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Microfluidics application note

Automate cell seeding in a microfluidic chip for dynamic cell culture

Dynamic cell culture

Automated cell seeding MFC7 cells in chip t4

In this application note, we present how to automate cell seeding for dynamic cell culture.

Microfluidic cell culture is the microscale culture of cells under flow to create a physico-mechanical environment that is more similar to that seen by cells and tissues in the body compared to static culture conditions. Cells naturally sense and respond to flow and shear forces introduced by blood circulation, interstitial flow and lymphatic drainage. Microfluidic perfusion replicates the physiological process of fluid transport, important for the exchange of nutrients, removal of waste, and application of shear stress.

Interested in dynamic cell culture? Watch the webinar by our researcher Lisa Muiznieks:

Advantages of this setup for cell biology:

In this application note we describe how to seed cells for dynamic cell culture. Seed with the automated OB1 pressure controller and hold your cells perfectly still in your perfusion chamber. Visually confirm seeding density and get reliable attachment. No longer worry about introducing bubbles between seeding and perfusion! Read also our application notes about perfusion and cell staining for dynamic cell culture.

Some main applications of dynamic cell culture include:

  • Live cell imaging (e.g. calcium imaging, FISH)
  • Drug screening
  • Shear stress
  • Cell rolling-adhesion assay
    • Immune response
    • Cancer invasion and metastasis
  • Models of physiology and disease
    • Organs on chip
    • Blood vessel formation & occlusion (atherosclerosis)
    • Bone homeostasis and disease (osteoporosis)
    • And many more!

List of components for automated cell seeding

Automated cell seeding microfluidic chip for microfluidic perfusion and stain cells e1599224620295

Microfluidic chamber designed for perfusion
(IBIDI µSlide I Luer)

Automated cell seeding setup diagram

Automated cell seeding setup diagram for perfusion and stain cells

HARDWARE:

  • Pressure & Flow controller: Impose a given pressure in order to create a stable and pulseless flow.
  • Reservoirs: Contain your medium, buffer, stains, cell suspension or samples. Various cap sizes are available, from Eppendorf to bottles.
  • Rotation valve (MUX-Distributor): Select the injected liquid.
  • Flow Sensor: Monitor and control the flow rate in real time.
  • Bubble trap: Trap and remove air bubbles to avoid them entering your chip, disturbing flow and adhered cells.
  • Perfusion chamber or microfluidic chip: Contains your cells. Compatible with microscopy.
  • Computer: Control all the parameters with our software and automate your experiment by creating injection sequences.

REAGENTS:

  • Cells: MCF7 (1×106 cells/mL)
  • Medium: DMEM high glucose, 10% FBS, Penicillin/ Streptomycin (100 U/mL; 100 µg/mL)
  • Wash buffer: PBS
  • Live/ dead cell stains: Calcein/ Hoechst/ Propidium Iodide

Protocol for automated cell seeding

Automated cell seeding cell seeding flow

Assemble system: Connect all modules, calibrate the instrument, and prepare and connect all solutions.

Purge: Disconnect tubing from the inlet to the bubble trap. Purge all reservoir lines of air. Reconnect tubing.

Flow medium: Switch MUX-Distributor inlet to medium reservoir. Start flow until the perfusion chamber is filled with medium.

Flow cells: Switch MUX-Distributor inlet to cells and flow until perfusion chamber is filled with cells.

IMPORTANT: accurately count the cells and use a well-mixed suspension immediately.

Stop the flow: Switch MUX-Distributor inlet to dead-end to stop the flow.

TIP: It is helpful to monitor the channel under a microscope during seeding.

Incubate: Give the cells time to adhere to the surface of the chip while no liquid is flowing. This can be up to 24 h depending on cell type.

Results

Automated cell seeding perfusion chamber

Perfusion chamber for automated cell seeding at microscope stage.

TIP: chip can be secured in a Petri dish for easier handling.

Automated cell seeding MFC7 cells in chip t2

MCF7 cells in chip, settling (t=2h).

Automated cell seeding MFC7 cells in chip t4

MCF7 cells in chip, beginning to adhere (t=4h).

Seeding video: Cells are flowed into a perfusion chamber filled with medium at a flow rate of 40 µl/min. Flow was stopped by switching the MUX-distributor valve to a dead-end block at t=18 s, then stopping the OBI. Note the instantaneous arrest of cells.

Hints and tips

Our team of experts can help you perform your experiment, improve the configuration of the setup for your specific application and tackle any issues you could face.
We can help you:

  • Incubation specifications
  • Easy flushing of remaining cells
  • Sterilizing the perfusion chamber
  • Cell counting
  • And a lot more!

CONTACT US

Application note written by Lisa MUIZNIEKS, Subia BANO, Camila BETTERELLI GIULIANO and Jessica AYACHE.

Acknowledgement: This work was done thanks to the funding of European Union’s Horizon 2020 research and innovation programme (PANBioRA project, grant agreement No 760921; MECH-LoC project, MSCA grant agreement No 793749; MTOAC project, MSCA grant agreement No 795754; Protomet project, MSCA grant agreement No 813873).

Want to run a similar experiment? Feel free to contact us at: contact@elveflow.com
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