Toxicity responses in human retinal pigment epithelium cells

Summary: An in vitro assay to help assess and predict drug-induced toxicity on the retinal pigment epithelium (RPE) cells.

Model description

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 

Fig. 1. Expression and localization of tight junction proteins, occludin (left panel), zonula occludens 1 (ZO-1, right panel) in novel RPE cells derived from human induced pluripotent stem cells (PCi-RPE1426). Scale bar 50 µm.
Fig. 1. Expression and localization of tight junction proteins, occludin (left panel), zonula occludens 1 (ZO-1, right panel) in novel RPE cells derived from human induced pluripotent stem cells (PCi-RPE1426). Scale bar = 50 µm. 
Fig. 2. : Transmission electron microscopy images of vertical cross sections of RPE cells showing the intracellular cell organelles, MV, microvilli; M, melanosome; N, nucleus.
Fig. 2. Transmission electron microscopy images of vertical cross sections of RPE cells showing the intracellular cell organelles, MV = microvilli; M = melanosome; N = nucleus. Scale bar = 2 µm. 
Fig. 3. : Novel RPE cells (PCi-RPE1426). Scale bar 100 µm.
Fig. 3. Novel RPE cells (PCi-RPE1426). Scale bar = 100 µm. 

Authors' picture