title: Making the cut: Innovative methods for optimizing perfusion-based migration assays.
abstract: Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell migration fundamental to the pathogenesis of many flow-mediated disorders and are applicable to other perfusion-based models where migration is of central importance. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Holt
authors.affiliation: Department of Engineering, East Carolina University, Greenville, North Carolina.
authors.forename: William E
authors.initials: WE
authors.lastname: Howard
authors.affiliation: Department of Biology, East Carolina University, Greenville, North Carolina.
authors.forename: Elizabeth T
authors.initials: ET
authors.lastname: Ables
authors.affiliation: Department of Engineering, East Carolina University, Greenville, North Carolina.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: George
authors.affiliation: Department of Internal Medicine, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Cindy A
authors.initials: CA
authors.lastname: Kukoly
authors.affiliation: Department of Engineering, East Carolina University, Greenville, North Carolina.
authors.forename: Jake E
authors.initials: JE
authors.lastname: Rabidou
authors.affiliation: Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: Jake T
authors.initials: JT
authors.lastname: Francisco
authors.affiliation: Department of Engineering, East Carolina University, Greenville, North Carolina.
authors.forename: Angel N
authors.initials: AN
authors.lastname: Chukwu
authors.affiliation: Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina.
authors.forename: David A
authors.initials: DA
authors.lastname: Tulis
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000187:drug effects*; D003599:Cytoskeleton / Q000648:ultrastructure; D006801:Humans; D010477:Perfusion; D013314:Stress, Mechanical; D014357:Trypsin / Q000494:pharmacology
chemical_list: D014357:Trypsin
keywords: 3D printing; cell migration; computational fluid dynamics; fluid shear stress; laser capture microdissection; smooth muscle cells