Patterning of Primary Retinal Cell Cultures on Multilayer Nanofilms

Abstract

Abstract: : Purpose: Cell growth can now be manipulated using microscale tools that provide high–definition, reproducible patterns of a variety of substrates. These microscale patterns will contribute to our understanding of cell–substrate attachment, factors controlling cell shape, and cell survival, migration, and neurite outgrowth. Because of the potential for retinal neurons to form networks in culture, we have constructed micropatterns of fluorescently labeled nanofilm scaffolds on glass substrates to investigate neuronal and glial patterning of retinal cells in vitro. Methods: Primary retinal cells were isolated from 1– to 3–day–old Sprague Dawley rat pups, plated at 200,000 cells/ml in culture medium containing 10% FBS, and after 1 day in vitro, switched to a defined, serum–free Neural Basal medium, in which the cells remained thereafter. Cells were plated onto glass coverslip substrates upon which two multilayer nanofilm scaffolds were fabricated: 1) secreted phospholipase A₂ labeled with fluorescein isothiocyanate (sPLA₂–FITC) and 2) poly–l–lysine labeled with tetramethyl rhodamine isothiocyanate (PLL–TRITC). The FITC (green) and TRITC (red) nanofilm scaffolds allowed us to distinguish discrete areas of cell attachment and growth using fluorescence microscopy. For comparative purposes, rat cortical neurons from Sprague Dawley embryos were plated onto identical coverslip substrates. Results: Retinal cells attached to both FITC and TRITC scaffolds, surviving in culture for at least one month. Cells of glial morphology attached preferentially to PLL–TRITC scaffolds. Cells of neuronal morphology attached in groups to both sPLA₂ and PLL scaffolds, and single cells formed fine, neurite–like processes preferentially on the sPLA₂–FITC scaffolds, consistent with parallel experiments using cortical neurons. In retinal cultures, the areas of greatest neuronal attachment were where glia were growing. This contrasted with brain cortical neurons, which were able to grow separately from glial groups. Conclusions: Multilayer nanofilm scaffolds of two or more compositions on the same cell–culture substrate could be a useful technology for selectively attaching different cell types from the retina. Having demonstrated here that retinal neurons and glia attach to fluorescently labeled sPLA₂ and PLL nanofilms, respectively, we are utilizing these micropattern arrays to optimize neuron–glia cell communication as a model to explore issues relevant to retinal development and disease.

Keywords: retinal culture • ganglion cells • retinal glia