DESIGN AND FABRICATION OF A PDMS MICROFLUIDIC PROBE AND PERFUSION CHAMBER FOR MICROFLUIDIC EXPERIMENTS WITH ORGANOTYPIC BRAIN SLICES Arthur Queval1, Cécile M. Perrault1, Mohammad A. Qasaimeh1, R. A. McKinney2 and David Juncker1 1
Biomedical Engineering Department, McGill University, CANADA Department of Pharmacology and Therapeutics, McGill University, CANADA Email:
[email protected] ABSTRACT Microfluidic systems are increasingly being used for the culture and study of dissociated cells because they facilitate drug screening and chemotaxis studies using minute amounts of chemicals. However, microfluidics have not been adopted for brain slices because of the challenges associated with the culture conditions necessary for maintaining the integrity of the slices. Here, we present a microfluidic system comprising a perfusion chamber compatible with confocal microscopy and a transparent microfluidic probe (MFP) for local microperfusion of organotypic brain slices for epilepsy studies. 2
KEYWORDS: Microfluidic probe, Organotypic slices, Superfusion. INTRODUCTION Organotypic brain slices are widely used for in vitro experiments since they maintain the architecture and circuitry of the central nervous system [1], and allow the study of neuronal circuits and synapse formation in an in vivo-like setting. To enable long term studies of brain slices, a special perfusion chamber (Fig. 1) was developed to maintain physiological conditions, be compatible with the MFP, and be mounted onto an inverted (confocal) microscope. The chamber comprises a perfusion inlet/outlet, a vacuum line, and 100 m thin metal sheets used as a coverslip holder that does not interfere with high magnification objective lenses. Each brain slice is attached on a thin coverslip serving as substrate, which is hermetically sealed to the chamber with a vacuum. The vacuum ensures liquid tight seal while media is flowed through the chamber with a peristaltic pump. By using this chamber, the sample is visualized directly through a thin coverslip (100 m) allowing the use of high magnification objectives for visualization of individual synapse formation.
Figure 1: Exploded view of the perfusion chamber for inverted microscope. 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©2008CBMS
1663
THEORY The principle of the MFP is to inject a solution in an aperture while completely aspirating it by another, creating a hydrodynamically-shaped flow pattern [2]. Here, we present a new MFP with six apertures made entirely of polydimethylsiloxane (PDMS-MFP) by using soft lithography techniques and SU8 molds (Fig. 2a). PDMS has the advantages of being a soft, low cost and optically transparent material. The new MFP is made of a thin spin-coated PDMS layer sandwiched between two thick layers with three microchannels with widths ranging from 15 m to 50 m. Upon bonding of the three layers of PDMS, the channels are cut perpendicularly with a high precision carbon steel razor blade. The resulting plane provides the surface of the probe bearing the apertures. With this simple fabrication method, a 2dimensional matrix with 3 x 2 holes can be made within two hours in a standard lab environment.
Figure 2: a) Microfabrication process of the PDMS-MFP. b) Cross section of the perfusion chamber. c) Microperfusion setup comprising of the perfusion chamber and the PDMS-MFP mounted on an inverted microscope. RESULTS AND DISCUSSION Each aperture can be connected to a high-precision syringe pump and used either for the aspiration or the injection of the solution, offering 62 possible injection/aspiration configurations. Furthermore, each aperture can be individually fed, allowing different solutions to be injected at the same time during the experiment (Fig. 3). The injected solutions contain fluorescein for visualization, whereas the immersion media is water. In figure 3, different configurations are shown: a single
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA
1664
(f), double (d, e, g) and triple (c, h) solution is injected while a single (c, d, e, f and g) and triple (h) holes aspirate back the solution. The injection is performed at 10 nl.s-1 while the aspiration is set to 100 nl.s-1.
Figure 3: (a) Overview of the PDMS-MFP (scale bar: 5 mm). (b) Enlarged view of the 2-dimensionnal array of holes. (c-h) Fluorescent micrographs of a PDMSMFP and different injection/aspiration configurations (scale bar: 40 m). The PDMS-MFP is used to microperfuse hippocampus brain micro-slices (Fig. 2b and 2c). Figure 4a and 4d show a single injection/aspiration configuration of the PDMS-MFP, where fluorescein dye, dissolved in Tyrode solution, is flowed through the tissue. The size of the perfused area can be modified, by adjusting the aspiration/injection flow rate ratio, to match the desired area (Fig. 4c). The MFP is mounted on a stage with submicrometric precision and then can be positioned where desired for localized perfusion with drugs in the slice.
Figure 4: (a-d) Local microperfusion of a thin (100 m) mouse hippocampus brain slice using the PDMS-MF (scale bar: 40 m). CONCLUSIONS Using this simple fabrication process, the design of the PDMS-MFP can be easily modified, allowing generation of customized gradients and therefore create new perpective for growth cone guidance at high spatio-temporal resolution. REFERENCES
[1] B.H. Gähwiler et al, Organotypic slice cultures: a technique has come of age, TINS, 20, 10 (1997). [2] D. Juncker, Heinz Schmid and E. Delamarche, Multipurpose microfluidic Probe, Nature materials, 4, 622 (2005). Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA
1665
