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Microfluidics application note

Unidirectional medium recirculation for microfluidic cell culture

Dynamic cell culture

medium recirculation microscope picture

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.

Watch the webinar by our researcher Lisa Muiznieks about dynamic cell culture!

Advantages of this setup for cell biology:

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:

  • 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 a microfluidic medium recirculation setup

Microfluidic chip for medium recirculation e1599666930652

Microfluidic chamber designed for perfusion
(IBIDI µSlide I Luer)

Medium recirculation setup diagram

medium recirculation setup e1602667927679

MUX-Injection: a close-up

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.

MUX injection medium recirculation close up
Medium recirculation POS 1
Medium recirculation POS 2

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.
  • MUX-Injection valve: Recirculate one liquid between two reservoirs, unidirectionally through the perfusion chamber.
  • Bubble trap: Trap and remove air bubbles to avoid them entering your chip, disturbing flow and adhered cells.
  • Flow sensor: Monitor and control the flow rate in real time.
  • 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)
  • Stain reagents: Hoechst 33342 (10 µg/mL), rhodamine phalloidin (1.5 µL/mL), PBS, paraformaldehyde (4%), BSA (1%), Triton X (0.1%)

Medium recirculation protocol

Recirculating medium visual protocol e1600261820431

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.

Results

Microscope picture of medium recirculation cells

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%).

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:

  • Automate the process with the ESI scheduler
  • Taking advantage of the flow shear stress
  • Easily purge air and the remaining cells from your system
  • Control perfusion by pressure or by flow rate
  • Improve cells lifespan
  • Recirculation using only microfluidic valves (no MUX-injection setup)
  • And a lot more!

CONTACT US

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).

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

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