Microfluidic Technologies for Cellular Reconstitution
Transcript of Microfluidic Technologies for Cellular Reconstitution
Microfluidic Technologies for
Cellular Reconstitution
Michael D. Vahey
Fletcher Lab
University of California, Berkeley
“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?
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
Cellular Reconstitution
Building biological functions from the bottom-up
Determining Size Changing Shape
Generating force
& movement
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
Outline
• Overview of encapsulation techniques
– Droplet microfluidics
– Inverted emulsions
• Microfluidic jetting
• Acoustic streaming
“Traditional” (PDMS)
microfluidics
Techniques to create
transient, micron-scale
inertial flows
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
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
Developmental Stages
Droplet microfluidics &
reconstitution: organelle scaling
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
Droplet microfluidics &
reconstitution: organelle scaling
Compartment size is sufficient to scale spindle
dimensions
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
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
Inverted emulsions: microfluidic
approaches
• Create aqueous
droplets in oil
• Flow droplets
into an ethanol
solution to
remove organic
solvent Lee et al., Biomicrofluidics 2011
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
Microfluidic jetting
• Create a droplet bilayer
• Deliver a jet of liquid to deform the bilayer into
spherical vesicles
Microfluidic jetting
Jetting capabilities
Jetting viscous liquids
Jetting relies on balance between inertial forces, shear
forces, and membrane tension:
Jetting cytoplasmic extracts
Inside the jet: E. coli extract
Inside the chamber: Plasmid DNA
Solutions mix during encapsulation
Automating and increasing
throughput
Replace the nozzle with an ultrasonic
transducer: acoustic jetting
Acoustic jetting
Acoustic jetting
Scale Bar: 200µm
Acoustic lens design
Increasing the
numerical aperture
increases resolution
and decreases depth
of field
Acoustic lens design
Future directions
Encapsulating biological solutions in lipid bilayers
has applications beyond cellular reconstitution
Acknowledgements
Dan Fletcher
The Fletcher Lab
• Matt Good
• Arunan Skandarajah
• Eva Schmid
• Ann Hyoungsook
Ruth L. Kirschstein National
Research Service Award