PARYLENE TECHNOLOGY FOR MECHANICALLYROBUST NEURO-CAGES
Ellis Meng1, Yu-Chong Tai1, Jon Erickson2, and Jerome Pine3
Departments of Electrical Engineering1, Bioengineering2, and Physics3
California Institute of Technology, Pasadena, CA 91126

ABSTRACT
We present a novel process to produce parylene cages for the in vitro study ofcultured neural networks. For the first time, a neuro-cage fabrication technology isdemonstrated that is scalable to high density cage arrays and able to withstand thechemical and mechanical rigors of supporting cellular cultures for long-term study.
Keywords: parylene, mechanical anchoring, neuro-cage
1. INTRODUCTION
Hippocampal neurons are known to play a role in learning and memory, thus it isinteresting to study the development and plasticity of hippocampal neuron networks.
Previously, it has been difficult to study these populations in detail. In vivo
measurements are ideal for observing neurons in their natural environment; however, it isextremely difficult to obtain and understand the complex recordings from tissue.
Additionally, observations of individual neuron behavior are difficult. One strategy toinvestigate neurons is to use in vitro techniques such as patterned extracellular electrodearrays [1, 2]. Even so, neuron mobility and the lack of specific connections between
neurons and electrodes severely limit our ability to study these systems. To enable longterm investigation of complex neuronal interactions, neuro-wells were designed [3, 4].
Each well contains a single dissociated neuron that is held in close proximity to an
extracellular electrode. While this device drastically improved neural researchtechnology, difficulties encountered in fabrication and scaling the design have inhibitedfurther device development. Parylene devices have been fabricated that are uniquely
suited to solving these problems (Figure 1). Here, improvements to mechanicalanchoring, first explored in [5], over cages developed in [6] are described in detail.
2. EXPERIMENTAL
4×4 arrays of cages arranged on 100 µm centers have been fabricated as a test bed forhippocampal neuron cultures (Figure 2a). This spacing is selected to match the range ofaxonal growth in cultures and neural networks of this size are known to produce
connections of sufficient complexity to warrant detailed study. Each parylene cage
consists of a loading hole (15 µm in diameter) and 8 tunnels (15 µm long, 5 µm wide, and
0.5 or 1 µm high) radiating from the cage base (30 µm in diameter and 15 µm high)(Figure 2b & c). These tunnels allow for the outgrowth of axons and dendrites. Parylene
cages are anchored to the substrate and sit on a SiO2 surface.

Figure 1. Neuro-cage concept showing (a) a loaded embryonic neuron and (b) a mature, trapped neuron

Figure 2. SEM images of (a) a neuro-cage array, (b) top view of a cage, (c) tilted view of a cage

Several technological innovations were required for fabricating neuro-cages. Theoverall cage structure is depicted in Figure 3. First, in order to provide robust mechanicalanchoring of the cages to the substrate, XeF2 gas phase etching was used to create
anchoring structures with large exposed surface area. Shallow pits measuring 0.5 µm
deep are created by etching and undercutting the silicon substrate through a SiO2 mask.
Then, these structures are filled with parylene. The resulting mushroom-shape ofstructures is seen in Figure 4 and the etched silicon surface in Figure 5. Thus, theparylene neuro-cages are able to root themselves solidly into the silicon substrate throughmechanical interlocking. This enables cage survival through sterilization and cell culture
treatments.

Figure 3. 3D cross section renderings of cages through (a) anchors and (b) tunnels

In addition, a high-aspect ratio etching technique using a modified Bosch-like process
was adapted to pattern the parylene structural material. Oxygen plasma etching alone isonly able to produce structures with a 1:1 aspect ratio. Aspect ratios of 2:1 or greater are
possible using ICP (inductively coupled plasma) -assisted etching techniques.

Figure 4. SEM images of (a)-(c) released parylene anchor structures

Figure 5. SEM images of (a) etched anchors in Si, (b) etched surface roughness, and (c) upside-down cage

3. RESULTS AND DISCUSSION
The surface roughness and undercut that result from XeF2 etching are extremely
important in keeping the neuro-cages firmly attached to the substrate during sterilizationand culturing. Cages survive the Scotch tape test and removal of the cages by pressure
sensitive adhesive film can only be accomplished when the SiO2 overhang is etched away.
A cage removed using this technique is shown in Figure 5. The quality of cage adhesion
to the substrate by mechanical anchoring far surpasses that achieved by chemicaladhesion promotion techniques.
The compatibility of neuronal cultures with parylene neuro-cage arrays has been
demonstrated in [6] and is seen in Figure 6. Here, cultures have been sustained for up to3 weeks. Cleaning techniques are now being developed to remove old neural cultures
such that the arrays can be reused for repeated culture experiments.
4. CONCLUSIONSThe successful growth and development of rat hippocampal neurons on parylene
surfaces has been demonstrated. Mechanical robustness in terms of culture support andsurvivability in chemical treatments has been verified. Currently, neuro-cages are beingfurther optimized for neural cultures. The next step is to integrate platinized gold
electrodes for non-destructively recording and stimulating trapped neurons. In thismanner, specific cells can be targeted for study without interfering with cell growth. It ishoped that the device can aid in the design of future in vivo studies. In addition, similar
structures can be used to trap and investigate the long term behavior of other types ofindividual cells or cellular networks.

Neurons
Figure 6. (a) Optical image of partially loaded cage array and (b) Nomarski image of 3 week old culture

ACKNOWLEDGEMENTS
This work was funded in part by the Engineering Research Centers Program of theNSF under Award Number EEC-9402726 and the NIH under Award Number R01
NSO44134. We would like to thank Mr Trevor Roper, Mr. Qing He, and Ms. Angela
Tooker for assistance in fabrication and Mr. Tuan Hoang for help with proofreading.
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