CO2 incubator-friendly microfluidic perfusion system for cell culture
Writer
Celeste Chidiac, PhD
Keywords
Microfluidic Devices, Intelligent Microfluidics, Artificial Intelligence, Machine Learning
Author
Lisa Muiznieks, PhD
Eve-Line Bancel, PhD
Publication Date
July 25, 2025
Keywords
Intelligent Microfluidics
Deep Learning
Microfluidic Devices
Artificial Intelligence
Machine Learning
CO2 incubator-friendly perfusion
Cell culture
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Introduction
Cellular models of health and disease are standard tools used for research and preclinical drug discovery. However, traditional static models in culture flasks or dishes lack the range of physical cues relevant to the in vivo microenvironment. The addition of medium flow over cells in culture in a microfluidic chip offers the benefits of controlled nutrient exchange, a known and reproducible flow rate, flow profile, and precise shear stress (or avoidance of mechanical stress), as required by the assay.
Methods for adding perfusion can be based on various working principles, from gravity-driven approaches (e.g., rocking platforms or hydrostatic pressure) to pumping systems (e.g., syringes, peristaltic pumps, or pressure-driven flow controllers). Pressure-driven flow controllers enable precise and highly reproducible flow rate control, which is particularly important for maintaining low flow rates (down to 1 µl/min) for perfusion experiments that last days to weeks.
Here, we introduce a pump specifically adapted to cell perfusion and provide a detailed protocol on how to add flow to cells in culture. Our Caterpillar perfusion system for cell culture has been designed for straightforward setup, while ensuring high flow stability and facilitating parallelization. Its small volume means that it can be placed inside the cell culture CO2 incubator (or next to a live cell imaging microscope) for long-term experiments. This means the entire fluidic pathway can be kept inside the CO2 incubator, minimizing the addition of variables other than flow for your experiment.
This application note demonstrates the setup and use of the Caterpillar perfusion cell culture system in a CO2 incubator for 8 days, reporting the results of real-time measured flow rates and cell viability staining.
Applications
Cell perfusion on the microscale has wide applications in fundamental biological research, disease modelling, and the medical and pharmaceutical industry, such as:
- Live cell imaging (2D, 3D cultures)
- Microphysiological systems; organ-on-a-chip
- Shear stress studies
- Molecular transport
- Drug screening
The Caterpillar perfusion system for cell culture offers specific benefits of flow control, including:
- Stable control of flow rates down to 1 µl/min
- Full compatibility for use inside the CO2 incubator or a live cell imaging microscope chamber
- Features an integrated flow rate sensor
- Only needs a power supply and computer connection (no compressed air source is required)
- Snap together up to 6 modules for experiment parallelisation
- Snap-in liquid reservoir connection for ease of assembly
Setup
In this setup, a Caterpillar perfusion system for cell culture was used to push the medium in one direction through a microfluidic chip seeded with cells. This pump is combined with an integrated flow sensor that regulates the flow rate to maintain a stable profile inside the cell culture CO2 incubator. The pressure and flow rate can be recorded during the experiment using the dedicated software interface.
Materials
Hardware:
- Caterpillar pump (supplied with 1 quick-connect reservoir cap, 1 integrated flow sensor, and 1 piece of resistance tubing adapted to the targeted flow rate range)
- Microfluidic chip (e.g. µ-Slide I Luer, ibidi ®)
- Tubings (PTFE, 1/16” outer diameter; OD), fittings (see for details of connectors)
- 2 ✕ 50 mL Falcon tubes (optional: 1 ✕ standard reservoir cap)
- Blunt end metal needle (14G ✕ 5 cm, female Luer connection)
Reagents:
- Cells (e.g. U-251 MG GFP; 0.6-0.8 × 106 cells/ mL)
- Medium: DMEM ([+] 4.5g/L D-Glucose; with 10% FBS, Penicillin/ Streptomycin (100 U/mL; 100 µg/mL)
- Phosphate buffered saline (PBS)
- Propidium iodide (PI) cell stain (75 µM)
Design of the chip
This experiment used a straight channel chip (µ-Slide I Luer 0.4; ibidi ®). The simple design and large internal geometry of the chip make it straightforward to fill, seed, and connect to the circuit. Its polymer coverslip base and microscope slide outer dimensions are ideally adapted for imaging, either as part of an endpoint staining protocol or for live cell imaging to monitor cell proliferation and morphology during perfusion.
µ-Slide I Luer 0.4 (ibidi ®) | Features |
Interface type | Female Luer |
Chamber volume | 100 µl |
Channel l ✕ w ✕ h | 50 ✕ 5 ✕ 0.4 mm |
Surface treatment | ibiTreat tissue culture treated |
Slide base | #1.5 polymer coverslip |
Quick start guide
Instrument connection and filling
This protocol first describes the assembly of the perfusion system for cell culture instrument in a Biosafety hood to maintain sterility. The setup is then transferred to the cell culture CO2 incubator, and the perfusion pump is connected to a power source and a computer to launch the software and start flow.
1. Select the appropriate size of falcon tube as the medium reservoir (e.g. 15 or 50 mL) and the corresponding tube cap. This experiment used a 50 mL Falcon tube.
2. Connect a sterilized needle to the underside of the quick connect reservoir cap in a Biosafety hood to maintain sterility (see image below: Quick connect reservoir cap assembly).
3. Fill the reservoir with medium in a Biosafety hood and carefully screw on the quick connect reservoir cap with needle. Connect the quick connect reservoir cap to the Caterpillar perfusion pump via the quick connect port.
4. Attach tubing (1/16” ID PTFE) to the top of the quick connect reservoir cap with a ¼”-28 threaded connector and ferule and the other end to the inlet of the Caterpillar flow sensor.
5. Connect the outlet of the flow sensor to the resistance tubing. To achieve the 1 µL/min target of this experiment, we used a 50 cm piece of tubing with 100 µm inner diameter.
6. Connect the outlet of the resistance tubing to a ¼”-28 threaded union, and add a length of 1/16” ID PTFE tubing to the outlet of the union.
7. Connect the free end of tubing to a female Luer union (in place of the seeded chip for initial system filling) and connect the collection/ waste container (see image: Full fluidic setup).
8. Transfer the setup on a tray to the cell culture CO2 incubator.
9. Connect the pump module to a Caterpillar mother module and a power supply.
10. Connect the pump to a computer. Open the software and connect to the interface (see the User Guide for further details).
11. Start the Caterpillar pump and completely fill the system with medium.
Chip preparation and seeding
1. Add surface coating, if desired. Note that a commercially purchased “cell culture treated” surface is suitable for cell attachment without additional treatment, e.g., hydrophilized, collagen, ibiTreat, etc.
2. Prepare cell suspension as per standard protocols. Ensure all cell clumps are gently but well dissociated and count carefully. Use the suspension immediately.
3. Seed a µ-Slide I Luer 0.4 chip with a cell density of 0.6-0.8 ´ 106 cells/mL using a pipette. Position the pipette tip at the base of the inlet right at the entrance to the channel and apply slow and gentle pressure to avoid generating air bubbles.
4. Cover inlets/ outlets and leave 6-18 h in the CO2 incubator for cells to attach, or as needed for the application.
Chip connection to fluidic circuit
1. Tape the chip to a sterile petri dish for maximum stability and mark on it the direction of flow.
2. Remove the female Luer union. Connect the chip by touching the small droplet of medium at the tip of the connector to the meniscus of the medium at the chip inlet, to avoid trapping air.
3. Connect tubing to the outlet of the chip. Carefully wipe away any medium from the outside of the chip. Perform a visual inspection about 1 h after starting the experiment to ensure there are no leaks.
Experiment
1. Set desired flow rate on the Caterpillar perfusion pump interface, e.g., 1 µl/min.
2. Start flow and leave for the desired time, e.g., 8 days
3. Analyze results of the cell perfusion experiment, e.g. stain and image cells in the chip or perform downstream analysis of collected aliquots of medium as desired.
Results
Cells were cultured in a microfluidic chip with constant perfusion (1 µl/min) using the Caterpillar perfusion system. After 8 days, cells were stained in the chip with propidium iodide (Fig. 1). The accuracy and flow profile of the perfusion pump were measured using the integrated flow sensor controlled via the interface.
The profile obtained was very stable, with amplitude peaks corresponding to external interventions (such as opening the cell culture CO2 incubator door).
More tips included in the Application Note PDF!
Acknowledgements
This application note has received funding from the European Union’s EIC Pathfinder Open program under the HORIZON-EIC-2022-PATHFINDEROPEN-01 Programme, grant agreement no. 101099719 (THOR).
This application note was written by Lisa Muiznieks, PhD, and Eve-Line Bancel, PhD.
Published on July 20255
Contact: Partnership[at]microfluidic.fr
