CellASIC™ ONIX
Microfluidic Platform
The next generation of live cell imaging
Erwin Swart
Cellular Specialist BeNeLux
Merck Millipore
[email protected]
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
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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
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?
?
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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.
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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
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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
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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.
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Long Term Primary Neuron Culture
6 Days
20 Days
15 Days
Rat Cortical Neurons
on poly-lysine
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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.
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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
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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.
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Cellular Response to Solution
Change
Drug Dose/Response
Cancer Migration
Neutrophil Chemotaxis/Migration
Gene Expression Dynamics
Synthetic Biology
Neural Progenitor Cell Culture
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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
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Questions
Merck Millipore
Booth 7C021 – Hal 7