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 % CO2
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% CO2)
References
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.