STUDENT PAPER AWARD

Biocompatible
Parylene Neurocages© ARTVILLEDeveloping A Robust Methodfor Live Neural Network Studies

BY ANGELA TOOKER,
ELLIS MENG, JON ERICKSON,
YU-CHONG TAI,
AND JERRY PINEe present a refined method and design forstudies. While greatly aiding the study of live neural netfabricating parylene neurocages for in vitro
works, the fabrication and scaling complexities of the neustudies of live neural networks. Parylene
rowells limited their continued development. In addition, theneurocages are biocompatible and very
neurons in the neurowells tended to be pulled away from therobust, making them ideally suited forbottom of the well, and hence from the electrode, by the neustudying the synaptic connections between individual neurons
rites growing out through the channels on top of the well.
to gain insight into learning and memory. The neurocage fabTo address these problems, our group developed surface
rication process is significantly less complex than earlier vermicromachined parylene neurocages [5], [6]. Unlike in thesions. Previous neurocage designs achieved limited neuronal
neurowells, neurites grow out the bottom of the neurocagesoutgrowth; however, the long-term cell survival rate was<25%. As outlined here, the incorporation of new materials
and different anchoring techniques, in addition to somedesign modifications, have improved the long-term cell survival rate to >50%.
Neurons play an important role in many of our biologicaland cognitive functions. Many studies concentrate on theproperties of neurons and the neural networks they form;
unfortunately, it is difficult to study these networks in vivo.
Initial in vitro techniques used patterned extracellular electrode arrays [1], [2], but neuron mobility and lack of neuronto-electrode specificity limit the use of these arrays, especiallyin long-term studies.
Our strategy counteracts this difficulty by using micromachined structures to physically trap individual neurons inFig. 1. SEM of neuro-well cross section.
close proximity to electrodes without inhibiting their growth.
The first implementation was theneurowell [3], [4]. This concept
involved etching wells in bulk silicon and then adding a nitride
Full-Grown Cell
NeuriteFull-Grown Cell
canopy to cover the top. The canopycontained openings to allow the outNeurite
growth of neurites, while at the sametime trapping a neuron in close
proximity to an electrode (Figure1). Arrays of neurowells permitted
the neurites from different neurons
to form connections, thereby allowing the development of neural netGold Lead
Platinum Black
Electrical Leadworks. With these neurowells,individual neurons in live neural(a)(b)
networks could be reliably stimulated and recorded from for long-term Fig. 2. (a) The neuron loaded in the neuro-well and (b) the neurocage.

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(a)

(b)

(c)

(d)

Fig. 3. SEMs showing top views of (a) 4 × 4 array of neurocages with 40-µm tunnel
lengths and 10-µm tunnel widths; (b) a neurocage with 40-µm tunnel lengths and 10µm tunnel widths; (c) 4 × 4 array of neurocages with 4-µm tunnel lengths and 10-µmtunnel widths; and (d) a neurocage with 4-µm tunnel lengths and 10-µm tunnel widths.

(a)

(b)

(Figure 2), pulling the neuron closerto the electrode.
Parylene was chosen to be thestructural material in this application
because it is biocompatible, nontoxic, extremely inert, and resistant tomoisture and most chemicals.
Hence, parylene is well suited forlong-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 are loaded 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%.
Methodology
Design

The neurocage consists of a chimney
30 µm in diameter and 4 µm highwith a 15-µm-diameter inlet hole atthe top for loading neurons.
Extending out from the chimney are
six tunnels for neuron outgrowthinterleaved with six anchors formechanical stability. The tunnels are
1.5 µm high and either 5 µm or 10µm wide. They extend for either 40µm or 4 µm (the thickness of thedeposited parylene, effectively creating a slot in the side of the chimney
rather than a tunnel). The neurocage
array (4 × 4) consists of 16 neurocages, each designed to hold a single neuron, centered within a 440 µm
× 440 µm square (Figures 3 and 4).
Fabrication(c)(d)

Fig. 4. SEMs showing tilted views of: (a) SEM of a 4 × 4 array of neurocages with 40-µm
tunnel lengths and 10-µm tunnel widths; (b) an SEM of a neurocage with 40-µm tunnel
lengths and 10-µm tunnel widths; (c) an SEM of a 4 × 4 array of neurocages with 4-µmtunnel lengths and 10-µm tunnel widths; and (d) an SEM of a neurocage with 4-µm tunnel lengths and 10-µm tunnel widths.

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The process flow for creating theneurocages is shown in Figure 5.
First, a thin layer of oxide, approximately 500 nm thick, is grown on asilicon substrate. The anchors for theparylene neurocage are then patterned, and the oxide is etched using
buffered oxide etch (BHF).
A partial exposure method uses
two separate exposures with different masks to define the chimneys
and tunnels using only a single layerof photoresist (AZ4400). Afterdeveloping, both of these features
are created.
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Cross Section Through Anchors

Cross Section Through Tunnels
Pattern andEtch Oxide forAnchors
PatternPhotoresist for
Chimneys (4 µm)
and Tunnels(1.5 µm)
Etch Silicon for
Anchors UsingDRIE

DepositParylene(4 µm)

Pattern andEtch Parylene
and Release
Chimneys andTunnels
SiliconOxide

Photoresist

Fig. 5. The fabrication process flow.

Parylene

Subsequently, a single layer ofparylene is deposited then patterned and etched using O2 plasma
to create the neurocages. The previous fabrication process required
two depositions of parylene. Thesacrificial photoresist defining thechimneys and tunnels are releasedusing acetone. Finally, the neurocages are cleaned using piranha(5:1:1 H2 SO4 :H2 O2 :H2 O) at 120◦
C for 10 min followed by a 10-shydrofluoric acid (HF) dip.
Cell Culture

After sterilization with UV light,
the neurocages are covered with
95% EtOH. The EtOH is thenexchanged for water. Five percent
poly-ethylene-imine (PEI) is added
to promote cell adhesion to the substrate. PEI is rinsed out of the dishand subsequently exchanged forneurobasal medium. Neurons arethen plated at a density of 30,000
K/cm2 . Cells are loaded manually
into the neurocages with a pressuredriven micropipette. The first signsof neuron growth usually appear
within 12–24 h of loading.
Results

Neurocages produced using this new
fabrication process are mechanicallyrobust and able to withstand various
cleaning procedures, including acetone and piranha, with no deformation or delamination. In addition,
initial studies have shown that neurocages can survive for long periodsof up to 80 days in saline at 30 ◦ Cwith no visible deformation ordelamination. (The study was concluded after 80 days; therefore, nodata is available for longer periods.)
Based on these studies, the neurocages should suffer no adverse
effects when placed in the neu(a)(b)
robasal medium for long periods.
Successful growth of live neuralFig. 6. (a) Nomarski picture of neurons and neurite outgrowth inside the neurocages
networks has been achieved using 4with 40-µm tunnel lengths and 10-µm tunnel widths. (b) Nomarski picture of neurons and× 4 arrays of neurocages (Figure 6),
neurite outgrowth inside neurocages with 4-µm tunnel lengths and 10-µm tunnel widths.
thereby proving that the neurocages
are biocompatible. Neuron outgrowth
The anchors are then etched into the silicon using a deephas been achieved in neurocages with tunnel widths of both 5µm and 10 µm and lengths of either 40 µm or 4 µm. No signifreactive iron etch (DRIE) process developed in our groupfor mechanically securing parylene to a substrate [7]. The
icant differences in neuronal survival rate and outgrowth have
DRIE uses a modified Bosch process: 50 loops of a stanbeen noted due to the different combinations of tunnel lengths
dard Bosch process to make an anisotropic trench withand widths.
nearly vertical sidewalls and a subsequent 30-s SF 6
In addition, these neurocages can be cleaned of all neuron
isotropic etch to create a mushroomlike bottom. The
debris using piranha and HF for reuse in growing live neuralanchors are 10–50 µm deep.
networks (Figure 7).
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Discussion

The current neurocage design and process, while similar to previous versions, dramatically increases the long-term cell survival
rate. In previous designs, the chimney was 15 µm high, the tunnel
heights varied from 0.3–2 µm (depending on the fabrication
process used), and the tunnel length was 30 µm. In the current
design, the chimney height is 4 µm, the tunnel height is 1.5 µm,
and the tunnel lengths are 4 µm and 40 µm. Clearly, the biggest
difference between the current design and previous designs is thechimney height. The reduced chimney height in the current
design seems to be a primary cause for the increased survival rate.
Another potential cause for the increased cell survival rateis the fabrication process. Previous neurocage designs used
two separate lithography processes to build the tunnels andchimneys. These tunnels were formed by sputtered silicon,
hardbaked photoresist, or thermally evaporated aluminum,
while a thick layer of photoresist (AZ9260) formed the chimneys. As a result, more drastic release methods wererequired: BrF3 or XeF2 gas etching for the sputtered silicon,
ST-22 photoresist stripper for the hardbaked photoresist, orAl etchant for the thermally evaporated aluminum. With
these methods, it was not always possible to ensure that thematerials used to form the tunnels had been completelyremoved. If any of this material remained, it could block thetunnel, thus preventing neuronal outgrowth, or, as in the caseof the hardbaked photoresist, it could kill the neurons (photoresist is toxic to neurons). With the current fabrication
process, the tunnels can be released using acetone, and it iseasier to make certain that all photoresist has been removed.
Although impoved long-term cell survival is achieved
with the new neurocage design, it is not clear whether thesuccess is attributed to the reduced chimney height or to thefabrication process. To definitively answer this question, it
would be necessary to fabricate neurocages with reduced
chimney height using the previous process flow.
(Limitations of the partial exposure method prevent it frombeing used with 15-µm-high chimneys.)Previous designs used BrF3 or XeF2 to etch the anchors. With
these methods, however, it was not possible to accurately control
the undercut associated with the isotropic nature of the etchingprocess. The undercut caused the size of the anchors to increase,
thereby shrinking the area available for the tunnels, and in somecases, eliminating the tunnels. With the DRIE process used forthe 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 toensure that the photoresist is completely removed from theanchors during the development process. Remnants of photoresist left in the anchors counteract the ability of the anchors tofirmly secure the parylene neurocages to the surface, often
causing them to release during subsequent cleaning procedures.
Conclusions

The design and process presented here for parylene neurocages
can be used for in vitro studies of live neural networks. Thisfabrication process is less complex than previous neurocage andneurowell fabrication processes. Biocompatible and robust neurocages can be created that achieve significantly higher
neuronal survival and outgrowth rate than previous versions.
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(a)

(b)

Fig. 7. (a) A neurocage array after neural network growth.
Note the debris in and around the cages. (b) A neurocage
array after cleaning in 10 min piranha, followed by 15-s HF dip.

The next step is to incorporate platinized gold electrodes intothe neurocages to stimulate and record from individual andgroups of neurons.
Acknowledgments

This work was funded by the the National Institutes of Health(R01 NSO44134). We would like to thank Trevor Roper,
Qing He, and Angelique Johnson for assistance withfabrication and testing.
Angela Tooker is a graduate research student in theDepartment of Electrical Engineering at the California
Institute of Technology, Pasadena, California.
Ellis Meng is an assistant professor of biomedical engineering
at the University of Southern California, Los Angeles,California.
Jon Erickson is a graduate research student in theDepartment of Bioengineering at the California Institute ofTechnology, Pasadena, California.
Yu-Chong Tai is a professor of electrical engineering at theCalifornia Institute of Technology, Pasadena, California.
Jerry Pine is a professor of physics at California Institute ofTechnology, Pasadena, California.
Address for Correspondence: Angela Tooker, California
Institute of Technology, M/C 136-93, Pasadena, CA 91125
USA. Phone: +1 626 395 2267. Fax: +1 626 584 9104. Email: atooker@mems.caltech.edu.
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