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Copyright © 2007 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 92, Issue 1, L04-L06, 1 January 2007

doi:10.1529/biophysj.106.096743

Biophysical Letters

Localized Functional Chemical Stimulation of TE 671 Cells Cultured on Nanoporous Membrane by Calcein and Acetylcholine

Susanne Zibek^*, Alfred Stett^*, Peter Koltay^†, Min Hu^†, Roland Zengerle^†, Wilfried Nisch^* and Martin Stelzle^*, ,  

^* Natural and Medical Sciences Institute, D-72770 Reutlingen, Germany
^† Institute of Microsystem Technology IMTEK, D-79110 Freiburg, Germany

Address reprint requests and inquiries to Dr. Martin Stelzle, Natural and Medical Sciences Institute, Tel.: 49-7121-5153075; Fax: 49-7121-5153062.

Electrostimulation has been the method of choice for almost all neurostimulation devices in use to date. As it relies solely on membrane depolarization by electric fields to induce a cellular response, it lacks, however, the cell type selectivity and spatial resolution in principle achievable by chemical stimulation. Several microfabricated drug delivery systems for controlled release of biochemically active compounds have been proposed 1,2,3. Earlier attempts to use microfluidic devices for chemical stimulation of cells suffered from the problem of uncontrolled leakage from open microapertures into the medium and consequently undefined concentration conditions 4,5. In contrast, our novel “air gap”-scheme 6 (Fig. 1) avoids such leakage completely. It facilitates the precise positioning of the application spot directly under or at a lateral distance with respect to cells of interest. Either calcein-AM or acetylcholine was used for stimulation. Concentration gradients develop along the surface of the cell layer eliciting specific cellular responses.

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Figure 1

Experimental setup for localized chemical stimulation of cells that grow on a nanoporous membrane forming the bottom of a tissue chamber. Either a single or a series of microdroplets may be generated and shot at the bottom face of this membrane by a bubble jet device at <10μs temporal resolution. The location of the application spot (diameter typically ≈ 60μm at 25 pl droplet volume) is adjusted by an xy-stage. The fluid penetrates the nanopores and either directly stimulates a cell growing on these pores or diffuses in the medium to cells in the periphery and eventually elicits cellular responses that are monitored by fluorescence microscopy.

Tissue chambers consisting of a glass ring with a Nucleopore membrane (Whatman International, Maidstone, UK) with pore sizes ranging from 50nm to 1μm glued to their bottom were homemade and treated in an air plasma (30s) before cultivation of TE 671 muscle sarcoma cells. Dulbecco's modified Eagle's medium (4.5 g/l glucose) supplemented with 10% fetal calf serum, 2mM glutamine, penicillin, and streptomycin was used. A commercially available bubble jet print head was used to apply single or multiple droplets of ∼25 pl volume to the bottom face of the membrane (Fig. 1) 6.

Mixtures of calcein-AM (0.25mM, nonfluorescent) and eosin Y dye (0.1mM) and of acetylcholine (1mM) and eosin Y dye (10μM) were used. Eosin Y served to indicate the distribution and the diffusive transport of the active, yet invisible substance (calcein-AM or acetylcholine, respectively) in the medium. Cellular response was recorded using a Zeiss Axiophot fluorescence microscope (Zeiss, Göttingen, Germany) fitted with a Hamamatsu C2400-08 video camera (Hamamatsu Photonics, Herrsching, Germany) employing 485nm excitation and 515 - 585nm emission filters. Calcein AM becomes fluorescent after enzymatic hydrolysis of ester residues after its incorporation into cells. To monitor excitation by acetylcholine, cells were first loaded with Ca²⁺-sensitive fluo-4-AM dye (Invitrogen, Karlsruhe, Germany) by incubation for 30min at a concentration of 2.3μM. Subsequently the medium was changed to remove extracellular fluo-4-AM dye and cells were incubated for another hour at 37°C. Upon excitation, intracellular Ca²⁺ concentration increases resulting in an increase of fluorescence intensity. The latter is determined using Image J software in regions of interest of 10μm diameter manually specified for each cell (cf. Fig. 3, t=5s).

Application of calcein-AM/eosin Y: TE 671 cells were cultivated on nanoporous membranes (pore size, 1μm) and stained with Hoechst 33342 dye (nucleus staining) to track cell location in the membrane (Figure 2A). The calcein-AM/eosin Y mixture (5 droplets, 125 pl total volume) was applied resulting in a transient fluorescence signal indicating the spot size (≈60μm) where substance was actually delivered to the cell layer. Control experiments with eosin Y did not show any uptake of this dye into TE 671 cells. Rather, eosin Y fluorescence vanishes over a period of several minutes to invisibility due to diffusion in the medium (Figure 2D). After this time period, only cells appear fluorescent, which is attributed to the incorporation and enzymatic hydrolysis of calcein-AM by the cells (Figure 2D). At the concentration employed here (0.25mM), only cells located directly above the application spot were sufficiently loaded by calcein to show appreciable fluorescence. Variation of concentration and droplet volume thus determines effective spatial resolution achievable by this chemical stimulation scheme.

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Figure 2

Chemical stimulation by calcein-AM / eosin Y. (A) Micrograph of cell culture before stimulation in Hoechst 33342 fluorescence. (B) Eosin Y fluorescence 1s after deposition of droplets. (C) Eosin Y diffuses in the medium and fluorescence intensity decreases. (D) After 4min, intracellular calcein fluorescence is retained, whereas eosin Y fluorescence has decayed.

Acetylcholine stimulation: single droplets of a mixture of acetylcholine (1mM) and eosin Y (10μM) were applied to the bottom of a nanoporous membrane (pore size, 50nm). Fig. 3 shows a panel of fluorescence micrographs of the cell culture after the application of the droplet (at t=0s). The eosin Y fluorescence indicates the location of the application spot (center). Subsequently, fluorescence of TE 671 cells appears and shows a wave of excitation progressing from the application spot to the periphery in a circular symmetry. In the figure inset, the effective radius of excitation is given as indicated by arrows in the micrographs. After 30s, fluorescence has almost completely decayed. Fluorescence intensity traces of the cells indicated by circles in Fig. 3 (t=5s) were analyzed in detail (Fig. 4, Table 1). The delay, τ, of cellular response with respect to droplet delivery varies proportional to the square of the distance, d, between the rim of the application spot and the location of the cell under investigation as is expected for diffusive transport (Fig. 4inset, Table 1).

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Figure 3

Panel of micrographs showing the application of a single droplet of acetylcholine (1mM)/eosin Y (10μM), the dilution of eosin Y as a result of diffusion, and the evolution of a circular excitation pattern in the cell layer visualized by Ca²⁺ imaging. The small circles in the graph at t=5s indicate the location of cells whose fluorescence intensity is plotted as a function of time in Fig. 4. Additional data is provided in Table 1.

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Figure 4

Temporal evolution of the fluorescence intensity (fluo-4-AM dye) in cells as indicated in Fig. 3 (traces of only three cells plotted). The inset shows the delay, τ, in the response of cells as a function of the distance, d, between the rim of the application spot and the cell for all cells indicated in Fig. 3.

The apparent lag time of 1.5s between stimulation and onset of excitation of cells (inset, Fig. 4) is attributed to intracellular signaling in agreement with earlier reports 7. The low level of fluorescence intensity at t<1.5s confirms that contribution of eosin Y to overall fluorescence is negligible outside the application spot. Interestingly, cells showed different maximum intensities, even if located at similar distances d. This may be due to different degrees of loading with fluo-4-AM dye. The origin of the modulation in some of the fluorescence signals (for example, cell 5, Fig. 4) is yet unknown.

The response time as it may be determined from the initial slope (from 10% to 50% of the final fluorescence signal intensity seems to show a tendency toward an increase with increasing d). This would point to a dose/response relationship as acetylcholine concentration is expected to decrease with increasing d. However, further study in cell cultures with a larger field of view will be necessary to confirm this preliminary finding. At lower concentration of acetylcholine, excitation is limited to a smaller area or even precisely to the application spot (data not shown). This finding is as expected, since half spherical diffusive transport of acetylcholine in the space above the cell layer will result in progressive dilution of the neurotransmitter. At a certain distance from the application spot, the concentration will be lower than the threshold level required to induce excitation of cells. Also, the use of smaller droplets would further enhance spatial resolution.

Functional chemical stimulation of adherent cells may be achieved in a well-controlled and versatile fashion by utilizing nanoporous membranes as support and localized substance application by bubble jet technology. By variation of concentration and/or volume of the fluid, spatial resolution and range of excitation may be controlled. Future research will be directed toward numerical simulation of concentration profiles along the surface of the cell layer considering the application spot as extended drainable reservoir and the measurement of complete dose/response curves from experiments as demonstrated in this Letter. As the cellular response reflects the local concentration, this approach should allow for rapid determination of dose/response curves in a single experiment as opposed to the multi-well plate approach commonly used today.