Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine

Abstract

Acetylcholine sensitive TE 671 cells were cultured on nanoporous membranes and chemically stimulated by localized application of i), calcein-AM and ii), acetylcholine, respectively, onto the bottom face of the membrane employing an ink jet print head. Stimulus correlated response of cells was recorded by fluorescence microscopy with temporal and spatial resolution. Calcein fluorescence develops as a result of intracellular enzymatic conversion of calcein-AM, whereas Ca(2+) imaging using fluo-4 dye was employed to visualize cellular response to acetylcholine stimulation. Using 25 pl droplets and substance concentration ranging from 10 microM to 1 mM on Nucleopore membranes with pore diameters between 50 nm and 1 microm, a resolution on the order of 50 microm was achieved.

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 <10 μs temporal resolution. The location of the application spot (diameter typically ≈ 60 μm 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.

FIGURE 2

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

FIGURE 3

Panel of micrographs showing the application of a single droplet of acetylcholine (1 mM)/eosin Y (10 μM), 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 = 5 s 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.

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.