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Michael Jochner, Stephan Reichl; In vitro model of the outer blood-retina barrier for testing of novel drugs and drug formulations. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4593.
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The outer blood-retina barrier (BRB) formed by retinal pigment epithelium cells (RPE) is a strong barrier for drugs from bloodstream targeting retinal diseases. The aim of this study was to establish appropriate isolation and cultivation protocols for porcine primary RPE cells as first step towards in vitro BRB models for preclinical drug testing. The primary model was compared with the human ARPE-19 cell line as a commonly used BRB model.
Primary RPE cells were isolated form freshly enucleated porcine eyes using enzymatic and mechanical methods. For cultivation of primary RPE or ARPE-19 cells different media compositions, serum contents and coatings were evaluated. Cell characterization was performed by microscopical examinations and immunostaining as well as measurements of biological barrier integrity via transepithelial resistance (TEER) studies.
Highest cell purity (RPE65-immunostaining positive) was obtained by using a sucrose gradient method. Optimal cultivation medium was DMEM:F12 (1:1) supplemented with 365.3 mg/L L-glutamine and 1 % FCS. Cultures of primary cells were naturally pigmented in contrast to ARPE-19 cells. We observed that primary RPE cells lose their pigmentation over time. Both ARPE-19 and primary cells formed polar monolayers, but only primary cells exhibited the typical cobblestone morphology. However, in contrast to ARPE-19 cells, primary cells could only be carried up to a few passages (2-3) and lose their properties and viability thereafter. In accordance to previous reports, ARPE-19 cells exhibited TEER values not higher than 100 Ωcm2. For primary cells TEER values ranging from 175 ± 9 Ωcm2 to 320 ± 25 Ωcm2 were determined after 28 days in culture, depending on isolation methods and culture conditions.
Primary RPE cells have been successfully isolated and cultivated. Plating density was detected as critical parameter to obtain polar, well organized monolayers of hexagonal RPE cells and to avoid their de-differentiation. ARPE-19 cells are easy to culture and express some characteristics of RPE cells. However, they lack major barrier properties. In order to combine positive aspects of primary porcine RPE cells and immortalized cells, in a next step we are focusing on establishing a new cell line of lentivirally immortalized RPE cells with improved characteristics.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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