Search

Since it was first introduced in 2011, three-dimensional (3D) “Sasai” method for retinal differentiation became a strategy of choice for retinal tissue and neuron production. It is based on the recapitulation of retinal development and requires several stages: aggregate formation, neuroectoderm induction, and eye field induction, followed by retinal maturation. To achieve the consistency of retinal differentiation needed for drug discovery and cell transplantation, we have attempted to improve spheroid formation as well as approach xeno-free conditions. In this study we compared the effect of cell culture plate shape and material, medium viscosity, lipid, and bovine serum albumin concentrations on aggregate formation from mouse embryonic stem (mES) cells. We have also assessed the possibility of substituting Matrigel with the synthetic vitronectin-mimicking oligopeptide. RX-GFP mES cell line was used for experiments. The dose–response of synthetic extracellular matrix (ECM) has been assessed and quantified by live fluorescence microscopy, immunohistochemistry, flow cytometry, and quantitative polymerase-chain reaction (qPCR) for early retinal development genes (Rx, Pax6, Lhx2, Sox2, Six6). The comparison of seeding conditions at 24 h postseeding showed the dose-dependent effects of lipids (lipid concentration of 2% resulted in 100% efficiency of aggregate formation and significant increase in size to 532.8 ± 31.87 μm, p< 0.05); and viscosity (methylcellulose concentration of 0.06% in optic vesicle medium showed 100% efficiency and increase in aggregate size 532 ± 19.23 μm, p< 0.01). The addition of synthetic matrix resulted in retinal differentiation (34.47% of RX as detected by flow cytometry compared with 33.8%, observed with Matrigel). The early retinal genes expression at day 7 was confirmed by qPCR. We present the optimized conditions for 3D retinal differentiation, including the option of xeno-free ECM. These defined medium conditions significantly decrease the variability within and between batches and allow substantial scale up of retinal tissue and cell production for drug discovery, disease modeling, and transplantation purposes.Impact StatementThe development of retinal regenerative therapies relies on the reproducible and renewable source of retinal neurons for drug discovery and cell transplantation. Three-dimensional approach for retinal differentiation from pluripotent cells recently emerged as the robust strategy for retinal tissue differentiation. In this work, we present the combination of optimized conditions and techniques for three-dimensional retinal differentiation from mouse embryonic cells that improves reproducibility and efficiency of retinal differentiation in organoid cultures. We also show that the retinal induction can be achieved with the synthetic oligopeptide instead of Matrigel that allows to approach xeno-free conditions for cell production.