  Purpose: The purpose of the current study was to investigate the effect of
microchannel geometry on neuronal cultures and to maintain these cultures for
long period of time (over several weeks) inside the closed microchannels of
cellular scale dimensions.
  Methods: The primary hippocampal neurons from E-18 rat were cultured inside
the closed polydimethylsiloxane (PDMS) microchannels of varying sizes. The
effect of the channel geometry on the spatial and the temporal variations in
the neural microenvironment was investigated by studying neural maturation and
variation in the media osmolality respectively. The cultures were maintained
for longer time spans by PDMS device pretreatment, control on media evaporation
(by using hydrophobic ethylene propylene membrane) and an effective culture
maintenance protocol. Further, the devices were integrated with the planar
microelectrode arrays (MEA) to record spontaneous electrical activity.
  Results: A direct influence of channel geometry on neuron maturation was
observed with cells in smaller channels maturing faster. The temporal variation
in the microenvironment was caused by several fold increase in osmolality
within 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 height
and width for over 1 month in serum free media condition and the time varying
spontaneous electrical activity was measured for up to 5 weeks using the MEA.
  Conclusions: The understanding of the effect of the culture scale on cellular
microenvironment and such long-term culture maintenance will be helpful in
studying neuronal tissue development; therapeutic drug screening; and for
network level neuronal analysis.
