Methodology for investigating the duration of intracellular calcium expression in response to mechanical stimulation in a single cell Jong Heon Jeon, Ok Chan Jeong ⇑ Dept. Biomedical Eng., Inje University, Gimhae 621-749, South Korea article info Article history: Available online 20 July 2012 Keywords: Cell chip Intracellular calcium Shear stress abstract This report describes an effective methodology for investigating the duration of intracellular calcium expression in response to mechanical stimulation in a single cell using a microfluidic platform. A micro cell chip was constructed using the sequential processes of micro contact printing for surface patterning with fibronectin on the cell culture substrate, plasma bonding of the cell substrate and the fluidic chan- nel, and cell seeding. All structures such as the micro stamp for the micro contact printing, the cell sub- strate, and the fluidic channel were fabricated with polydimethylsiloxane (PDMS). Micro stamps having a cell-matrix with various cell densities were fabricated to determine the proper conditions for cell seeding on the substrate. We found that a distance of 65 lm between cell sites was preferable for the patterning of MG-63 cells (human osteoblast-like bone cell) using our micro stamp. While the pressure-driven shear stress in the microchannel was applied using a computer-controlled pneumatic system, the intracellular calcium response of a single cell was measured using a laser-scanning microscope. Based on the temporal response of fluorescence intensity under the steady fluid flow, the average period of multiple calcium peaks in the cell was approximately 128 ± 4 s at 1 Pa and 164 ± 20 s at 2 Pa. This work provides a useful method for fabricating a micro cell chip with a patterned surface for cell culture and for measuring the duration of the intracellular calcium response in a single cell. Ó 2012 Elsevier B.V. All rights reserved. 1. Introduction In the field of microfluidics, the rapid development of new fabri- cation methods based on soft-lithography with polydimethylsilox- ane (PDMS) [1] and different kinds of essential components such as micro channels, valves [2], mixers [3], and pumps [4] enables the fabrication of a broad range of subsystems and fully integrated functional systems [5] for various applications. There is growing interest in using micro system technology to study cell biology because the dimensions of the cells attached on the substrate (typically 10–100 lm) are well suited for control using microfluidic PDMS devices. The material choice of PDMS can provide excellent optical transparency, low toxicity, and high permeability for oxygen and carbon dioxide. Thus, PDMS has been used widely as a suitable material for the functional components of cell chip systems. To date, many types of micro cell handling devices (e.g., for culture [6], sorting [7], and traps [8]) have been fabricated for the investiga- tion of various facets of cell biology. Another interesting research topic is the use of micro fluidic chips for investigating mechanotransduction in bone cells; mecha- notransduction is the mechanism by which extracellular physical stimulation is converted into cellular responses [9]. For example, in vivo, the lacunar–canalicular network is filled with interstitial fluid and flows in response to mechanical deformation of the bone tissue [10]; thus, pressure-driven fluid flow was identified as one of the strongest mechanical stimuli on bone cell behavior in vitro [11]. However, there were marked variations in the patterns of mechanical stimulation because the effects of the magnitude and frequency of the fluid-flow-induced shear stress on cells were not well understood. Additionally, cells may require their own proper patterns of mechanical stimulation. Therefore, a simple and effec- tive method is needed for determining the optimum stimuli pat- terns for a specific single cell using the microfluidic platform. This paper describes a methodology for investigating the proper duration of mechanical stimulation for a single cell using a micro- channel-type cell chip with a stable and uniform microenviron- ment. The micro contact printing method [12] was used for surface patterning in order to control cell density in the micro- channel. To overcome the bulky methods typically used for these types of experiments [13], which require a large number of cells/ media, time, and cost, the micro cell chip was fabricated using a replica molding process with PDMS, and shear-stress-induced intracellular calcium expression in MG-63 cells (human osteo- blast-like bone cell line) was measured for investigating the effects of mechanotransduction. 0167-9317/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2012.07.050 ⇑ Corresponding author. E-mail address: memsoku@inje.ac.kr (O.C. Jeong). Microelectronic Engineering 98 (2012) 642–646 Contents lists available at SciVerse ScienceDirect Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee