TE671 cells comprise two different kinds of acetylcholine receptors, namely muscle type nicotinic (ionotropic) and muscarinic (metabotropic). This cell line is used as a model of human medulloblastoma (Stratton et al., 1989). The stimulation of nicotinic receptors leads to influx of sodium through the membrane channels and depolarization (Fryer and Lukas, 1999; Reitstetter et al., 1999; Fitch et al., 2003). Through a signaling cascade (IP3-receptors and ryanodine receptors) the stimulation of muscarinic receptors leads to the release of Ca²⁺ from the reservoirs into the cytoplasma (Bencherif and Lukas, 1991; Grassi et al., 1993; Giovannardi et al., 1994; Edahiro et al., 2004). This allows for the observation of the response of a large number of cells located within the field of view area by calcium imaging techniques.

TE671-cells were thawed and cultivated in Dulbecco's Modified Eagle's Medium (DMEM, 4.5 g/l glucose) supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin and streptomycin at 37°C and 5% CO₂. After 24 h the medium was exchanged. For cell transfer medium was removed, adherent cells were washed and subsequently incubated with buffer containing trypsin (0.5 mg/ml) and EDTA (0.22 mg/ml) to release cells from the substrate. Cells were transferred to a centrifuge vessel, sedimented at 1200 rpm and resuspended in warm culture medium. Cells were then seeded at a density of 3 × 10⁵/ml in a T75 flask and cultivated for 4 days. A maximum number of 35 passages were performed before a new lot of cells was retrieved from the cryo stock.

Membranes with pore diameters ranging from 50 to 820 nm were obtained from Whatman (Table 1) and were used for construction of cell culture vessels. SEM images of membranes (not shown) were taken in order to confirm manufactures data with respect to pore size and porosity.

Prior to cell seeding the membranes were treated with air plasma for 60 s (Harrick plasma cleaner) and subsequently coated by incubation in a 25 μg/ml laminin solution for 3 h (Zibek et al., 2007). Cells were seeded on the top side of nano- or microporous membranes.

In Figure 1 the experimental setup is shown. Cells are cultured in a vessel consisting of a glass ring (diameter 25 mm) with the porous membrane glued to its bottom. During a stimulation experiment, the cell culture vessel is mounted above an inkjet printhead with a bubble jet actuator allowing for the application of droplets (V_droplet = 20 pL) to the bottom face of the membrane. The distance between the inkjet apertures and the membrane was 2–3 mm, providing for an air space (Hu et al., 2007; Zibek et al., 2007) which effectively prevents any leakage of the stimulant from the printhead into the cell culture vessel. The printhead chip (Olivetti), comprising 208 nozzles and multiplexing electronics was integrated into a homemade electronics board and operated using a digital I/O board (National Instruments) controlled by a LabView^® application. This setup allowed for the arbitrary choice of a particular nozzle as well as for the number of droplets applied and the droplet frequency. The relative position of printhead and cell culture dish was adjusted manually. The density of pores is high and their lateral spacing small (Table 1) compared to the size of a droplet (diameter approx. 60 μm). As a result even in case of the largest pore size a large number of pores are always moistened. Thus, no particular alignment between pores and printhead is required.

The setup was operated at ambient temperature and humidity.

We modeled the substance transport from a sessile droplet underneath the membrane into the culture vessel via a porous membrane and the spatio-temporal distribution within the vessel and simulated it numerically (Figure 2A). Thus, a detailed understanding of the processes involved in substance delivery was gained. Also, quantitative data of substance transport and spatio-temporal distribution that could be related to experimental data on cellular response was obtained.

Mass transfer from the bottom side through the pores of the membrane into the cell chamber side is denoted as transmembrane flux J_sum according to Figure 2A and Eq. 1.

J_sum comprises two main contributions, namely diffusion and pressure driven flow, J_D and J_P, respectively, as is outlined in Section “Introduction” and “Materials and Methods” below. In addition, evaporation of the sessile droplet is considered in Section “Results and Discussion.”

Transport processes and spatio-temporal distribution within the chamber were calculated numerically employing a combined diffusion and convection approach (Figure 2A; Bruus, 2008). Velocity fields were calculated using Navier Stokes equations for incompressible fluids. FlexPDE^® software was used for all calculations.

The excitation of TE671 cells upon stimulation with a mixture of acetylcholine and Eosin Y was monitored via Ca²⁺-imaging. Eosin Y was added to indicate the position of the application spot. The dye does not stain or stimulate the cells as independent measurements confirmed. The increase of the fluorescence intensity observed within the cells is solely due to intracellular Ca²⁺ release which occurs as a result of the stimulation. A typical example of how the excitation progresses within the cell culture from the center (application spot) to the periphery is shown in Figure 6. In this case the pore diameter was 820 nm, acetylcholine concentration was 1 mM and Eosin Y concentration was 1 μM. In the graph at t = 3 s, some ROI which were used to calculate the integrated fluorescence intensity of cells are indicated (white circles). These data were retrieved from the frames of a video recording of the stimulation experiment and plotted as a function of time according to Eq. 7 chapter 2.4.

Figure 7A shows that the slope of the intensity plot toward the excitation maximum generally decreases with increasing distance of the respective cell from the application spot. Also, an increase of the delay between stimulation and cellular response is observed with increasing distance. This delay reflects the diffusion of the stimulant from the application spot to the site of the cell. The intensity plots It∗, can be related to c(r, t) obtained from the numerical simulation of the stimulant concentration (Figure 7B). The onset of the excitation (at 10% of the maximum value of the fluorescence intensity trace) of the cellular response coincides with a common threshold concentration of approximately ∼0.01 μM acetylcholine in all four cells considered in Figure 7A. For the sake of clarity, results of only four cells located at largely different distances from the application spot are displayed in this graph. An excitation level larger than 10% of the maximum value of the fluorescence was considered above threshold according to the general observation, that at this level the signals were well beyond noise contributions and excitation could be determined in a robust manner. This result was also verified in independent experiments with different membranes (not shown). As was expected and indeed observed in the experiments, the threshold concentration is independent of pore size and location of the cells. A threshold concentration value of ∼0.01 μM acetylcholine for TE671 cells was obtained in all cases.

These experiments demonstrate how the stimulation threshold concentration may be determined from a single chemical stimulation experiment by evaluation of Ca²⁺ signals obtained from a number of cells located at different distances from the application spot.

The dose related response of TE671 cells upon stimulation with acetylcholine was determined from the slope of the excitation plots. Data obtained from multiple experiments (at least three independent experiments on each membrane with three different droplet concentrations from 0.01 to 1 mM) on membranes with different pore sizes were combined (Figure 8). For each data point, the threshold concentration and the peak concentration at the site of a particular cell were calculated employing c(r, t) as previously obtained from numerical simulation. The slope of the excitation curve was retrieved from the measurement of the fluorescence intensity It* of that cell. Even though considerable scatter of data points is observed, a sigmoidal response as a function of stimulus can be fitted to these data yielding an EC50 value of 27 ± 4 μM. So far, there are no independent literature reports available concerning threshold concentration and EC50 value for the muscarinic acetylcholine receptors (mAChR) of TE671 cells after stimulation by acetylcholine. However, mAChR have been investigated in different cell types, namely chick embryo cells (Oettling et al., 1985) and human melanoma cells (Noda et al., 1998). In these cell types somewhat lower EC50 values ranging from 3.4 to 4.3 μM acetylcholine were observed.

We consider this result a first proof of principle for the measurement of dose–response curves employing this chemical stimulation setup. However, these data also show that while there are many data points at very low stimulant concentrations, there is somewhat a lack of data points at or even beyond saturation concentration. This is due to the fact that stimulant concentration decays approximately according to 1/r² with increasing distance from the application spot. On the other hand, the number of cells experiencing a certain concentration, c(r, t), increases according to r² with increasing distance assuming constant density of cells on the membrane. As a consequence relatively few cells that have been subjected to high concentration of the stimulant are recorded in any given experiment. In conclusion, in an optimized experimental protocol stimulant concentrations in excess of the saturation concentration have to be applied in order to determine saturation concentration.

In addition to the transient stimulation of cells discussed above where threshold and peak concentration of the stimulant determine the response, there are cellular processes which rely on the accumulation of the stimulant so that cells respond according to the integral of the concentration c(r, t):

As a model system, cellular uptake of calcein-AM and its conversion to fluorescent calcein was studied. Calcein is retained in the cells, i.e., no outflux needs to be considered. In these experiments, we assumed that the uptake rate of calcein-AM (Zibek et al., 2007) would be proportional to the concentration at the surface of the cell. Fluorescence micrographs in Figure 9 show a typical stimulation result. Figure 10A displays the fluorescence intensity of individual cells measured as a function of time and distance with respect to the application spot. In Figure 10B, fluorescence intensity of individual cells measured at six different times after application of calcein is plotted as a function of distance from the application spot. The lines in these plots are guides for the eye, connecting fluorescence intensity values recorded at the time indicated. Since the function c(r, t) is available from the simulation (Figure 10D), the integral, i.e., the total stimulus, may be calculated for any given location on the membrane (Figure 10C). Figure 10C shows that saturation of the stimulus is almost reached within less than a minute. On the other hand, the onset of fluorescence is delayed with respect to the stimulation signal. This may be explained by the enzymatic conversion of calcein-AM to fluorescent calcein by unspecific esterases. Here, several acetoxymethyl groups have to be esterified. Also, the cell culture was kept at room temperature during the experiment probably slowing down cell metabolism. The detailed origin of this discrepancy between calculation and experimental observations is subject to further investigation.

In this study the feasibility for both threshold gated stimulation and accumulated dose–response by localized droplet application was demonstrated employing a novel concept for localized chemical stimulation of cells adhering on a porous membrane.

Numerical simulation of substance transport showed that the kinetics of the stimulus may be tailored to a particular application by variation of pore size. Small pore size provides for a longer, continuous stimulation whereas larger pore size produces transient stimuli with higher initial stimulation amplitude and faster decay. In addition, the concentration of the stimulant in the solution can be adjusted as well: chemical stimulation at a given flow yet with varying kinetic pattern becomes feasible. Limitations exist, however, for the following reasons: firstly, as the solubility of compounds will always be limited, the maximum flow achievable by delivery through small pores will never reach the flow rates accessible when using large pores. Secondly, upper pore size limit is dominated by surface tension considerations, as a pore diameter which is too large will result in leakage of medium through the membrane rendering the system useless.

The degree of lateral resolution of chemical stimulation that is achievable not only depends on the size of the application spot but also on the particular stimulation mechanism, the concentration of the stimulant and the threshold concentration. For instance, in case of acetylcholine the spatial resolution of the stimulation is determined by the distance from the application spot at which the stimulant concentration has decayed below the stimulation threshold of the cells.

Numerical models were established providing quantitative predictions of chemical stimuli. Dose–response curves were obtained for stimulation of TE671 cells with acetylcholine. The EC50 values were compared to the available literature data of EC50 values obtained from different cell types which were nevertheless of comparable magnitude.

For further validation of this chemical stimulation scheme experiments employing substances with well-known response characteristics are needed. There, it will be important to employ sufficiently high concentrations of the stimulant solution in order to obtain an adequate number of responses from cells close or beyond the saturation concentration.

Future research will also be directed toward the development and integration of gateable microapertures with low leakage in microsystems for possible application in neural prostheses.