A MICROFLUIDIC PLATFORM FOR FOCALCHEMICAL STIMULATION OF CELLS
G. Mallén-Ornelas, L. Chang, P. Y. Li, T. Hoang, L. J. Ho,
K. Swertfager, and E. Meng
Viterbi School of Engineering, University of Southern California, USAABSTRACT
A new microfluidic platform for focal stimulation of cells and tissue has beendeveloped. The device consists of a 100 μm x 4 μm x 6 mm Parylene microchannel
with a 5, 10, or 20 μm diameter pore. First results of real time focal chemical stimulation of cell cultures are presented.
KEYWORDS: Microfluidics, Focal Chemical Stimulation, Parylene, PC12INTRODUCTION
Focal delivery of chemicals at cellular and sub-cellular resolution enables understanding biological responses of cells and tissue, and provides a means of interfacingwith the nervous system beyond electrical stimulation [1-4]. A new microfluidic
platform has been developed for focal delivery of chemicals to cell cultures and tissue. For the first time, fluid intake as well as ejection and passive diffusion are possible. This platform is easily scaled to an array with multiple individually-addressed
stimulation sites. First results of real time focal chemical stimulation of cell cultures
are presented.
EXPERIMENTAL
The basic unit of the platform is a surface micromachined Parylene C microchannel (100 μm x 4 μm x 6 mm) perforated with a single central pore (20 μm diameter) (Fig. 1). The device has integrated platinum thermal flow sensors and SU-8
microfluidic interconnects at each end. The Parylene microchannel wall is 2 μm
thick, supported by posts, and selectively reinforced with a 75 μm thick layer of SU8 to prevent buckling and deformation of the channels and interconnects. Custom
packaging connects the platform to a gas-tight syringe (Fig. 2). A syringe pumpcontrols fluid flow in the microchannel.

Figure 1. Microfluidic platform photographs showing the microchannel, pore location, thermal flow sensors, SU-8 reinforcement and inlets/outlets (circle and funnel at either end). Right figure is a magnified view of the pore and surrounding
thermal flow sensing elements.

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
978-0-9798064-1-4/µTAS2008/$20©2008CBMS613Figure 2. Packaged devices: left panel shows a die with three microchannels inan acrylic jig and the right panel shows a close up of a die in an Ultem jig.

Figure 3. Testing setup: left panel shows a die with cells cultured on top of microchannel and the right panel shows cells attached to an inverted glass cover slip.
Rat pheochromocytoma cells (PC12) [5] were cultured overnight on a device pretreated with polyethyleneimine (PEI) to promote cell adhesion [6]. After loadingwith fluo-4 dye, a pulse of a 10 mM bradykinin solution was delivered through thepore (Fig. 3). Fluo-4 is a fluorescent Ca2+ indicator dye that enables spatiotemporal
tracking of intracellular Ca2+ [7]. Bradykinin induces a concentration-dependent release of intracellular Ca2+ stores [8], which was observed by fluorescence microscopy. The time evolution of average cell brightness as a function of distance fromthe pore is shown in Figure 4.

Figure 4. Comparison of mean light curves from 6 concentric 150-pixel thick annuli at different distances from the pore after focal delivery of a burst of bradykinin.
RESULTS AND DISCUSSION
The peak brightness and the timing of bradykinin-induced Ca2+ fluorescencechanges as a function of distance from the site of delivery, as shown in Figure 4.
Cells closest to the pore have the brightest, narrowest peak and show the earliest rise
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA614

in brightness. Cells furthest from the pore have the faintest peak brightness, broadest peak, and latest increase in brightness. This spectrum of bradykinin-induced responses demonstrates a decreasing bradykinin concentration with increasing distancefrom the pore. In a second test used to simulate tissue, PC12 cells were attached to aPEI-coated glass chip which was inverted and suspended 75 μm above the pore (Fig.
3). Continuous delivery of ~30 mM Rhodamine B clearly showed a slow radial progression of Rhodamine uptake by the cells as a function of time for a given flow ratefrom the pore (15 nL/min) (Fig. 5).

Figure 5. Time sequence of continuous ~30 mM Rhodamine B delivery to a culture of PC12 cells spanning eight minutes. Each frame measures 1 x 1.3 mm, andthe location of the pore is indicated by the arrow. White cells are fluorescing after
treatment with fluo-4, whereas black cells have taken up the Rhodamine B.
CONCLUSIONS
Focal chemical delivery and stimulation from a microchannel-addressed pore has
been demonstrated. This approach is scalable to a high density pore platform inwhich each pore is individually-addressed such that specific cell populations can betargeted. Integration of electrodes will enable a multi-modal neural interface.
ACKNOWLEDGEMENTS
This work was supported by NSF CAREER grant number EEC-0547544. G.M.O. is supported by a USC Provost Fellowship.
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