Summary: An in vitro assay to help assess and predict drug-induced toxicity on the retinal pigment epithelium (RPE) cells.
An in vitro assay that allows for the assessment and prediction of study compound-induced toxicity on the retinal pigment epithelium (RPE) cells. RPE is a tight cell monolayer between retina and choroidal blood. RPE forms the outer blood-retinal barrier (oBRB) restricting the drug entry to retina after systemic administration.
RPE cell monolayers represent tight paracellular barrier and several characteristics of RPE (Hakkarainen et al. 2016), thus, this in vitro cell model can be used reliably to screen drug candidates in early phase of drug development.
|Cells||RPE cells derived from human induced pluripotent stem cells (PCi-RPE, Phenocell, France; Maruotti et al. 2013)|
|Viability assessment||PCi-RPE1426 cells are grown on 96-well plates. The cells can be exposed to testing molecules or formulations for selected times (e.g. 4h, 24h). Cytotoxicity is measured using resazurin reduction assay (Nociari et al., 1998). A cell permeable redox indicator resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) is used to measure oxidation-reduction reactions in viable cells. The non-fluorescent resazurin is irreversibly reduced by metabolically active cells to highly fluorescent metabolite resorufin (7-hydroxy-3H-phenoxazin-3-one). Resorufin formation is measured by using Cytation 3 multi-mode reader (BioTek Instruments, Winooski, VT, USA). The quantity of resorufin produced is proportional to the number of viable cells.|
|Positive control||tert-butyl hydroperoxide (tBHP) at concentrations 0-30 mM|
|Read-outs||1. Cell viability (%)|
2. IC50 value (50% cell viability) for positive control
3. IC50 value is calculated for study compound if applicable
Outcomes and Read-Outs
Characterization of hRPE cells
- Hakkarainen JJ, Maruotti J, Seppänen, Onteniente B, Kalesnykas G, Reinisalo M. hiPSC-derived RPE cells: Characterization of blood-retinal barrier properties and drug permeability. ARVO2016 Poster. Investigative Ophthalmology & Visual Science September 2016, Vol.57, 268.
- Maruotti J, Wahlin K, Gorrell D, Bhutto I, Lutty G, Zack DJ. A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. Stem Cells Transl Med. 2013,2(5):341-354. doi: 10.5966/sctm.2012-0106
- Nociari et al. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods. 1998; 213(2):157-167. PMID: 9692848 DOI: 10.1016/s0022-1759(98)00028-3