@article {1445329,
	title = {Optimized conditions and use of synthetic matrix for retinal differentiation from pluripotent cells},
	journal = {Tissue Eng Part C Methods},
	year = {2019},
	month = {2019 Jun 14},
	abstract = {PURPOSE: Since it was first introduced in 2011, three-dimensional "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. In order 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. METHODS: 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 cells. We have also assessed the possibility of substituting Matrigel with the synthetic vitronectin-mimicking oligopeptide. RX-GFP mES cell line used for experiments. The dose-response of synthetic ECM has been assessed and quantified by live fluorescence microscopy, immunohistochemistry, flow cytometry and qPCR for early retinal development genes (Rx, Pax6, Lhx2, Sox2, Six6). RESULTS: The comparison of seeding conditions at 24hr. post seeding showed the dose-dependent effects of lipids (lipids concentration of 2\% resulted in 100\% efficiency of aggregate formation and significant increase in size to 532.8 {\textpm} 31.87um, p\&lt; 0.05); and viscosity (methylcellulose concentration of 0.06\% in OV medium showed 100\% efficiency and increase in aggregate size 532{\textpm}19.23 um, p\&lt;0.01). The addition of synthetic matrix resulted in retinal differentiation (34.47\% of RX as detected by flow cytometry compared to 33.8\%, observed with Matrigel). The early retinal genes expression at day 7 was confirmed by qPCR. CONCLUSIONS: We present the optimized conditions for 3D retinal differentiation including the option of xeno-free extracellular matrix. 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.},
	issn = {1937-3392},
	doi = {10.1089/ten.TEC.2019.0053},
	author = {Perepelkina, Tatiana and Kegeles, Evgenii and Baranov, Petr Y}
}
