A drop array culture for patterning adherent mouse embryonic stem cell-derived neurospheres.

New therapeutic approaches for repairing an injured or degenerating nervous system have accelerated the development of methods to generate populations of neurons derived from various stem cell sources efficiently. Many of these methods require the generation of neurospheres. Here a simple technique is described 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 3 days to prepare from an mESC source; they can be maintained in 15 μl drops of medium, and exhibit extensive neurite elaboration after 8 days of cultivation. Additionally, the potential of treating the adherent neurospheres in selected drops of an array is demonstrated with a variety of differentiation-inducing reagents and subsequently individually analysing such neurospheres for gene expression, protein levels and morphological development.