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Direct Access and Control of the Intracellular Solution Environment in Single Cells Jessica Olofsson,† Helen Bridle,† Aldo Jesorka,† Ida Isaksson,† Stephen Weber,‡ and Owe Orwar*,† Department of Chemical and Biological Engineering, and Microtechnology Centre, Chalmers University of Technology, SE-412 96 Go¨teborg, Sweden, and Department of Chemistry, Chevron Science Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Methods that can control and vary the solution environment around single cells are abundant. In contrast, methods that offer direct access to the intracellular proteome and genome in single cells with the control, flexibility, and convenience given by microfluidic methods are both scarce and in great demand. Here, we present such a method based on using a microfluidic device mounted on a programmable scanning stage and cells onchip permeabilized by the pore-forming glycoside digitonin. We characterized the on-chip digitonin poration, as well as the solution exchange within cells. Intracellular solution exchange times vary with the dose of exposure to digitonin from less than a second to tens of seconds. Also, the degree of permeabilization obtained for cells treated with the same dose varies considerably, especially for low doses of digitonin exposure and low permeabilities. With the use of the presented setup, the degree of permeabilization can be measured during the permeabilization process, which allows for “on-line” optimization of the digitonin exposure time. Using this calibrated permeabilization method, we demonstrate the generation of intracellular oscillations, intracellular gradients, and the delivery of substrate to initiate enzymatic reactions in situ. This method holds the potential to screen and titrate intracellular receptors or enzymes or to generate intracellular oscillations, useful in the study of signaling pathways and oscillation decoding among other applications. Since the cell membrane is impermeable to many substances, it is difficult to control the intracellular environment. Therefore, the supply of ligands and substrates to study and identify intracellular receptors and enzymes, for example, is a challenge. While different techniques, including photoactivation of caged compounds,1 the use of pore-forming agents2,3 or membranepermeability promotors,4,5 electroporation,6,7 and various membrane* Corresponding author. Owe Orwar, e-mail:
[email protected]. Phone + 46 (0) 31 772 30 60. Fax + 46 (0) 31 772 61 20. † Chalmers University of Technology. ‡ University of Pittsburgh. (1) Ellis-Davies, G. C. R. Nat. Methods 2007, 4, 619–628. (2) Geelen, M. J. Anal. Biochem. 2005, 347, 1–9. (3) Schulz, I. Methods Enzymol. 1990, 191, 280–300. (4) Dolmetsch, R. E.; Xu, K. L.; Lewis, R. S. Nature 1998, 392, 933–936. (5) Zweifach, A.; Lewis, R. S. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6295– 6299.
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permeable agents,8-11 have been developed to allow access to the interior of the cell, there is currently no method allowing for the control of intracellular environments such that, e.g., complex chemical waveforms could be applied to single cells. This paper describes a method for controlling the chemistry inside single cells. The technique combines the use of digitonin as an agent to increase the membrane permeability2,3 with a system, comprising a microfluidic device and a programmable, motorized scanning stage, for rapid solution exchange. When digitonin binds to cholesterol present in the cell membrane, a complex is formed yielding a membrane-spanning pore. It has also been shown that digitonin can permeabilize cholesterol-free membranes, but in those cases, the dose required is higher.12 It is thus thought that digitonin at low concentrations permeabilizes the outer plasma membrane but, generally, not organelles that contain less cholesterol.3,13,14 Digitonin has previously been used to study many different biochemical processes including import and export of nuclear proteins,15 nuclear permeability in apoptosis,16 the influence of calcium ions upon catecholamine secretion,17,18 and intracellular enzyme kinetics,2,19-21 e.g., tyrosine hydroxylase activity and phosphorylation22,23 and the conversion (6) Olofsson, J.; Nolkrantz, K.; Ryttsen, F.; Lambie, B. A.; Weber, S. G.; Orwar, O. Curr. Opin. Biotechnol. 2003, 14, 29–34. (7) Fox, M. B.; Esveld, D. C.; Valero, A.; Luttge, R.; Mastwijk, H. C.; Bartels, P. V.; van den Berg, A.; Boom, R. M. Anal. Bioanal. Chem. 2006, 385, 474–485. (8) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 4888–4894. (9) Di Carlo, D.; Aghdam, N.; Lee, L. P. Anal. Chem. 2006, 78, 4925–4930. (10) Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Chem. Biol. 2003, 10, 123–130. (11) Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Nature 2001, 411, 1016–1016. (12) Gogelein, H.; Huby, A. Biochim. Biophys. Acta 1984, 773, 32–38. (13) Comte, J.; Maisterrena, B.; Gautheron, D. C. Biochim. Biophys. Acta 1976, 419, 271–284. (14) Korn, E. D. Annu. Rev. Biochem. 1969, 38, 263. (15) Adam, S. A.; Marr, R. S.; Gerace, L. J. Cell Biol. 1990, 111, 807–816. (16) Roehrig, S.; Tabbert, A.; Ferrando-May, E. Anal. Biochem. 2003, 318, 244– 253. (17) Peppers, S. C.; Holz, R. W. J. Biol. Chem. 1986, 261, 14665–14669. (18) Wilson, S. P.; Kirshner, N. J. Biol. Chem. 1983, 258, 4994–5000. (19) Martins, A. M.; Mendes, P.; Cordeiro, C.; Freire, A. P. Eur. J. Biochem. 2001, 268, 3930–3936. (20) Martins, A. M.; Cordeiro, C. A.; Ponces Freire, A. M. FEBS Lett. 2001, 499, 41–44. (21) Ponces Freire, A. M.; Martins, A. M.; Cordeiro, C. Biochem. Educ. 1998, 26, 161–163. (22) Goncalves, C. A.; Hall, A.; Sim, A. T.; Bunn, S. J.; Marley, P. D.; Cheah, T. B.; Dunkley, P. R. J. Neurochem. 1997, 69, 2387–2396. 10.1021/ac802081m CCC: $40.75 2009 American Chemical Society Published on Web 02/05/2009
Figure 1. (A) The microfluidic device comprises 16 microchannels which individually connect solution reservoirs to an open volume. (B) Schematic illustration of the channel outlets (the area outlined in red in part A). The 16 microchannels exit as a tightly packed array generating a patterned laminar flow in an open volume. Note that diffusion acts to broaden each stripe in the flow pattern so that further out from the channel outlets the boundary between the separate solution environments is less sharp (this has not been included in this schematic). The flow pattern is controlled by the loading pattern applied to the solution reservoirs. A cell, held with a holding pipet, can be scanned through the flow pattern and exposed to the different solutions. The panel illustrates the cell as it is scanned from channel 1 to 11. Initially, the cell excludes the green substance from channel 2 and maintains its intracellular environment intact and unchanged. On exposure to digitonin in channel 4, pores are formed in the cell membrane and the intracellular solution is exchanged. Subsequent exposures result in uptake separated by washing in buffer.
of hydroquinone to benzoquinone catalyzed by peroxidase.24 The use of digitonin for complex control of intracellular solution environments has not been previously reported. This requires, in addition to a pore-forming agent, a technique facilitating the application of multiple solution exposures in sequence. The microfluidic system used here generates a patterned laminar flow containing a large number of different solution environments in an open volume.25 Scanning of a probe, such as a cell or cell fragment, through the flow pattern results in the probe experiencing a series of sequential exposures with millisecond exchange times between them.26,27 This microfluidic system has previously been applied to screen for ion channel functionality,28,29 to prepare receptor populations in different conformational distributions,30 to investigate memory effects of ion channels,27,30 and to generate chemical waveforms,26 mimicking physiological oscillations. The microfluidic system could alternatively be combined with other methods of increasing the permeability of the cell membrane, e.g., thapsigargin for the generation of calcium oscillations,5 other pore forming agents such as saponin,3 or electroporation.6 With the method reported in this paper, one obtains a high degree of control over the environment inside a single cell and sequential exposures to different solutions can be performed using the same cell, generating a wide result space. Further, sequential exposure algorithms makes it possible to control the degree of (23) Cheah, T. B.; Bobrovskaya, L.; Goncalves, C. A.; Hall, A.; Elliot, R.; Lengyel, I.; Bunn, S. J.; Marley, P. D.; Dunkley, P. R. J. Neurosci. Methods 1999, 87, 167–174. (24) Gao, N.; Wang, W.; Zhang, X.; Jin, W.; Yin, X.; Fang, Z. Anal. Chem. 2006, 78, 3213–3220. (25) Olofsson, J.; Pihl, J.; Sinclair, J.; Sahlin, E.; Karisson, M.; Orwar, O. Anal. Chem. 2004, 76, 4968–4976. (26) Olofsson, J.; Bridle, H.; Sinclair, J.; Granfeldt, D.; Sahlin, E.; Orwar, O. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8097–8102. (27) Granfeldt, D.; Sinclair, J.; Millingen, M.; Farre, C.; Lincoln, P.; Orwar, O. Anal. Chem. 2006, 78, 7947–7953. (28) Sinclair, J.; Pihl, J.; Olofsson, J.; Karlsson, M.; Jardemark, K.; Chiu, D. T.; Orwar, O. Anal. Chem. 2002, 74, 6133–6138. (29) Pihl, J.; Sinclair, J.; Sahlin, E.; Karlsson, M.; Petterson, F.; Olofsson, J.; Orwar, O. Anal. Chem. 2005, 77, 3897–3903. (30) Sinclair, J.; Granfeldt, D.; Pihl, J.; Millingen, M.; Lincoln, P.; Farre, C.; Peterson, L.; Orwar, O. J. Am. Chem. Soc. 2006, 128, 5109–5113.
plasma membrane permeabilization. This is done by measuring the degree of permeabilization during the permeabilization process and by adjusting the time of digitonin exposure to the requirements of each individual cell. This single-cell calibration procedure minimizes the exposure of intracellular structures to digitonin and is also minimally disruptive as the exposure to digitonin more precisely can be interrupted when a sufficient degree of plasma membrane permeability has been obtained. As shown in this article, the degree of permeabilization after a certain exposure to digitonin differs markedly between cells. The method can be used to create complex chemical waveforms in situ with the obvious limitation that the chemicals applied must be small enough to allow passage through the pores created by digitonin.3,31 The time required to equilibrate the intracellular concentration of a substance small enough to pass through the pores, with the extracellular concentration (i.e., the speed of the intracellular solution exchange), is the main factor which limits the resolution of the waveforms that can be created. Solution exchange times are modeled theoretically and investigated experimentally in this article. Applications of the system reported here include mimicking of physiological oscillations to study intracellular signaling and oscillation decoding as well as the pharmacological characterization and identification of intracellular receptors and enzymes. EXPERIMENTAL SECTION The Microfluidic System. All experiments were performed using the Dynaflow 16 microfluidic chip (Cellectricon AB, Gothenburg, Sweden) in which the normal bottom glass slide had been exchanged for a coverslip with thickness 130 µm in order to be able to utilize high-magnification, high-aperture optics with short working distances. The commercially available device comprises 16 microchannels (width 50 µm, height 60 µm, and length 50 mm) which originate in individual solution reservoirs and exit as a tightly packed array (separated by walls with a thickness of 22 µm at the point of exit) into an open volume (Figure 1A). The (31) Dourmashkin, R. R.; Harris, R. J. C.; Dougherty, R. M. Nature 1962, 194, 1116.
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Figure 2. FEM simulations of intracellular solution exchange. The cell is modeled as a sphere with a radius of 15 µm. The solution surrounding the sphere is instantaneously switched from 0 to 1 at time 0 and the subsequent concentration profiles along a cross-section through a sphere are plotted for three different cases: (A) a cell without a cell membrane, i.e., having no barrier to diffusion; (B) a cell with a membrane permeability P ) 9.612 µm/s; (C) a cell with a membrane permeability P ) 4.581 µm/s. In part A, the lowest blue line shows the concentration profile for t ) 40 ms and in parts B and C the lowest blue lines show the concentration profile for t ) 200 ms. The lines show the subsequent concentration profiles for a series of times with ∆t ) 40 ms in part A and ∆t ) 200 ms in parts B and C.
Figure 3. Part A shows how fluorescence intensity varies over time during an experimental series where the following exposure pattern is repeated six times: the cell is exposed to digitonin for 20 s, buffer solution for 20 s, fluorescein solution for 30 s, and buffer solution for 30 s. The blue curve shows the average intensity variation inside a region of interest inside the cell. The green curve shows the average intensity change in a region of interest outside the cell. Both regions of interest are marked as black circles in parts B-D. (B-D) Pairs of bright field images and fluorescence micrographs for three different times. The times are marked with color coded stars in part A.
volume of the 16 solution reservoirs is 80 µL each, and the dimensions of the open volume are 20 mm × 35 mm × 4 mm. The fluid exciting the microchannels couples viscously in the open volume to form a patterned laminar flow (Figure 1B).25 Cells scanned through this flow experience sequential solution exposure with typical solution exchange times of ∼10 ms. Prior to experiments, the device was loaded with different solutions, as indicated in the figure texts, using a micropipet. A 1 mm thick polycarbonate lid was attached over the solution reservoirs using double adhesive tape to create a closed system. The lid was attached using PE tubing to an electronic pressure controller (Cellectricon AM, Gothenburg, Sweden), which was used to compress the air enclosed by the lid to initiate a welldefined pressure-driven flow within the channels, forming a patterned laminar flow in the open volume. The flow rates used during measurements were 1-3 mm/s, depending upon the experiment. Cell Culture and Permeabilization. NG108-15 cells were cultivated in dishes 1-4 days prior to experiments in the medium (DMEM, high glucose with L-glutamine) supplemented with fetal calf serum (10%). All chemicals used in the cell culturing were from PAA Laboratories (PAA Laboratories, GmbH, Pasching, 1812
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Austria). Before experiments, the cells were washed and detached in extracellular buffer (ECB) containing (in millimolar) 155 NaCl, 2 CaCl2, 4.5 KCl, 3 MgCl2, 0.1 ZnCl2, 10 D-glucose, and 5 HEPES, pH adjusted to 7.2 using NaOH. A portion of the cell solution was added to the open volume of the chip and allowed to settle. During this period of the experiment, a low flow velocity (
