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.

Chemical Stimulation of Adherent Cells by Localized Application of Acetylcholine from a Microfluidic System

“…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.…”

“…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.…”

“…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.…”

Discrete Chemical Release From a Microfluidic Chip

“…In our previous work [18], localized functional chemical stimulation of adherent TE 671 cells was demonstrated by release of calcein AM and acetylcholine through a modular setup of bubble-jet print head. In this paper, we present an integrated approach for precisely controlled delivery of biochemical agents like neurotransmitters to specified regions within a cell culture.…”