Simultaneous generation of chemical concentration and mechanical shear stress gradients using microfluidic osmotic flow comparable to interstitial flow† Joong Yull Park, ab Sung Ju Yoo, ac Chang Mo Hwang ad and Sang-Hoon Lee * a Received 8th December 2008, Accepted 9th April 2009 First published as an Advance Article on the web 27th April 2009 DOI: 10.1039/b822006a Cells are very sensitive to various microenvironmental cues, including mechanical stress and chemical gradients. Therefore, physiologically relevant models of cells should consider how cells sense and respond to microenvironmental cues. This can be accomplished by using microfluidic systems, in which fluid physics can be realized at a nanoliter scale. Here we describe a simple and versatile method to study the generation of chemical concentration and mechanical shear stress gradients in a single microfluidic chip. Our system uses an osmotic pump that produces very slow (<a few mm/s) and controlled flow, allowing a wide and stable diffusion of specific chemical concentration. We also established a shear stress gradient passively via a circular channel in the interstitial level. For evaluation of the system, we used L929 mouse fibroblast cells and simultaneously exposed them to a mechanical stress gradient and a chemical nutrient gradient. The interstitial shear stress level clearly affected cell alignment, mobility velocity, and attachment. At the same time, cell proliferation reflected nutrient concentration level. Our system, which enables continuous and long-term culture of cells in a combined chemical and mechanical gradient, provides physiologically realistic conditions and will be applicable to studies of cancer metastasis and stem cell differentiation. Introduction During development of multicellular organisms, exposure of cells to specific microenvironments can affect external appearance and internal anatomy. Recent morphogenesis studies have focused on the mechanisms (degeneration or regeneration) that control cell fate and the formation of multicellular tissues and organs. Understanding the basis of morphogenesis is particularly important for the treatment of diseases, such as cancer and certain hereditary conditions, and for regenerative medicine. During morphogenesis, cells are affected by numerous micro- environmental cues, such as geometrical constraints, 1 mechanical stress, 2–4 matrix elasticity, 5 and gradients of morphogenetic and chemotactic proteins. 6–9 Interstitial flow, which exists to some extent in all tissues, is involved in nutrient transport through tissues, regulation of tissue development, and organ and tissue maintenance and remodeling. 10 Small convective interstitial flows yield small shear stresses at the cell surface, but can cause large changes in morphogen distribution, even when convection does not domi- nate diffusion. 11 Interstitial flow has two direct effects on cells: 12 (a) small fluid shear with normal force on the cell surface and (b) redistribution of pericellular proteins that bind to cell receptors. Many previous studies of interstitial flow and morphogenesis have examined fibroblast alignment and matrix remodeling, 13,14 myofibroblast differentiation, 15 and interstitial flow as mor- phoregulators. 12,16 Therefore, understanding how cells sense and respond to the physical and chemical cues that underlie inter- stitial flow is a great challenge for developmental and cellular biologists. 17 Microfluidic technology can provide physiologically relevant models of interstitial flow because this approach considers fluid physics at the nanoliter scale and provides a controlled envi- ronment for the in vitro study of cells. 18–21 Many microfluidic approaches have been developed to study mechanical or chem- ical stimulation of cells. 2,7,19,22 Mechanical shear stresses play important regulatory roles in various physiological and patho- logical processes. 23 Physiological studies that have utilized microfluidic technology have examined the effect of quantitative shear stress on cell mechanics, 2 the effect of fluid mechanical stresses on lung cells (human airway epithelia), 24 cytoplasmic stiffening, 23 and pulsatile compression mechanical stimulation of human mesenchymal stem cells (hMSCs). 25 However, the level of shear stress used in these studies was far higher than that caused by interstitial flow. In addition, chemical effects are also a Department of Biomedical Engineering, College of Health Science, Korea University, Jeongneung-dong, Seongbuk-gu, Seoul 136-703, Republic of Korea. E-mail: dbiomed@korea.ac.kr; Fax: +82-2-921-6818; Tel: +82-2- 940-2881 b Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA c Department of Chemistry, College of Advanced Science, Dankook Univeristy, Anseo-dong, Cheonan 330-714, Republic of Korea d Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA † Electronic supplementary information (ESI) available: The transient of gradient generation in the system was recorded (Movie 1); mesh generation for computer simulation (Fig. S1); development and stabilization of chemical gradient (Fig. S2); the angle of each cell was measured from the reference line perpendicular line to the flow stream, and changed from 0  to 90  , implying that the average value should be 45  (Fig. S3); cell distribution in the main channel (phase contrast image) (Fig. S4). See DOI: 10.1039/b822006a 2194 | Lab Chip, 2009, 9, 2194–2202 This journal is ª The Royal Society of Chemistry 2009 PAPER www.rsc.org/loc | Lab on a Chip