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Capture and Labelling of cancer cells using Aria

This application note describes the use of the new Fluigent Aria – a software-assisted instrument capable of delivering of up to 10 different solutions – for the automation of 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.

We have demonstrated the use of Aria and its related software for the automated delivery different liquids for the capture and labelling of breast cancer cells using a complex microfluidic setup. We also demonstrate how Aria can adapt to specific protocols by making use of its specific software features, allowing for the optimization of protocols.

Aria-setup

This study has been made in collaboration with Emile Lakis, post doctorate at Institut Curie, Team MMBM (Macromolecules and Microsystems in Biology and Medicine)

MATERIAL AND METHODS

SYSTEM SETUP
Aria is connected to a pressure source and a computer for real-time flow control. The microfluidic chip is connected to Aria. The microfluidic chip is placed in a water-cooled electro-magnetic coil (Figure 3) and the microfluidic channels are visualized using an optical microscope in bright field or fluorescence mode.

EPHESIA: A MICROFLUIDIC DEVICE FOR CANCER CELLS CAPTURE AND ANALYSIS
To achieve sufficient sensitivity and sensibility, Institut Curie has developed the “EPHESIA” microfluidic device. CTC capture is based on the specific EpCam antigen-antibody reaction. EpCam coated magnetic beads are arranged into a sieve of columns inside the chip to increase the probability of contact with CTC. The sample to analyze is pumped through the chip and the CTC’s captured on the columns are counted and analyzed.

Aria Software Application Note

PROTOCOL STEPS

Aria software was used to automate each injection step :

  1. Surface treatment: the chip is flushed with ethanol to wet the microfluidic channels and create a sterile environment for cell manipulation. The system is next flushed with distilled water. A surface treatment is then performed to prevent non-specific adsorption of beads, cells or proteins. In Aria’s software the function “Wait” was used to set 1 hour incubation time before automatically restarting. A washing step follows: PBS + 1% BSA is injected.
  2. Beads injection and column self-assembly: Anti-EpCAM antibodies-coated beads are injected in the microfluidic chip. A magnetic 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 made of magnetic bead columns.
  3. Cells injection and capture: A solution containing MDA-MB-231 cells is subsequently injected. Injection flow rate has been optimized to avoid destruction of the sieve of magnetic beads.
  4. Fixation, permeabilization and staining: Paraformaldehyde is injected in the chip to fix captured cells. After a programmed incubation, a permeabilization step follows. Cells are labelled to stain the nucleus, and to visualize actin filaments. Cells were previously stained to identify DNA damage within the nucleus. After an incubation step, antibodies are injected. Cells were finally washed.

Partial RESULTS

CELL CAPTURE AND LABELLING
Once columns are formed within the microfluidic chip, the solution containing MDA-MB-231 cells was subsequently injected. We can observe in the figure on the left below cells that bind to the EpCAM coated columns, exhibiting green, blue and orange luorescence. Note that the columns formed by the magnetic beads display a weak orange fluorescence, as Phalloidin binds to EpCAM. The figure on the right shows a composite micrograph of cells attached to a column using higher magnification. We can distincly observe orange fluorescence indicating stained actin filaments, blue fluorescence indicating stained nucleus and green fluorescence indicating damaged DNA within the nucleus. These results confirm the proper function of the staining injection protocol.

Aria Results Application Note Fig2a
Aria Results Application Note Fig2b

Composite image of the three micrographs from the same area using blue, orange and green fluorescent filters

Composite image of the three micrographs from the same area using blue, orange and green fluorescent filters

Conclusion

We have demonstrated the use of Aria and its related software for the automated delivery different liquids for the capture and labelling of 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 adapt to specific protocols by making use of its specific software features, allowing for the optimization of complex protocols.

Aria

ARIA

Fluigent introduces Aria our automated solution for cellular perfusion or timed injection protocols. Aria allows one to automate delivery up to 10 different solutions into a chamber or microfluidic chip by following user defined protocols.

BENEFITS

  • Save time with automation
  • No manipulation to reduces contamination or changes to cell conditions
  • Higher reproducibility compared to manual methods for reliable results

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