Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes.

An array of PDMS microchambers was aligned to an array of sensor electrodes and stimulating microelectrodes, which was used for the electrochemical monitoring of the metabolic activity of single isolated adult ventricular myocytes inside the chamber array, stimulated within a transient electric field. The effect of the accumulation of metabolic byproducts in the limited extracellular volume of the picolitre chambers was demonstrated by measuring single muscle cell contraction optically, while concomitant changes in intracellular calcium transients and pH were recorded independently using fluorescent indicator dyes. Both the amplitude of the cell shortening and the magnitude of the intracellular calcium transients decreased over time and both nearly ceased after 20 min of continuous stimulation in the limited extracellullar volume.

Regional electroporation of single cardiac myocytes in a focused electric field.

There is now a significant interest in being able to locate single cells within geometrically defined regions of a microfluidic chip and to gain intracellular access through the local electroporation of the cell membrane. This paper describes the microfabrication of electroporation devices which can enable the regional electroporation of adult ventricular myocytes, in order to lower the local electrical resistance of the cell membrane. Initially three different devices, designed to suit the characteristic geometry of the cardiomyocyte, were investigated (all three designs serve to focus the electric field to selected regions of the cell).

Local regional stimulation of single isolated ventricular myocytes using microfluidics.

The regional manipulation of the microenvironment surrounding single adult cardiac myocytes in a microfluidic structure is described. The flow rates of laminar streams were adjusted such that the fluid interface between an injection flow and a perfusion flow was manipulated laterally to stimulate regions of the cell surface. Using this general principle, we were able to selectively expose defined regions of the cell to test solutions, with predefined pulse durations and frequencies.

Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

Influence of hydrodynamic conditions on quantitative cellular assays in microfluidic systems.

This study demonstrates the importance of the hydrodynamic environment in microfluidic systems in quantitative cellular assays using live cells. Commonly applied flow conditions used in microfluidics were evaluated using the quantitative intracellular Ca2+ analysis of Chinese hamster ovary (CHO) cells as a model system. Above certain thresholds of shear stress, hydrodynamically induced intracellular Ca2+ fluxes were observed which mimic the responses induced by chemical stimuli, such as the agonist uridine 5'-triphosphate tris salt (UTP).

Microfluidic systems to examine intercellular coupling of pairs of cardiac myocytes.

In this paper we describe a microfluidic environment that enables us to explore cell-to-cell signalling between longitudinally linked primary heart cells. We have chosen to use pairs (or doublets) of cardiac myocyte as a model system, not only because of the importance of cell-cell signalling in the study of heart disease but also because the single cardiomyocytes are both mechanically and electrically active and their synchronous activation due to the intercellular coupling within the doublet can be readily monitored on optical and electrical recordings. Such doublets have specialised intercellular contact structures in the form of the intercalated discs, comprising the adhesive junction (fascia adherens and macula adherens or desmosome) and the connecting junction (known as gap junction).

Microfluidic partitioning of the extracellular space around single cardiac myocytes.

This paper describes the partitioning of the extracellular space around an electrically activated single cardiac myocyte, constrained within a microfluidic device. Central to this new method is the production of a hydrophobic gap-structure, which divides the extracellular space into two distinct microfluidic pools. The content of these pools was controlled using a pair of concentric automated pipets (subsequently called "dual superfusion pipet"), each providing the ability to dispense (i.

Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform.

A device based on five individually addressable microelectrodes, fully integrated within a microfluidic system, has been fabricated to enable the real-time measurement of ionic and metabolic fluxes from electrically active, beating single heart cells. The electrode array comprised one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an electrochemical lactate microbiosensor, that were used to measure the amounts of lactate produced by the heart cell. The device also allowed simultaneous in-situ microscopy, enabling optical measurements of cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+.

Extracellular recordings of field potentials from single cardiomyocytes.

Open microfluidic channels were used to separate the extracellular space around a cardiomyocyte into three compartments: the cell ends and a central partition (insulating gap). The microchannels were filled with buffer solution and overlaid with paraffin oil, thus forming the cavities for the cell ends. The central part of the cardiomyocyte rested on the partition between two adjacent microchannels and was entirely surrounded by the paraffin oil.

Characterisation of spatial and temporal changes in pH gradients in microfluidic channels using optically trapped fluorescent sensors.

This paper demonstrates the use of micron sized beads, modified with fluorescent dyes, as non-invasive sensors to probe the local changes in pH, within a microfluidic channel. To achieve this, amine modified polystyrene spheres (either 3 microm or 6 microm in diameter) were functionalised with the pH sensitive fluorochrome SNARF-1 to produce point sensors. The modified beads were trapped at defined positions close to a pair of integrated planar gold microelectrodes within the channel, using optical tweezers.

Stimulation of isolated ventricular myocytes within an open architecture microarray.

This paper is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca2+ and cell shortening, a series of microfluidic chambers (50 microm wide, 180 microm long, 400 microm high, 500 microm pitch) and connecting channels (200 microm wide, 5000 microm long, 50 microm high, 500 microm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8.

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction.

We asked to what extent Ca(2+) signals in two different domains of Paramecium cells remain separated during different stimulations. Wild-type (7S) and pawn cells (strain d4-500r, without ciliary voltage-dependent Ca(2+)-channels) were stimulated for trichocyst exocytosis within 80 ms by quenched-flow preparation and analysed by energy-dispersive X-ray microanalysis (EDX), paralleled by fast confocal fluorochrome analysis. We also analysed depolarisation-dependent calcium signalling during ciliary beat rerversal, also by EDX, after 80-ms stimulation in the quenched-flow mode.

Stimulation of single isolated adult ventricular myocytes within a low volume using a planar microelectrode array.

Microchannels (40- microm wide, 10- microm high, 10-mm long, 70- microm pitch) were patterned in the silicone elastomer, polydimethylsiloxane on a microscope coverslip base. Integrated within each microchamber were individually addressable stimulation electrodes (40- microm wide, 20- microm long, 100-nm thick) and a common central pseudo-reference electrode (60- microm wide, 500- microm long, 100-nm thick). Isolated rabbit ventricular myocytes were introduced into the chamber by micropipetting and subsequently capped with a layer of mineral oil, thus creating limited volumes of saline around individual myocytes that could be varied from 5 nL to 100 pL.

Ultra-low-volume, real-time measurements of lactate from the single heart cell using microsystems technology.

The fabrication of microelectrodes integrated within ultra-low-volume microtiter chambers for the amperometric determination of metabolites continues to be of interest in the subject of single-cell and high-throughput screening. The microsystem described in this paper consists of a two-microelectrode sensor with a microfluidic dispensation technology, which is able to deliver both very low titers (6.5 pL) and single heart cells into a low-volume microphotoelectrochemical cell.