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.
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:
Cell and biology pack
Microfluidic chamber designed for perfusion (IBIDI µSlide I Luer)
Flow controller OB1 Mk3+
Mux-distributor
Microfluidic bubble trap
Flow sensor
Manifold 9 ports
Tubing, fittings and reservoirs
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.
Perfusion chamber for automated cell seeding at microscope stage.
TIP: chip can be secured in a Petri dish for easier handling.
MCF7 cells in chip, settling (t=2h).
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.
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).
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