Forces such as tension, compression and shear stress are physiologically important regulators of cell responses in vivo. Differences in blood vessel diameter and geometry across the arterial tree account for a wide physiological range of flow rates, flow profiles and shear stress in the circulatory system. Interstitial flow through porous tissue networks creates pressure and shear stress gradients. Fluid flow over cells cultured in microfluidic perfusion chambers provides key mechanical stimuli that are largely absent from traditional culture methods. Importantly, parameters such as flow rate and shear stress can be carefully controlled.
In this application note we describe how to recirculate medium for dynamic cell culture, use the OB1 pressure controller to maintain a precise flow rate of medium over cells in the perfusion chamber and hook up a MUX-Injection valve to enable recirculation of medium between two reservoirs unidirectionally over your cells. Recirculation enables higher flow rates to be maintained for multiple days, medium conditioning and studies of shear stress. Read also our application notes about automated cell seeding, microfluidic perfusion and cell staining for dynamic cell culture.
Some applications of dynamic flow in cell culture include:
Cell and biology pack
Microfluidic chamber designed for perfusion (IBIDI µSlide I Luer)
Flow controller OB1 Mk3+
Mux-Injection
Microfluidic bubble trap
Flow sensor
Tubing, fittings and reservoirs
Alternate between MUX-Injection valve POS 1 and POS 2. Liquid flows from one reservoir (pressurised) to the other (not pressurised). Flow is unidirectional through the chip.
Seed cells manually using a pipette into a microfluidic chip. Leave 6-24 h to attach.
Set valve: Set MUX-Injection valve to position 1.
Flow: Start flow from reservoir 1 (pressurise using OB1 channel 1). Reservoir 2 will collect the flow-through from the chip. Perfuse for desired time.
TIP: Both reservoirs will need to contain medium at the beginning: fill reservoir 1 (initial liquid source) with about 70% of the total volume and reservoir 2 (initial liquid collector) with 30%. Leave some volume in reserve in reservoir 1 before switching liquid sources to avoid introducing air into your perfusion chamber.
Switch valve: Switch MUX-Injection valve to position 2.
Flow: Start flow in reservoir 2 (pressurise using OB1 channel 2, depressurise reservoir 1). Reservoir 1 will collect the flow-through from the chip. Perfuse for desired time.
Repeat: Continue cycling between MUX-Injection valve positions to perfuse cells in culture for desired overall time.
MCF7 cells grown with continuous medium recirculation (10 µL/min, 24 h). Stained with Hoechst/ rhodamine phalloidin (fixed with PFA, 4%, permeabilized with BSA, 1%/ Triton X, 0.1%).
Application note written by Lisa MUIZNIEKS, Subia BANO, Camila BETTERELLI GIULIANO and Jessica AYACHE.
This work was done with the support from the French Agence Nationale de la recherche (ANR) in the frame of ERA-NET JPco-fuND 2019 (Orgtherapy).
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