The pigmented layer of retina or retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells, and is firmly attached to the underlying choroid and overlying retinal visual cells.
Cell Culture
On receipt, intact globes are rinsed in antibiotic
antimycotic solution (diluted to 10X; cat. no. 15240-096; Invitrogen)
plus gentamicin (1 mg/mL) for 3 to 5 minutes.
1. After incubation antibiotics are rinsed off twice with medium such as HBSS or PBS.
2. One eye globe at the time is transferred to 10 cm Petri dish with coated with Sylgard-184 (WPI) and secured with 27G needles.
3. Using ClearCut sideport knife (Alcon) an incision is made through the sclera below the ciliary body (1/3 of the distance from eye equator to the anterior surface).
4. This incision is used to start a circular cut for removal of the anterior eye portion.
5. This cut is made using a tungsten- carbide coated curved iris scissors with one blade serrated (FST).
6. Prior to the removal of the anterior part of the eye, a cut is made through the vitreous body to avoid detaching the retina from the RPE at the posterior pole.
7. After the anterior portion of the eye is removed, the posterior pole is incubated with dispase-I solution (2 U/mL, cat. no. 04942086001; Roche Diagnostics, Indianapolis, IN) in 5% serum containing medium for 40-60 minutes in 37°C-5% CO2.
8. After dispase treatment, the posterior poles are transferred to a HBSS in Petri dishes with silicon padding (Sylgard 184; Dow Corning, Midland, MI) and dissected into quadrants or larger pieces to sufficiently flatten tissues.
9. Then the retina is gently removed with forceps. Single-cell RPE layers were peeled off in sheets and collected directly into cold trypsin-EDTA (Gibco, #25200-056) solution.
10. After the RPE are collected, tubes with tissues in trypsin-EDTA are sealed and transferred into water bath for 10-15 mins at 37°C.
11. After 10 mins of incubation, the tubes are vigorously shaken to separate RPE into small clusters.
12. If the separation is not complete, the tubes are placed back into water bath for another 5 mins. After trypsin-EDTA incubation, the tubes are inspected for possible un-dissolved mixed cell clusters.
13. Any observed clusters are removed using fine tipped glass Pasteur pipette.
14. After spinning down (1.4 rpm on clinical centrifuge for 4 mins), hfRPE cells are re-suspended in 15% RPE media and then put into Primaria flasks (example: cat. no. 08-772-45; Fisher Scientific, Pittsburgh, PA).
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