T3P.20

SURFACE TREATMENT STRATEGIES FOR MICROFLUIDIC DEVICESTOWARDS LONGITUDINAL PC12 NEURONAL CELL STUDIES

T.Q. Hoang1, L. J. Ho1, K. Swertfager1, A. Gill2, K. Malhotra3, C. Jones1, J. Chen1, and E. Meng1
1
University of Southern California, Los Angeles, CA, 90089 USA
2
Massachusetts Institute of Technology, Boston, MA, 02139 USA
3
Cornell University, Ithaca, NY, 14850 USA
ABSTRACTWe demonstrate polypeptide surface treatments strategies for the promotion of PC12 cell adhesion,proliferation, and differentiation on several MEMS substrates likely to be incorporated into a microfluidic neuralinterface platform. In particular, a method to successfully clean and reuse microfluidic devices without damaging
microfabricated structures following cell culture experiments is described.

KEY WORDS: Surface Treatment, Cell Adhesion, PC12, Biocompatibility
INTRODUCTION
We presented microfluidic MEMS devices that provide focal chemical gradients to targeted cells and localized
regions of whole tissue slices [1]. The fabricated cell interface platforms consist of Parylene C microchannels,
integrated thermal flow sensors, and SU-8 fluidic interconnects. This microdevice allows study of neuronal cells
and tissue subjected to stimulation, differentiation, and recording. Longitudinal cell studies on reusable microfluidic
devices require biocompatible surfaces that are durable to conditions of cleaning, culturing, and assessment (pH,temperature cycling, and soaking). For microfluidic flow studies where shear stress may be applied to cells, thesurface treatments to promote cell adhesion are necessary, especially when working with non-adherent cell lines [2].
The pheochromocytoma (PC12) cell line is a non-adherent cell type which does not attach well to mostmaterials. It is the most widely used model of neuronal cell function and its differentiation is induced by nerve
growth factor (NGF). Its adhesion can be promoted using non-animal derived surface treatments including
polyethylenimine (PEI) and poly-D-lysine (PDL) that contain no NGF. Animal-derived treatments (i.e. Matrigel)
containing NGF are avoided as they may confound NGF-induced PC12 differentiation studies. Thus, surface
treatment strategies using PEI and PDL were investigated on coupons of common MEMS materials and microfluidicdevices. PC12 cells were incubated from 1 to 4 days to enable longitudinal studies of focal chemical gradients.

METHODS AND RESULTS
Test coupons consisted of cell culture substrata (glass and polystyrene (PS)) and MEMS materials (Si, Parylene
C on Si (Pa), Parylene on SiO2 (PaO), Sylgard 184, and medical-grade silicone (MDX4-4210)). For each coupon,four regions on the surface were prelabeled for repeated measurements. Coupon surfaces and microfluidic devices
were cleaned in DI water and sonicated for 5 min.s. Coupons/devices were baked dry (37˚C, 30 min.s) and stored atroom temperature. Contact angle measurements were performed at each stage of the surface treatment study.
Coupons were separated into 6-well plates and incubated (37˚C, 1 hr) in either PEI (100 µM/mL) or PDL (100µg/mL) [3]. All coupons were triple rinsed with PBS, baked dry (37˚C, 30 min.s), and stored at room temperature.
Varying storage temperatures (4, 25, 37 °C) did not significantly change contact angles over a 3 day period (data notshown). PEI treatment resulted in a reduced contact angle (more hydrophilic) compared to those of PDL- and pretreated for PS, Pa, PaO, and Si. Sequential treatments of PEI or PDL were moderately effective (Fig.1 – only PS
data shown). No significant contact angle changes were observed in the silicones or glass for either treatment.

Figure 1: Contact angle data showing (a) stabilization of surface coating following sequential coating treatmentsand (b) surface coating removal on PS treated surfaces following sequential cleaning treatments.

0-9743611-4-3/MMB2009/$20©09TRF-0001212

Proceedings of 2009 International Conference on
Microtechnologies in Medicine and Biology
Québec City, Canada, April 1-3, 2009

PC12 cells were thawed from frozen stock and cultured in RPMI culture media supplemented with 7.5% donor
horse serum, 7.5% fetal bovine serum, and 0.5% streptomycin/penicillin. Cells were incubated (37˚C, 5% CO2) in
75 cm3 culture flasks. PC12 cells (100,000 cells/mL) were then incubated on corresponding polypeptide-coatedcoupons with or without NGF. NGF (50ng/mL) was added upon plating and every other day for the duration of theexperiment. Cell viability was assessed from Day 1-4 using either trypan blue (dead cells) or a fluorescent doublestaining assay with Calcein AM (live cells) and propidium iodide (dead cells). Cell viability data was obtained
under reflected brightfield and fluorescence microscopy and analyzed using NIH ImageJ. An additional vigorous
rinsing step to remove loosely attached cells, which emulates microfluidic agitation generated by focal delivery inthe device, dramatically decreased cell density (data not shown). NGF-treated cell cultures were more viable
compared to non-NGF-treated cells in all materials tested (Fig.2 – only PaO data shown). Cell densities on bothsilicones were substantially higher compared to the other materials. Low concentrations of NGF did not induce
differentiation and high concentrations (>100 ng/mL) induced differentiation within 22 hrs (data not shown).
Localized differentiation by focal delivery of NGF using the microdevice is currently being investigated.
NGFNo NGF

Figure 2: Count of viable PC12 cells labeled with Calcein AM and propidium iodide (PDL-coated, PEI-coated, anduncoated PaO surfaces) (a) with and (b) without NGF treatment.
To clean previously-used coupons/devices covered with PC12 cells and cell debris, coupons/microdevices weretreated with trypsin EDTA and incubated for 20 min.s. Coupons/devices were then washed twice with PBS andsonicated in DI water for 5 min.s at a time and repeated sonication as needed (Fig. 3). Device structures survived
repeated sonication up to 1 hr total duration before noticeable damage. Passive soaking in DI water or 1M HCl wasineffective in removal of cells and debris compared to active mechanical agitation. Contact angles were restored tonative values in all materials that were rendered more hydrophilic by the treatment (Fig 1B – only PS data shown).

(a)

(b)

(c)

(d)

Figure 3: PC12 cells grown on PEI-coated glass coupon (a) and untreated microfluidic devices (sensor contactpads shown) (c). Same surfaces shown after cleaning (b,d). A PC12 soma is ~10 Pm in diameter.

CONCLUSIONSPEI treatment created a more hydrophilic surface that promoted greater PC12 cell adhesion compared to PDL
treatment. Priming cell cultures with low concentrations of NGF facilitated adhesion and proliferation of PC12 cells
without dramatic induction of differentiation. Cells and debris were successfully removed from test coupons anddevices without harsh chemicals or inducing mechanical damage. Methods developed here will enable longitudinalstudies of neuronal cultures on microfluidic focal stimulation platforms, especially targeted NGF differentiation.

REFERENCES[1][2][3]

L. Chang, P.Y. Li, L. Zhao, T. Hoang, E. Meng, “Integrated Flow Sensing for Focal Biochemical
Stimulation”, IEEE NEMS Conference, pp. 921-6, 2008.
A. Tooker, E. Meng, J. Erickson, Y.C. Tai, J. Pine, “Biocompatible Parylene Neurocages”, IEEE
Engineering in Medicine and Biology Magazine, vol. 24, pp. 30-3, 2005.
G. Mallén-Ornelas, L. Chang, P.Y. Li, T. Hoang, L.J. Ho, K. Swertfager, E. Meng, “A Microfluidic
Platform for Focal Chemical Stimulation of Cells”, MicroTAS Conference, pp. 613-5, 2008.

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