The main disadvantage of monolayer methods is the lack of spatial organization that is found in the brain. Moreover, monolayer approaches confer highly unphysiological mechanical and adhesive properties to differentiating neurons, which alter neuronal morphology and gene expression. As a result, the quantity and types of neurons produced are highly dependent upon subtle variations in cell-to cell contacts and interactions, which are in turn influenced by the initial plating density of the neuronal progenitors, the adhesive substrate, and the exogenous factors added to the preparation.
