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).

15. This medium is replaced after 1 day with 5% serum-containing RPE medium, and subsequent changes were made every 2 to 3 days.

16. After 3 to 4 weeks, the cells became confluent and uniformly pigmented.

17. They are then trypsinized in 0.25% trypsin-EDTA for 10 to 15 minutes, re-suspended in 15% serum-containing RPE cell culture medium, and seeded onto clear cell culture inserts at 150 to 200K cells per well (Transwell; Corning Costar, Corning, NY), using 12-mm diameter inserts, 0.4-um pores, polyester membranes (example: cat. no. 07-200-161, Fisher Scientific).

18. Before seeding, the wells were coated with human extracellular matrix (10 μg in 150 μL HBSS per well, cat. no. 354237; BD Biosciences, Franklin Lakes, NJ) and cured with UV light in the hood for 2 hours.

19. In some cases, the trypsinization procedure was repeated for a second time, to collect the cells that did not detach after the first trypsinization.

20. The same protocol (excluding coating with ECM) was used to culture cells on the flasks to generate the P1 population of cells.

21. These cells were used in experiments when they had a total tissue resistance of ≥ 200 Ω•cm2 and were uniformly pigmented.

Step by Step Procedures
1. Prepare 12-well plate with multiple solutions (for each eye 4 wells needed):
a. add 10x antibiotic antimycotic solution - 1 well/eye
b. add PBS/HBSS solution for rinse - 2 wells/eye
c. add dispase solution - 1 well/eye

2. Prepare dissecting Petri dish by unpacking 5 fixation needles and filling it with HBSS

3. Unpack eyes and place them into antibiotic-antimycotic solution for 3-5 mins (prepared in step 1)

4. Rinse eyes in two wells (prepared in step 1) with PBS/HBSS, then transfer them to dissecting dish.

5. Trim excessive muscle and connective tissues around the eye

6. Using 27G needles secure (pin down to silicon base) eyes in dissecting dish aligning them in a way where cornea faces up.

7. Using sideport knife make incision below cornea, where circular cut will begin

8. Using iris scissors make cut around the eye, then using same scissors cut through vitreous and lift anterior portion of the eye away.
Note: in steps 3 to 8 avoid any excessive mechanical pressure to eyeball.

9. Transfer open eye into dispase solution (prepared in step 1)

10. Incubate eye cup for 40-60 minutes at 37°C with 5 % CO^₂

11. Replace HBSS solution in dissecting dish with fresh one.

12. Transfer eye from dispase solution to dissecting dish.

13. Position eye in Petri dish (eyeball cup facing up) and secure with two 27G needles.

14. Gently lift partially separated retina and using retinal scissors cut retina away from the optic nerve. Discard retina.

15. Using iris scissors make one incision from periphery of the eye towards optic nerve.

16. Using all five 27G needles flatten eye making RPE layer nicely stretched.

17. Using retinal scissors make circular cut around optic nerve separating RPE layer from attachment to optic nerve.
Note: steps 13-17 can be done with low magnification or without stereo microscope

18. Adjust stereomicroscope to 250x magnification or more and find edge of RPE sheet close to optic nerve along the cut made by iris scissors.

19. Using two forceps separate RPE-Bruch's membrane from the choroidal tissue layer. It may require a few attempts to find area along the edge where the connection of RPE and choroid is weakest.

20. Place RPE sheets into cold trypsin-EDTA solution in 15 mL tube

21. After the RPE are collected, cap and transfer tubes with tissues in trypsin-EDTA into water bath for 10-15mins at 37°C.

22. After 10 mins of incubation, vigorously shake 15 mL tubes to separate RPE into small clusters. If the separation is not complete, place tubes back into water bath for another 5 mins.

23. Inspect the tubes for possible un-dissolved mixed cell clusters. Any observed clusters should be removed using fine tipped glass Pasteur pipette.

24. Spin down (1.4 rpm on clinical centrifuge for 4 mins) hfRPE cells, remove supernatant and re-suspend cells in 15% RPE media (9 mL total)

25. Put 3 mL of cell suspension into Primaria flasks add 2 mL of fresh 15% RPE media, place flask into incubator until next day (37°C, 5% CO^₂)

References
1. Cassin, B. and Solomon, S. (2001). Dictionary of eye terminology. Gainesville, Fla: Triad Pub. Co. ISBN 0-937404-63-2.
2. Boyer MM, Poulsen GL, Nork TM. "Relative contributions of the neurosensory retina and retinal pigment epithelium to macular hypofluorescence." Arch Ophthalmol. 2000 Jan;118(1):27-31.
3. http://www.jove.com