RESUMO
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). We demonstrate that one of these three devices revealed the sequence of cellular responses to field strengths of increasing magnitudes, namely, cell contraction, hypercontraction, and lysis. This same device required a reduced threshold voltage for each of these events, including in particular membrane breakdown. We were not only able to show the gradual regional increase in the electric conductivity of the cell membrane but were also able to avoid changes in the local intra- and extracellular pH (by preventing the local generation of protons at the electrode surface, as a consequence of the reduced threshold voltage). The paper provides evidence for new strategies for achieving robust and reproducible regional electroporation, a technique which, in future, may be used for the insertion of large molecular weight molecules (including genes) as well as for on-chip voltage clamping of the primary adult cardiomyocyte.
Assuntos
Eletroporação/instrumentação , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Condutividade Elétrica , Eletrodos , Eletroporação/métodos , Concentração de Íons de Hidrogênio , Técnicas Analíticas Microfluídicas , Miócitos Cardíacos/química , CoelhosRESUMO
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. The intracellular pH decreased gradually during 20 min of continuous stimulation after which a dramatic pH drop was observed, indicating the breakdown of the intracellular buffering capacity. After continuous stimulation, intracellular lactate was released into the microchamber through cell electroporation and was detected electrochemically at a lactate microbiosensor, within the chamber. A mitochondrial uncoupler was used to mimic ischaemia and thus to enhance the cellular content of lactate. Under these circumstances, intracellular lactate concentrations were found to have risen to approximately 15 mM. This array system has the potential of simultaneous electrochemical and optical monitoring of extracellular and intracellular metabolites from single beating heart cells at a controlled metabolic state.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Miócitos Cardíacos/metabolismo , Análise Serial de Tecidos/métodos , Análise de Variância , Animais , Técnicas Biossensoriais/métodos , Cálcio/metabolismo , Células Cultivadas , Estimulação Elétrica , Concentração de Íons de Hidrogênio , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ácido Láctico/análise , Ácido Láctico/metabolismo , Miócitos Cardíacos/química , CoelhosRESUMO
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. We demonstrate the transient depolarisation of the cardiomyocyte through the regional chemical stimulation of localized areas of the cell with elevated potassium concentrations (100 mM). The results show that chemical stimulation at frequencies < or = 0.25 Hz evoked Ca(2+) transients and cell shortening, comparable to those induced by electrical (field) stimulation. At higher frequencies the membrane potential failed to recover sufficiently from the depolarisation with the high K(+) solution, possibly because of the slow clearance of the ion from the t-tubular system. To test this hypothesis, the clearance of fluorescently labeled 10 kDa dextran from the t-system was measured and found to be approximately 0.5 s delayed compared to that of the bulk extracellular space, indicating the slow diffusion inside the confined space of the tubular membrane invaginations.
Assuntos
Ventrículos do Coração/metabolismo , Potenciais da Membrana , Microfluídica/instrumentação , Microfluídica/métodos , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Análise de Injeção de Fluxo , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Potássio , CoelhosRESUMO
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). The latter structure enables adjacent heart cells to share ions, second messengers and small metabolites (<1 kDa) between them and thus provides the structural basis for the synchronous (syncytical) behaviour of connected cardiomyocytes. Using the unique environment provided by the microfluidic system, described in this paper, we explore the local ionic conditions that enable the propagation of Ca(2+) waves between two heart cells. We observe that the ability of intracellular Ca(2+) waves to traverse the intercalated discs is dependent on the relative concentrations of diastolic Ca(2+) in the two adjacent cells. These experiments rely upon our ability to independently control both the electrical stimulation of each of the cells (using integrated microelectrodes) and to rapidly change (or switch) the local concentrations of ions and drugs in the extracellular buffer within the microfluidic channel (using a nanopipetting system). Using this platform, it is also possible to make simultaneous optical recordings (including fluorescence and cell contraction) to explore the effect of drugs on one or both cells, within the doublet.
Assuntos
Canais de Cálcio/fisiologia , Junções Intercelulares/fisiologia , Microfluídica/métodos , Miócitos Cardíacos/fisiologia , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/fisiologia , Estimulação Elétrica , Eletrofisiologia , Junções Intercelulares/ultraestrutura , Modelos Biológicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Óptica e Fotônica , Coelhos , Gravação em VídeoRESUMO
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+. Initial experiments aimed to create a metabolic profile of the beating heart cell, and results show well defined excitation-contraction (EC) coupling at different rates. Ca2+ transients and extracellular pH measurements were obtained from continually paced single myocytes, both as a function of the rate of cell contraction. Finally, the relative amounts of intra- and extra-cellular lactate produced during field stimulation were determined, using cell electroporation where necessary.
Assuntos
Técnicas Biossensoriais/instrumentação , Microfluídica/instrumentação , Microfluídica/métodos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Aerobiose , Anaerobiose , Animais , Técnicas Biossensoriais/métodos , Cálcio/análise , Técnicas de Cultura de Células , Células Cultivadas , Estimulação Elétrica , Eletroporação , Enzimas Imobilizadas/química , Desenho de Equipamento , Ventrículos do Coração/citologia , Concentração de Íons de Hidrogênio , Ácido Láctico/biossíntese , Microeletrodos , Microscopia , Oxigenases de Função Mista/metabolismo , Contração Muscular/fisiologia , CoelhosRESUMO
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. Both transient and steady-state electrochemical potentials were applied to the microelectrode pair in order to generate changes in the local pH, associated with electrolysis. The functionalised beads indicated the pH changes in the channel, measured as a change in the fluorescence signal, generated by the immobilised pH sensitive dye. Responses were measured with temporal resolutions of between 1 and 200 ms, whilst the spatial resolution of the pH gradients was limited by the size of the beads to 3 microm.
Assuntos
Benzopiranos/química , Corantes Fluorescentes/química , Técnicas Analíticas Microfluídicas/métodos , Naftóis/química , Óptica e Fotônica/instrumentação , Rodaminas/química , Eletroquímica/métodos , Concentração de Íons de Hidrogênio , Microeletrodos , Técnicas Analíticas Microfluídicas/instrumentação , Poliestirenos/químicaRESUMO
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. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 microm long, 20 microm wide, 0.1-10 microm thick, 180 microm apart) patterned on a microscope coverslip base, thus defining cavities of approximately 4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and > or = 4 microL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca2+ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for approximately 3 min in the 4-nL volume and approximately 20 min for the 4 microL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.
Assuntos
Cálcio/metabolismo , Estimulação Elétrica/instrumentação , Citometria de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência/instrumentação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Potenciais de Ação/fisiologia , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Estimulação Elétrica/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Citometria de Fluxo/métodos , Ventrículos do Coração/citologia , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência/métodos , Contração Miocárdica/fisiologia , Coelhos , Função VentricularRESUMO
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. EDX and fluorochrome analysis enable to register total and free intracellular calcium concentrations, [Ca] and [Ca(2+)], respectively. After exocytosis stimulation we find by both methods that the calcium signal sweeps into the basis of cilia, not only in 7S but also in pawn cells which then also perform ciliary reversal. After depolarisation we see an increase of [Ca] along cilia selectively in 7S, but not in pawn cells. Opposite to exocytosis stimulation, during depolarisation no calcium spill-over into the nearby cytosol and no exocytosis occurs. In sum, we conclude that cilia must contain a very potent Ca(2+) buffering system and that ciliary reversal induction, much more than exocytosis stimulation, involves strict microdomain regulation of Ca(2+) signals.
Assuntos
Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Paramecium/citologia , Paramecium/metabolismo , Transdução de Sinais/fisiologia , Animais , Cílios/metabolismo , Exocitose/fisiologiaRESUMO
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.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Animais , Células CHO , Cálcio/análise , Cálcio/química , Cátions Bivalentes/análise , Cátions Bivalentes/química , Adesão Celular , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Ligantes , Técnicas Analíticas Microfluídicas/métodos , Transdução de Sinais , Uridina Trifosfato/metabolismoRESUMO
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.e., the source, inner pipet) and aspirate (the sink, outer pipet) a buffer solution (perfusate) into each of the two pools. For rapid solution switching around the cell, additional dual superfusion pipets were inserted into the microchannel for defined time periods using a piezostepper, enabling us to add a test solution, such as a drug. Three distinct areas of the cell were manipulated, namely, the microfluidic environment, the cellular membrane, and the intracellular space. Planar integrated microelectrodes enabled the electrical stimulation of the cardiomyocyte and the recording of the evoked action potential. The device was mounted on an inverted microscope to allow simultaneous sarcomere length and epifluorescence measurements during evoked electrical activity, including, for example, the response of the stimulated end of the cardiac myocyte in comparison with the untreated cell end.
Assuntos
Espaço Extracelular , Microfluídica/métodos , Miócitos Cardíacos/citologia , Potenciais de Ação , Animais , Soluções Tampão , Membrana Celular , Fluorescência , Humanos , Microeletrodos , Técnicas Analíticas Microfluídicas , Perfusão , SarcômerosRESUMO
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). This effect is of significance given the increasing application of microfluidic devices in high-throughput cellular analysis for biophysical applications and pharmacological screening.
Assuntos
Cálcio/análise , Células Imobilizadas , Microfluídica/métodos , Animais , Células CHO , Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Cricetinae , Cricetulus , Corantes Fluorescentes/química , Microfluídica/instrumentação , Estresse MecânicoRESUMO
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. This arrangement increased the extracellular electrical resistance to > 20 MOmega and facilitated the recording of the time course of the change in extracellular voltage and current during subthreshold and suprathreshold stimuli. The waveform of the extracellular current and voltage in response to an extracellular depolarizing stimulus comprised an initial monophasic signal followed by a biphasic signal with a delay of 2-15 ms. The latter was associated with a transient contraction and therefore caused by an action potential. The biphasic signal became monophasic after the depolarization of one cell end by raised extracellular [K+]. This form of differential recording revealed the repolarization phase of the action potential. At rest, the sarcomere length within the gap was 12% +/- 4.8% longer than outside the gap, but intracellular Ca2+ transients occurred to the same extent as that observed in the outer pools. This data demonstrate the feasibility of the use of a microfluidic bath design to limit the extracellular resistance between two ends of an isolated cardiomyocyte.
Assuntos
Potenciais de Ação , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Cálcio/metabolismo , Forma Celular , Estimulação Elétrica , Técnicas In Vitro , Microeletrodos , Microfluídica , Coelhos , Sarcômeros/fisiologiaRESUMO
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. Excitation contraction coupling was studied by monitoring myocyte shortening and intracellular Ca(2+) transients (using Fluo-3 fluorescence). The amplitude of stimulated myocyte shortening and Ca(2+) transients remained constant for 90 min in the larger volume (5 nL) configuration, although the shortening (but not the Ca(2+) transient) amplitude gradually decreased to 20% of control within 60 min in the low volume (100 pL) arrangement. These studies indicate a lower limit for the extracellular volume required to stimulate isolated adult cardiac myocytes. Whereas this arrangement could be used to create a screening assay for drugs, individual microchannels (100 pL) can also be used to study the effects of limited extracellular volume on the contractility of single cardiac myocytes.
Assuntos
Dimetilpolisiloxanos/química , Miócitos Cardíacos/metabolismo , Silicones/química , Animais , Cálcio/química , Cálcio/metabolismo , Eletrodos , Eletrofisiologia , Ventrículos do Coração/citologia , Humanos , Microscopia Confocal , Óleo Mineral , Miocárdio/metabolismo , CoelhosRESUMO
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. Devices were fabricated using photolithography and liftoff giving reproducible sensors integrated within high aspect ratio titer chambers (with a volume of 360 pL), made of the photoepoxy SU8. In this paper, the determination of lactate was optimized using an enzyme-linked assay based upon lactate oxidase, involving the amperometric determination of hydrogen peroxide at +640 mV versus an internal Ag/AgCl pseudoreference. The microsystem (including the microfluidic dispensers and structures as well as the microsensor) was subsequently used to measure the lactate content of single heart cells. Dynamic electrochemical measurements of lactate during cell permeabilization are presented. We also show the use of respiratory uncouplers to simulate ischemia in the single myocyte and show that, as expected, the rate of lactate production from the hypoxic heart cell is greater than that within the normoxic healthy myocyte.