Semi-Automated Approach for Retinal Tissue Differentiation.

Abstract

PurposeThree-dimensional strategy for the differentiation of pluripotent stem cells to the retina has been widely used to study retinal development, although the cell production and drug discovery applications are limited by the throughput. Here we attempted to scale up the protocol using a semiautomated approach.MethodsFor the experiments we used the Rx-GFP mouse embryonic stem cell (mES) reporter cell line, specific for early retinal development and human embryonic stem cell line Brn3b-tdTomato, specific for retinal ganglion cells. To increase the throughput, we implemented automated media exchange using Thermo WellWash Versa with Thermo RapidStack robot. To analyze the rate of retinal differentiation in mouse stem-cell derived organoids we imaged the plates at day 10 of differentiation using Life Technologies EVOS Fl Auto. The automated image analysis of fluorescent images was performed with custom Python OpenCV script.ResultsThe implementation of a semiautomated approach significantly reduced the operator time needed: 34 minutes versus two hours for 960 organoids over the course of 25 days without any change in differentiation pattern and quantity of retinal differentiation. Automated image analysis showed that Forskolin treatment starting from day 1 leads to a significant increase in retinal field induction efficiency.ConclusionsSemiautomated approach can be applied to retinal tissue differentiation to increase the throughput of the protocol. We demonstrated that automated image analysis can be used to evaluate differentiation efficiency, as well as for troubleshooting and to study factors affecting retinal differentiation.Translational RelevanceUsing robotic approach reduces the risk of human error and allows to perform all cycle of cell production in enclosed conditions, which is critical for GMP cell manufacture.