RPE Cell Culture Protocol
1. Isolate eyes from chick embryos on embryonic days 7, 10, or 14 (E7, E10, or E14).
2. Remove anterior structures, vitreous body, and neural retina.
3. Transfer eyecups to PBS containing 0.54mM EDTA (E7 or E10) or 1.0mM EDTA (E14) and
incubate for 30 min at 37C.
4. Peel RPE from the choroid and incubate for 10 min at 37C in PBS containing 100ug/ml trypsin,
120ug/ml collagenase and 60 ug/ml DNAse.
5. Rinse cells with DME containing 2.5% fetal calf serum and incubate for 2 hr -- then transfer to
either a serum-free medium (SF2), or a 2.5-15% FCS medium.
6. Freshly coat 12mm Transwell filters (Corning Costar Co., Cambridge, MA) with laminin (5
ug/cm2) according to the manufacturer's directions (Collaborative Biomedical Products, Bedford,
MA). [*Cultures fail to form on commercially coated filters or filters on which the laminin is allowed
7. Plate cells on coated filters at a density of 5-7 x 10e5 cells/cm2.
8. For some cultures, the medium in the apical medium chamber is replaced by E14 retinal
conditioned medium, which was prepared by culturing neural retinas that were isolated from E14
embryos in SF2 (12-14 retinas/15ml). The SF2 was supplemented with 1.0mM glutathione and 50
uM ascorbic acid, and the cultures were maintained with gentle agitation for 5hr at 37C in a
humidified atmosphere of 10% CO2 and 40% O2.
Special points to consider:
-- E7 chick embryos showed better results than E10 or E14.
-- Transferring to a 10% FCS at step 5 showed the best results relative to 2.5%, 5% or 15% FCS.
-- Most of the cultures showed clumping, rather than a uniform layer, after a few days of growth.
Prof. Larry Rizzolo has suggested that this may be connected to interbatch differences in laminin.
-- With regard to dissections, according to Arvydas Maminishkis,
"It is very important to remove all visible choroid pieces. Basically, use of stereo-microscope is
absolutely necessary. Usually during dissection I have two tubes for collecting dissected RPE
tissues. One of them is for "Premium" - without any choroid contamination and another for
"second grade" tissue with possibility of some choroid cells. We do dissections in cell culture
room, not in the hood. It is slight chance of contamination because that, but it does not happen
often. Changing media after one day reduces contamination possibility. Another level of sterility
can be added by having Bunsen burner close to the microscope. From microbiology classes - it's
creates sterile area with the radius 20-30 cm. And of course using sterilized instruments."
LT suggests trying as further developmental step, using E7 embryos, a serum-free medium at
Step 5, a new batch of laminin, and dissection with a stereo microscope according to Arvydas'
Stored in 1.5ml Eppendorf Tubes in the -70C freezer in a box labeled "Larry" in 42ul aliquots @
Prof. Lawrence Rizzolo
-- Using chick RPE cultures in differential gene expression experiments.
Prof. Larry Hjelmeland
-- Created the ARPE-19 cell line.
Dr. Arvydas Maminishkis
Section on Epithelial and Retinal Physiology and Disease, NEI/NIH
-- Expert on RPE physiology