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RESEARCH PROFILES Right on target: Controlled dosing of cells Dumping a reagent over a layer of cultured cells and watching their collective reaction can only tell you so much. In many cases, it’s more informative to direct reagents to particular areas of a culture and observe how organelles or subcellular regions respond. In this issue of Analytical Chemistry (pp 5987–5993), Jason Shear and Rex Nielson at the University of Texas, Austin, describe a microfluidic device in which cells were monitored as they responded to laminar streams of reagent that flowed over targeted regions. This work is not the first to apply microfluidics to studies of cultured cells. For instance, George Whitesides and colleagues have previously demonstrated that laminar streams of reagents can be flowed over regions of cells inside a microfluidic device (Nature 2001, 411, 1016). Nielson explains that the Whitesides approach has potential limitations because the interface between laminar streams is formed at a Y junction of a microfluidic device. “Where the adherent cell [gets] dosed depends on its location in relation to this Y junction, and that doesn’t allow you very much flexibility in targeting any particular cellular region,” he says. Shear and Nielson’s device gave them the freedom to precisely dose any part of a cell or region of a culture that was of interest. The device consisted of several parts. Cells were grown on a polycarbonate or Mylar membrane. The membrane with the cells was then sandwiched between two PDMS channels so that the cells faced the bottom channel. The top PDMS channel carried the reagent; the bottom channel carried media for the cells. The device was placed over a microscope objective lens through which a femtosecond laser beam was focused onto the membrane. The investigators picked a region of the cell culture that was of interest and positioned the laser beam upstream of that region. The energy of the laser beam broke down the membrane, creating a micrometer-diame-
ter hole. Reagent from the top PDMS channel Top flow channel flowed through the hole Pore Dosed cell and over the chosen reMembrane Plume gion in a thin, defined plume. The process could Cell Bottom flow channel be repeated so that multiple plumes flowed over Drain the culture in a laminar fashion. Top flow channel “Relative to other miMembrane crofluidic methods, we Bottom flow channel Feed believe our approach has Cover glass greater flexibility in the number and orientation Objective of flow streams that can be created for dosing cells,” says Shear. “Dozens of streams could be In the cell-dosing device, cells grown on a membrane are created in parallel, a capaselectively exposed to plumes of a reagent that enter through bility that is likely to be holes in the membrane. useful in various studies, while fluid is present in the top PDMS such as stimulation of cellular networks.” channel, a bubble forms and blocks the As proof of a concept, Shear and hole. The investigators now get around Nielson cultured neuroblastoma glioma that problem by keeping the top PDMS cells on the membranes and selectively channel empty during the ablation and dosed particular cells and subcellular rethen filling it up with reagent, but Nielgions with 5-carboxyfluorescein diacson says they are working to redesign etate, a dye that’s converted into its the channels and the laser setup to see fluorescent form by enzymes inside the cells, and MitoTracker Green, a dye that whether the ablation process can be carried out in the presence of fluid. localizes with different cellular strucAnother limitation at the moment is tures. They also showed they could disthat “we can form a plume, but it can rupt particular cells within a culture by only be in a longitudinal direction down dehydration with ethanol. the laminar flow of the channel,” says Furthermore, the investigators Nielson. The team is working on a new demonstrated that the holes in the design in which multiple channels that membrane could be plugged up to stop a laminar stream of reagent. They intro- run in different directions intersect; with this arrangement the plume can be duced a solution of bovine serum albusteered in different directions. min (BSA) and a photosensitizer, such Shear and Nielson also want to study as flavin adenine dinucleotide. When the the different types of cells, especially laser was focused at a hole, a plug of keratocytes and neutrophils, which are cross-linked BSA formed in
