Proceedings of the 26th Annual International Conference of the IEEE EMBS
San Francisco, CA, USA • September 1-5, 2004

DEVELOPMENT OF BIOCOMPATIBLE PARYLENE NEUROCAGESAngela Tooker1, Ellis Meng2, Jon Erickson3, Yu-Chong Tai1, and Jerry Pine4
1

Department of Electrical Engineering, California Institute of Technology, CA, USA
Department of Mechanical and Aeronautical Engineering, University of California, Davis, CA, USA
3
Department of Bioengineering, California Institute of Technology, CA, USA
4Department of Physics, California Institute of Technology, CA USA

2Abstract—We present a refined method and designfor building parylene neurocages for in vitro studies oflive neural networks.
Parylene neurocages arebiocompatible and very robust, making them ideallysuited for studying the synaptic connections between
individual neurons to gain insight into learning andmemory.
The neurocage fabrication process is
significantly less complex than earlier versions. Previous
neurocage designs achieved limited neuronal outgrowth;
however, the long-term cell survival rate was <25%.
The incorporation of new materials and different
anchoring techniques, in addition to some designmodifications, as outlined here, have improved the longterm cell survival rate to >50%.
Keywords—neurocage, neuron, neuro-well, parylene

I. INTRODUCTION
Neurons play an important role in many of our
biological and cognitive functions.
Many studies
concentrate on the properties of neurons and the neuralnetworks they form; unfortunately, it is difficult to study
these networks in vivo. Initial in vitro techniques usedpatterned extracellular electrode arrays [1,2], but neuron
mobility and lack neuron-to-electrode specificity limit theuse of these arrays, especially in long-term studies.
Our strategy counteracts this difficulty by using
micromachined structures to physically trap individual
neurons in close proximity to electrodes, without inhibiting
their growth. The first implementation was the neuro-well[3,4]. This concept involved etching wells in bulk silicon,Fig. 1. SEM of a neuro-well.

0-7803-8439-3/04/$20.00©2004 IEEE

Fig. 2. Neuron loaded in neuro-well (left) and neurocage (right).

and then adding a nitride canopy to cover the top. The
canopy contained openings to allow the outgrowth ofneurites, while at the same time trapping a neuron in closeproximity to an electrode (Fig. 1). Arrays of neuro-wells
permitted the neurites from different neurons to form
connections, thereby, allowing the neurons to develop into
neural networks.
With these neuro-wells, individual
neurons in live neural networks could be reliably stimulated
and recorded from for long-term studies. While greatly
aiding the study of live neural networks, the fabrication andscaling complexities of the neuro-wells limited theircontinued development. In addition, the neurons in theneuro-wells tended to be pulled away from the bottom of thewell, and hence from the electrode, by the neurites growing
out through the channels on top of the well.
To address these problems, our group developed
surface micromachined parylene neurocages [5,6]. Unlikein the neuro-wells, neurites grow out the bottom of theneurocages (Fig. 2), pulling the neuron closer to theelectrode.
Parylene was chosen to be the structural material in thisapplication because it is biocompatible, non-toxic,
extremely inert, and resistant to moisture and mostchemicals. Hence, parylene is well suited for long-term cell
culture experiments. Its conformal deposition makes it easy
to fabricate three-dimensional structures like the neurocage.
In addition, parylene is transparent; thus when neurons areloaded into neurocages, they can easily be seen.
The initial neurocage design achieved some neuron
outgrowth, but long-term cell survival was low (<25%).
The new neurocage process and design presented here,
while preserving several elements of the previous designs,
increases the long-term cell survival rate to >50%.

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II. METHODOLOGY
A. Design
The neurocage consists of a chimney, 30µm indiameter and 4µm high, with a 15µm inlet hole at the top for
loading neurons (Fig. 3). Extending out from the chimney
are 6 tunnels, for neuron outgrowth, interleaved with 6anchors, for mechanical stability. The tunnels are 1.5µm
high and either 5µm or 10µm wide. They extend for either
40µm or 4µm (the thickness of the deposited parylene,effectively creating a slot in the side of the chimney rather
than a tunnel). The neurocage array (4 x 4) consists of 16
neurocages, each designed to hold a single neuron, centered
within a 440µm x 440µm square (Figs. 4-5).

etched using O2 plasma to create the neurocages. The
previous fabrication process required two depositions ofparylene. The sacrificial photoresist defining the chimneys
and tunnels are released using acetone. Finally, theneurocages
arecleanedusingpiranha(5:1:1
H2SO4:H2O2:H2O) at 120°C for 10 minutes followed by a 10
second HF dip.

B. Fabrication
The process flow for creating the neurocages is shownin Fig. 6. First, a thin layer of oxide, approximately 500nm
thick, is grown on a silicon substrate. The anchors for theparylene neurocage are then patterned, and the oxide isetched using BHF.
The partial exposure method uses two separate
exposures with different masks to define the chimneys andtunnels using only a single layer of photoresist (AZ4400).
After developing, these features are created.
The anchors are then etched into the silicon using a
DRIE process developed in our group for mechanicallysecuring parylene to a substrate [7]. The DRIE uses a
modified Bosch process: 50 loops of a standard Boschprocess to make an anisotropic trench with nearly verticalsidewalls, and a subsequent 30 second SF6 isotropic etch tocreate a mushroom-like bottom. The anchors are 10µm50µm deep.
Subsequently, a single layer of parylene is deposited,covering the whole surface, which is then patterned andADBECF

Fig 4. Optical Pictures of Neurocages (A-C) 40µm tunnel length.
(A) 4x4 Array, 10µm tunnel width. (B) 10µm tunnel width. (C)5µm tunnel width. (D-F) 4µm tunnel length. (D) 4x4 Array, 10µmtunnel width. (E) 10µm tunnel width. (F) 5µm tunnel width.

ABCAC
B

D

TunnelTunnelAnchorAnchorTunnelFig 5. SEM Pictures of Neurocages, 4µm tunnel length. (A) 4x4Array, 5µm tunnel width. (B) 10µm tunnel width. (C) 10µmtunnel width.

Fig 3. 3D Neurocage Model (A) 40µm tunnels. (B) Cross-Section with
40µm tunnels. (C) 4µm tunnels. (D) Cross-Section with 4µm tunnels.

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In addition, these neurocages can be cleaned of all
neuron debris using piranha and HF, for re-use in growing
live neural networks (Fig. 8)
IV. DISCUSSION
The current neurocage design and process, while
similar to previous versions, dramatically increases thelong-term cell survival rate. In previous designs, thechimney was 15µm high, the tunnel heights varied from
0.3µm to 2µm (depending upon the fabrication processused), and the tunnel length was 30µm. In the current
design, the chimney height is 4µm, the tunnel height is1.5µm, and the tunnel lengths are 4µm and 40µm. Clearly,
the biggest difference between the current design andprevious designs is the chimney height. The reduced
chimney height in the current design seems to be a primarycause for the increased survival rate.
Another potential cause for the increased cell survival
rate is the fabrication process. Previous neurocage designs
used two separate lithography processes to build the tunnels
and chimneys. These tunnels were formed by sputteredFig 6. Process Flow.

C. Cell Culture
After sterilization with UV light, the neurocages arecovered with 95% EtOH. The EtOH is then exchanged forwater. 5% PEI (poly-ethylene-imine) is added to promote
cell adhesion to the substrate. PEI is rinsed out of the dishand subsequently exchanged for neurobasal medium.
Neurons are then plated at a density of 30K/cm2. Cells areloaded manually into the neurocages with a pressure-driven
micropipette. The first signs of neuron growth usuallyappear within 12-24 hours of loading.

AIII. RESULTS
Neurocages produced, using this new fabrication
process, are mechanically robust, able to withstand various
cleaning procedures, including acetone and piranha, with nodeformation or delamination. In addition, initial studies
have shown that neurocages can survive for long periods, upto 80 days, in saline at 30°C with no visible deformation ordelamination. (The study was concluded after 80 days;
hence, no data is available for longer periods.) Based onthese studies, the neurocages should suffer no adverse
effects when placed in the neurobasal medium for longperiods.
Successful growth of live neural networks has beenachieved using 4 x 4 arrays of neurocages (Fig. 7), thereby
proving that the neurocages are biocompatible. Neuron
outgrowth has been achieved in neurocages with tunnel
widths of both 5µm and 10µm, and lengths of either 40µmor 4µm. No significant differences in neuronal survival rate
and outgrowth have been noted due to the different
combinations of tunnel lengths and widths.

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BFig 7. Nomarski Pictures of neurons inside neurocages, withneurite outgrowth. (A) 40µm tunnel length, 10µm tunnelwidth. (B) 4µm tunnel length, 10µm tunnel width. Arrows
denote neurite outgrowth.

AV. CONCLUSION

BFig 7. (A) Neurocage array after neural network growth. Note debris in andaround the cages. (B) Neurocage array after cleaning (10 minutes piranha,followed by 15 second HF dip).

silicon, hardbaked photoresist, or thermally evaporated
aluminum, while a thick layer of photoresist (AZ9260)
formed the chimneys. As a result, more drastic releasemethods were required: BrF3 or XeF2 gas etching for the
sputtered silicon, ST-22 photoresist stripper for the
hardbaked photoresist, or Al etchant for the thermallyevaporated aluminum. With these methods, it was notalways possible to ensure that the materials used to form thetunnels had been completely removed. If any of thismaterial remained, it could block the tunnel, thus preventing
neuronal outgrowth, or, as in the case of the hardbaked
photoresist, it could kill the neurons (photoresist is toxic toneurons). With the current fabrication process, the tunnels
can be released using acetone, and it is easier to makecertain that all photoresist has been removed.
Although good long-term cell survival can be achieved
with the new neurocage design, it is not clear whether thesuccess can be attributed to the reduced chimney height orto the fabrication process. To definitively answer thisquestion, it would be necessary to use the previous
fabrication processes with the reduced chimney height.
(Limitations of the partial exposure method prevent it frombeing used with 15µm high chimneys.)Previous designs used BrF3 or XeF2 to etch the anchors.
With these methods, however, it was not possible toaccurately control the undercut. The undercut caused thesize of the anchors to increase, thereby shrinking the area
available for the tunnels, and in some cases, eliminating thetunnels. With the DRIE process used for the current
neurocages, the undercut can be reliably controlled.
In the current process, the anchors are not etched into
the silicon as part of the initial step because the subsequent
lithography step, to create the tunnels and chimneys, allows
photoresist to flow into the anchors. Since the anchors
cover such a small surface area and are comparatively deep,
it is not possible to ensure that the photoresist is completely
removed from the anchors during the development process.
Remnants of photoresist left in the anchors, counteract theability of the anchors to firmly secure the parylene
neurocages to the surface, often causing them to releaseduring subsequent cleaning procedures.

The design and process presented here for parylene
neurocages can be used for in vitro studies of live neuralnetworks. This fabrication process is less complex thanprevious neurocage and neuro-well fabrication processes.
Biocompatible and robust neurocages can be created that
achieve significantly higher neuronal survival andoutgrowth rate than previous versions. The next step is toincorporate platinized gold electrodes into the neurocages tostimulate and record from individual neurons.
ACKNOWLEDGMENT
This work was funded by the NIH under Award
Number R01 NSO44134. We would like to thank Mr.
Trevor Roper, Mr. Qing He, and Ms. Angelique Johnson forassistance with fabrication and testing.

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