OPEN-CHAMBER FOCAL STIMULATION DEVICE FOR BIOMIMETIC STUDY OF THE NEUROMUSCULAR JUNCTION

T. Chang1*, N. Bhattacharjee and A. FolchUniversity of Washington, USA

ABSTRACT In this paper we present a novel microfluidic device designed for the patterning and culturing of myotubes in an open-chamber format, and the subsequent stimulation of sub-cellular regions. The device is assembled using three poly(dimethylsiloxane) (PDMS) layers and consists of an array of 32 identical focal stimulation jets. Each jet consists of a single stimulant channel and two flanking channels used to hydrodynamically focus the stimulant. These focal stimula-tion jets can be formed in an open-chamber format, allowing the cells to be cultured using conventional techniques, which significantly improves cellular viability and enhances protein expression for studying Neuromuscular Junction.

KEYWORDS: Open-Chamber, Hydrodynamic Focusing, Neuromuscular Junction

INTRODUCTION The mimicking of neuromuscular junction (NMJ) formation in a microfluidic cell culture system can be used in order to understand the formation and development of synapses. In previous studies, we used hydrodynamic focusing in a closed device to focally stimulate myotubes, which were patterned orthogonally to the direction of flow and fed conti-nuously with fresh tissue culture media through a network of perfusion channels [1-3]. However, the specialized opera-tion conditions necessary for a closed microfluidic device makes the platform difficult for use by biologists. Moreover, we observed (unpublished results) that basal acetylcholine receptor expression in myotubes grown in closed chambers is lower than that of ones grown in open Petri dishes using conventional culture techniques. Therefore, this novel device allows for long term cell viability similar to open-chamber culture, while generating stable focal streams with three-inlet plugs.

THEORY In a chemical synapse, the presynaptic neuron releases neurotransmitters focally, which bind to receptors located in the postsynaptic cell, usually embedded in the plasma membrane. In our design for mimicking a chemical synapse in vi-tro, the pattern of myotubes is perpendicular to the direction of fluid flow (Figure 1a). The device is assembled using three PDMS layers and consists of an array of 32 identical focal stimulation jets. The cell-culture trenches on the bottom layer of the device shields the myotubes from flow-induced shear. A focal plume is generated across the cell-laden trench by gravity-driven flow of stimulants through a 20 x 20 �m aperture and simultaneous vacuum aspiration through a 50 x 50 �m aperture on the other side of the trench. This system consumes very low amounts of reagents (80 �L/hour for all 32 apertures) despite being driven by hydrostatic pressure. Flanking channels (green in Figure 1a) aid in the hydrody-namic focusing of the stimulant. The fluid flow is mainly driven by a vacuum aspiration channel, which prevents the stimulant from diffusing into the medium bath. Depending on the depth of the trenches [4] where the myotubes are formed, the stimulant molecules can be transported to the cells by both convection and diffusion as shown in Figure 1b. For a quantitative study, the flow can be controlled by a syringe pump instead of hydrostatic flow and the vacuum aspira-tion can be adjusted with a precise digital gauge and a tuning valve.

Figure 1: a) 3D schematic: Based on a combination of flow injection and vacuum aspiration, the fluid flows over the patterned myotubes along the trench perpendicularly and in a focal manner. b) Cross section of a single jet of the device: The cells are seeded and shielded in the trenches of the open-chamber device for long term culturing. The flow is ejected from one channel and collected by a vacuum aspiration channel in order to create a focused stream. The molecules in the stream diffuse to the cells below, so the cells are chemically stimulated, but not exposed to large amounts of shear stress.

978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 869 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences3 - 7 October 2010, Groningen, The Netherlands

EXPERIMENTAL Device Fabrication. The device was fabricated with a 10:1 mixture of PDMS and curing agent using soft photolithogra-phy, replica molding and exclusion molding techniques. The three layers of PDMS were assembled using oxygen plas-ma treatment (22 s at 600 W and 0.75 Torr). The PDMS device was then bonded onto a glass slide using oxygen plasma before being fixed to a cell culture dish using PDMS. Operation of the Open-Chamber Microfluidic Device. The device contains three ports, two inlets for injection and one vacuum aspiration port, in order to generate an array of 32 identical focal stimulation jets (Figure 2). Bovine Serum Albumin (BSA) conjugated with Alexa Fluor 488 (Invitrogen) was used to visualize and pattern focal plumes on the PDMS substrate and thus ensure the delivery of the molecules to the bottom of the trench. This was also used to deter-mine the reliability and stability of the focal streams (Figure 3a). Before loading cells, the channels are filled with media in order to avoid air bubbles clogging the channels, especially the vacuum aspiration channels. Surface Coating. A PDMS insert was fabricated with a 10:1 mixture of PDMS and curing agent using standard soft photolithography, then coated with 1% filtered Pluronic F127 (Invitrogen) at room temperature for an hour. The open-chamber device was treated with oxygen plasma to form hydroxyl groups [-OH] on the glass-bottom surface of the trenches. The open chamber was then enclosed with a PDMS insert to allow the introduction of 100 µm/mL poly-D-lysine (PDL) by vacuum aspiration, which was coated at room temperature for an hour. The PDL-coated trenches were then gently washed with PBS three times before coating with a 1:5 mixture of growth factor-reduced Matrigel Matrix(BD Biosciences, Bedford, MA) in Dulbecco’s Modified Eagle’s Medium, which was allowed to adsorb overnight at 4°C. Finally, the PDMS insert can be removed easily with tweezers because of the repellent layer of Pluronic F127 present. Cell Seeding. The murine skeletal muscle cell line C2C12 was purchased from the American Type Cell Culture (Manas-sas, VA). Those myoblasts were seeded into the cell-culture trenches previously coated with PDL and Matrigel. After the cells reach 80% confluence (the cells need to attach along the trenches and form contacts with each other in order for cell fusion to occur), the normal high-serum medium (containing 10% fetal bovine serum) is replaced with low-serum medium (2% horse serum) in order to initiate differentiation into myotubes [5], which remain in open Petri dish condi-tions until the moment of focal delivery. The myotubes can also be embedded in Matrigel, resulting in 3D cultures close-ly mimicking in vivo conditions.

Figure 2: The device generates an array of 32 identical plumes which allows for high throughput data acquisition. The focal plumes are shown in blue while the focusing channels are shown in yellow.

Figure 3: a) BSA-conjugated with Alexa Fluor 488 patterning (no flow) b) Linescan of the focal stream (blue dash line in Figure 3a). The focal length is approximately 20 µm across the cell seeding trench.

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RESULTS AND DISCUSSION Open-Chamber Culture Device. Myotubes have been successfully fused in the device, while the focal stream was ap-plied perpendicularly across the myotube as showed in Figure 4a. As the cell culture chamber is open in the design of the device and the device is fixed onto a cell culture dish, long-term culture only requires exchanging media, similar to standard cell culture techniques. Accordingly, this open-chamber format eliminates the doubt of cell viability while cul-turing cells in the microfluidic system. Cell Tracker Experiment. A Cell Tracker experiment was performed on myoblasts in order to ensure the focal ability as shown in Figure 4b and c. The further Cell Tracker experiments will be performed in order to quantitatively measure the focal area on the myotubes. Cell Loading vs. Dimension of the Trench. The biggest challenge in operating this device is the cell loading process. Different dimensions of the trenches cause the resistance to change significantly while loading cells. Hence, the cells may be either difficult to pull through the enclosure trench or not distributed evenly throughout the loading area. Thus, surface modification may be needed in order to create cell repellent layers for loading cells from the top without using the PDMS insert. In further studies, 3D culture might be a superior solution for solving the problem of uneven cell loading. The cells can be well-mixed into the gel (preferably Matrigel due to it’s composition of 60% laminin which has the functionality of in-ducing acetylcholine receptor clustering ) before loading into the cell culture trenches. Furthermore, the system would more closely mimic in vivo conditions.

Figure 4: a) Myotubes are cultured in a 50 µm wide and 100 µm deep trench in open-Petri dish conditions for five days. The focal stream is produced above the trench perpendicularly. b and c) Fluorescence and overlay images of the Cell Tracker experiment. Myoblasts were seeded in a 60 µm wide and 30 µm deep trench. After culturing for 24 hours, Cell Tracker was delivered through the central fluid channels (bottom) for 1 hr before stopping the flow and applying vacuum from the vacuum aspiration channels (top) in order to produce focused streams.

CONCLUSIONS We have demonstrated that our open-chamber microfluidic device enables long-term culturing of muscle cells and supports muscle cell in a manner similar to conventional open-dish culturing. Cells can be grown perpendicular to the fluid direction by restricting their location within the device and the process of cell fusion can be geometrically con-trolled, resulting in well-defined myotubes. The focal streams can be reliably generated in the open-chamber format with the simple two inlet and one outlet device, even when the device has been submerged in cell culture medium for weeks. The role of agrin in the development of the neuromuscular junction will be investigated using this device.

ACKNOWLEDGEMENTS The research was funded by National Institutes of Health (NIH) Grant 2R01 EB001474.

REFERENCES [1] Tourovskaia, A., Figueroa-Masot, X. and Folch, A., "Differentiation-on-a-chip: A Microfluidic Platform for Long- Term Cell Culture Studies", Lab on a Chip 5, 14 (2005) [2] Tourovskaia, A., T.F. Kosar, and Folch, A. "Local Induction of Acetylcholine Receptor Clustering in Myotube Cul- tures Using Microfluidic Application of Agrin", Biophysical Journal 90, 2192 (2006). [3] Tourovskaia, A., Li, N., and Folch, A., "Localized acetylcholine receptor clustering dynamics in response to micro fluidic focal stimulation with agrin", Biophys. J. 95, 3009 (2008) [4] Manbachi, A., et al., Microcirculation within grooved substrates regulates cell positioning and cell docking inside mi- crofluidic channels. Lab on a chip, 2008. 8(5): p. 747. [5] C. Neville,N. Rosenthal, M.McGrew, N. Bogdanova and S. Hauschka, “Methods Cell Biol”, 1997, 52, 85–116.

CONTACT *T. Chang, tel: +1-206-6059489; tim7252@u.washington.edu

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