Gradient generation by an osmotic pump and the behavior of human mesenchymal stem cells under the fetal bovine serum concentration gradient{ Joong Yull Park, a Chang Mo Hwang, ab Soon Hyuck Lee c and Sang-Hoon Lee* a Received 13th July 2007, Accepted 23rd August 2007 First published as an Advance Article on the web 6th September 2007 DOI: 10.1039/b710777c This paper describes a method to generate a concentration gradient using an osmosis-driven pump, without the need for bulky peripheral devices, such as an electric syringe pump or a pneumatic pump. By the osmosis, the flow in the microfluidic channel can be controlled even to a very slow speed (nanolitre scale), which enables its application to generate the stable and wide (width = 4 mm) concentration gradient profile, even within a short flow path. A computational simulation was also performed to predict the local distribution of the solute concentration and velocity-pressure profile in the microfluidic chip. The performance of the osmosis-driven pump was evaluated by culturing human mesenchymal stem cells within the concentration gradient of fetal bovine serum. The effects of the gradient on attachment, viability and morphology of the cells were analyzed and quantified. The cell density in a higher serum concentration region was twice greater than that in the pure culture media. The compact, cost-effective, self-powered and osmosis-based gradient generation system can be useful for biomedical and chemical applications. Introduction In the field of cell biology, it is a well-known fact that gradients of soluble factors influence biological developmental and physiological processes. 1–7 The traditional method for experi- mental cell assays uses a glass micropipette or a Boyden chamber; 8 these techniques are widely used despite several shortcomings. Recently, microfluidic technology has provided a good alternative approach for generating a gradient of biological factors, such as chemokines and growth factors. Unlike the traditional glass micropipette assay, the micro- fluidic gradient generation has several advantages, including reproducibility, high-throughput, easy-to-use, stability, and controllability. 9 Microscale flow-based gradient generation devices, in which diffusion mixing at the interface of laminar streams occurs, have been successfully used to study chemotaxis by excellent control over the concentration gradient. One of the first promising microgradient generators was proposed by Jeon et al. 10 They proposed a simple method of generating gradients based on controlled diffusive mixing of species in solutions by laminar flow. Their method was applied to a device for the generation of overlapping gradients composed of different species 11 as well as neutrophil chemotaxis. 12 Although this was a useful method, a noticeable shearing effect limited its use as discussed by Walker et al.; 13 they quantified the effects of flow on cell migration during chemotaxis in a microfluidic device, and showed that a minimal shearing effect is essential for accurate study of cellular behaviors. Therefore, the mechanical stress must be considered in the design of flow-based gradient generators. In addition to that, most of the flow-based gradient generators have common weaknesses; first, they require a large volume of expensive reagents to maintain an adequate gradient band width in a channel, and second, the continuously flowing streams of fluid carry away the secreted factors, which may lead to an inaccurate interpretation of cell behavior. 5 There have been two prior studies using only diffusion phenomena to generate a concentration gradient. Chung et al. developed a microfluidic multi-injector (MMI) device that can stably generate temporal and spatial con- centration gradients in an opened chamber. 9 They applied diffusion phenomena so that no flow was observed in the MMI, except for the periodic injections of fluid into the mixing chamber. However, this technique has the limitations of short duration, small size of the gradient chamber, and frailty with a thermal gradient; in addition it requires valves and pneumatic pumps. The other study using a porous membrane for gradient generation was proposed by Abhyankar et al.; 5 the use of diffusion for the creation of a stable static chemical gradient was successfully described and its application to the study of cell–cell signaling showed promising results. However, the change of sample solution during the process seems to be impossible and a concentration shift appears unavoidable as the sources of two solutions were mixed and diluted gradually. In most of microfluidic devices, including the above- mentioned devices, concentration gradients were created by a diffusion-based or flow-based mechanism, and each method has their own advantages and disadvantages. Therefore, a Department of Biomedical Engineering, College of Medicine, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-705, Republic of Korea b Korea Artificial Organ Center, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-705, Republic of Korea c Department of Orthopedic Surgery, Korea University Anam Hospital, Anam-dong, Seongbuk-gu, Seoul 136-705, Republic of Korea { Electronic supplementary information (ESI) available: Supple- mentary information Fig. S1, Fig. S2 and movie clips. See DOI: 10.1039/b710777c PAPER www.rsc.org/loc | Lab on a Chip This journal is ß The Royal Society of Chemistry 2007 Lab Chip, 2007, 7, 1673–1680 | 1673