mL of balanced salt solution (BSS Plus, Alcon, USA), giving a concentration of 1 : 100000. The solutions were prepared at the start of surgery by the surgeon (A-Yong Yu), given intracamerally just after cornea incision, and stayed at anterior chamber for 1 minute. It took approximately 3 minutes to complete the whole procedure mentioned above. No placebo eye drops were used in the intracameral group. The contralateral eye, as the topical group, received 3 drops of topical mydriatics (Mydrin, Santen, China), comprising a mixture of tropicamide 0.5% and phenylephrine 0.5%, given topically at 5-minute intervals 30 minutes before surgery. Irrigating solution with 0.4 mL of epinephrine hydrochloride 0.01% in 500 mL balanced salt solution was used in both groups.

Frozen cryovials were maintained in liquid nitrogen for at least 1 day and up to 15 months. To thaw, cryovials were placed in a 37 °C water bath and REPS were removed from cryovials using fine-tipped forceps. Thawed REPS were rinsed in two sequential volumes (2 mL and 4 mL, respectively) of Normal Saline (Quality Biological), Lactated Ringers (Hospira), BSS (Alcon), or BSS PLUS (Alcon) at room temperature for approximately 20–30 s for the first rinse and approximately 1 min for the second rinse. Following the second rinse, REPS were transferred to X-VIVO 10 culture medium and maintained in a 37 °C incubator at 5% CO₂.

iPSC-RPE cells were injected into the subretinal space of Mertk^–/– mice (129 genetic background) and BALB/cJ albino mice. Cultures of iPSC-RPE cells were washed thoroughly with PBS before enzymatic dissociation with TrypLE™. BSS PLUS™ (0065080050; Alcon Laboratories) was added to create a suspension at a concentration of 50,000 cells/μl. Mice (postnatal day (P) 10–16) were anesthetized by isoflurane inhalation. Their pupils were dilated with a drop of 1% (w/v) Atropine Sulfate ophthalmic solution (17478-215-02; Akorn Pharmaceuticals), and the corneas were kept moist with Hypromellose ophthalmic demulcent 2.5% solution (51394-315-15; Wilson Ophthalmic Corp.). A 1-μl suspension of iPSC-RPE cells was injected into the subretinal space of each eye, under a Zeiss Stemi 2000 microscope, as described previously [26 (link)]. Ophthalmic ointment (Neomycin & Polymyxin B sulfates and Dexamethasone, 61314-631-36; Falcon Pharmaceuticals) was applied to each eye immediately following injection. Cyclosporine (200 mg/l, 0078-0109-61; Novartis) was added to the drinking water of the dam from 1 day prior to the injection until the pups were weaned at P28. Mice were kept on a 12-h dark/12-h light cycle. For experiments concerning phagosomes, they were killed between 15 and 30 min after lights on.