Medium recirculation using microfluidic valves

Microfluidic cell culture set-up with valves

Dynamic cell culture using microfluidic recirculation valves adds important stimuli that bring in vitro experiments closer to the complexity of physiological conditions. In a living organism, cells are constantly perfused by body fluids, such as blood and interstitial fluids. Therefore, controlling the flow rate of media helps recreate a dynamic environment and allows for precise control of shear stress, nutrient delivery and waste removal. Microfluidic instruments allow to expose the cells to different flow profiles and different substrates.

Advantages of this setup for medium recirculation

This application note illustrates how to set-up a recirculation protocol for dynamic cell culture using the OB1 pressure/flow controller, the MUX wire valve controller in combination with two low pressure 3-way valves. This set-up, controlled by the proprietary ESI software, allows long-term automated and unidirectional cell perfusion with a predefined amount of medium. This will save cell culture medium and simultaneously allow the accumulation of desired cell products and offers an alternative to the microfluidic set-up using the MUX-injection valve.

Some applications of recirculation in cell culture include:

• Tissue on chip models requiring the analysis of molecules produced in low concentration
• Cell culture experiments supplemented with expensive media compounds
• Experiments requiring high amounts of media, including long term cell culture, and the effect of medium to high shear stress
• And many more!

List of components for a microfluidic medium recirculation setup with valves

Cell and biology pack

Microfluidic chamber designed for perfusion
(IBIDI µSlide I Luer)

Flow controller OB1 Mk3+

Schematic of the medium recirculation set-up with valves

MUX-WIRE: a close-up

The above image shows the two circuits in a single image. The media flow in the system can be broken up in two circuits for easier understanding, each diagram depicting one flow direction (see below):

Circuit 1:

Circuit 2:

TIPS

• Connect the valves to the reservoirs and to the T-junctions as shown in the figure. This way, when setting up a sequence in the ESI software, you will be able to save two configurations as follows: one where both valves are set to N.O. (normally open) and one where they are both set to N.C. (normally closed)
• Fill ALL tubing with liquid. Recirculate from reservoir 1 into reservoir 2 and back to reservoir 1, paying special attention to remove the air from the tubing between the T-junctions and the valves.

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. (15ml)
• 3/2 Valves (3-way valve): Switches between two positions to control the direction of the flow. In the default position the port of the valve defined as N.O. is open.
• Valve controller (MUX wire): Controls position of valves.
• T-junction (passive splitter): Allows intersection of three to passively split the flow.
• 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: HeLa (1.5×10⁶ cells/mL; aim for seeding density of ~75% confluence after 24H of attachment)
• Medium: DMEM high glucose, 10% FBS, Penicillin/ Streptomycin (100 U/mL; 100 µg/mL)
• Wash buffer: PBS
• Stain reagents: Calcein-AM 2 µM, Propidium Iodide 75 µM

Protocol for medium recirculation with valves

Step 1: Manually fill chip with media without introducing air bubbles.

Step 2: Seed cells into the microfluidic chip using a pipette.

Step 3: Incubate for 6-24 h to allow cells to attach.

Step 4: Connect the chip to the set-up without introducing air bubbles.

TIP: start with 20 µL\min and do not attach the chip until a small droplet is present at the end of the tubing that needs to be attached to the inlet of the channel

Step 5: Set both valves on N.O. using ESI software.

Step 6: Start flow from reservoir 1 (pressurize using OB1 channel 1). Reservoir 2 will collect the flow-through from the chip. Perfuse for desired amount of time.

TIP: start with a ramp if shear stress is high (based on type of cells used)

Step 7: Set both valves on N.C. using ESI software.

Step 8: Start flow from reservoir 2 (pressurize using OB1 channel 2). Reservoir 1 will collect the flow-through from the chip. Perfuse for desired amount of time.

Results

HeLa cells grown under dynamic perfusion with recirculating medium (50h after attachment, 20 µL/min, phase contrast image).

Live/dead staining of HeLa cells grown under dynamic perfusion with recirculating medium (50h after attachment, 20 µL/min). Live cells are stained with calcein (green), dead cells with Propidium Iodide (red).

Application note written by Francesca Romana BRUGNOLI – Acknowledgement: This work was done thanks to the funding of European Union’s Horizon 2020 research and innovation programme NeuroTrans (H2020-MSCA-ITN-2019-Action “Innovative Training Networks”, Grant agreement number: 860954) and the support from the French Agence Nationale de la recherche (ANR) in the frame of ERA-NET JPco-fuND 2019 (Orgtherapy).