Dynamic Full-Field OCT to image RPE cell cultures

Abstract

Purpose : Dynamic Full-Field OCT (D-FFOCT) is able to discriminate cells with different metabolic activity without contrast agents. Retinal pigment epithelium (RPE) is involved in many retinal diseases, including Age-related Macular Degeneration (AMD). Here, we used RPE cell cultures as a model to study behavior in the basal state and in response to acute damage by live imaging with D-FFOCT.

Methods : D-FFOCT is a non-invasive micrometric live imaging technique whose contrast is created by intrinsic subcellular organelle movement. Human induced pluripotent stem cell (hiPSC)-derived RPE and primary porcine RPE cell cultures, grown on polycarbonate membranes, were analyzed. The dynamic intracellular signal was recorded with 0.5µm transverse x 1.5µm axial resolution over periods of several hours. Immunochemistry was performed in order to validate results and decipher the subcellular structures generating the D-FFOCT signal. We performed scratch assays with a scalpel blade on RPE cultures and observed and quantified the dynamic response behavior of cells.

Results : The porcine RPE closed with a speed of ~20 µm/h, while for hiPSC derived RPE, we measured ~5 µm/h. The difference in speeds between porcine and human samples may be related to their different origin (hiPSC cells versus primary culture). Interestingly, if the membrane on which the RPE is grown was cut, or if the wound was too large (>40µm), after failing to close the wound through migration, both parts retracted, increasing the wound size. D-FFOCT allowed us to quantify the increase in intracellular activity around the damaged area.

Conclusions : D-FFOCT seems a convenient tool to image live RPE cell cultures, to measure both metabolic cell activity and speed of wound closing following acute damage. D-FFOCT allowed us to document behavior of healing RPE cells, showing that large wounds tend to retract rather than close, hence mimicking atrophic AMD expansion.

This is a 2020 ARVO Annual Meeting abstract.

 

Timepoints before (top), just after (middle) and 1-2 hours after (bottom) scratch assays on porcine (left) and hiPSC-derived (right) RPE cell cultures. The wound on porcine RPE closed in almost one hour, at ~20 µm/h, whereas in human RPE, closing was slower (~5 µm/h). Colorbar indicates metabolic activity level (warm/bright = active).