The microfluidic lighthouse: an omnidirectional gradient generator.

Studies of chemotactic cell migration rely heavily on various assay systems designed to evaluate the ability of cells to move in response to attractant molecules. In particular, the development of microfluidics-based devices in recent years has made it possible to spatially distribute attractant molecules in graded profiles that are sufficiently stable and precise to test theoretical predictions regarding the accuracy and efficiency of chemotaxis and the underlying mechanism of stimulus perception. However, because the gradient is fixed in a direction orthogonal to the laminar flow and thus the chamber geometry, conventional devices are limited for the study of cell re-orientation to gradients that move or change directions. Here, we describe the development of a simple radially symmetric microfluidics device that can deliver laminar flow in 360°. A stimulant introduced either from the central inlet or by photo uncaging is focused into the laminar flow in a direction determined by the relative rate of regulated flow from multiple side channels. Schemes for flow regulation and an extended duplexed device were designed to generate and move gradients in desired orientations and speed, and then tested to steer cell migration of Dictyostelium and neutrophil-like HL60 cells. The device provided a high degree of freedom in the positioning and orientation of attractant gradients, and thus may serve as a versatile platform for studying cell migration, re-orientation, and steering.

Analysis of single-cell differences by use of an on-chip microculture system and optical trapping.

A method is described for continuous observation of isolated single cells that enables genetically identical cells to be compared; it uses an on-chip microculture system and optical tweezers. Photolithography is used to construct microchambers with 5-microm-high walls made of thick photoresist (SU-8) on the surface of a glass slide. These microchambers are connected by a channel through which cells are transported, by means of optical tweezers, from a cultivation microchamber to an analysis microchamber, or from the analysis microchamber to a waste microchamber. The microchambers are covered with a semi-permeable membrane to separate them from nutrient medium circulating through a "cover chamber" above. Differential analysis of isolated direct descendants of single cells showed that this system could be used to compare genetically identical cells under contamination-free conditions. It should thus help in the clarification of heterogeneous phenomena, for example unequal cell division and cell differentiation.

On-chip culture system for observation of isolated individual cells.

To investigate the properties of isolated single cells with their environment, we developed the differential analysis method for single cells using an on-chip microculture system. The advantages of the system are, (i). continuous cultivation of a series of isolated single cells or a group of cells under contamination free conditions, (ii). continuous observation and comparison of those cells with 0.2 microm spatial resolution by a phase-contrast/fluorescent microscopy system with digital image processing. The core of the system is an n x n (n = 20-50) array of chambers, where each is 20-70 microm in diameter and 5-30 microm deep holes etched into a biotin-coated 0.17 mm thick glass slide. The biotin-coated glass slide is covered with the streptavidin coated cellulose semipermeable membrane, which is fixed on the surface of the glass slide by streptavidin-biotin attachment, separating those holes from the nutrient medium circulating through a 'cover chamber' above. A single cell or group of cells can thus be isolated from environment perfused with the same medium, and the medium in each chamber can be changed within the diffusion time (<1/30 s). In addition, the microchamber volumes of specific cells or cell groups can be controlled by the sizes of the chambers. By using this system we found that the length of isolated Escherichia coli increased at 0.06 microm min(-1) between cell divisions regardless of the chamber volume, and that the cell concentration reached 10(12) cells ml(-1) under contamination free conditions. The system is thus particularly useful for one cell level analysis because the direct descendants of single cells can be cultured and compared in the isolated microchambers, and the physical properties of the cells in each microchamber can be continuously observed and compared.