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Purpose: The purpose of the current study was to investigate the effect ofmicrochannel geometry on neuronal cultures and to maintain these cultures forlong period of time (over several weeks) inside the closed microchannels ofcellular scale dimensions.  Methods: The primary hippocampal neurons from E-18 rat were cultured insidethe closed polydimethylsiloxane (PDMS) microchannels of varying sizes. Theeffect of the channel geometry on the spatial and the temporal variations inthe neural microenvironment was investigated by studying neural maturation andvariation in the media osmolality respectively. The cultures were maintainedfor longer time spans by PDMS device pretreatment, control on media evaporation(by using hydrophobic ethylene propylene membrane) and an effective culturemaintenance protocol. Further, the devices were integrated with the planarmicroelectrode arrays (MEA) to record spontaneous electrical activity.  Results: A direct influence of channel geometry on neuron maturation wasobserved with cells in smaller channels maturing faster. The temporal variationin the microenvironment was caused by several fold increase in osmolalitywithin 2-3 days due to rapid media evaporation. With our culture methodology,neurons were maintained in the closed channels as small as 50 microns in heightand width for over 1 month in serum free media condition and the time varyingspontaneous electrical activity was measured for up to 5 weeks using the MEA.  Conclusions: The understanding of the effect of the culture scale on cellularmicroenvironment and such long-term culture maintenance will be helpful instudying neuronal tissue development; therapeutic drug screening; and fornetwork level neuronal analysis.

Neuronal micro-culture engineering by microchannel devices of cellular scale dimensions