Microelectrode Array in Evaluation of RPE Functionality

Abstract

Purpose: : Damage or malfunction of retinal pigment epithelium (RPE) is a cause of many eye diseases, thus there is demand for methods to replace damaged RPE tissue. We have developed a method that can be used to evaluate the functionality of the RPE cells e.g., differentiated from human embryonic stem cells (hESCs). The method is based on in vitro electroretinography (ERG) of co-culture of mice retina with hESC derived RPE cells. To our best knowledge this has not been attempted before. In this work we present the development of our measurement system.

Methods: : ERG signal from mice retinas and from retina/hESC derived RPE cell co-culture is measured with micro electrode array (MEA) system in vitro (Multi Channel Systems MCS GmbH). Custom made light stimulator (LED light with a wavelength of 490 - 520 nm, intensity 6000 - 26000 lux) was used to stimulate the co-culture and retinal condition and effect of RPE cells were evaluated by ERG response. The cultures were kept in the incubator between the ERG measurements.

Results: : We can reproduce successful preparations and measure ERG responses (a and b wave) from mice retinas. We showed that using either KO-DMEM (15% KO-SR without bFGF) or Ames HEPES medium during the ERG measurement doesn’t affect the measured responses. Long lasting light stimulus does not change the ERG response indicating that ERG originates from the cone response - light sensitive rods have been bleached out during the preparation even though the preparation is carried out under dim red light. The co-culture stopped responding to the light stimuli about seven hours after preparation. Co-culturing the retina together with hESC-RPE cells doesn’t prolong the time the retinas were able to respond to light stimuli.

Conclusions: : Retina and RPE may require longer co-culturing period than reached with the current setup - providing enough nutrition and oxygen for the retina hESC-RPE cultures during the MEA measurements and culturing set up challenges. However, the setup has a potential to reveal RPE cell functionality since the retinal recovery from an intense light exposure causing photopigment bleaching is impossible without the presence of a functional RPE. Thus, dark adaptation could be used as an indicator of the co-culture and thus RPE functionality.

Keywords: electroretinography: non-clinical • retinal culture • retinal pigment epithelium