3-hydroxycholest-7-ene-14-carbaldehyde

Research corneas were incubated in three washes of antibiotic/antimycotic solution in PBS, 15-minute each. Primary cultures of hCECs were established as described. Primary hCECs were isolated using a two-step, peel-and-digest method. Corneoscleral rims were placed endothelial-side-up on a disposable cornea vacuum punch (Ripon, England), and mildly stabilized by the vacuum suction created (Figure 2A). A brief 30 seconds treatment with Trypan Blue solution (0.2%) was used to delineate Schwalbe's line. The DM-endothelial layer was carefully stripped off, approximately 1 mm anterior to the Schwalbe's line (away from the trabecular meshwork) from the posterior stroma under the dissecting microscope (Nikon, Japan). Paired DM-endothelial layers obtained were pooled and digested enzymatically in collagenase A (2 mg/ml) for at least 2 hours and up to 6 hours. This allowed full detachment of the CE from the DM, which tended to conglomerate into tightly-packed hCEC clusters. The hCEC cultures were rinsed once in PBS and further dissociated in TE for 5 minutes. Cell pellets collected after a mild centrifugation (800 g for 5 minutes) were plated equally into organ-culture dishes coated with FNC coating mixture, in four culture conditions coded as M1, M2, M3, and M4 (Table 1). All incubation and cultivation of hCECs were carried out in a humidified incubator at 37°C containing 5% CO₂. Fresh media were replenished every two days.
After primary cultures of hCECs reached confluence at P0, cells were dissociated using TE, and sub-cultured on FNC-coated culture dishes at a matched plating density of 5,000 cells/cm². Subsequent passages of hCECs (P1 through to P3) were also dissociated using TE. During the course of the study, cultures with insufficient cell numbers for subsequent passage were excluded. A Nikon TS1000 microscope with a Nikon DS-Fi1 digital camera was used to capture phase contrast images during expansion and at confluence to document general hCEC morphology. Variation in hCEC size (polymegathism) and the variation in cell shape (pleomorphism) of confluent cultures at P0 were assessed using Nikon NIS-Elements basic research software (Nikon, Japan). In each culture condition, the mean and standard deviation of cultivated corneal endothelial cell sizes were calculated, from which a coefficient of variation index in cell area (SD/mean cell area X 100) was calculated. The closer the calculated index value was to zero, the more uniform the overall cell sizes were and vice versa [22] (link).

The monkey corneal endothelial dysfunction model was created by scraping the corneal endothelium completely from the Descemet’s membrane with a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan) while the animal was under general anesthesia, as described previously8 (link). The MCEC injection experiments were conducted on the following 3 groups: 1) MCECs (5.0 × 10⁵ cells) were suspended in 200 μl of DMEM supplemented with 100 μM of Y-27632 (ROCK inhibitor; Wako Pure Chemical Industries, Ltd.) and injected into the anterior chamber (n = 6), (2) MCECs (5.0 × 10⁵ cells) were suspended in 200 μl of DMEM and injected into the anterior chamber (n = 2), and (3) no MCECs were injected (n = 2). The HCEC injection experiments were conducted on the following 4 groups:1) HCECs (5.0 × 10⁵ cells) were suspended in 200 μl of OptiMEM-I supplemented with 100 μM of Y-27632 (Wako Pure Chemical Industries, Ltd.) and injected into the anterior chamber (n = 8), (2) HCECs (5.0 × 10⁵ cells) were suspended in 200 μl of OptiMEM-I and injected into the anterior chamber (n = 2), (3) human pre-cut donor corneas were transplant using a DSAEK procedure (n = 2), and (4) no HCECs were injected(n = 2). The eyes were kept in the face-down position for 3 hours under general anesthesia, except for the eyes of monkeys that underwent DSAEK. The corneal transparency and thickness of the anterior segments were evaluated by slitlamp microscopy. A Pentacam^® (OCULUS Optikgeräte GmbH, Wetzlar, Germany) instrument was used to visualize the corneal shape. Corneal thickness was determined with an ultrasound pachymeter (SP-2000; Tomey, Nagoya, Japan), and the mean of 10 measured values was calculated (up to a maximum thickness of 1200 μm, the instrument’s maximum reading). Intraocular pressure was determined by a Tonovet^® (icare Finland, Vantaa, Finland) instrument. The corneal endothelium was evaluated by non-contact specular microscopy (FA-3809, Konan Medical, Nishinomiya, Japan). Eyes that exhibited clinical features such as the presence of keratic precipitates, progression of corneal edema, and conjunctival injection were diagnosed as having undergone graft rejection.

Cellular ROS production was measured using a DCFDA assay kit according to manufactory’s protocol. DCFDA (2’,7’-dichlorofluorescein diacetate), a cell-permeable fluorogenic dye, is deacetylated by cellular esterases to a non-fluorescent compound, and later oxidized by ROS into highly fluorescent 2’,7’-dichlorofluorescein (DCF), which measures hydroxyl, peroxyl and other ROS activity within the cell. HCECs were grown on 96-well plates or 8-chamber slides. When reached confluence, the cells were washed twice with phosphate-buffered saline (PBS) and then incubated with 25 μM DCFDA in essential medium with 10% FBS in 37°C incubator for 45 min. After PBS washed twice, the cells were exposed to hyperosmotic media (350, 400 or 450 mOsM) by adding NaCl (19, 44 or 69 mM, respectively) for different time periods (30–180 min). Cells images were taken under fluorescent microscope. Cell fluorescence in 96-well plates was measured at 488 nm excitation and 525 nm emission using Tecan Infinite M200 Multimode Microplate Reader (Tecan US, Inc. Morrisville, NC) after adding NaCl for 15–120 mins. Relative changes of DCF fluorescence were expressed as fold increase over untreated cells.

HCEC-B4G12 (human corneal cell line) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) and cultured according to the recommended conditions. These cells were maintained in a complete medium, which consisted of 89% Dulbecco’s modified eagle medium, 10% FBS, and 1% penicillin/streptomycin. All cell cultures were incubated at 37 °C in a humidified environment with 5% CO₂. Next, the five types of Mt (Na-Mt, H-Na-Mt, C-H-Na-Mt, Ca-Mt, and MMt) were respectively dispersed in phosphate-buffered saline (PBS) to prepare stock solutions (50 mg/mL). The stock solutions were diluted to different concentrations within a culture medium containing 2% FBS and 1% penicillin/streptomycin, just before the cells’ treatment. The cells were adjusted to a concentration of 1 × 10⁵ cells/mL, in a volume of 100 μL per well in 96- well plates, for the below toxicity assays.

The production of intracellular ROS in HCEC-B4G12 cells was detected by H₂DCF-DA staining as described previously [30 (link)]. Briefly, H₂DCF-DA (10 μM) was used to treat HCEC-B4G12 cells for 30 min in a cell culture incubator. To remove the unbound dye, the cells were washed three times with PBS and then exposed to Na-Mt and C-H-Na-Mt of various concentrations (3.13–50 μg/mL) with a phenol red-free medium. Subsequently, the cells were incubated continuously and the oxidation of H₂DCF-DA was later detected by CLSM (Zeiss LSM 800, Germany) at 2, 6, 12, and 24 h. In vivo, the levels of corneal ROS were measured via DHE staining of 8-μm-thick corneal cryosections. Briefly, cryosections were fixed with 4% paraformaldehyde at room temperature for 60 min. Then, the sections in 10 μM DHE were incubated for 30 min in the dark, after which DAPI was added to counterstain and seal each after rinsing with PBS for corneas. Three sections of each rat were analyzed and photographed using CLSM (Zeiss LSM 800, Germany).

Using a dose of 50 μg/mL, the five types of Mt (Na-Mt, H-Na-Mt, C-H-Na-Mt, Ca-Mt, and MMt) were respectively used to treat HCEC-B4G12 cells for 24 h, after which the cells were seeded in 6-cm-diameter dish and cultured overnight at 37 °C in a 5%-CO₂ incubator. The cells were washed three times with PBS and collected, then fixed overnight with 2.5% glutaraldehyde solution at 4 °C. After undergoing gradient dehydration and embedding, the sample cells were sectioned by ultrathin slicer, stained and dried, and examined with transmission electron microscope (TEM; Hitachi H7650, Japan).