Topologically Defined Neuronal Networks Controlled by Silicon Chips

CHAPTER 2. NETWORKS OF DEFINED TOPOGRAPHY
Figure 2.6: Schematic illustration of the patterning procedure.
2.3.1 Patterns of growth-promoting proteins
Tracks of intact extracellular matrix proteins (ECM) surrounded by UV-inactivated areas have shown
to reliably guide growing leech neurites [35]. This technique has been adapted to the requirements of
Lymnaea neuronal cell culture by A. Prinz [79]. Instead of ECM, substrate-adsorbed material (SAM)
is patterned with UV light. The procedure involves the following steps, which are also illustrated in fig.
2.6:
– deposit a layer of SAM on glass coverslips as described in section 2.2.2
– remove conditioning brains, supernatant and flexiPERM adhesive culture chambers (in vitro Systems
& Services, Osterode, Germany) and dry the coated coverslips in a sterile flow hood
– 20min exposure to the full spectrum of a 200W mercury lamp (Osram HBO 200, intensity of the
366nm-line approximately 170mW/cm 2 ) through a lithographic mask in direct contact with the
proteinacious layer
– remove the mask and rinse with defined medium to wash away denatured protein from exposed
areas
– attach flexiPERM chamber and refill with DM or a 1:1 mixture of DM and supernatant
– place neurons on the patterned substrate and cultivate for two to three days
Masks consist of 1mm-2mm thick silica plates transparent to UV light. They are coated with a thin but
nontransparent layer of aluminum into which the desired patterns are etched [35].
To avoid any contamination, the entire procedure, including UV exposure, is carried out under a sterile
flowhood and the masks are immersed in a solution of 70% ethanol and 30% water for 30min prior to
use.
2.3.2 Topographic structures
The first topographic structures were made from the n-type polyimide photoresist HTR3-200 (Olin
GmbH, Munich, Germany) processed onto glass coverslips and pieces of Si-wafers. Process parameters
and steps are similar to those described in [129]. Although compatible with cell culture and resistant
to all cleaning procedures, including the short application of piranha solution (see 2.2.3), structures
made from polyimide are not satisfactory. Resolution and feature accuracy is poor, especially for deep,
narrow grooves with aspect ratios of 2 or higher (ratio of structure height to width). Also polyimide
layers of 10µm thickness or more detach frequently at the sides of the grooves, leaving a gap between
substrate and resist into which neurites can grow; as shown in fig. 2.7. Due to these limitations, HTR3-
200 is not used any more for making topographic structures directly in contact with cell culture.
Nevertheless, thin polyimide layers with well resolved features are ideally suited as sacrificial masks
for etching the structure directly into the substrate underneath. However, the RIE80 Plasmalab etcher
(Oxford Instruments) available in the departmental cleanroom facility cannot etch silicon anisotropically.
Walls of grooves are not vertical but inclined, reducing the directional information drastically,
with many neurites leaving the guidance structure. Etching topographic structures directly into the substrate
was not followed any further, even though there are ways to process vertical walls into silicon,
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