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Microfluidic Technologies for
Cellular Reconstitution
Michael D. Vahey
Fletcher Lab
University of California, Berkeley
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“Top-down” and “bottom-up” biology
Top-Down: Genetic Screens
• Study protein(s) in the context of the cell to deconstruct a specific process
What molecules are necessary for a process?
Bottom-up: Reconstitution
• Study protein(s) in isolation to reconstruct a specific process
What molecules are sufficient for a process?
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Commercial applications
Polymerase Chain Reaction (PCR)
• Reconstituted enzymes for DNA amplification
• Central to many sequencing technologies (e.g. Illumina)
In vitro expression systems
• Kits to synthesize proteins outside of the cell
Our focus: Developing technologies to advance more complex cellular reconstitutions
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Cellular Reconstitution
Building biological functions from the bottom-up
Determining Size Changing Shape
Generating force
& movement
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Cellular Reconstitution
Proteins need a suitable platform for their self-
organization:
• Control over the encapsulated solution
• Control over membrane composition
• Control over timing
Microfluidics offer precise techniques for controlling
initial conditions and boundary conditions in
cellular reconstitutions
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Outline
• Overview of encapsulation techniques
– Droplet microfluidics
– Inverted emulsions
• Microfluidic jetting
• Acoustic streaming
“Traditional” (PDMS)
microfluidics
Techniques to create
transient, micron-scale
inertial flows
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Microfluidic encapsulation
Creating and manipulating droplets has become a
leading application of microfluidic technology
Aqueous
•Biochemically resembles a
membrane for many applications
•More stable and mechanically
robust than bilayer membranes
Well-suited for studying confinement: how
volume affects biological processes
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Developmental Stages
Droplet microfluidics &
reconstitution: organelle scaling
How is organelle size
regulated during embryo
development?
Example: the Xenopus laevis
mitotic spindle decreases
~4× in length during the
first 8 cell divisions
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Developmental Stages
Droplet microfluidics &
reconstitution: organelle scaling
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Droplet microfluidics &
reconstitution: organelle scaling
• Encapsulate Xenopus
egg cytoplasm and
chromosomes in
droplets of varying size
• Quantify spindle size as
a function of droplet
size
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Droplet microfluidics &
reconstitution: organelle scaling
Compartment size is sufficient to scale spindle
dimensions
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Converting monolayers to bilayers
Inverted Emulsions (Weitz et al. PNAS 2003)
Droplet Interface Bilayers (Bayley et al. JACS 2007)
Many reconstitutions require a bilayer membrane
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Inverted emulsions: microfluidic
approaches
Paegel et al., JACS 2011
• Create aqueous droplets
in oil
• Use a physical barrier to
force droplets across a
second lipid monolayer
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Inverted emulsions: microfluidic
approaches
• Create aqueous
droplets in oil
• Flow droplets
into an ethanol
solution to
remove organic
solvent Lee et al., Biomicrofluidics 2011
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Inverted emulsions: microfluidic
approaches
Creation of the bilayer is the most challenging step
• Bilayer formation is not instantaneous
Too fast: bilayer breaks or becomes contaminated with oil
Too slow: sacrifice control over reaction timing
Alternative approach: create the bilayer
first, then mix and encapsulate
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Microfluidic jetting
• Create a droplet bilayer
• Deliver a jet of liquid to deform the bilayer into
spherical vesicles
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Microfluidic jetting
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Jetting capabilities
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Jetting viscous liquids
Jetting relies on balance between inertial forces, shear
forces, and membrane tension:
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Jetting cytoplasmic extracts
Inside the jet: E. coli extract
Inside the chamber: Plasmid DNA
Solutions mix during encapsulation
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Automating and increasing
throughput
Replace the nozzle with an ultrasonic
transducer: acoustic jetting
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Acoustic jetting
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Acoustic jetting
Scale Bar: 200µm
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Acoustic lens design
Increasing the
numerical aperture
increases resolution
and decreases depth
of field
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Acoustic lens design
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Future directions
Encapsulating biological solutions in lipid bilayers
has applications beyond cellular reconstitution
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Acknowledgements
Dan Fletcher
The Fletcher Lab
• Matt Good
• Arunan Skandarajah
• Eva Schmid
• Ann Hyoungsook
Ruth L. Kirschstein National
Research Service Award