Purchase this article with an account.
Stephanie Proulx, Rémi Parenteau Bareil, Olivier Rochette-Drouin, Solange Landreville; Isolation Of Several Choroidal Cell Types And Retinal Pigmented Epithelial Cells From A Single Rabbit Eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4719.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Melanocytes, fibroblasts and endothelial vascular (EV) cells from the choroid as well as retinal pigment epithelial (RPE) cells are required to generate functional tissue-engineered choroidal substitutes. The purpose of this study was to develop an isolation protocol that enables the establishment of pure cultures of choroidal and RPE cells.
Enucleated rabbit eyes were cut in a half just below the ora serrata. The retina and the optic nerve were then carefully removed. A 20 min dispase treatment (2.5%) and/or gentle pipetting were used to separate small sheets of RPE that were then put in culture. After a complete removal of the RPE, the choroid was peeled out of the sclera and incubated overnight in collagenase H (0,125U). The resulting cell suspension was centrifuged and incubated 10 min in trypsin-EDTA (0,05%-0,01%). The cell suspension was then filtered with a cell strainer. Dynabeads and anti-CD31 antibody were used to sort choroidal EV cells from the cell suspension. The residual mixture was equally plated in two selective growth media: a non-restrictive medium allowing fibroblast proliferation and a melanocyte growth medium containing G418 selection agent for eradication of contaminating fibroblasts. Cells were seeded onto glass coverslips and purity was assessed by phase contrast microscopy and immunofluorescence (K8/18, CD31, HMB45, vimentin).
The method described herein allowed for the isolation of four ocular cell types. RPE cells expressing K8/18 were successfully isolated. EV cells positive for CD31 were expended for at least three passages without any loss of phenotype. Pure cultures of HMB45-positive melanocytes were obtained after a two-week culture period in G418 selective medium. Finally, cultures of fibroblasts positive for vimentin and negative for the other markers were established after the third passage. At low passages (P1), less than 1% of melanocytes were present in the fibroblast cultures. Melanocyte contamination was absent at higher passages (P3 and over).
Our results show the feasibility of obtaining pure cultures of four ocular cell types required to develop functional tissue-engineered choroidal substitutes. These substitutes may be used as allogeneic sub-retinal grafts in preclinical studies for the treatment of retinal diseases.
This PDF is available to Subscribers Only