Fluorescein

Scanning laser ophthalmoscopy (SLO), FA, ICG and OCT were performed using a Spectralis™ HRA+OCT (Heidelberg Engineering, Heidelberg, Germany) device modified for use with rats based on protocols from other studies (Huber et al., 2009 (link); Fischer et al., 2009 (link)). FA and ICG dyes [42 µl fluorescein (Alcon 10%), 208 µl 0.9% NaCl, 250 µl Indocyanine Green (5 mg/ml, Diagnostic Green)] were injected in the tail vein, and in vivo imaging was performed using 488 and 795 nm lasers, respectively. Late phase angiograms were acquired after about 10–20 min. The GFP expression was visualized by imaging in the FA mode before the injection of the dyes.

After the induction of anesthesia and pupil dilation of the animals, the vascular leakage was analyzed with the FA mode of the Spectralis^TM HRA/OCT device. For this 100 µL of 2.5% fluorescein (Alcon, Großwallstadt, Germany) diluted in 0.9% sodium chloride was administered intraperitoneally. Images were acquired 10 min after fluorescein administration (late phase). Vascular leakage at the CNV area was analyzed by quantifying the pixel intensity in two regions of interest (ROI) within and one ROI outside each laser spot using the program ImageJ (National Institutes of Health (NIH), Bethesda, MD, USA), and then the background pixel intensity was subtracted from the laser spot values. All 3 laser lesions were averaged to give one experimental value per eye.

Two weeks after the photocoagulation (day 14), the mice were anesthetized by an intramuscular injection of ketamine and xylazine. After dilation of the pupils and intravenous administration of 0.1 mL of a tenfold saline dilution of 10% fluorescein (Alcon Japan), fundus fluorescein angiography (FFA) was performed with a Micron IV Retinal Imaging Microscope (Phoenix Research Laboratories). The grade of leakage was assigned as described below: 1, “no leakage,” faint hyperfluorescence or mottled fluorescence; 2, “questionable leakage,” hyperfluorescent lesion without a progressive increase in intensity or size; 3, “leaky,” hyperfluorescence increasing in intensity but not in size; 4, “pathologically significant leakage,” hyperfluorescence increasing in both intensity and size.

Pluronic F-127 was provided by Funakoshi Co., Ltd. (Tokyo, Japan), and Tra powder and CA-TRA were kindly donated by Kissei Pharmaceutical Co., Ltd. (Nagano, Japan). A Rat TNF-α Quantikine ELISA Kit was purchased from Bio-techne (Seattle, WA, USA), and fluorescein was obtained from Alcon (Tokyo, Japan). Pluronic F68, Evans blue (EB), isoflurane, ethyl p-hydroxybenzoate, and D-mannitol (Man) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Benzalkonium chloride (BAC) was provided by Kanto Chemical Co., Inc. (Tokyo, Japan), and MC was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). HPβCD and Carbopol (Carbopol^® 934) were obtained from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and Serva (Heidelberg, Germany), respectively. Cell Count Reagent SF was purchased from Nacalai Tesque Inc. (Kyoto, Japan), and penicillin, Dulbecco’s Modified Eagle’s Medium/Ham’s F12, heat-inactivated fetal bovine serum (FBS), and streptomycin were provided by GIBCO (Tokyo, Japan). A Bio-Rad Protein Assay Kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals and organic solvents were of analytical grade.

Thirty microliters of 1% CLZ_nano was instilled into the right eyes of STZ rats twice a day (9:00 a.m. and 7:00 p.m.) for six weeks (repetitive instillation). The eyes were kept open for about 1 min after instillation to prevent the 1% CLZ_nano from being washed out. The wound area (corneal epithelial damage) was stained with 1% fluorescein (Alcon Japan, Tokyo, Japan) and measured using a TRC-50X fundus camera (Topcon, Tokyo, Japan) equipped with a digital camera (the instillation of fluorescein can stain the corneal epithelium damage). The image was obtained 5 h after the instillation (2:00 p.m.).

Whole HROs were imaged with a Zeiss spinning disk confocal microscopy system based on an inverted Axio.Observer Z1 stand with a Yokogawa CSU-X1 and a cage incubator (Supplementary Data 3; Plan Apo 20×/0.8 objective; imaging frequency four frames per hour; total stack sizes 130−200 µm; slice distance 2 µm). For en-face imaging (Fig. 3b) of whole living HROs, the optical plane was positioned at the PIS level and imaged from above at an orthogonal angle (en-face). To capture cell extrusion out of the apical retinal border and ectopic cells positioned on the outside organoid surface, we performed live-imaging by taking optical image stacks acquired with a retinal cross-sectional focus plane (Fig. 3c and Supplementary Movies 2–4). Samples were incubated during live-imaging (phenol red-free N2 + FBS + DMEM/F12 medium, µ-dishes (ibidi), 37 °C, 5% CO₂). The nuclear live-dye SiR-DNA (150 nM, tebu-bio) was added two hours before imaging. At the end of time-lapse imaging, cell ectopy was verified by adding sodium-Fluorescein (Fluorescein Alcon 10%; 1:10,000) to the cell-culture media to differentiate the apical organoid boundary from the surrounding space (Supplementary Movie 4).