figure 8 shows that the peak brightness and the timing of bradykinin-induced ca 2+ fluorescence...
TRANSCRIPT
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:
• 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
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.
References:[1] L Chang, PY Li, L Zhao, T Hoang, and E Meng. 3rd IEEE
International Conference on NEMS, Sanya, China, 921-926 (2008).
[2] AR Vancha, S Govindaraju, KVL Parsa, M Jasti, M Gonzalez-Garcia, 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).
3. Experimental Setup1. Introduction
A MEMS Microfluidic Platform for Focal Chemical StimulationG. Mallén-Ornelas, L. Chang, P. Y. Li, T. Hoang, L. J. Ho, K. Swertfager, E. Meng
Department of Biomedical Engineering, USC, Los Angeles, CA
5. Discussion
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.
6. Conclusion and Future Work
Figure 1: Microfluidic platform photographs.
2. Platform Components
Figure 8: Comparison of mean light curves from 6 concentric 150-pixel thick annuli at different distances from the pore (indicated in the legend) after focal delivery of a burst of bradykinin.
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.
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.
microfluidicinterconnect
1 mm
microchannel
pore
SU-8
flow sensors
pore
flow sensors
microchannel support posts
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]
Custom packaging is used to connect the platform to a gas-tight
syringe (Fig. 3). A syringe pump controls fluid flow in the
microchannel.
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.
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).
4. PC12 Cell ExperimentsExperimental 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).
• 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.
Focal chemical delivery and stimulation from a
microchannel-addressed pore has been demonstrated. In
the future, integration of electrodes will enable a multi-
modal neural interface.
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.
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.
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.
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.
In a second test used to simulate tissue, PC12 cells were attached to a PEI-coated glass chip which was inverted and suspended 75 m
above the pore. Continuous delivery of a ~30 mM Rhodamine B solution (Fig. 4, right panel) clearly showed a slow radial progression of
Rhodamine uptake by the cells as a function of time for a flow rate from the pore of 15 nL/min (Fig. 7).
This spectrum of bradykinin-induced responses shows
there is a decreasing bradykinin concentration with
increasing distance from the pore.
200 m200 m 100 m
1 mm
microchannel
SU-8 32 41
200 m 200 m 200 m200 m
100 m