Schmitz, K.

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Prof. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationMicrosystems to study leukocyte chemotaxis –from gradient generators to micropatterned surfacesInstitute of Organic Chemistry (IOC)Dr. Katja SchmitzMerida, 14.12.10KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association www.kit.eduOverviewCell-based applications of MEMSThe importance of cell migrationTraditional migration assaysGradient generatorsMicrostructured surfaces3D chemotaxis assaysOutlookInstitute of Organic Chemistry2 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisAdvantages of MiniaturizationReduction of total sample volume (ml → nl) ⇒ Cost effectivenessMaximization of usable surface area for reaction/interaction⇒ Acceleration‘‘lab-on-a-chip’’:low volume liquid handling devices + biochip platformsSequential steps performed in one deviceHi h t t f ll li tiInstitute of Organic Chemistry3 14.12.10g  ex en  o  para e za onCell sized-devices to observe individual cell responsesDr. Katja Schmitz - Microsystems to study leukocyte chemotaxisCell-based assays in microsystemsHigh-througput cell-based screensCell-based BiosensorsMonitoring cell behaviour in microfludic devicesInstitute of Organic Chemistry4 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxis1536well / 3456well platesProf. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationMonitoring cellular behaviorBacteria: Biofilm formation, chemotaxisFungi: Dyctiostelium discoideum behaviorPlants: Settling of algae, Animals: stem cell differentiation, tumor developmentInstitute of Organic Chemistry5 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxiswww.planet-schule.de www.transdeath.uni-graz.debacteria slime moldCell migration in humansLeukocyte migrationNeural patterningMetastasis formationFertilizationEmbyonal developmentInstitute of Organic Chemistry6 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisWound healingChemotaxis in inflammatory processesSite of inflammationEndothelial cells of blood vessel wallBlood flowAdhesion Rolling and activation Extravasation Chemoattractant release ⇒transmigrationInstitute of Organic Chemistry7 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisClues that influence leukocyte chemotaxisConcentration of chemoattractantGradient of chemoattractantMix of chemoattractantsSurrounding matrix (elasticity, composition…)Blood flow (shear forces)Pathogenic: fMLPEndogenousInterleukin-8Institute of Organic Chemistry8 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisProf. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationLeukocytes are good to observe:Institute of Organic Chemistry9 14.12.10 Dr. Katja Schmitz - Microfluidic Systems for Cell-Based ApplicationsChallenges in the design of chemotaxis assaysChemoattractant gradientStability over timeStability in spaceDefined profileMulti-compound gradientsRealistic settingLining of the walls with cell adhesion moleculesElasticity of the mediumSh t llInstitute of Organic Chemistry10 14.12.10ear s ress on ce sMimicking extravasation (blood-vessel → tissue)channel size, flow speed, temperature, cell loading…Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisTraditional approaches: Boyden chamber (1962)Transwell Assay+ easy quantitative evaluationleukocytesporous membranereservoir of chemoattractantCount:cells in insertcells in reservoirInstitute of Organic Chemistry11 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxis- chemokine gradient is not stable ⇒ time dependence- large numbers of cells required- no single cell responses- no well defined gradientBoyden, S. J. Exp. Med., 1962Traditional approaches: Dunn chamber (1991)chemoattractant leukocytes+ stable over several hours+ predictable gradient behaviorObserve:Individual cells on „bridge“Institute of Organic Chemistry12 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxis   + reproducible thickness of gradient layer- no fast gradient changes- only linear gradientsZicha, J. Cell Sci, 1991Prof. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationTraditional approaches: Agarose assayChemoattractant (fMLP)Cells (neutrophils)agarose gel+ stable over several hours+ 3D environment - gradient takes long to form- no fast gradient changeInstitute of Organic Chemistry13 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisHeit, J. Cell Sci., 2005   - only linear gradients- no control over gradient- defined channel geometry ⇒ defined chemical gradient- channel in cellular dimensions- large reservoirs to maintain gradientdifferentiatedHL-60 cellsDiffusion-based gradients in microchannelsMigration towards channel entrancegradient cell migrationVelocities:Reservoir:2.5 µm/minChannel:55 µm/minInstitute of Organic Chemistry14 14.12.10NeutrophilsAttractant(fMLP)Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisA. Rosenhahn, University of Heidelberg‘‘Y’’ type microfluidic gradient generatorContinuous laminar flow for generating gradients+ rapid evolution of gradient T-cell velocities:Institute of Organic Chemistry15 14.12.10    + stable gradient in observation region+ muticompound mixing possible+ slow flow rate ⇒ low shear stress⇒ little effect on directionDr. Katja Schmitz - Microsystems to study leukocyte chemotaxisLin and Butcher, Lab Chip, 2006 CCL19: 6.2  ±1.0 mm/minCXCL12: 7.4 ± 0.2 mm/min Pyramidal networks for complex gradient shapescontrolled diffusive mixing of laminar flow fluids by repeated splitting, mixing, and recombination of fluid streams+ t d t t s ea y s a es+ dynamic gradients+ gradients of different profiles and resolution+ complex gradient shapesInstitute of Organic Chemistry16 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisJeon, Langmuir, 2000Prof. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationPyramidal networks for complex gradient shapesInstitute of Organic Chemistry17 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisDertinger, Anal. Chem. 2001Neutrophil response to IL-8 gradient profiles+ spatially and temprorarycontrolled gradients+ single cell tracking+ quantification of chemotactic flux and chemotactic coefficient- limit: gradient smearingInstitute of Organic Chemistry18 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisJeon, Nat. Biotechnol. 2002Temporal gradient controlTwo gradient generating networksMicrostructured membranes as Institute of Organic Chemistry19 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisIrimia, Lab Chip, 2006⇒ fast and accurate switches between any two stable gradientsvalvesNeutrophil responses to IL8-gradient changesInitial response Step downInstitute of Organic Chemistry20 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisGradient flip Irimia, Lab Chip, 2006Prof. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationNon-linear migration tracksCan leukocytes find the shortest way to the gradient origin?Institute of Organic Chemistry21 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisAmbravaneswaran, Integr. Biol., 2010 Oriented chemokines in 2D micropatternsHypothesis: Binding of chemokines to glycosaminoglycans orients them for improved receptor binding Idea: Immobilize chemokines so that receptor binding site faces away from surfaceInstitute of Organic Chemistry22 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisSkelton, Structure, 1999Chemokine surface patterns as 2D-gradientsParameters to explore:Influence of pattern on responseR l f b k d l lEven distribution Gradient pattern High-density patchesInstitute of Organic Chemistry23 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisGradient patterns microcontact printingo e o  ac groun  mo ecu esThreshold concentration for migrationMicrofluidics meets microcontact printing Institute of Organic Chemistry24 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisVon Philipsborn, Science STKE, 2007Prof. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationHow to make chemotaxis assays more life-like?confluent layer of endothelial cells on coated channelcontinuous flow of cells soft gel scaffolds (agarose, ECM-like matrices)3D structureschemokine gradient released from tissues through endotheliumInstitute of Organic Chemistry25 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisToetsch, Integr. Biol., 2009Boyden chamber coated with endothelial cellsLadder Chamber: Gel Chemotaxis & Microfluidicsneutrophilsinterleukin-8„reservoir“IL 8 diffuses through collagen matrixcollagen-filledgroovesInstitute of Organic Chemistry26 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxis-     cells move perpendicular to central flow⇒ „extravasation“Saadi, Biomed. Microdevices, 2007Neutrophil migration in the Ladder ChambercollagenInstitute of Organic Chemistry27 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisSaadi, Biomed. Microdevices, 2007SummaryChemotaxis is important to many biological processesTraditional diffusion-based chemotaxis assays may be too simpleMicrofluidic gradient generators forstable gradients of one or two compoundscomplex profilestemporal controlMicropatterned surface to study effects of adherent chemoattractantsInstitute of Organic Chemistry28 14.12.10Hydrogels as 3D-matrices for migrationCell-coated devices as life-like environmentDr. Katja Schmitz - Microsystems to study leukocyte chemotaxisProf. Dr. Max Mustermann | MusterfakultätKIT – University of the State of Baden-Wuerttemberg and National Laboratory of the Helmholtz AssociationConclusions and ConsiderationsMicro electromechanical systems have brought cell-based experiments to the next levelMore „life-like“ = more parameters to control  Every biological question poses a new set of challengesThe suitability of an approach depends on the biological question adressedInstitute of Organic Chemistry29 14.12.10Easy-to-operate systems are more likely to be used in life scienceDr. Katja Schmitz - Microsystems to study leukocyte chemotaxisConclusions and ConsiderationsLife scientists and engineers need to communicate:the requirements of biological systems the available technological means  LInstitute of Organic Chemistry30 14.12.10 Dr. Katja Schmitz - Microsystems to study leukocyte chemotaxisEngineer – Life ScientistLife Scientist - Engineer

Microsystems to study leukocyte chemotaxis - from gradient generators to micropatterned surfaces

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Microsystems to study leukocyte chemotaxis - from gradient generators to micropatterned surfaces

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