Chemical stimulation of adherent cells by localized application of acetylcholine from a microfluidic system

Abstract

Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup microdroplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution traveled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose-response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively. Numerical modeling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 μm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 μM acetylcholine independent of pore size were determined.

Keywords: acetylcholine; calcium imaging; chemical stimulation; microfluidic system; nanoporous membrane; neurotechnology; numeric modeling.

Figure 1

(A) Schematic depiction of setup and mode of operation. A cell culture vessel consisting of a glass ring with a porous membrane glued to its bottom is placed above an inkjet printhead allowing for the application of droplets containing a chemical stimulant to the bottom face of the membrane via an air space. (B) Distribution of stimulus and stimulated cell. (C) Closeup of the setup.

Figure 2

(A) Schematic depiction of the various contributions considered in the model of substance transport across porous membranes and within the chamber. (B–G) Example of calculated distribution of stimulant within the culture vessel at three different times after application of droplet to the bottom face of the membrane. Cross-section in the x–z-plane are shown, dimensions are in μm. Concentration of the stimulant in droplet was 1 mM, pore diameter was 50 nm (left column) or 820 nm (right column), respectively. Note the distinctly different kinetic and level of delivery of the stimulant to the culture vessel in both cases.

Figure 3

(A–D) Calculation of the contributions of diffusive and convective flow to the overall volume flow through the pores of the membrane as a function of time. The contact area wetted by the droplet was A = 2826 μm², concentration of stimulant within the droplet was 1 mM. Pore diameters ranged from 50 to 820 nm.

Figure 4

Droplet lifetime on membranes with different pore diameter (PD). Volume loss is caused by pressure driven flow due to surface tension and by evaporation, respectively. Evaporation of the droplet (V = 20 pL) is dominant in case of very small pore diameters (see also Figure 3). Complete evaporation of a droplet takes about 2 s according to Eq. 5. This value also reflects the maximum pulse duration achievable under the given experimental conditions. A repeated application was considered after achieving 1 pL of the preceding droplet volume. On the other hand, in case of micropores, evaporation is practically negligible and pressure driven flow is dominant thus allowing for very short stimulation pulses.

Figure 5

Validation of the model. The amount n(x, t) (volume-integral of concentration) of the stimulant in a volume element on the top surface of the membrane in the cell chamber was both calculated and determined experimentally. Eosin Y dye (droplet concentration 1 mM) was used as fluorescent tracer. Data was recorded for three different distances (40, 50, and 60 μm) with respect to the application spot and four types of membranes. Pore diameters: (A) 50 nm, (B) 100 nm, (C) 170 nm, (D) 820 nm. The volume element had a quadratic base area (side length l = 2 μm), its height spanning the fluid height in the cell culture vessel.

Figure 6

Series of fluorescence micrographs of stimulated TE671 cells cultivated on a porous membrane (pore size 820 nm) after application of a 20 pL droplet of acetylcholine (1 mM)/Eosin Y (1 μM). The dye indicates the position of the application spot (wetted area schematically shown in picture t = 0 s). Eosin Y is not taken up by the cells as was confirmed in separate experiments (not shown). Fluorescence observed within cells is solely due to a change of intracellular Ca²⁺ concentration as a result of excitation of the cells and the related change in Fluo-4 fluorescence. White circles indicate the approximate range of chemical stimulation at the respective time.

Figure 7

(A) Time course of stimulation related fluorescence intensity change (Ca²⁺-imaging) obtained from the ROI of cells located at distances between 70 and 140 μm from the center of the application spot (Figure 6). (B) Stimulant concentration as a function of time obtained from numerical simulation. The delay in the onset of the cellular response of cells at different distances is in agreement with the respective concentration functions. (C) A common threshold concentration of ∼0.01 μM can be retrieved from the combination of measurement and simulation (stimulant concentration: 100 μM acetylcholine, PD = 50 nm).

Figure 8

Dose–response curve obtained by fitting a sigmoidal curve to data obtained from stimulation of TE671 cells using an acetylcholine concentration ranging from 0.01 to 1 mM. An EC50 value of 27 ± 4 μM was obtained.

Figure 9

Accumulated dose–response: cell response after application of 20 droplets (400 pL) of calcein-AM to the bottom of the membrane PD = 820 nm. The first image at t = 0 s also displays the application spot (circle), subsequent images show increasing uptake and conversion of calcein-AM to fluorescent calcein over time. Brightness of cells decreases with distance to the application spot. Diameter of the stimulated region is >150 μm (see also Figure 10B). Application of only 10 droplets resulted in a response of cells only within the area wetted by the droplet (application spot A = 2826 μm²) (data not shown).

Figure 10

Experimental parameters as in Figure 9. (A) Fluorescence intensity I* as a function of time recorded in cells located at different distances from the application center. Peak brightness attained is higher for cells located closer to the application spot. (B) Fluorescence intensity distribution (i.e., response) as a function of distance from the application spot. For each cell I* is plotted at different times after stimulation. Lines are guides for the eye. (C) Integral of c(r, t) calculated from numerical modeling results indicating the total dose applied to cells at the given distance. (D) Relative concentration distribution calculated as fraction of maximum concentration achievable obtained from numerical modeling data.