Transcript Document

A MEMS Microfluidic Platform for Focal Chemical Stimulation
G. Mallén-Ornelas, L. Chang, P. Y. Li, T. Hoang, L. J. Ho, K. Swertfager, E. Meng
Department of Biomedical Engineering, USC, Los Angeles, CA
3. Experimental Setup
1. Introduction
Focal delivery of chemicals at cellular and sub-cellular
resolution enables understanding biological responses of
cells and tissue, and provides a means of interfacing with
the nervous system beyond electrical stimulation. A new
microfluidic platform has been developed for focal
delivery of chemicals to cell cultures and tissue [1]. For
the first time, fluid intake as well as ejection and passive
diffusion are possible. First results of real time focal
chemical stimulation of cell cultures are presented here.
Custom packaging is used to connect the platform to a gas-tight
syringe (Fig. 3). A syringe pump controls fluid flow in the
microchannel.
5. Discussion
For cell stimulation tests, a cell culture was grown either on the
device itself, or on a glass cover slip that was inverted and
suspended above the pore (Fig. 4).
Figure 8 shows that the peak brightness and the timing of
bradykinin-induced Ca2+ fluorescence changes as a
function of distance from the site of delivery:
• Cells closest to the pore have the brightest peak and
are the first to increase in brightness.
• Cells furthest from the pore have the faintest peak
brightness, and are the last to increase in brightness.
Figure 3: Left panel shows a die with three microchannels in an acrylic jig
and the right panel shows a die in an Ultem jig.
Figure 4: Cell experiment setup: left panel shows a die with cells cultured
on top of the microchannel and the right panel shows cells attached to an
inverted glass cover slip.
This spectrum of bradykinin-induced responses shows
there is a decreasing bradykinin concentration with
increasing distance from the pore.
4. PC12 Cell Experiments
2. Platform Components
•
Parylene C microchannel (100 μm x 4 μm x 6 mm)
•
Single central pore (5, 10, or 20 μm diameter)
•
Integrated platinum thermal flow sensors
•
SU-8 microfluidic interconnects
The 2 μm thick Parylene wall is reinforced with a 75 μm
thick layer of SU-8
•
microchannel
microfluidic
interconnect
flow sensors
Experimental procedure
1. The device was treated with polyethyleneimine (PEI) to
promote cell adhesion [2].
2. Rat pheochromocytoma cells (PC12) [3] were cultured
overnight directly on the device.
3. Cells were loaded with fluo-4 fluorescent Ca2+ indicator dye [4].
4. A pulse of a 10 mM bradykinin solution was delivered through
the pore (Fig. 4, left panel).
5. Bradykinin induces a concentration-dependent release of
intracellular Ca2+ stores [5]. A focal increase of fluorescence
was clearly visible (Fig. 5).
Data analysis
1. Cells were identified automatically in the first image using
astronomical software packages IRAF1 and DAOPHOT [6] (Fig.
6).
2. The brightness of each detected cell was measured on every
image and divided by the brightness of the same cell in the first
image.
3. The cells were grouped in concentric annuli centered on the
pore.
4. The mean light curve of all the cells in each annulus was
computed and is shown in Figure 8.
Figure 8: Comparison of mean light curves from 6 concentric 150pixel thick annuli at different distances from the pore (indicated in
the legend) after focal delivery of a burst of bradykinin.
SU-8
1 mm
pore
microchannel support posts
6. Conclusion and Future Work
pore
200 mm
flow sensors
Figure 1: Microfluidic platform photographs.
200 mm
Figure 5: The left panel shows a device with a culture of PC12 cells that
were treated with fluo-4 dye to allow tracking of Ca2+. The right panel shows
the same device during the delivery of a burst of bradykinin through the
pore. Each frame measures 1 x 1.3 mm.
100 mm
100 mm
Figure 6: Left: a multi-layer culture of PC12 cells (bright green objects).
Right: the same image with added pink circles showing objects automatically
identified by astronomical software package DAOPHOT.
In a second test used to simulate tissue, PC12 cells were attached to a PEI-coated glass chip which was inverted and suspended 75 mm References:
above the pore. Continuous delivery of a ~30 mM Rhodamine B solution (Fig. 4, right panel) clearly showed a slow radial progression of [1] L Chang, PY Li, L Zhao, T Hoang, and E Meng. 3rd IEEE
International Conference on NEMS, Sanya, China, 921-926
Rhodamine uptake by the cells as a function of time for a flow rate from the pore of 15 nL/min (Fig. 7).
(2008).
[2] AR Vancha, S Govindaraju, KVL Parsa, M Jasti, M GonzalezGarcia, RP Ballestero. BMC Biotechnol., 4, 23 (2004).
[3] LA Greene, JM Aletta, A Rukenstein, SH Green. Method.
Enzymol., 147, 207-216 (1987).
[4] KR Gee, KA Brown, WNU Chen, J Bishop-Stewart, D Gray, I
Johnson. Cell Calcium, 27(2), 97-106 (2000).
[5] KC Appell, DS Barefoot. Biochem. J., 263, 11-18 (1989).
[6] P Stetson, PASP 99, 191 (1987).
microchannel
1
2
SU-8
3
4
1 mm
200 mm
Figure 2: 3D illustration of the device with a detail shown in the inset.
The microchannel is partially removed to show the cross-sectional
structure. [1]
Focal chemical delivery and stimulation from a
microchannel-addressed pore has been demonstrated. In
the future, integration of electrodes will enable a multimodal neural interface.
200 mm
200 mm
200 mm
Figure 7: Time sequence of continuous delivery of a ~30 mM Rhodamine B solution to a culture of PC12 cells treated with fluo-4. Each frame measures 1 x
1.3 mm, and the 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
infused Rhodamine B. The interval between frames 1 and 4 is eight minutes.
1. IRAF is distributed by the National Optical Astronomy Observatory, which is operated by the Association of Universities for Research in Astronomy (AURA) under cooperative agreement with the National Science Foundation.
Acknowledgments: This work was supported by NSF CAREER
grant number EEC-0547544. G.M.-O. is supported by a USC
Provost Fellowship. G.M-O. wishes to thank the members of the
Biomedical Microsystems Lab at the University of Southern
California, especially Mei Li Nickles for her contribution to the
fabrication of the device packaging.