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