Single-cell encapsulation using a droplet-microfluidic array

Seleipiri Charles, Nora Safabakhsh and Adam Melvin

Department of Chemical Engineering, Louisiana State University

Abstract
The tradi.onal approach of studying cell-behavior involves performing bulk analysis capable of only average measurements of how an en.re popula.on of cells responds to external treatments. Results obtained from such analysis
can o?en be inaccurate because they do not account for the behavior of individual cells that o?en .mes respond dierently from other cells in a heterogeneous popula.on. Droplet microuidics is one way of bypassing the
complica.ons that arise from such cellular heterogeneity. In this project, we sought to design, fabricate and op.mize a two-phase droplet microuidic device capable of isola.ng individual cells in picoliter-sized droplets. The
underlying principle of the device is to encapsulate cells in discrete aqueous droplets in a con.nuous oil phase. The droplet generators were fabricated using poly(dimethyl siloxane) (PDMS) by established so? lithography and PDMS
replica.on techniques. The uidic channels were treated using an Aquapel solu.on to alter the surface proper.es of the PDMS to make it more hydrophobic. Succeeding error correc.on, the ow rates of both the inlet aqueous and
oil streams were op.mized to con.nuously produce droplets of reliable size. Cell encapsula.on rates were performed using HeLa cells to ensure single cell trapping in each droplet. Finally, the system was modied to include a droplet
trapping array downstream of the ow focusing junc.on. The array was designed to trap the aqueous droplets by taking advantage of the dierence in uid densi.es and the surface tension of the drops to perform the .me-
dependent analysis of cellular behavior, an aspect of droplet microuidics that is currently limited. The results presented here are the rst step in the development of a new microuidic plaRorm capable of quan.fying intracellular
kine.cs at mul.ple .me points in intact single cells in a high throughput manner.

Droplet Genera.on

Introduc.on
The underlying principle
involves
compartmentalizing
picoliter-sized
monodisperse aqueous
droplets in an inert
con.nuous oil phase
This can be applied in cell
sor.ng, cell culturing,
chemical synthesis, single
cell analysis, and high-
throughput screening
High throughput screening (HTS) of single cells can be u.lized to
overcome cellular heterogeneity commonly associated with
tumors
The ul.mate goal is to incorporate a long lived, the cell
permeable biosensor in order to directly measure enzyme
ac.vity in intact single cells in a high-throughput manner

Materials/Methods
Microudic devices are fabricated by a combina.on of so?
lithography and PDMS replica.on
Fluidic channel geometry is fabricated on to a silicon wafer with
a photoac.ve polymer (SU-8)
PDMS replicas are a nega.ve relief of the silicon master which
are permanently bonded to glass using plasma oxygen
PDMS is pre-treated with the commercially-available water
repellent Aquapel to render the channels hydrophobic
Flow is introduced into the device via Tygon tubing connected
to syringe pumps

Es.mated droplet genera.on rate of ~10 droplets/sec

Droplet geometry is directly controlled by ow rate
Droplet volume was calculated from droplet diameters
Droplet diameter was determined by comparison to a
scale bar on the device using ImageJ (NIH)
Large droplet forma.on
Flow rate: 90 L/hr water and 250 L/hr oil
Average diameter = 276 m
Average volume = 13.8 nL
Small droplet forma.on
Flow rate: 90 L/hr water and 180 L/hr oil
Average diameter = 67.6 m
Average volume = 165 pL

Droplet Trapping Array

Modied the tradi.onal droplet generator to allow for on-chip incuba.on

and visualiza.on of the encapsulated cells
Fabrica.on was adjusted to a two-layer treatment to account for channels
of varying heights
Immiscibility of the two phases will force the droplets from con.nuous
phase into traps
Droplet trapping array consisted of 60 m circles imprinted 40 m into the
PDMS above the trapping channel

Cell Encapsula.on

Single cells can be encapsulated in droplet, but cell

density must be op.mized to increase the rate
single versus mul.ple cell encapsula.on
Drop size is related to the ow rate of the oil phase
Microuidic droplet array is able to trap single
droplets for real .me measurement
Volumes of drops produced were calculated from
the diameters of the drops
The diameters were derived by comparing the
dimensions of the drops with that of a scale bar on
the images by using an image edi.ng so?ware such
as ImageJ (NIST)
The surfactant ensures stable droplet forma.on and
prevents droplet coali.on. Hence, a surfactant that
is compa.ble with the carrier oil is necessary for
stable droplet forma.on

Future Work
Refabrica.ng the trapping array in order to improve
trapping eciency
Improving cell encapsula.on rates by increasing the
cell density of the aqueous stream in future
experiments
Performing cell viability studies to assess the
longevity of cells on-chip
Op.mized system will be used to analyze the uptake
of a commercially available cell penetra.ng pep.de
(cpp) in a heterogeneous cancer cell popula.on

Le?: Trapped aqueous droplets (brown circle) generated from 5.3M 5,6-
carboxyuoroscein and NOVEC 7500 uorinated oil
Right: Poten.al cell encapsula.on (orange circle) in trapping array
Current trapping eciency is approximately 2% as a result of defects from
device fabrica.on

So? Lithography

Droplet Merging Challenges

A. Fluidic channel geometry for the droplet generator

was generated using AutoCAD dra?ing so?ware
B. Flow focusing junc.on where two phases meet
Inlet oil stream is uorinated oil (Novec 7500, 3M)
with a 0.1% w/w uorosurfactant (FC 4430, 3M) to
prevent droplet aggrega.on
C. Single HeLa cell encapsula.on in aqueous droplet
(* marks a HeLa cell)

Conclusions

Above: Permeability assay of OWRWR (a

pep.de used in the lab that has been shown to
be cell penetra.ng [data not shown]) on cancer
cells is measured
Le?: Fluorescence given o by cells incubated
with OWRWR
Right: Control; cells not incubated with OWRWR

Acknowledgements
Aqueous inlet contained HeLa cells (2x106 cells/mL) in
extracellular buer supplemented with 15% (v/v) Op.-
Prep to prevent density gradients in the syringe
Flow rate: 50 L/hr water and 100 L/hr oil
Average droplet diameter: 67.6 m
Average droplet volume: 163pL

Droplet merging due to incompa.ble

carrier oil and surfactant (1% w/w FC
4430). Surfactant could not dissolve in
oil

Steady and stable droplet genera.on due

to compa.ble carrier oil and surfactant (2%
w/w 008-FluoroSurfactant). Surfactant
dissolves completely in oil a?er mixing

The authors will like to thank Gavin Pappas for his

assistance in this project and the Louisiana Board of
Regents for support