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Sarah Decembrini, Nathalie Brandenberg, Sylke Hoehnel, Yvan Arsenijevic, M.P. Lutolf; Micro-culture arrays to control the generation of retinal organoids. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1146.
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© ARVO (1962-2015); The Authors (2016-present)
The feasibility to recapitulate mammalian retinogenesis in vitro by growing self-organizing retinal organoids from pluripotent stem cells was recently demonstrated by our and other laboratories. We observed that retinal organoids are not completely synchronized in culture, resulting in retinas with different sizes, shapes, and apical-basal orientation suggesting the need to improve the adopted cell culture conditions.
We attempt here to standardize an approach, based on microwell arrays, that can be used to screen compounds to reproducibly generate retinal organoids meanwhile saving time, space and materials. Different biomaterials were tested as culture substrates of microwell arrays. The stiffness, quantity of hydrogels, well shape and size, volume of the reaction and medium composition as well as the effect of cell number were evaluated in respect to the efficiency to generate retinal organoids.
Different qualities and stiffness of hydrogels were tested in order to establish the best culture substrate supporting retinal organoid generation. Agarose, noble agarose, polyethylene-glycol (PEG) and polydimethylsiloxane (PDMS) were assessed. The results highlight the higher propensity of 5% w/vol PEG, to generate retinal organoids in comparison to all the other tested materials. AggreWellTM (Stemcell Technologies) served in primis as template to construct the desired well topography. Flat- or V- bottom wells, round or square with a diameter of 400 or 800 µm were tested. The shape and size of the microwells tested were not able to support the generation of the retinal organoids. A standard photolithography was developed to generate an unlimited well size and geometry. The optimal microwell size and shape was identified in 1.5mm height (1.7 max), 1.7mm width, U-bottom wells. Such microwells allow the aggregate formation in less than 6 hours. Different medium conditions, supplements, matrigel concentrations and surviving factors were assessed to fine-tune the retinal organoid production. The results suggest the requirement of factors to induce retinal organoids different than those utilized with plastic wells revealing some distinctive properties of the microwells that are currently under investigation.
We conclude that the microwell plates support the survival and differentiation of retinal organoids and can be used to screen morphogens aimed to improve the development of retinal organoids.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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