Anal. Chem. 2007, 79, 1205-1212
Microfluidic Partitioning of the Extracellular Space around Single Cardiac Myocytes Norbert Klauke,*,† Godfrey L. Smith,‡ and Jonathan M. Cooper*,†
Department of Electronics, University of Glasgow, Glasgow G12 8LT, Scotland, and Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland
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. Microfluidic channels are becoming increasingly popular for the culture and subsequent study of different types of isolated cells and cocultures thereof.1 In macroscaled culture chambers, the bulk of the buffer solution and the continuous convection negate against the formation of extracellular microdomains or local concentration gradients of signalling molecules within the extracellular fluid. Within microfludic systems, the volume of the extracellular space more closely matches the cell volume and the possibility therefore arises to precisely control the local environment around the cell, including creating well-defined concentration gradients of drugs and ions.2 * To whom correspondence should be addressed. E-mail:
[email protected]. † Department of Electronics. ‡ Institute of Biomedical and Life Sciences. (1) Huh, D.; Gu, W.; Kamotani, Y.; Grotberg, J. B.; Takayama. S. Physiol. Meas. 2005, 26, R73-R98. (2) Yu, H.; Meyvantsson, I.; Shkel, I. A.; Beebe. D. J. Lab Chip 2005, 5, 10891095. (3) Klauke, N.; Smith, G. L.; Cooper, J. M. Biophys. J. 2003, 85, 1766-1774. 10.1021/ac061547k CCC: $37.00 Published on Web 12/30/2006
© 2007 American Chemical Society
The study of isolated heart cells has a significant history, particularly for the recordings of electrical signals using the patchclamp technique. With the recent advent of microfluidic systems and lab-on-a-chip for cell studies, the topic has taken on a new importance, providing a series of new tools that can be used to investigate both normal physiology and pathophysiology associated with disease states. The techniques also have the potential to create new, high-throughput tools to screen for new medicines or to test for drug toxicology. The importance of the development of such tools for the study of the cardiomyocte is underlined by the fact that heart disease remains one of the most significant causes of mortality and morbidity in the developed world. In our previous work, we continuously stimulated cardiomyocytes using extracellular microelectrodes in microfluidic arrays and observed normal cell shortening even in the absence of cell superfusion with the buffer solution.3 We also demonstrated the use of the integrated microelectrodes for the extracellular recording of the action potential associated with the electrical activation of the single cardiomyocyte and the development of novel microbiosensor formats for measuring metabolites from the cell.4-7 We now describe a superfusion system for the rapid switching of buffer solutions around separate regions of the single adult ventricular myocyte. The microchannels, which confine the extracellular space to a picoliter volume, were split into two compartments or pools, each associated with one end of the cell. The 20-µm-wide gap between the pools, associated with the central portion of the cell, was defined by a bridge structure, created using polymer soft lithography and sealed with mineral oil. Although this structure prevents bulk cross-contamination between the two aqueous pools on either side of the gap, the residual space of
