CellASIC™ ONIX Microfluidic Platform The next generation of live cell imaging Erwin Swart
CellASIC™ ONIX Microfluidic Platform The next generation of live cell imaging Erwin Swart Cellular Specialist BeNeLux Merck Millipore erwin.swart@merckgroup.com What is the CellASIC™ ONIX Microfluidic Platform? • CellASIC™ ONIX Microfluidic Platform uses microfluidic technology to enable continuous live-cell imaging with media flow • Allows cells to be exposed to different solutions and conditions via pressurized flow channels controlled by user–specified time intervals and flow rates Overview Current Technologies – Static dish culture is not representative of natural state – Advance of systems biology requires accurate cell phenotypes ONIX™ Microfluidic Perfusion System: How does it work – Result of 10 years of microfluidic cell culture research – Innovative micro-chamber, perfusion dynamics, temperature/gas control Applications for Live Cell Imaging – Use with standard inverted microscopes – Easy to adapt to existing experiment methods Progress in other Areas The need for a better way to address the cell environment 1900’s Cells Today 4 Current culture methods are inadequate We have great imaging techniques- but how can we keep the cells happy? Prerequisites for Cell Health? Difficult to Achieve with Dish Culture • Temperature: 37ºC • Local temp/gas environment changes constantly • Controlled gas and humidity • Constant accumulation of waste is not in-vivo like • Good delivery of nutrients • Humidity hard to maintain, evaporation causes cell death ? ? ? ? ? ? Do we actually know what the environment is at the cell-level? What is the ONIX Microfluidic Platform? ONIX System Microfluidic Plate & Manifold Unique Features: Automated perfusion system provides environment that more closely resembles in vivo like conditions Your Microscope ONIX System ONIX Software Unmatched control of introduction & removal of stimuli from cells for more accurate phenotypes Improved stability of live cell assays relative to alternatives: Uniform design Software controlled Easy-to-use More Predictive Cell Culture In vivo Microfluidic Approach • The CellASIC™ ONIX Microfluidic Platform is modeled after the dynamics of an in vivo tissue • Perfusion barrier recreates diffusive transport of nutrients and gas. 7 CellASIC enables dynamic experiments Maintain temperature or perform dynamic temperature changes Simply plug in your choice of gas condition for hypoxia, anaerobic, or 5% CO2 Add or remove media, drugs, or other perturbatives accurately with ONIX Create a PREDICTIVE live cell model 8 Multiple Plate Types Enable Many Applications for CellASIC Technology Plate Design Plate Code M04S Application Cell Type Function Cell response to solution change, drug exposure, and many more Adherent or Nonadherent mammalian cells cultured on glass Standard mammalian plate for long-term live cell imaging. (Up to 4 media conditions) Chemotaxis/ Cell Migration Adherent or nonadherent mammalian cells cultured on glass Create and change stable spatial chemogradients for over 10+ hours Tissue/Spheroid Culture, 3D Cell Culture Large aggregates, cells or tissues > 50 um Open-top cell chamber Bacteria single cell imaging Bacteria, 0.7-3.0 um Cells trapped in single monolayer Yeast single cell imaging Yeast, 3.5 - 4.5 um Cells trapped in single monolayer Yeast single cell imaging Yeast, 5 - 7 um Cells trapped in single monolayer Algae single cell imaging Algae, 3-5 um Cells trapped in “perfusion” pockets M04G M04L B04A Y04C Y04D C04A Microfluidic Chamber Design Culture chamber schematic 1 2-5 6 7-8 Actual chamber Highly-magnified chamber Inlet for gravity perfusion Flow inlets for presser driven flow Inlet for cell loading Waste outlets Diffusion through barrier • Perfusion barriers enable fluid dynamics which mimic in vivo diffusion conditions – no shear stress. • New medium is continuously perfused in and waste is perfused out. Capillary Driven Cell Loading Cell In Cell Out • Moves cells automatically from inlet well into microfluidic chamber • Capillary driven, no pump needed • Gentle, stress-free method Capillary Driven Cell Loading Loading of MCF-7 cells (2.5 million cells/ml) into the M04S microfluidic culture chamber via capillary flow. Note that as the cells enter, they settle onto the glass surface. Perfusion Cell Culture Day 0 Day 1 Day 2 Day 3 Day 4 ›Cancer: HeLa, PC-3, MCF-7, MCF-10A ›Neurons: Primary retinal ganglion, Hippocampal, Cortical, PC-12 ›Traditionally difficult cell culture is easy with the ONIX: Embyronic Stem Cells, Primary Cells ›Suspension cells: Lymphocytes, Platelets, Yeast, Bacteria ›Coat with ECM: Poly-d-lysine, laminin, fibronectin, and more 13 Long-term cell culture of MCF7 cells 24h 96 h 168 h Live/dead staining of long-term cultures. Even after 168 hours of culture, cells showed high viability. 14 Long Term Primary Neuron Culture 6 Days 20 Days 15 Days Rat Cortical Neurons on poly-lysine 15 Green: MAP2; Red: Synapsin Neuronal Stem Cells under Hypoxia Results 48 hours 24 hours Laminin coating, 3% oxygen After day 4, cells formed 3D “sphere-like structures,” termed neurospheres. Staining for Nestin (green) and Sox2 (red) revealed the tight 3D mass contained only bright spots of Sox2, while outer ring exhibited both Nestin and Sox2. 72 hours M04G: Spatial Gradient Control Top Inlets Perfusion Barrier Culture Chamber Bottom Inlets • • • • • Four chambers (4.0x0.5x0.1 mm) The volume is 100 nl Stable diffusion gradient between top and bottom streams Two switchable solutions for each stream Perfusion barrier separates cells from flows Gradient (M04S/M04G) FITC-Dextran 3kDa, Texas Red Dextran-20kDa, 16hrs • Stable, continuous flow, laminar diffusion gradient (available in both • • • • M04S and M04G microfluidic plates) Dynamic control- turning gradients on and off, and toggling between gradient and single solution exposure Stable profile for > 2 days Works in 2D and 3D culture Application: Chemotaxis/Migration or directed growth (i.e. Axon Growth) for > 2 days Neutrophil migration in chemo-gradient Chemotaxis of differentiated neutrophil-like HL-60 cells in a stable gradient of formyl-Met-Leu-Phe. Note the cell movement downwards as the attractant was added midway through the movie. Courtesy Jason Park, Wendell Lim Lab, UCSF Cancer Cell Migration to Stimulus No gradient (0/0) Gradient (10/0) No gradient (10/10) Gradient (0/10) X/Y migration plots highlight the impact of the FBS gradient on MDA-MB-231 cell movement. 20 Exposure to FBS gradients stimulated active migration of MDAMB-231 cells towards the high concentration sink. 3D Culture: MCF10A cells in Matrigel 2 Day Perfusion: 2D 2D Static Least In-vivo Like 2D Perfusion 2 Day Perfusion: 3D Matrigel 3D ECM 3D ECM w/ perfusion Most in-vivo Like MCF10A in Matrigel imaged (M04L plate) MCF10A Breast cancer cells cultured in 3D (Matrigel) in the M04L Plate. Cells organize into tumor spheroids of day 1. On-Chip Transfection MCF10A Tubulin HT1080 Tubulin Cells cultured in micro-chamber, then exposed to live cell transfection agent for imaging Live-cell Imaging Autophagosome formation and degradation with LentiBrite™ GFP-p62 and CellASIC ONIX 0 min 20 min 0 min Media → starve 40 min 60 min Starve 60 min 80 min 24 120 min Starve → media 180 min media 100 min Host-Pathogen Disease Model (M04S) M. Smegmatis infecting rat macrophages, Courtesy Stanley and Riley Lab, UC Berkeley (40x mag) 1 hour post infection, HT29 cells; Bacteria courtesy Tim Lu, MIT Controlled exposure/washout of virus/pathogen during live cell imaging Bacterial infection monitoring over days Pathogen/virus introduced to chambers through 4um barriers via solution inlets 25 Y04: Yeast Cell Imaging Plate • • • • • Four chambers (3.0x3.0 mm) Three trap heights per chamber (e.g. 3.5, 4.0, 4.5 um) The volume is 36 nl Cells held by elastic ceiling for long term imaging Six switchable solutions for each chamber Cell Trapping c) S. pombe in 3.5 µm trap (40X phase). d) RFP nuclear expression in cerevisiae. Yeast Perfusion Culture S. Cerevisiae grown in CSM medium in Y04C plate. DIC images acquired using monochromatic light illumination with Zeiss Plan-Apochromat 150x immersion objective. Courtesy of Jan Wisniewski (National Cancer Institute, NIH) B04A Microfluidic Plate • Allows long-term live cell imaging of bacteria • Cells remain in single focal plane during perfusion • Chamber heights of 0.7, 0.9, 1.1, 1.5, 2.0, 4.0 microns • The volume 6.3 nl. • Solution exchange (5 inlets) during imaging • 4 culture chambers per plate B04A traps a variety of bacteria types a) b) c) d) e) f) Schematic of elastic trap chamber E. coli in 0.9um trap (100X phase objective) After 24 hours growth, baclight stain (0.9 um trap) Caulobacter Mycobacterium E. Coli film after 24 hours All scale bars = 10 um Bacteria Response to Antibiotic E. coli cell growth in the presence of ampicillin to visualize antibiotic effects on the cell membrane. Cells grow and divide normally until ampicillin is added, causing them to burst. Images every 10 minutes for 5 hours @ 100X Courtesy of Sonia Singhal, Rob Egbert, and Eric Klavins (University of Washington, Seattle) Induction • E. coli induced with arabinose at t=30 min • Turns on GFP expression Courtesy of Lu Lab, MIT Applications Platform design is flexible to a wide range of applications for dynamic, time-lapse studies. › › › › › › › Cellular Response to Solution Change Drug Dose/Response Cancer Migration Neutrophil Chemotaxis/Migration Gene Expression Dynamics Synthetic Biology Neural Progenitor Cell Culture › › › › › › › › Cell Cycle and Mitosis DNA Damage (Long-term) Cell Polarization 3D Live Cell Imaging GFP Linked Nuclear Trafficking Cell Starvation and Recovery Gene Expression Host Pathogen Interactions Summary Microfluidics for Live Cell Imaging Stability and control of cell environments Powerful tool for dynamic perfusion experiments with time-lapse imaging Easy-to-Use Growing number of application specific designs Less than 10 minutes to start collecting data, 5 minutes to clean up Compatible with existing cell culture workflow Flexible Technology for New Applications ASIC concept allows reach into broad base of applications We’re committed to working with customers to address research needs 34 Questions Merck Millipore Booth 7C021 – Hal 7
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