A Drop Array Culture for Patterning Adherent Mouse Embryonic Stem Cell-Derived Neurospheres

摘要

New therapeutic approaches for repairing an injured or degenerating nervous system have accelerated the development of methods to efficiently generate populations of neurons derived from various stem cell sources. Many of these methods require the generation of neurospheres. Here we describe a simple technique for creating an array of adherent mouse embryonic stem cell (mESC)-derived neurospheres using a conventional plastic culture dish and a patterning template. mESC-derived neurospheres are confined to circular (4 mm-diameter), gel-coated regions within an array. The adherent neurosphere arrays require three days to prepare from an mESC source; they can be maintained in 15 µL drops of medium, and exhibit extensive neurite elaboration after eight days of cultivation. Additionally, we demonstrate the potential of using treating the neurospheres in selected drops of an array with a variety of differentiation-inducing reagents and/ subsequently individually analyzing such neurospheres for protein, gene expression and morphological development.