title: Planar microfluidic chamber for generation of stable and steep chemoattractant gradients.
abstract: The extracellular availability of growth factors, hormones, chemokines, and neurotransmitters under gradient conditions is required for directional cellular responses such as migration, axonal pathfinding, and tissue patterning. These responses are, in turn, important in disease and developmental processes. This article addresses critical barriers toward devising a chemotaxis assay that is broadly applicable for different kinds of cancer cells through the design of a microfluidic chamber that produces a steep gradient of chemoattractant. Photolithography was used to create microchannels for chemoattractant delivery, flow diversion barriers/conduits, and small outlets in the form of apertures. The 1-microm apertures were made at the active surface by uncapping a thin (1.5 microm) layer of AZ1518. This process also created a vertical conduit that diverted the flow such that it occurred perpendicularly to the active, experimental surface where the gradients were measured. The other side of the vertical conduit opened to underlying 20-microm deep channels that carried microfluidic flows of tracer dyes/growth factors. Modeled data using computational fluid dynamics produced gradients that were steep along the horizontal, active surface. This simulation mirrors empirically derived gradients obtained from the flow analyses of fluorescent compounds. The open chamber contains a large buffer volume, which prevents chemoattractant saturation and permits easy cell and compound manipulation. The technique obviates the use of membranes or laminar flow that may hinder imaging, rinsing steps, cell seeding, and treatment. The utility of the chamber in the study of cell protrusion, an early step during chemotaxis, was demonstrated by growing cancer cells in the chamber, inducing a chemoattractant gradient using compressed air at 0.7 bar, and performing time-lapse microscopy. Breast cancer cells responded to the rapidly developed and stable gradient of epidermal growth factor by directing centroid positions toward the gradient and by forming a leading edge at a speed of 0.45 microm/min.
authors.affiliation: Australian Microscopy and Microanalysis Research Facility, Australian Key Centre for Microscopy and Microanalysis, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Sandra
authors.initials: S
authors.lastname: Fok
authors.forename: Peter
authors.initials: P
authors.lastname: Domachuk
authors.forename: Gary
authors.initials: G
authors.lastname: Rosengarten
authors.forename: Norbert
authors.initials: N
authors.lastname: Krause
authors.forename: Filip
authors.initials: F
authors.lastname: Braet
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Eggleton
authors.forename: Lilian L
authors.initials: LL
authors.lastname: Soon
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002468:Cell Physiological Phenomena / Q000187:drug effects*; D002630:Chemotactic Factors / Q000008:administration & dosage* / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods
chemical_list: D002630:Chemotactic Factors