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PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing
Biosensors & Bioelectronics 17 (2002) 87 – 93 www.elsevier.com/locate/bios
PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing Pierre Thie´baud, Lars Lauer, Wolfgang Knoll, Andreas Offenha¨usser * Max Planck Institute for Polymer Research in Mainz, Ackermannweg 10, 55128 Mainz, Germany Received 27 November 2000; received in revised form 14 August 2001; accepted 13 September 2001
Abstract A microfluidic device in polydimethylsiloxane (PDMS) consisting of an eight lines micro-injection array integrated in a base flow channel has been realized. The device is assembled from multiple PDMS parts, which have been moulded using notably micromachined masters in SU-8 photoresist. In contact with a planar substrate, up to eight independent laminar flow lines with cross-sections of 100 ×200 mm2 can be generated. Dedicated for the application of pharmaceutical compounds to electrogenic cells in vitro, this device was tested with a neuronal cell line, Mz1-cells. These were cultured on lines of laminin deposited onto polystyrene substrates by microcontact printing. We were able to inject into this culture multiple lines of coloured PBS in parallel to the orientation of cellular growth. No mixing between the individual flow lines did occur. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Microinjector array; Polydimethylsiloxane; SU-8 photoresist; Microcontact printing; PCC12 Mz1 neuroblastoma cells.
1. Introduction For extracellular signal recording from nerve cells in vitro, the use of microfabricated microelectrode arrays (MEAs) is of increasing interest. These devices employ glass or silicon substrates, onto which electrode arrays made of gold, platinum or indium tin oxide are fabricated. With such systems, simultaneous recordings from multiple sites of electrogenic cultures have been reported (Thomas et al., 1972; Pine, 1980; Gross et al., 1985; Novak and Wheeler, 1988; Fromherz et al., 1991; Bove et al., 1998; Egert et al., 1998; Thie´baud et al., 1999; Spro¨ssler et al., 1999). Following this achievement, methods were developed, able to guide neuronal growth to the individual electrodes. Indeed, sufficient electrical coupling between the cell and the electrode for extracellular signal recording is achieved only when a neuron is located directly on top of the electrode (Buitenweg et al., 2000). In order to manipulate the * Corresponding author. Present address: Institute for Thin Films and Interfaces, Research Center Ju¨lich, 52425 Ju¨lich, Germany. Tel.: +49-2461-61-2330; fax: +49-2461-61-2333. E-mail address: [email protected] (A. Offenha¨usser).
growth of neuronal cells, photolithographic techniques can be applied to produce guiding structures on the device surface. These structures can either control cellular growth by their topography (Connoly et al., 1990; Jimbo et al., 1993) or by their chemical properties modulating cellular adhesion. In this latter approach, one can either suppress cell adhesion around specific areas by deposition of non-adhesive layers like teflon® (Makohliso et al., 1998) or increase cell adhesion on specific areas by deposition of extracellular matrix (ECM) proteins such as laminin, fibronectin or collagen (Stenger and McKenna, 1994). A very convenient way to structure substrates with ECM proteins is microcontact printing (Singhvi et al., 1994). In this technique polymer-based microstamps, moulded from photolithographically structured masters are used to directly stamp patterns of ECM proteins to a substrate surface (James et al., 1998; Branch et al., 2000). Besides the improvement of coupling from the cell to the electrode, another opportunity arises when cells are arranged on the surface of a recording device in a defined pattern. In combination with a microfluidic system, producing laminar flow of different agents along the substrate surface, exposure of specific cells in
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P. Thie´baud et al. / Biosensors & Bioelectronics 17 (2002) 87–93
one culture to different pharmaceutical compounds becomes possible. When simultaneous extracellular recordings of the electrical activity are performed during drug delivery, a perturbation produced by the applied drugs can individually be analyzed in comparison to unexposed cells, or in comparison to cells exposed to another drug within the same culture. The study of signal transmission between cells exposed to different drugs is one of many other opportunities (e.g. studies of beating cardiac myocytes, observation of induced morphological changes, staining) provided by such an experimental setup. In this paper we present a microfluidic system which allows such a controlled application of drugs to cell cultures as described above. Its design and its fabrication will be presented in detail and the results of first experiments with patterned neuronal cells will be given.
2. Materials and methods In order to guarantee normal conditions for the cell culture and to maintain full accessibility of the substrate surface for cell plating, the microfluidic device is detachable from the culture substrate. Only when the experiment is started, a laminar flow carrying the agents of interest is generated in the culture by pressing the device to the substrate surface. This concept is illustrated in Fig. 1. A base flow is formed in a small
channel of 2500× 100 mm2 cross-section created between the top plate of the microfluidic device and the planar substrate surface. Drugs are added to this base flow by the second part of the device, the microinjector array. This part consists in an array of eight channels with 100× 150 mm2 cross-sections with a separation distance of 100 mm, through which the drugs are injected in the base flow. The specific dimensions of the device were chosen in order to fit the patterned microflow to typical arrangements of recording spots on the surface of MEA’s. As laminar flow is easily obtained inside microchannels of the employed dimensions (Kenis et al., 1999), mixing of the drugs does not occur. Thus it is possible to create up to eight different lines of pharmaceutical compounds flowing over the culture substrate and reaching the cells at specific spots. When switchable drug delivery pumps are used, also the timing of drug delivery can be controlled with the device. The entire microfluidic device is fabricated from polydimethylsiloxane (PDMS), a commercially available silicone based polymer (Sylgard® 182/184, Dow Corning, Germany). This silicone is a well known and widely employed material for the fabrication of microfluidic systems (McDonald et al., 2000; Delamarche et al., 1997; Effenhauser et al., 1997 Armani et al., 1999; Jo and Beebe, 1999). Apart from unwanted drug absorption by the material in certain circumstances, the qualities of PDMS lie in a high biocompatibility and
Fig. 1. Photograph of three injected flows of coloured solution (15 ml/min each) in a base flow (250 ml/min) (A). Ordered growth of nerve cells on a laminin patterned culture substrate at day 3 in culture (B). Scheme for the combination of patterned cells to patterned microflows (C).
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favourable mechanical properties, such as extreme flexibility and stability. In addition, post-curing assembly of different PDMS pieces is possible after oxygen plasma treatment.
3. Experimental
3.1. Patterned cell culture Structured cellular growth in vitro has been achieved by controlled transfer of laminin (1243217, Boehringer Mannheim GmbH, Germany) to hydrophobic petri dishes of polystyrene (Greiner Labortechnik, Germany) with the method of microcontact printing as described in detail before (Scholl et al., 2000; Lauer et al., 2001). For this purpose, a microstamp with line structures (parallel lines of 6 mm, separated at a distance of 100 mm) was fabricated by curing PDMS in 10 ml eppendorf tubes upside down on a master stamp produced by photolithography. Patterning was performed by first inking the microstamp for 30 s in 25 mg/ml laminin dissolved in PBS. Then drying the stamp surface in a stream of nitrogen, and immediately pressing the stamp to the substrate for 10 s with a force of 50 g/cm2 (Jeon et al., 1997; Libioulle et al., 1999). PCC7-Mz1 mouse embryonic carcinoma cells (Berger et al., 1997; Herget et al., 1998; Lang et al., 1989) were cultured on the patterned substrates and differentiated as described before (Jostock et al., 1998). Fig. 1B shows a phase contrast micrograph of the cell culture at day 3. The cells are ordered in lines following the laminin traces on the substrate surface. Individual size of the cell bodies typically was 10–15 mm in diameter.
3.2. De6ice fabrication Fig. 2 shows in which way the whole device is assembled from 4 components: As illustrated in Fig. 2A, the first two components form a microinjector. A base flow channel is composed by the other two components. The entire device is assembled by insertion of the microinjector into the base flow channel. A schematic cross-section of the assembled device is shown in Fig. 2B. All components are fabricated by pouring PDMS in specific moulds, and subsequently curing it in an oven. The temperatures applied for curing are in the range of 25– 150 °C according to the type of silicone and the material of the mould. Before pouring, the PDMS is systematically degassed in a vacuum chamber to remove bubbles. The first component, the microinjector top plate, contains eight grooves forming the individual injection channels of 100× 150 mm2 cross-section. The mould used for its production was fabricated on a silicon substrate by structuring SU-8 photoresist (MicroChem
Fig. 2. Cross-sectional view of the individual components of the device (A) and of the assembled setup (B).
Corp.) with photolithographic methods. SU-8 photoresist allows for the production of thick structures up to more than 1 mm (Lee et al., 1995; Despont et al., 1997; Dellmann et al., 1997), and thus has already been used for PDMS moulding in previous fluidic applications (Duffy et al., 1998). The grooves of this component are covered and sealed by a flat PDMS piece, the microinjector bottom plate, which is formed from a second mould. Attachment of top plate to bottom plate is achieved by exposing both components to a short oxygen plasma treatment and then connecting both pieces. Plasma treatment was performed for 15 s with 100 W at 0.1 mbar oxygen pressure. To obtain sharp edges at the channel openings of the microinjector array, the outlet side of the device is cut with a scalpel. Fig. 3 illustrates the entire fabrication process of the microinjector array in a cross-sectional view.
Fig. 3. Fabrication process of a microinjector: a microinjector top plate (1) moulded from a SU-8 photoresist master is bonded to a bottom flat plate (2) by oxygen plasma treatment (1 & 2).
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Fig. 4. Photograph of the device compared to a syringe needle.
For the production of the base flow channel moulds, no microlithography has been used as the dimensions of the channel (2.5 mm width and 100 mm depth) formed by this part are rather macroscopic. A simple metal mould, fabricated by normal fine mechanical means, has been used. In this mould, the previously formed microinjectors array is inserted such, that the eight microchannel outlets are pushed on the base of the mould. In this configuration, PDMS is poured in the mould. In order to prevent that the silicone, driven by capillary forces, enters the injection channels, these are prefilled with photoresist which is later dissolved by acetone after the polymerization has taken place. Finally, as shown in Fig. 2, the base flow channel is covered partly with a flat PDMS sheet by using the oxygen plasma method described before. The base flow top plate is fixed in such way to the base flow bottom plate, that the last 5 mm of the channel remain uncovered. Later in the experiment, these open 5 mm of the channel are pressed against the cell culture substrate to form a laminar flow in the resulting tube between channel and substrate surface. Connections to the microinjector array are made by inserting syringe needles, 600 mm in diameter, in the inlets of the device, which consist of 100 mm wide and 900 mm long slots. The base flow channel is directly connected to a plastic tube, 1.5 mm in diameter, by insertion of the tube into the channel. Each connection is sealed with silicone to avoid leakages.
4. Results and discussion The resulting device, shown in Fig. 4, consists of two integrated PDMS parts: an array of microinjectors (formed by eight channels with outlets sizes of 100× 150 mm, separated by 100 mm) is inserted in a base flow channel of 2.5 mm width and 100 mm depth. The injection takes place 10 mm before the end of the base flow channel. The last 5 mm of the base-flow channel are open to the substrate surface. Hence the channel is continued in this area between the substrate surface and the PDMS cover. The external width of this cover is 4 mm. Thus application of the device on any substrate surface greater than 4×5 mm2 is feasible. The moulding method applied for the fabrication of the device components allows the realization of well-defined and mechanically flexible fluidic paths. Fig. 5 shows a micrograph of the microinjector top plate where the eight microchannels come together to form the outlet array. It can be observed that the stress of the SU-8 photoresist induces a slight tilt angle to the structures. In total, a complex three-dimensional integrated setup has been produced from PDMS, which is entirely sealed and allows the application of laminar microfluidic flows to planar cell culture substrates. For experiments with cultured cells, the device was positioned relative to the cell culture substrate by means of a three axis micromanipulator setup (Mini 25, Luigs & Neumann, Germany). With this micromanipu-
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lator, the device was aligned parallel to the patterned cellular growth direction and then pressed to the substrate surface. Any leakage of the seal between device and substrate resulted in a strong deformation of the flow geometry. To avoid this effect, additional mechanical pressure was applied on top of the base-flow channel with a second micromanipulator arm to achieve a tight seal between microfluidic device and substrate surface. In this configuration, tests were performed using a four channel peristaltic pump. The first pump channel, filled with phosphate buffer solution (PBS), was connected to the base flow. Channels two, three and four were filled with trypan blue solution (L6323, Sigma, Germany) and connected to three adjacent micro-injectors from the eight channel microinjector array. The resulting coloured laminar flow on the cell culture substrate surface was observed through a phase contrast microscope. A typical micrograph of the resulting laminar flow lines is shown in Fig. 1A: the three injected flow lines, 15 ml/min each, embedded in the base flow of 250 ml/min, can clearly be distinguished. They appear with individual widths of 200 mm separated by 80 mm. Best results were achieved with base flow rates between 250 and 750 ml/min. At lower base flow rates, diffusion effects prevented a clear separation of the flow lines. Significantly higher base flow rates removed the cells from the substrate surface. In order to carry the injected liquids down to the substrate surface, the speed of the injection flow had to be chosen sufficiently high according to the speed of base flow. In
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addition to the injection itself, diffusion of molecules from the injected liquid may be responsible for them reaching the substrate surface. When magnification is increased, Mz1 cells aligned on the patterned lines of laminin can be distinguished under the flow lines as shown in Fig. 6. The flow lines are aligned in parallel to the cell pattern such that only cells on specific lines of the pattern are exposed to the blue dye. Trypan blue is a common dye for the identification of dead cells. After a short exposure time, dead cells exposed to the injected trypan blue solution became coloured, whereas staining of cells outside the injected lines was not observed. This demonstrates that different drugs could be specifically applied to distinct lines of the pattern without any mixing. However, further experiments need to be carried out in order to quantify the relationship between base flow and injection speed and the final drug concentration present at the cells on the substrate.
5. Conclusion In summary, this paper describes the realization of a microfluidic device for localized drug application to cell cultures. The principle of the system is based on the low diffusion properties of laminar flows in microchannels. The device is fabricated using PDMS technology. It is assembled from two parts, an array of micro-injectors and a base-flow channel. Independent and sharply
Fig. 5. Micrograph of the PDMS microinjector top plate. The groves of the eight injection channels of the device are highlighted with arrows. The location of the picture frame in respect to the entire device is shown in the insert.
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Fig. 6. Micropatterned flow of trypan blue in three lines applied to a culture of patterned neuronal cells.
defined lines of liquids were obtained without overlapping. Especially dedicated to cells cultures, this device was tested with neuronal cells patterned in a line geometry by means of microcontact printing. The results demonstrate that application of different pharmaceutical components to cells of only one culture and on only one single substrate can now be performed.
Acknowledgements The authors would like to thank Professor Alfred Maelicke and Dr Christoph Klein for providing the neuronal cell culture used in the experiments. The financial support of the Ministerium fu¨ r Bildung, Wissenschaft, Forschung und Technologie (BMBF) project no. 0310895 is gratefully acknowledged.
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Buitenweg, J.R., Rutten, W.L.C., Marani, E., 2000. Geometry based dynamic modeling of single neuron recording and stimulation. Abstracts of the International Meeting on Substrate-Integrated Microelectrode Arrays, Reutlingen, Germany, p. 21. Connoly, P., Clark, P., Curtis, A.S.G., Dow, J.A.T., Wilkinson, C.D.W., 1990. An extracellular microelectrode array for monitoring electrogenic cells in culture. Biosens. Bioelectron. 5, 223 –234. Delamarche, E., Bernard, A., Schmid, H., Michel, B., Biebuyck, H., 1997. Patterned delivery of immunoglobulins to surfaces using microfluidic networks. Science 276, 779 – 781. Dellmann, L., Roth, S., Beuret, C., Racine, G.-A., Lorenz, H., Despont, M., Renaud, P., Vettiger, P., de Rooij, N.F., 1997. Fabrication process of high aspect ratio elastic structures for piezoelectric motor applications. Proceeding of the 9th International Conference on Solid-State Sensors and Actuators: TRANSDUCERS’97, pp. 641 – 644. Despont, M., Lorenz, H., Fahrni, N., Brugger, J., Renaud, P., Vettiger, P., 1997. High-aspect-ratio, ultrathick, negative-tone near-UV photoresist for MEMS applications. Proceedings of IEEE Micro Electro Mechanical Systems: MEMS ‘97, pp. 518 – 522. Duffy, D.C., McDonald, J.C., Schueller, J.A., Whitesides, G.M., 1998. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal. Chem. 70, 4974 – 4984. Effenhauser, C.S., Bruin, G.J.M., Paulus, A., Ehrat, M., 1997. Integrated capillary electrophoresis on flexible silicone microdevices: analysis of DNA restriction fragments and detection of single DNA molecules on microchips. Anal. Chem. 69, 3451 – 3457. Egert, U., Schlosshauser, B., Fennrich, S., Nisch, W., Fejtl, M., Knott, T., Mu¨ ller, T., Ha¨ mmerle, H., 1998. A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays. Brain Res. Protoc. 2, 229 – 242. Fromherz, P., Offenha¨ usser, A., Vetter, T., Weis, J., 1991. A neuron – silicon junction: a retizus cell of the leech on an insulated -gate field-effect transistor. Science 252, 1290 – 1293. Gross, G.W., Wen, W.Y., Lin, J.W., 1985. Transparent indium – tin oxide electrode patterns for extracellular, multisite recording in neuronal cultures. J. Neurosci. Methods 15, 243 – 252.
P. Thie´ baud et al. / Biosensors & Bioelectronics 17 (2002) 87–93 Herget, T., Specht, H., Esdar, C., Oehrlein, S.A., Maelicke, A., 1998. Retinoic acid induces apoptosis-associated neural differentiation of a murine teratocarcinoma cell line. J. Neurochem. 70 (1), 47– 58. James, C.D., Davis, R.C., Kam, L., Craighead, H.G., Isaacson, M., Turner, J.N., Shain, W., 1998. Patterned protein layers on solid substrates by thin stamp microcontact printing. Langmuir 14, 741 – 744. Jeon, N.L., Finnie, K., Branshaw, K., Nuzzo, R.G., 1997. Structure and stability of patterned self-assembled films of octadecyltrichlorosilane formed by contact printing. Langmuir 13, 3382 – 3391. Jimbo, Y., Robinson, H.P.C., Kawana, A., 1993. Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture. IEEE Trans. Biomed. Eng. 40, 804 – 810. Jo, B.-H., Beebe, D.J., 1999. Fabrication of three-dimensional microfluidic systems by stacking molded polydimethylsiloxane (PDMS) layers. SPIE Conference on Microfluidic Devices and Systems, vol. 3877, pp. 222 – 229. Jostock, R., Rentrop, M., Maelicke, A., 1998. Cell fate specification in an in vitro model of neuronal development. Eur. J. Cell Biol. 76, 63 – 76. Kenis, P.J.A., Ismagilov, R.F., Whitesides, G.M., 1999. Microfabrication inside capillaries using multiphase laminar flow patterning. Science 285, 83 – 85. Lang, E., Marizauric-Stu¨ ker, M.L., Maelicke, A., 1989. States of developmental commitment of a mouse embryonal carcinomal cell line differentiating along a neuronal pathway. J. Cell Biol. 109, 2481 – 2493. Lauer, L., Klein, C., Offenha¨ usser, A., 2001. Spot compliant neuronal networks by structure optimized micro contact printing. Biomaterials, 22, 1925 –1932. Lee, K.Y., LaBianca, N., Rishton, S.A., Zolgharnain, S., Gelorme, J.D., Shaw, J., Chang, T.H.-P., 1995. Micromachining applications of a high resolution ultrathick photoresist. J. Vac. Sci. Technol. B 13, 3012 –3016.
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Libioulle, L., Bietsch, A., Schmid, H., Michel, B., Delamarche, E., 1999. Contact-inking stamps for microcontact printing of alkanethiols on gold. Langmuir 15, 300 – 304. Makohliso, S.A., Giovangrandi, L., Le´ onard, D., Mathieu, H.J., Ilegems, M., Aebischer, P., 1998. Application of teflon-AF® thin film for bio-patterning of neural cell adhesion. Biosens. Bioelectron. 13, 1227 – 1235. McDonald, J.C., Duffy, D.C., Anderson, J.R., Chiu, D.T., Wu, H., Schueller, O.J.A., Whitesides, G.M., 2000. Review on fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21, 27 – 40. Novak, J.L., Wheeler, B.C., 1988. Multisite hippocampal slice recording and stimulation using a 32 element microelectrode array. J. Neurosci. Methods 23, 149 – 159. Pine, J., 1980. Recording action potentials from cultured neurons with extracellular microcircuit electrodes. J. Neurosci. Methods 2, 19 – 31. Scholl, M., Spro¨ ssler, C., Denyer, M., Krause, M., Nakajima, K., Maelicke, A., Knoll, W., Offenha¨ usser, A., 2000. Ordered networks of rat hippocampal neurons attached to silicon oxide surfaces. J. Neurosci. Methods, J. Neuroscience. Meth. 104, 65 – 75. Singhvi, R., Kumar, A., Lopez, G.P., Stephanopulos, G.N., Wang, D.I.C., Whitesides, G.M., Ingber, D.E., 1994. Engineering cell shape and function. Science 264, 696 – 698. Spro¨ ssler, C., Denyer, M., Britland, S., Knoll, W., Offenha¨ usser, A., 1999. Electrical recordings from rat cardiac muscle cells using field-effect transistors. Phys. Rev. E 60, 2171 – 2176. Stenger, D.A., McKenna, T.M., 1994. Enabling Technologies for Cultured Neural Network. Academic Press, San Diego. Thie´ baud, P., Beuret, C., Koudelka-Hep, M., Bove, M., Martinoia, S., Grattarola, M., Jahnsen, H., Rebaudo, R., Balestrino, M., Zimmer, J., Dupont, Y., 1999. An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices. Biosens. Bioelectron. 14, 61 – 65. Thomas, C.A., Springer, P.A., Loeb, G.E., Berwald-Netter, Y., Okun, L.M., 1972. A miniature microelectrode array to monitor the bioelectric activity of cultured cells. Exp. Cell Res. 74, 61 –66.
PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing Pierre Thie´baud, Lars Lauer, Wolfgang Knoll, Andreas Offenha¨usser * Max Planck Institute for Polymer Research in Mainz, Ackermannweg 10, 55128 Mainz, Germany Received 27 November 2000; received in revised form 14 August 2001; accepted 13 September 2001
Abstract A microfluidic device in polydimethylsiloxane (PDMS) consisting of an eight lines micro-injection array integrated in a base flow channel has been realized. The device is assembled from multiple PDMS parts, which have been moulded using notably micromachined masters in SU-8 photoresist. In contact with a planar substrate, up to eight independent laminar flow lines with cross-sections of 100 ×200 mm2 can be generated. Dedicated for the application of pharmaceutical compounds to electrogenic cells in vitro, this device was tested with a neuronal cell line, Mz1-cells. These were cultured on lines of laminin deposited onto polystyrene substrates by microcontact printing. We were able to inject into this culture multiple lines of coloured PBS in parallel to the orientation of cellular growth. No mixing between the individual flow lines did occur. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Microinjector array; Polydimethylsiloxane; SU-8 photoresist; Microcontact printing; PCC12 Mz1 neuroblastoma cells.
1. Introduction For extracellular signal recording from nerve cells in vitro, the use of microfabricated microelectrode arrays (MEAs) is of increasing interest. These devices employ glass or silicon substrates, onto which electrode arrays made of gold, platinum or indium tin oxide are fabricated. With such systems, simultaneous recordings from multiple sites of electrogenic cultures have been reported (Thomas et al., 1972; Pine, 1980; Gross et al., 1985; Novak and Wheeler, 1988; Fromherz et al., 1991; Bove et al., 1998; Egert et al., 1998; Thie´baud et al., 1999; Spro¨ssler et al., 1999). Following this achievement, methods were developed, able to guide neuronal growth to the individual electrodes. Indeed, sufficient electrical coupling between the cell and the electrode for extracellular signal recording is achieved only when a neuron is located directly on top of the electrode (Buitenweg et al., 2000). In order to manipulate the * Corresponding author. Present address: Institute for Thin Films and Interfaces, Research Center Ju¨lich, 52425 Ju¨lich, Germany. Tel.: +49-2461-61-2330; fax: +49-2461-61-2333. E-mail address: [email protected] (A. Offenha¨usser).
growth of neuronal cells, photolithographic techniques can be applied to produce guiding structures on the device surface. These structures can either control cellular growth by their topography (Connoly et al., 1990; Jimbo et al., 1993) or by their chemical properties modulating cellular adhesion. In this latter approach, one can either suppress cell adhesion around specific areas by deposition of non-adhesive layers like teflon® (Makohliso et al., 1998) or increase cell adhesion on specific areas by deposition of extracellular matrix (ECM) proteins such as laminin, fibronectin or collagen (Stenger and McKenna, 1994). A very convenient way to structure substrates with ECM proteins is microcontact printing (Singhvi et al., 1994). In this technique polymer-based microstamps, moulded from photolithographically structured masters are used to directly stamp patterns of ECM proteins to a substrate surface (James et al., 1998; Branch et al., 2000). Besides the improvement of coupling from the cell to the electrode, another opportunity arises when cells are arranged on the surface of a recording device in a defined pattern. In combination with a microfluidic system, producing laminar flow of different agents along the substrate surface, exposure of specific cells in
0956-5663/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 2 7 9 - 2
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P. Thie´baud et al. / Biosensors & Bioelectronics 17 (2002) 87–93
one culture to different pharmaceutical compounds becomes possible. When simultaneous extracellular recordings of the electrical activity are performed during drug delivery, a perturbation produced by the applied drugs can individually be analyzed in comparison to unexposed cells, or in comparison to cells exposed to another drug within the same culture. The study of signal transmission between cells exposed to different drugs is one of many other opportunities (e.g. studies of beating cardiac myocytes, observation of induced morphological changes, staining) provided by such an experimental setup. In this paper we present a microfluidic system which allows such a controlled application of drugs to cell cultures as described above. Its design and its fabrication will be presented in detail and the results of first experiments with patterned neuronal cells will be given.
2. Materials and methods In order to guarantee normal conditions for the cell culture and to maintain full accessibility of the substrate surface for cell plating, the microfluidic device is detachable from the culture substrate. Only when the experiment is started, a laminar flow carrying the agents of interest is generated in the culture by pressing the device to the substrate surface. This concept is illustrated in Fig. 1. A base flow is formed in a small
channel of 2500× 100 mm2 cross-section created between the top plate of the microfluidic device and the planar substrate surface. Drugs are added to this base flow by the second part of the device, the microinjector array. This part consists in an array of eight channels with 100× 150 mm2 cross-sections with a separation distance of 100 mm, through which the drugs are injected in the base flow. The specific dimensions of the device were chosen in order to fit the patterned microflow to typical arrangements of recording spots on the surface of MEA’s. As laminar flow is easily obtained inside microchannels of the employed dimensions (Kenis et al., 1999), mixing of the drugs does not occur. Thus it is possible to create up to eight different lines of pharmaceutical compounds flowing over the culture substrate and reaching the cells at specific spots. When switchable drug delivery pumps are used, also the timing of drug delivery can be controlled with the device. The entire microfluidic device is fabricated from polydimethylsiloxane (PDMS), a commercially available silicone based polymer (Sylgard® 182/184, Dow Corning, Germany). This silicone is a well known and widely employed material for the fabrication of microfluidic systems (McDonald et al., 2000; Delamarche et al., 1997; Effenhauser et al., 1997 Armani et al., 1999; Jo and Beebe, 1999). Apart from unwanted drug absorption by the material in certain circumstances, the qualities of PDMS lie in a high biocompatibility and
Fig. 1. Photograph of three injected flows of coloured solution (15 ml/min each) in a base flow (250 ml/min) (A). Ordered growth of nerve cells on a laminin patterned culture substrate at day 3 in culture (B). Scheme for the combination of patterned cells to patterned microflows (C).
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favourable mechanical properties, such as extreme flexibility and stability. In addition, post-curing assembly of different PDMS pieces is possible after oxygen plasma treatment.
3. Experimental
3.1. Patterned cell culture Structured cellular growth in vitro has been achieved by controlled transfer of laminin (1243217, Boehringer Mannheim GmbH, Germany) to hydrophobic petri dishes of polystyrene (Greiner Labortechnik, Germany) with the method of microcontact printing as described in detail before (Scholl et al., 2000; Lauer et al., 2001). For this purpose, a microstamp with line structures (parallel lines of 6 mm, separated at a distance of 100 mm) was fabricated by curing PDMS in 10 ml eppendorf tubes upside down on a master stamp produced by photolithography. Patterning was performed by first inking the microstamp for 30 s in 25 mg/ml laminin dissolved in PBS. Then drying the stamp surface in a stream of nitrogen, and immediately pressing the stamp to the substrate for 10 s with a force of 50 g/cm2 (Jeon et al., 1997; Libioulle et al., 1999). PCC7-Mz1 mouse embryonic carcinoma cells (Berger et al., 1997; Herget et al., 1998; Lang et al., 1989) were cultured on the patterned substrates and differentiated as described before (Jostock et al., 1998). Fig. 1B shows a phase contrast micrograph of the cell culture at day 3. The cells are ordered in lines following the laminin traces on the substrate surface. Individual size of the cell bodies typically was 10–15 mm in diameter.
3.2. De6ice fabrication Fig. 2 shows in which way the whole device is assembled from 4 components: As illustrated in Fig. 2A, the first two components form a microinjector. A base flow channel is composed by the other two components. The entire device is assembled by insertion of the microinjector into the base flow channel. A schematic cross-section of the assembled device is shown in Fig. 2B. All components are fabricated by pouring PDMS in specific moulds, and subsequently curing it in an oven. The temperatures applied for curing are in the range of 25– 150 °C according to the type of silicone and the material of the mould. Before pouring, the PDMS is systematically degassed in a vacuum chamber to remove bubbles. The first component, the microinjector top plate, contains eight grooves forming the individual injection channels of 100× 150 mm2 cross-section. The mould used for its production was fabricated on a silicon substrate by structuring SU-8 photoresist (MicroChem
Fig. 2. Cross-sectional view of the individual components of the device (A) and of the assembled setup (B).
Corp.) with photolithographic methods. SU-8 photoresist allows for the production of thick structures up to more than 1 mm (Lee et al., 1995; Despont et al., 1997; Dellmann et al., 1997), and thus has already been used for PDMS moulding in previous fluidic applications (Duffy et al., 1998). The grooves of this component are covered and sealed by a flat PDMS piece, the microinjector bottom plate, which is formed from a second mould. Attachment of top plate to bottom plate is achieved by exposing both components to a short oxygen plasma treatment and then connecting both pieces. Plasma treatment was performed for 15 s with 100 W at 0.1 mbar oxygen pressure. To obtain sharp edges at the channel openings of the microinjector array, the outlet side of the device is cut with a scalpel. Fig. 3 illustrates the entire fabrication process of the microinjector array in a cross-sectional view.
Fig. 3. Fabrication process of a microinjector: a microinjector top plate (1) moulded from a SU-8 photoresist master is bonded to a bottom flat plate (2) by oxygen plasma treatment (1 & 2).
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Fig. 4. Photograph of the device compared to a syringe needle.
For the production of the base flow channel moulds, no microlithography has been used as the dimensions of the channel (2.5 mm width and 100 mm depth) formed by this part are rather macroscopic. A simple metal mould, fabricated by normal fine mechanical means, has been used. In this mould, the previously formed microinjectors array is inserted such, that the eight microchannel outlets are pushed on the base of the mould. In this configuration, PDMS is poured in the mould. In order to prevent that the silicone, driven by capillary forces, enters the injection channels, these are prefilled with photoresist which is later dissolved by acetone after the polymerization has taken place. Finally, as shown in Fig. 2, the base flow channel is covered partly with a flat PDMS sheet by using the oxygen plasma method described before. The base flow top plate is fixed in such way to the base flow bottom plate, that the last 5 mm of the channel remain uncovered. Later in the experiment, these open 5 mm of the channel are pressed against the cell culture substrate to form a laminar flow in the resulting tube between channel and substrate surface. Connections to the microinjector array are made by inserting syringe needles, 600 mm in diameter, in the inlets of the device, which consist of 100 mm wide and 900 mm long slots. The base flow channel is directly connected to a plastic tube, 1.5 mm in diameter, by insertion of the tube into the channel. Each connection is sealed with silicone to avoid leakages.
4. Results and discussion The resulting device, shown in Fig. 4, consists of two integrated PDMS parts: an array of microinjectors (formed by eight channels with outlets sizes of 100× 150 mm, separated by 100 mm) is inserted in a base flow channel of 2.5 mm width and 100 mm depth. The injection takes place 10 mm before the end of the base flow channel. The last 5 mm of the base-flow channel are open to the substrate surface. Hence the channel is continued in this area between the substrate surface and the PDMS cover. The external width of this cover is 4 mm. Thus application of the device on any substrate surface greater than 4×5 mm2 is feasible. The moulding method applied for the fabrication of the device components allows the realization of well-defined and mechanically flexible fluidic paths. Fig. 5 shows a micrograph of the microinjector top plate where the eight microchannels come together to form the outlet array. It can be observed that the stress of the SU-8 photoresist induces a slight tilt angle to the structures. In total, a complex three-dimensional integrated setup has been produced from PDMS, which is entirely sealed and allows the application of laminar microfluidic flows to planar cell culture substrates. For experiments with cultured cells, the device was positioned relative to the cell culture substrate by means of a three axis micromanipulator setup (Mini 25, Luigs & Neumann, Germany). With this micromanipu-
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lator, the device was aligned parallel to the patterned cellular growth direction and then pressed to the substrate surface. Any leakage of the seal between device and substrate resulted in a strong deformation of the flow geometry. To avoid this effect, additional mechanical pressure was applied on top of the base-flow channel with a second micromanipulator arm to achieve a tight seal between microfluidic device and substrate surface. In this configuration, tests were performed using a four channel peristaltic pump. The first pump channel, filled with phosphate buffer solution (PBS), was connected to the base flow. Channels two, three and four were filled with trypan blue solution (L6323, Sigma, Germany) and connected to three adjacent micro-injectors from the eight channel microinjector array. The resulting coloured laminar flow on the cell culture substrate surface was observed through a phase contrast microscope. A typical micrograph of the resulting laminar flow lines is shown in Fig. 1A: the three injected flow lines, 15 ml/min each, embedded in the base flow of 250 ml/min, can clearly be distinguished. They appear with individual widths of 200 mm separated by 80 mm. Best results were achieved with base flow rates between 250 and 750 ml/min. At lower base flow rates, diffusion effects prevented a clear separation of the flow lines. Significantly higher base flow rates removed the cells from the substrate surface. In order to carry the injected liquids down to the substrate surface, the speed of the injection flow had to be chosen sufficiently high according to the speed of base flow. In
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addition to the injection itself, diffusion of molecules from the injected liquid may be responsible for them reaching the substrate surface. When magnification is increased, Mz1 cells aligned on the patterned lines of laminin can be distinguished under the flow lines as shown in Fig. 6. The flow lines are aligned in parallel to the cell pattern such that only cells on specific lines of the pattern are exposed to the blue dye. Trypan blue is a common dye for the identification of dead cells. After a short exposure time, dead cells exposed to the injected trypan blue solution became coloured, whereas staining of cells outside the injected lines was not observed. This demonstrates that different drugs could be specifically applied to distinct lines of the pattern without any mixing. However, further experiments need to be carried out in order to quantify the relationship between base flow and injection speed and the final drug concentration present at the cells on the substrate.
5. Conclusion In summary, this paper describes the realization of a microfluidic device for localized drug application to cell cultures. The principle of the system is based on the low diffusion properties of laminar flows in microchannels. The device is fabricated using PDMS technology. It is assembled from two parts, an array of micro-injectors and a base-flow channel. Independent and sharply
Fig. 5. Micrograph of the PDMS microinjector top plate. The groves of the eight injection channels of the device are highlighted with arrows. The location of the picture frame in respect to the entire device is shown in the insert.
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Fig. 6. Micropatterned flow of trypan blue in three lines applied to a culture of patterned neuronal cells.
defined lines of liquids were obtained without overlapping. Especially dedicated to cells cultures, this device was tested with neuronal cells patterned in a line geometry by means of microcontact printing. The results demonstrate that application of different pharmaceutical components to cells of only one culture and on only one single substrate can now be performed.
Acknowledgements The authors would like to thank Professor Alfred Maelicke and Dr Christoph Klein for providing the neuronal cell culture used in the experiments. The financial support of the Ministerium fu¨ r Bildung, Wissenschaft, Forschung und Technologie (BMBF) project no. 0310895 is gratefully acknowledged.
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