Generation of microisland cultures using microcontact printing to pattern protein substrates

The capacity to isolate small numbers of neurons in vitro is an essential tool to study the cell biology of synapses and the development of neuronal networks by specific cell types. Microisland culture assays allow for single neurons, or simple neural networks, to be isolated on islands of glial cells; however, the techniques commonly used to produce microisland substrates are expensive, challenging to control, and typically result in many discarded substrates. Here, we used microcontact printing to pattern a glass surface with islands of extracellular matrix proteins known to support neural cell growth and differentiation. To promote segregation of the cells to the islands, the substrate surrounding the islands was backfilled with polyethylene glycol (PEG), forming a relatively non-permissive surface on which cell attachment is limited. Astrocytes, and subsequently hippocampal neurons, were then seeded onto the islands of patterned protein. Using this method, readily reproducible patterns of protein islands were produced that permit cell attachment, differentiation, and growth. The technique is a rapid, inexpensive, and reliable means to generate patterned substrates appropriate for microisland cultures.