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Tatiana Perepelkina, Petr Y Baranov; Optimization of xeno-free 3D culture method for retinal differentiation from pluripotent cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):566.
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© ARVO (1962-2015); The Authors (2016-present)
Since it was first introduced in 2011, three-dimensional “Sasai” method for retinal differentiation became a promising strategy for retinal neuron production. It is based on the recapitulation of retinal development and requires several stages: aggregate formation, neuroectoderm induction, eye field induction/retinal maturation. In order to achieve the reproducibility 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 concentration on aggregate formation from mouse embryonic mES. We have also studied the possibility of substituting Matrigel with the synthetic vitronectin-mimicking oligopeptide (Synthemax 2 SC) as a ligand for Integrin receptor activation, needed for eyefield induction. RX-GFP mES was used for experiments. The dose-response has been assessed and quantified by live fluorescence microscopy, immunohistochemistry, flow cytometry and qPCR for early retinal development genes (Rx, Pax6, Lhx2, Sox2, Six6).
The comparison of seeding conditions 24hr after seeding showed the dose-dependent benefit of lipids (lipids concentration of 2% in OV medium showed 100% efficiency of aggregate formation and significant increase in size 532.8 ± 31.87um, p< 0.05) and viscosity, controlled by methylcellulose (methylcellulose concentration of 0.06% in OV medium showed 100% efficiency and increase in aggregate size 532±19.23 um, p<0.01). The addition of Synthemax 2CS at concentrations from 0.005 mg/ml to 0.008 mg/mml resulted in retinal differentiation (40-55% of RX as detected by flow cytometry compared to 55-65%, observed with Matrigel). The early retinal gene expression at day 7 was confirmed by qPCR.
We present the optimized conditions for 3D retinal differentiation, including the option of xeno-free culture. These defined medium conditions will significantly decrease the variability within and between batches, decrease the risk of contamination while allowing substantial scale up of retinal tissue and cell production for drug discovery, disease modeling and transplantation purposes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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