Multilayer PDMS Microfluidic Brain Slice Chamber for...

CREATED USING THE RSC ARTICLE TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS

ARTICLE www.rsc.org/[journal] | [journal name]

This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1

Multilayer PDMS Microfluidic Brain Slice Chamber for

Controlling Slice Microenvironment

5

Blake AJ,*a Pearce TM,*a Rao NS, a Johnson SM,b and Williams JCa

Receipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT] Publication data [DO NOT ALTER/DELETE THIS TEXT] 10

DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]

Abstract. A novel microfluidic device has been created for clinical neurophysiology applications. In our study, we observe the respiratory rhythm generated from slice preparations of 15

neonatal rat medulla for up to 3 hours, in vitro in the multilayer microfluidic brain slice chamber. The construction process utilizes microfabrication techniques to create a three-layer microfluidic polydimethylsiloxane (PDMS) device with two fluid chambers that hold a tissue slice in place with microposts above 20

and below it, while also maintaining appropriate perfusate flow. The middle layer is designed to specifically fit the shape and thickness (530-700 µm) of a medullary brain slice taken from one to four week old neonatal rats. Fabrication permits rapid production of any planar form for the middle layer, thereby 25

allowing the method to be applied to brain slices of different shapes and sizes (e.g. hippocampal). This design is unique in that not only does one have independent control of flow rates for six inputs, but two separate fluid chambers allow optimal perfusion of the tissue slice on both the top and bottom. The laminar flow 30

exhibited by the microfluidic chamber allows controlled solutions to target specific areas of the tissue slice based on the input flow rates. We demonstrate this focused flow by directing a multitude of different solutions over specific areas of the tissue slice while monitoring the neuronal activity. The multilayer slice chamber 35

design integrates typical electrophysiology tools used to study tissue slice electrical activity, while maintaining the highest viability of the tissue slice preparation. Recording electrodes are applied to the tissue for prolonged periods of time to verify that the brain slice is indeed active. This is performed on both 500 µm 40

and 700 µm neonatal brain tissue slice preparations. This new perfusion chamber technology has much to offer for different experiments involving controlled drug delivery and can be further expanded by integrating microelectrode arrays (MEAs) onto the microfluidic device. 45

Introduction Viable, electrically active brain slices are widely used for

neurophysiological studies because they provide access to neural networks and permit analysis of underlying cellular and synaptic mechanisms. Maintaining brain slice viability is 50

critical to sustaining the electrophysiological properties of the neural network. Brain slices are typically kept alive in a perfusion chamber with oxygenated artificial cerebrospinal fluid (ACSF) passing over the slice (Dingledine et al., 1980). In selecting a tissue preparation and perfusion system, several 55

variables must be considered. For example, thicker slices allow for greater synaptic connectivity between neurons and better dendritic integrity, but the limitation is that tissue oxygen demand must not exceed the diffusion-limited oxygen delivery capacity of the perfusion bath (REF Haddad). Also, 60

chemicals or drugs are often added to the ACSF to alter neuronal activity and gain insight into various biological phenomena. In most of these experiments, however, the entire brain slice is exposed to the chemicals or drugs. There are few examples whereby the perfusion chamber design has been 65

modified to apply substances to specific regions of brain slices (Haas et al., 1979; Kelso et al. 1983; Koerner et al., 1983; Palovcik et al., 1986; Matthies et al., 1997). Recently, a modified version of the Haas type perfusion chamber employed some of the principles of microfluidics, which 70

allowed for multiple drug exposures (Passeraub et al., 2002). To address these variables, we developed a microfluidic brain slice chamber that can be used to apply solutions to specific slice regions while maintaining adequate slice viability. Microfluidics has emerged as a powerful technology 75

in the biological community (Walker et al., 2004; Atencia et al. 2005; Beebe et al., 2002, Kruger et al., 2002, Gravensen et al., 1993) because it offers many advantages over classical approaches. A reduction in the feature sizes gives control over fluid phenomena such as laminar flow, shear stresses, and 80

velocity profiles. These characteristics can be manipulated so as to promote a favorable brain slice environment, both for tissue health and experimental purposes. Control of the slice microenvironment is achieved by simply varying the velocity of multiple fluid inputs. Solutions can even be switched over 85

the same brain slice area within milliseconds. The material properties of this novel perfusion device are far superior than that of existing perfusion chmabers. Many of the existing perfusion chambers use common materials, such as Plexiglas, glassware, nylon mesh, and some times metallic 90

parts (Passeraub et al., 2003). While these materials are easily accessible, it is impossible to fine-tune the design on the microscale, as is possible with microtechnology. Thus, we were able to develop a microfluidic chamber that uses inexpensive, easily modified materials that provide maximum 95

experimental flexibility. The ability to alter the design on the microscale gives microfluidics a huge advantage over conventional approaches that provide little if any fine control over the microenvironment. In this paper, a novel multilayer perfusion chamber is 100

described that uses a combination of current microfabrication and replica-molding techniques (Tan et al., 2003) that has the following advantages: 1) Controlled laminar flow allowing multiple fluids to be

directed over the tissue slice for chemical studies. 105

a Address, Address, Town, Country. Fax: XX XXXX XXXX; Tel: XX XXXX XXXX; E-mail: [email protected] b Address, Address, Town, Country. Fax: XX XXXX XXXX; Tel: XX XXXX XXXX; E-mail: [email protected] † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See http://dx.doi.org/10.1039/b000000x/


2 | [journal], [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]

2) Controlled fluid exchange at the tissue-fluid interface for nutrient and waste management using ACSF.

3) Localized exposure on both top and bottom of the brain slice to optimize chemical delievery and viability through diffusion. 110

4) Compatibility with typical electrophysiology tools found in many laboratories.

5) Batch processing to produce inexpensive identical devices within hours. 6) Future integration of sensors and microelectrode arrays 115

(MEAs). 7) Adaptation to any brain tissue slice shape with short fabrication turnover times.

120 Fig. 1. Schematic displaying the side view of the general fluid velocity profiles for typical perfusion systems (A, B) and the novel method for controlled perfusion using a multilayered perfusion device (C).

Materials and Methods

Microfluidic Chamber Fabrication 125

Common photolithography and microfabrication techniques were implemented for microfluidic device construction. The three different layer patterns were drawn using image software (Adobe Illustrator 10.0) and then printed (Silverline Studio, silverlinestudio.com) on a transparency using a high 130

resolution printer (3600 dpi). This resolution ensures feature sizes down to 10 µm, which is more than adequate for our mask design. Silicon wafers with a diameter of 7.5 cm were used as the substrate material. A double-negative process was utilized in the construction 135

of the polydimethylsiloxane (PDMS) microfluidic channel layers. The procedure began with the fabrication of an SU-8 master that is a positive of the final PDMS chamber design (Fig. 2). The SU-8 master is used to cast a PDMS master mold (negative). The PDMS master is then used to cast the desired 140

PDMS microfluidic channel layer. The PDMS negative mold surface is silanized to allow newly cured PDMS microfluidic channel layers to be removed. A PDMS master was used due to the short fabrication times (2.0 h) compared to making multiple similar SU-8 masters (up to 10 h) for batch 145

processing. PDMS is advantageous since it is gas permeable and reduces the chance of small feature sizes not being

replicated due to gas pockets (e.g., gas trapped in the micropost portion of the mold). The continual heating and cooling required for curing PDMS increases the risk of stress 150

cracks or failure of a fragile SU-8 master. The PDMS master is more durable than using an SU-8 master. This double-negative fabrication process begins with curing a thin layer (30-40 µm) of negative photoresist (SU-8 50, MicroChem Inc.) to the surface of the silicon wafer. This 155

provides a base of common material that enhances adhesion of the microposts that will be deposited on the cured surface. The base layer was deposited on the silicon wafer using a spin coater set to 3000 rpm spin speed for a spin time of 25 s and acceleration/deceleration times of 2.3 s. The substrate was 160

then pre-baked on a hot plate for 5 min at 65ºC and for 15 min at 95ºC. After cooling the wafer down to 30ºC, the wafer was placed on a mask aligner and the entire area was exposed to UV light (25 mW/cm2 at 365 nm) for 12 s, thereby photopolymerizing the SU-8. Next, a 250 µm layer (SU-8 100, 165

MicroChem Inc.) was deposited using the spin coater set to 1000 rpm spin speed for a spin time of 25 s and acceleration/deceleration times of 2.3 s. The substrate was pre-baked for 30 min at 65ºC and for 90 min at 95ºC. After cooling the wafer down to 30ºC, it was placed on the mask 170

aligner with the channel layer mask in place and exposed to UV light (25 mW/cm2 at 365 nm) for 20 s. The substrate was post-baked for 2 min at 65ºC and ramped to 95ºC for 20 min. After cooling the wafer down to 30ºC, it was placed in SU-8 developer (MicroChem Inc.) until all unexposed SU-8 was 175

removed and all features of the design were observed. Isopropanol and acetone were used to remove any SU-8 left behind by the developer. The final product was baked on a hot plate for 1.0 h at 115ºC to ensure that the SU-8 was cured. Prepolymer PDMS (Sylgard 184, Dow-Corning) was 180

poured over the array of microposts and channel design, casting a negative of the positive SU-8 master using replica-molding (Chen et. al 2003). The wafer/PDMS mixture was placed on a hot plate for 2 h at 80ºC. The cured PDMS was removed from the SU-8 master after reaching room 185

temperature. This new PDMS negative master was made rigid by permanently adhering it to a glass slide via oxygen-plasma bonding. The surface of the PDMS master mold was silanated in a vacuum desiccator with 8 drops of trichloro(1H,1H,2H,2H-perfluorooctyl) silane (Aldrich) for 2 190

h. OS-20 (Dow Corning) was added to prepolymer PDMS to reduce the viscosity, thereby increasing micropost yield. Following the second PDMS casting step, the prepolymer solution was poured over the silanated PDMS master mold and set on a hot plate for 2 h at 80ºC to cure. The silanated 195

surface of the PDMS master mold resulted in easy removal of the newly cured PDMS microfluidic layers (Fig. 2A) from the master. The middle layer of the three-layer device was constructed using a 650 µm negative SU-8 master mold that was 200

fabricated to match the desired tissue slice shape and thickness. To construct the 650 µm master out of SU-8, a 500 µm layer was deposited first, and then a 150 µm layer was deposited (SU-8 100 has height limitations). The 500 µm layer of negative photoresist was deposited (SU-8 100, 205

MicroChem Inc.) using a spin coater set to 700 rpm spin



speed for a spin time of 40 s and acceleration/deceleration times of 2.3 s. The substrate was then pre-baked on a hot plate for 20 min at 65ºC and 2 h at 115ºC. After cooling the wafer down to 30ºC, a second layer of 150 µm (SU-8 100, 210

MicroChem Inc.) was deposited using the spin coater set to 2000 rpm spin speed for a spin time of 22 s and acceleration/deceleration times of 2.3 s. The substrate was pre-baked for 4 h at 95ºC. After the wafer cooled to room temperature it was placed on a mask aligner with the tissue 215

slice mask in place and exposed to UV light (25 mW/cm2 at 365 nm) for 32 s. The substrate was post-baked for 20 min at 65ºC and ramped to 95ºC for 4 h. After cooling the wafer down to 30ºC, it was placed in SU-8 developer until all unexposed SU-8 was removed. Isopropanol and acetone were 220

used to remove any SU-8 left behind by the developer. The final product was baked on a hot plate for 1 h to ensure that the SU-8 master was cured. Prepolymer PDMS was poured over the SU-8 tissue slice master and placed on a hot plate with an integrated 225

compression stand (constructed in house). The compression stand is used to press out the excess PDMS on top of the SU-8 master, thereby cutting out the tissue slice shape through the entirety of the PDMS. A thin transparency was placed on top of the SU-8 master/PDMS prepolymer to provide a flexible 230

interface to peel off after curing and since the PDMS does not cure in contact with the metal compression device. A block of polycarbonate was placed between the transparency and compression instrument to distribute pressure and reduce stress on the silicon wafer. The hot plate was set to 80ºC for 2 235

h and cooled to 30ºC before removing the PDMS layer from the SU-8 master. The result was a 650 µm layer shaped to house a tissue slice, as shown in Figure 2A, center. The inlets for the two fluid chamber layers were cut out using modified syringe tips. The three layers were aligned and 240

permanently bonded together (Fig. 2A-2B) using an oxygen-plasma treatment. The final device was permanently bonded

to a standard glass slide to provide a rigid substrate for mounting purposes. Fig. 2. Schematic of the PDMS 3-layer microfluidic slice chamber 245

construction. (A) Two masks with three distinct designs were used to pattern the three different PDMS layers. (B) The layers were assembled and cut to generate a flap that could be opened (C) to allow insertion of a brain slice (D). (E) Suction electrodes attached to nerve roots allowed recording of spontaneous respiratory motor output. The flap was removed 250

in (D) and (E) for clarity.

Modeling

Finite element analysis was utilized to gain a better understanding of the fluid interactions in the fluid chamber 255

layers of our multilayer perfusion chamber. Before simulations could be performed, the device design was drawn using Gambit (version 2.1.6, Fluent Inc.) and meshed using 80526 triangular face mesh elements. The following parameters were setup for the mesh: interval size of the face 260

mesh = 1 mm, boundary layer width = 10 µm, interval size of the edge mesh of the outer wall = 100 µm, interval size of the edge mesh of the post walls = 50 µm. Fluent UNS solver was used to define the boundary conditions (Fig. 3).

Fig. 3. Top and bottom layer of the multilayer perfusion chamber has the 265

following dimensions (in mm): Inlet circles diameter = 1.75; Width of inlet pipes = 1.025; Post diameter = 0.22; Post/Wall height= 0.250. The meshed device was imported into Fluent (Release 6.1.22, Fluent Inc.), a commercially available software for 270

computational fluid dynamics. Simulations were performed to analyze and compare the flow properties among the various designs (see Fig. 6). A multiphase VOF (Volume of Fluid) model was used. Two phases were defined, both being the same material (liquid water) with no surface tension 275

interaction between them and no wall adhesion forces. The pressure at the outlet was set to zero to indicate free flow uninhibited through the end of the chamber. The velocities of the inlets were varied in two different simulation trials (convergence was achieved in 53 iterations for each 280

simulation). The two outer inlets were always set to the same velocity (10 mm/s), while the middle inlet velocity was varied. These flow rates are comparable to those used in typical perfusion chambers (REF). 285

Medullary Slice Preparations

All procedures were approved by the Animal Care and Use Committee at the University of Wisconsin-Madison School of Veterinary Medicine. Sprague-Dawley rats (P1-P4) were anesthetized with isoflurane (5%, balance oxygen) and 290

decerebrated. The brainstem and cervical spinal cord were isolated in ice-cold standard solution and pinned onto a wax-covered plastic chuck. Standard solution contained (in mM): 120 NaCl, 26 NaHCO3, 20 glucose, 1.25 Na2HP04, 1.0 CaCl2, 2.0 MgSO4, 9.0 KCl (equilibrated with 95% O2 / 5% CO2 to 295

get pH~7.35). A vibratome (Leica VT1000S, Leica Microsystems Inc.) was used to serially section the rostral brainstem in the transverse plane to within 100-150 µm of the



pre-Bötzinger Complex (preBötC) rostral boundary. The preBötC is hypothesized to be a critical site for respiratory 300

rhythm generation (Onimaru et al., 2006). One transverse slice (530-700 µm thick) containing the preBötC (located bilaterally) was cut (Fig. 5A) and transferred to a microfluidic chamber. In microfluidic chambers, slices were bathed with standard solution at room temperature (flow rate = 2-3 305

ml/min). In one experiment, Na+-free solution was applied to one side of the slice (Fig. 5C, 5D). Na+-free solution consisted of (in mM): 120 choline chloride, 26 choline bicarbonate, 20 glucose, 1.25 K2HP04, 1.0 CaCl2, 2.0 MgSO4, 7.75 KCl (equilibrated with 95% O2 / 5% CO2 to get pH~7.35). To 310

record respiratory motor bursts, glass suction electrodes were attached to hypoglossal (XII) nerve rootlets (Fig. 5A, 5C). Signals were amplified (10,000x) and band-pass filtered (1-500 Hz) using a differential AC amplifier (model 1700, A-M Systems, Everett, WA) before being rectified and integrated 315

(time constant = 50 ms) using a moving averager (MA-821/RSP, CWE, Inc., Ardmore, PA) (Fig. 5). Burst analysis was performed using Clampfit 9.0 software (Axon Instruments, Foster City, CA). All measurements were averaged into 20-min bins and reported as mean ± S.E.M. 320

Results and Discussion

Design of Microfluidic Device

PDMS was chosen as the construction material because it offers many advantages over the classical approach, e.g. can be fabricated on the microscale, offers fine control of the fluid 325

microenvironment, and quickly fabricated to fit any planar tissue shape (rapid prototyping). The two fluid chambers were designed with 3 ~1mm diameter inlets. The chamber width is 5.5 mm to suffice the medullary brain tissue slice size. The design can be easily modified and fabricated to fit any brain 330

tissue slice dimension within days. The microposts cannot be too large in diameter or too closely spaced since this would inhibit diffusion of nutrients to the tissue slice, resulting in a higher rate of neural cell death. Neural cell death may cause changes in electrophysiology readings, such as a decrease in 335

rythmic burst times. The microposts should not be too small in diameter or spaced too far apart, to prevent indentation of the tissue slice by unequal distribution of its weight on the microposts. This could alter the mechanical integrity of the neural network residing in the tissue slice, leading to 340

distortions in the electrical recordings. After much trial and error, a design was achieved that met the appropriate standards for a tissue slice perfusion chamber. The microposts are arranged quincunx (diagonal-alternating pattern) (Fig. 3) to optimize flow distribution of oxygen/nutrients to the tissue 345

(Passeraub et al., 2003). The microposts are 250 µm high with a diameter of 200 µm, vertical spacing of 521 µm, and horizontal spacing of 456 µm, spanning the top and bottom area of the tissue slice. This configuration best supports the tissue slice, while maintaining adequate flow volumes and 350

distribution above and below it. The arrangement of the microposts in the chamber consists of a span of 150 µm in the center uninterrupted by microposts (Fig. 3). This layout of the microposts achieves a higher variation in fluid exchange rates

down the midline of the tissue slice compared to the area 355

consisting of posts. The uninterupted area in the center of the chamber enables the use of laminar flow to focus a specific solution down the tissue slice center at different flow speeds compared to that through the micrpost area, while still permiting fine control over the spatial resolution (Fig. 7). 360

Different micropost patterns and laminar flow demonstrate a means in which specific tissue slice areas can be targeted by solutions (Fig. 4, 7).

Brain Slice Flow Testing 365

Fluid tests were performed on the tissue slice chamber to observe the laminar flow and verify the mechanical stability of the tissue slice at different flow rates. Fluid flow rates reached as high as 1 ml/min for each of the three inlets in the top fluid chamber. Even at a combined inlet flow rate of 3 370

ml/min the mechanical integrity of the tissue was not compromised. The next step was to visually verify laminar flow over the tissue slice using a standard solution and distilled water containing florescent dye. Each of the outer inlets were set to an equal flow rate, 0.5 ml/min, and it was 375

observed that the two halves of the brain tissue slice of the neonatal rat were exposed to different solutions simultaneously (Fig. 4). Laminar flow can be further exploited to perform pulses or gradients of different solutions and monitor the changes in electrical activity of the targeted 380

neural network. More inlets may be added to the device to further increase combinations of solutions exposed over the tissue slice region, thereby increasing productivity in having multiple experimental environments within one tissue slice chamber that can be monitored. 385

Fig. 4. Distilled water containing concentrated fluorescent dye was used in conjunction with a standard solution to verify flow over the 650 µm brain slice slice in a microfluidic chamber. The two solutions maintained equal flows ranging from 0.5 to 1 ml/min, thereby exposing the two halves of the tissue slice to different environments. 390

Electrophysiology

Electrophysiology was performed after confirmation of both laminar flow and the tissue slice mechanical integrity at different flow rates. Slices (n=5) placed in the multilayer 395

PDMS microfluidic chamber began to spontaneously produce rhythmic motor bursts which reached a stable frequency and amplitude within 20 min (Fig. 5A). Burst frequency (baseline = 0.18 ± 0.02 Hz) and amplitude then decreased by 12 ± 1%



and 20 ± 5%, respectively, after 60 min (Fig. 5B). Two slices 400

were allowed to produce rhythmic motor bursts for > 3 hr. In a separate experiment (n=1), baseline activity was recorded from both left and right XII roots while the slice was bathed in standard solution (Fig. 5C). The fluid bathing one side of the slice was then switched to Na+-free solution to test 405

whether rhythmic activity could be blocked on one side of the slice. After 50 min, the XII root on the side bathed with Na+-free solution was silent while the other XII root continued to produce bursts (Fig. 5D).

410 Fig. 5 (A) Rythmic respiratory motor bursts from left and right XII roots during fluid flow in a microfluidic chamber. (B) Time-dependent changes in burst frequency and amplitude are small under these conditions. (C) Standard solution flowing simultaneously over separate halves of the tissue slice allowed simultaneous bursts to be recorded on both XII nerve 415

roots. Figure 5A and 5C display similar configurations with different time scales to distinguish separate experiments. (D) Superfusion of the right side of the slice with Na+-free solution showed that rhythmic XII activity was blocked on the right side while XII activity could be recorded on the left side, thereby showing that the slice microenvironment can be 420

selectively altered. These experiments clearly demonstrate how this novel perfusion device can be successfully used in complex electrophysiology experiments to monitor the electrical effects 425

of multiple solutions simultaneously flowing over a tissue slice. All experiments were carried out using common electrophysiology laboratory equipment.

Simulations 430

Simulations were used to quantify areas of stagnant flow and calculate the flow velocities through a transversal cross section of the brain tissue slice area (Fig. 6). Two different scenarios are depticted in Figure 6 where the fluid flow is 10 mm/s for all three inlets (10, 10, 10; Fig. 6B) as compared 435

with the outter two inlets maintaining a flow of 10 mm/s and no flow for the center inlet (10, 0, 10; Fig. 6C). The velocity of fluid through the uninterupted midline area ranged from 60.4 mm/s to 90.7 mm/s (Fig. 6C, 6B respectively). The fluid velocities through the micropost arrangement consistenty 440

varied within 13 mm/s for both scenarios (11-24.5 mm/s; 7.44-20.8 mm/s respectively). The design of the microposts

allows high flow rates for the midline, while maintaining a stable, non-stagnant flow in the microposts area, providing much more flexibility in altering the desired fluid exchange 445

rates than is possible in existing perfusion chamber systems. The technology can be extended to other experimental studies by altering the micropost design to a specific pattern to provide very flexible fluid exchange rates over specific areas of the tissue slice being studied and use focus flow to 450

concentrate a specific solution over the uninterupted area of the tissue slice.

Fig. 6. Modeling of the perfusion chamber design was done using Gambit 455

and Fluent (A-C). A plot of the fluid flow velocities (D) during inlet L fluid flow at 10 mm/s (B) and 0 mm/s (C), revealed that removing microposts from the midline achieves more spatial control down the midline of the tissue slice (shaded region of D) than through the micropost area. The velocity magnitude is determined by the length and 460

color intensity of the arrows. Other biological experiments are underway in which a focused flow is sent down the midline of the tissue slice in order to target the link between the bilateral rhythm 465

generation areas (pre Botzinger Complex) of the neural network found in the meduallary tissue slice (Fig. 7). The focus flow of a Na+-free solution serves to block communication between the left and right rhythm generators. In breaking this link, the rhythm generators should become 470

out of sync. Simply stopping the flow of Na+-free solution will reestablish the synchronicity. This clearly illustrates the fine control over the microfluidic environment that can provide new methods to study tissue slice preparations.

475

Fig. 7. Demonstration of a focus flow going through the midline of the chamber and tissue slice area without any interference from microposts.



Conclusions 480

A new multilayer perfusion chamber is demonstrated that perfuses brain tissue slices in a fine and controlled manner using multiple solutions simultaneously, while recording and monitoring the tissue’s electrical properties with common electrophysiological laboratory equipment. The microfluidic 485

environment promotes controlled laminar flow that offers fast localized exchange rates of solutions. The flexibility in bothe the fabrication process and flow control allows any user to have superior control over the microenvironment and the diffusion parameters they seek to expose to a specific tissue 490

slice area. Flow rates of up to 1 ml/min for each inlet do not alter the position of the brain slice due to the microposts holding the tissue slice in place, while maintaining the viability of the tissue slice as displayed in the electrical recordings. 495

Microfluidics has emerged as a useful tool for biological studies. The novel multilayer PDMS perfusion chamber design has much to offer to brain slice experimentation. The technology is highly adaptable to any brain tissue slice shape and offers superior control over the fluid microenvironment 500

that no other perfusion chamber in current literature can realize. Not only can multiple fluids target specific areas of the tissue slice but the widths of the fluid streams can be regulated by adjusting the flow rate accordingly. With short fabrication turnover times and the ability to construct feature 505

on the microscale, there are so many design applications awaiting. Microfluidics can be easily integrated with all types of instrumentation (e.g. MEA, thermal sensor) to further improve the precision of brain tissue slice experiments, and add to the simplification of the experimental setup and 510

quantification of results. The future automation possibilities added much interest into what the future hold for microfabricated perfusion systems.

Notes and references 1. Atencia J, Beebe DJ. Controlled Microfluidic Interfaces. Nature. 2005 515

Sep 29;437(7059):648-55. 2. Beebe DJ, Mensing GA, Walker GM. Physics and Applications of

Microfluidics in Biology. Annu Rev Biomed Eng. 2002;4:261-86. 3. Corrigall WA, Lucato RM. A Simple Modification to Permit Fast-Flow

Perfusion of Brain Slices. Brain Res Bull. 1980 Jul-Aug;5(4):481-2. 520

4. Croning MD, Haddad GG. Comparison of Brain Slice Chamber Designs for Investigations of Oxygen Deprivation in Vitro. J Neurosci Methods. 1998 Jun 1;81(1-2):103-11.

5. Duport S, Millerin C, Muller D, Correges P. A Metallic Multisite Recording System Designed for Continuous Long-Term Monitoring 525

of Electrophysiological Activity in Slice Cultures. Biosens Bioelectron. 1999 Apr 30;14(4):369-76.

6. Fagni L, Hugon M, Folco A, Imbert G. A Versatile Chamber for Microphysiologic Studies with Gas Mixtures Under High Pressure. Undersea Biomed Res. 1987 Mar;14(2):161-8. 530

7. Johnson SM, Smith JC, Feldman JL. Modulation of Respiratory Rhythm in Vitro: Role of Gi/o Protein-Mediated Mechanisms. J Appl Physiol. 1996 Jun;80(6):2120-33.

8. Kelso SR, Nelson DO, Silva NL, Boulant JA. A Slice Chamber for Intracellular and Extracellular Recording during Continuous 535

Perfusion. Brain Res Bull. 1983 Jun;10(6):853-7. 9. Koerner JF, Cotman CW. A Microperfusion Chamber for Brain Slice

Pharmacology. J Neurosci Methods. 1983 Mar;7(3):243-51.

10. Onimaru H, Homma I, Feldman JL, Janczewski WA. The para-facial respiratory group (pFRG)/pre Botzinger Complex (preBotC) is the 540

primary site of respiratory rhythm generation in the mammal. J Appl Physiol. 2006.

11. Palovcik RA, Phillips MI. A Constant Perfusion Slice Chamber for Stable Recording during the Addition of Drugs. J Neurosci Methods. 1986 Aug;17(2-3):129-39. 545

12. Passeraub PA, Almeida AC, Thakor NV. A microfabricated fluidic chamber for the perfusion of brain slices. [Engineering in Medicine and Biology, 2002. 24th Annual Conference and the Annual Fall Meeting of the Biomedical Engineering Society] EMBS/BMES Conference, 2002. Proceedings of the Second Joint , vol.3, no.pp. 550

2117- 2118 vol.3, 23-26 Oct. 2002 13. Passeraub PA, Almeida AC, Thakor, NV. Design, Microfabrication

and Analysis of a Nicrofluidic Chamber for the Perfusion of Brain Tissue Slices. Biomedical Microdevices. 2003;5(2): 147-155.

14. Stoppini L, Duport S, Correges P. A New Extracellular 555

Multirecording System for Electrophysiological Studies: Application to Hippocampal Organotypic Cultures. J Neurosci Methods. 1997 Mar;72(1):23-33.

15. Stopps M, Allen N, Barrett R, Choudhury HI, Jarolimek W, Johnson M, Kuenzi FM, Maubach KA, Nagano N, Seabrook GR. Design and 560

Application of a Novel Brain Slice System that Permits Independent Electrophysiological Recordings from Multiple Slices. J Neurosci Methods. 2004 Jan 30;132(2):137-48.

16. Tan JL, Tien J, Pirone DM, Gray DS, Bhadriraju K, Chen CS. Cells Lying on a Bed of Microneedles: An Approach to Isolate Mechanical 565

Force. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1484-9. 17. Taylor AM, Rhee SW, Jeon NL. Microfluidic Chambers for Cell

Migration and Neuroscience Research. Methods Mol Biol. 2006;321:167-77.

18. Walker GM, Zeringue HC, Beebe DJ. Microenvironment Design 570

Considerations for Cellular Scale Studies. Lab Chip. 2004 Apr;4(2):91-7.

‡ Footnotes should appear here. These might include comments relevant to but not central to the matter under discussion, limited experimental and 575

spectral data, and crystallographic data. 1 A. Name, B. Name and C. Name, Journal Title, 2000, 35, 3523; A.

Name, B. Name and C. Name, Journal Title, 2000, 35, 3523. 580



Single column figure/scheme (below)

Double column figure/scheme (below) 585

Single column image (no caption) (below)

Double column image (no caption) (below)

Single column numbered equation/reaction (below)

Single column table (below)

Double column table (below) 590

X Fig./Scheme XX Caption.

X Fig./Scheme XX Caption.

X

X

X (X)

Table XX Caption

X a Footnote text.

Table XX Caption

X a Footnote text.

Multilayer PDMS Microfluidic Brain Slice Chamber for...

Documents

Transcript of Multilayer PDMS Microfluidic Brain Slice Chamber for...