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AUTOMATION OF CAPTURE
AND LABELLING OF CANCERCELLS WITH ARIA
INTRODUCTION
AIM

This application note describes the use of Fluigent Aria – a software-assisted
instrument capable of delivering of up to 10 different solutions – for the automationof a complete protocol of capture and labelling of MDA-MB-231 breast cancer cells
including surface treatment, injection of antibodies-coated beads and cell suspension,and immunostaining steps.

CONTEXT

Cell capture and labelling are usually time consuming processes that can last forseveral hours, and often require exposure to multiple fluids. As an example, typicalstaining protocols usually require more than 5 consecutive solutions. Cell labelling, cellmanipulation, drug screening or perfusion also demand very precise injection volumes
with the use of controlled flow rates (ranging from 1 µL/min to 1 000 µL/min) in orderto perform reproducible experiments. Smooth fluid control is also recommended
as uncontrolled flow rates can create high shear stresses, subsequently affecting cellfunctional properties and damaging samples. Aria is the perfect instrument to addressthese concerns and automate fluid delivery protocols.
Specific needs for circulating tumor cell sorting: Metastases occur frequently and areresponsible for about 90% of cancer deaths1. They result from the breakout of malignantcells, named Circulating tumor cells (CTCs), from the primary tumour site. CTC enter
the blood through an intravasation process,disseminate into distant organs and initiate
new metastasis2 (Figure 1).
Because blood can be easily sampled andanalyzed, CTCs are particularly interesting as
metastasis-related biomarkers3. Further-more,
CTC count is used as a dynamic biomarker
correlated to treatment efficacy of breast
cancer patients4. However, the search for
CTCs in blood is a strong challenge as theirconcentration is extremely low: in 1 milliliter ofblood, about 1 CTC is found among ten million
white blood cells and billions of red blood cells1.

Figure 1: CTCs pathway, Capture and Analysisof Circulating Tumor Cells in Microfluidics: From
Conception to Clinica Validation, Julien Autebert,
Physico-Chimie Curie, UMR168, 2013

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APPLICATION NOTE
Various techniques for capture and analysis of CTCs have been proposed. Many capturedevices make use of antibodies directed against cancer cell membrane to capture
tumor cells. A commonly used capture molecule is the EpCAM (Epithelial Cell Adhesion
Molecule) antibody: a transmembrane glycoprotein mediating Ca2+-independent
homotypic cell–cell adhesion in epithelia. As EpCAM is overexpressed by CTC’s and not
expressed by white and red blood cells, it can be used as diagnostic marker for various
cancers in blood samples.

CHALLENGE

We describe the automation of a rare cell capture and detection method including
surface treatment, beads and cells solution injections, and immunostaining using
precise volume control using Aria and its related automation software. We use theEPHESIA microfluidic chip from Institut Curie and MDA-MB-231 breast cancer cell line asa demonstration of system function and utility.

MATERIALS AND METHODSSYSTEM SETUP

Figure 2: System setup. Aria and a valve areconnected to the EPHESIA microfluidic chip,
which is placed in a coil. Visualization of thechip channels is performed using an optical microscope in bright field or fluorescence mode

Aria is connected to a pressure source and a computer for real-time flow control. Aria
comes with a valve that directs the flow either to the chip or to the waste. The microfluidic
chip is connected to Aria via 1/16 in. transparent tubes with PEEK fittings. The tubing is
cut in order that the system is as close as possible tothe chip to reduce swept volume. The microfluidic
chip is placed in a water-cooled electro-magnetic
coil (Figure 3) and the microfluidic channels arevisualized using an optical microscope in bright fieldor fluorescence mode.
The fluidic delivery protocol of the experiment isentirely automated using the Aria Automation
Software: allowing to set injection parameters basedon time or volume, and desired flow-rate values.
Figure 3: Microfluidic chip placedin an electromagnetic coil andconnected to the tubing

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APPLICATION NOTEEPHESIA:A MICROFLUIDIC DEVICE FOR CANCER CELLS CAPTURE AND ANALYSIS

To achieve sufficient sensitivity and sensibility, Institut Curie has developed the “EPHESIA”
microfluidic device shown in Figure 5. CTC capture is based on the specific EpCamantigen-antibody reaction. EpCam coated magnetic beads are arranged into a sieveof columns inside the chip to increase the probability of contact with CTC (Figure 4).
The sample to analyze is pumped through the chip and the CTC’s captured on thecolumns are counted and analyzed. Another substantial added value of magnetic beads
is the possibility to retrieve the CTC’s captured at the end of the experiment to perform
additionnal analysis.
EPHESIA chips have a specific architecture.
The channel geometry
is a tree-likearchitecture that splits the flow into
multiple channels in order to increase
the flow velocity uniformity within mainchambers. The two-layered chip is made of
COC (cyclic olefin copolymer). The top layer
contains the microfluidic channels as wellas the inlet and outlet ports. The bottom
section is made of two capture zones whichcontain 48 000 holes that will containthe anchor magnetic beads. Each hole isfilled with approximately ten beads before
bonding the two layers of the chip together.
Anchored beads withstand hydrodynamic
flow and maintain column stability during
the capture and analysis process.

Figure 4: Schematic illustration of the capture ofcells in a self-assembled array of magnetic beads
guided by a magnetic pattern6

The microfluidic chip is placedin an electro-magnetic coil togenerate a uniform magnetic
field in the center of the chip. A
suspension containing EpCAM
coated magnetic beads is injected,
and magnetic columns areformed on the anchorage arrays.
It is then possible to inject cellsolutions. Cells expressing EpCAM
antigen will specifically bind to
EpCAM-coated beads (Figure 4).
Captured cells can be visualized
and counted using fluorescencemicroscopy.

Figure 5: Diagram and photography of the EPHESIA cell capture chip5

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APPLICATION NOTEIMMUNOFLUORESCENCE

Cells were labelled with Hoechst to stain the nucleus, and Phalloidin to visualize actin
filaments. Cells were previously genetically modified to express GFP in the area ofdamaged DNA within the nucleus.

AUTOMATED CELL CAPTURE AND LABELLINGThe complete fluidic delivery protocol of the experiment is presented in Figure 7.

VALVES

Figure 6: Scheme representing the microfluidic setup and the solutions injected during the protocolFigure 7: Sequential injection automated protocol using Aria Automation Software

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APPLICATION NOTE
Protocol Steps

Setup preparation and surface treatment: Before injecting the various liquids, a prefill
step using PBS is required to fill empty tubing of the system and calibrate Aria. Oncethe prefill is completed, the chip is flushed with ethanol using a flow-rate of 200 µL/
min for 2 minutes to wet the microfluidic channels and create a sterile environment forcell manipulation. The system is next flushed with distilled water using a flow-rate of200 µL/min for 1 minute. A surface treatment is then performed to prevent non-specificadsorption of beads, cells or proteins: Pluronic acid F-127 is injected in the chip using
a flow-rate of 200 µl/min for 1 minute and incubated for 1 hour. In Aria’s software thefunction “Wait” was used to set 1 hour incubation time before automatically restarting,as shown in Figure 8. A washing step follows: PBS + 1% BSA is injected using a flow-rateof 50 µL/min for 2 minutes
Beads injection and column
self-assembly: After the surface treatment is performed,
anti-EpCAM antibodies-coatedbeads are injected in the microfluidic chip using a flow-rateof 200 µL/min for 1 minute. A Figure 8: Software snapshot of the protocol showing the “Wait” featuremagnetic field perpendicular
to the chip bottom layer is turned on. The magnetic beads self-assemble into columns
that are spontaneously anchored on the magnetic patterns. The result is a sieve madeof magnetic bead columns.
Cells injection and capture: Once columns are formed within the microfluidic chip, a300 µL solution containing ~15 000 MDA-MB-231 cells is subsequently injected using
a flow-rate of 50 µL/min for 2 minutes. Injection flow rate has been optimized to avoid
destruction of the sieve of magnetic beads.
Fixation, permeabilization andstaining:
Paraformaldehyde
is injected in the chip to
fix captured cells. After aprogrammed incubation time of20 minutes, a permeabilization
step follows: Triton-X is injected
at 50 µL/min for 4 minutes. It is
possible to observe the stableflow-rate injection of Triton-X in
Figure 9.
Figure 9: Triton X injection step and related flow-rate control

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APPLICATION NOTE
We label cells using Hoechst to stain the nucleus,
and Phalloidin to visualize actin filaments. Cells werepreviously stained with GFP, allowing to identify DNAdamage within the nucleus. After an incubation
step of 20 minutes, antibodies are injected using aflow-rate of 50 µL/min for 4 minutes and anotherfinal incubation step of 20 minutes. Cells were finallywashed by injecting PBS + 1% BSA using a flow-rateof 50 µL/min for 4 minutes. Cells were observed
using fluorescent microscopy. As shown in Fig. 10, itis possible to use Aria in dark environments.

Figure 10: : Cell observation using
fluorescent microscopy

Adapt your biological protocol using Aria instrument

To maximize the cell capture and labelling protocol, some aspects should be taken into
consideration:» Some solutions might be relatively expensive, hence they should not be used in
the prefill step.
» To promote cell survival, cell suspensions must be prepared immediately before
injection in the chip and they should not be prepared in advance.
» The antibody solutions should also be prepared at the last moment as they requirepreservation at low temperature (4°C) and in the dark.
» Diffusion between two consecutive liquids may happen within the system, possibly
affecting experimental results.
As a consequence, it is highly recommended to:» Anticipate the number of reservoirs required for a specific protocol, and performthe prefill step using PBS or another relatively inexpensive buffer. Note that it ispossible to label solutions using the Aria Automation Software to facilitate protocol
step identification while running as shown in Figure 11.
» Add unstable solutions at the last moment: Additional useful software features
can be included in the automated protocol. The feature “Wait for user” allows theuser to pause the protocol, as shown in Figure 12. The user can prepare unstable
solutions during this step, add the reservoir containing the solution to Aria systemand then continue protocol execution. In our protocol, this feature is used for theinjection of beads and cells suspensions, and the antibody solutions.

Fig. 11: : Software snapshot of showing the reservoirs labelling Fig. 12: Software snapshot of the protocol showing the “Wait for user” feature» Inject a buffer solution between two consecutive liquids to avoid contamination
due to liquid diffusion. To avoid diffusion between cell suspension and PFA in thetubing, the ‘flush tubing’ function was used. As a result, the cell suspension was
first loaded in the tubing and injected in the chip. Then the tubing was flushedwith PBS which was discarded to waste. PFA was then loaded in the tubing.
Figure 13: Software snapshot of the protocol
showing the “Flush tubing” feature.

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APPLICATION NOTERESULTSBEADS INJECTION

After the surface treatment, anti-EpCAM antibodies-coated beads are injected throughthe microfluidic chip. As the magnetic field is turned on, beads self-assemble intocolumns that are spontaneously anchored on the magnetic patterns. Figure 14 a/ andb/ show respectively micrographs of coated beads attached to the columns with focus
on patterned columns or coated-beads, confirming the proper functionin of the beads
injection protocol.

a/b/

Figure 14: Micrographs of self-assembled injected beads on the patterned columns focused on a/ columns b/ beads

CELL CAPTURE AND LABELLINGOnce columns are formed within themicrofluidic chip, the solution containing
MDA-MB-231 cells was subsequently
injected. We can observe in Figure 15
several cells that specifically bind to theanti-EpCAM coated columns.
Finally, captured cells were stained withdifferent dyes following the protocol
from the automated cell capture andlabelling section. We can observe inFigure 16 cells that bind to the EpCAM
coated columns, exhibiting green, blue Fig. 15: Micrograph of MDA-MB-231 cells captured on the columns
and orange fluorescence. Note thatthe columns formed by the magnetic beads display a weak orange fluorescence, as
Phalloidin binds to EpCAM. Figure 17 shows a composite micrograph of cells attached
to a column using higher magnification. We can distincly observe orange fluorescenceindicating stained actin filaments, blue fluorescence indicating stained nucleus andgreen fluorescence indicating damaged DNA within the nucleus. These results confirm
the proper function of the staining injection protocol.

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Figure 16: Composite image of the three micrographsfrom the same area using blue, orange and green
fluorescent filters

Figure 17: Composite image of the three micrographsfrom the same area using blue, orange and green
fluorescent filters

CONCLUSION
We have demonstrated the use of Aria and its related software for theautomated delivery of different liquids for the capture and labellingof breast cancer cells using a complex microfluidic setup. This
protocol included 10 different liquid injections, surface treatment,beads injection, cells injection, capture and labelling in a sequential
and automated manner. We also demonstrate how Aria can adaptto specific protocols by making use of its specific software features,
allowing for the optimization of complex protocols.
Aria provides key features for performing this application as it brings
to the user:» Reduced manipulation time and timed exposure to antibodies,
fluorophores, DNA probes with automated and timed protocols» Reduced handling for minimal contamination< or changes tocell conditions» Higher reproducibility compared to manual methods, hence
more reliable results

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APPLICATION NOTEREFERENCES
1. Autebert, J. et al. 2015 High purity microfluidic sorting and analysis of circulatingtumor cells: towards routine mutation detection. Lab Chip 15, 2090–2101 (2015).
2. Kim, M. Y. et al. Tumor Self-Seeding by Circulating Cancer Cells. Cell 139, 1315–1326(2009).
3. Cabel, L. et al. Clinical potential of circulating tumour DNA in patients receiving
anticancer immunotherapy. Nat. Rev. Clin. Oncol. 15, 639–650 (2018).
4. Bidard, F. C. et al. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: A pooled analysis of individual patient data. Lancet Oncol.
15, 406–414 (2014).
5. Saias, L., Autebert, J., Malaquin, L. & Viovy, J.-L. 2011 Design, modeling and characterization of microfluidic architectures for high flow rate, small footprint microfluidicsystems. Lab Chip 11, 822–32 (2011).
6. Bernacka-wojcik, I. 2014 Design and development of a microfluidic platform foruse with colorimetric gold nanoprobe assays. (Universidade Nova de Lisboa, 2014)

This study has been made in collaboration withEmile Lakis, PhD from Institut Curie, Team
Pierga, Circulating Cancer Biomarkers

For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com