Fraction collector for microfluidic cell culture
Introduction
Microfluidic perfusion is a powerful addition to cells in culture. Medium and nutrients can be exchanged, waste can be removed and non-adherent cells can be circulated. Flow can also be used to add mechanical stimuli such as shear stress to the cell environment. These cues are absent in static flasks or well plates, and increase the physiological relevance of culture models. Not least, flow unlocks the ability to follow changes over time.
Collecting fractions from the culture medium using perfusion offers the great advantage of capturing time-resolved biochemical molecular snapshots for analysis of dynamics, and measuring cell responses to physiological drug residence times (pharmacokinetics/ pharmacodynamics, PK/PD). These dynamics can arise from metabolism (catabolism/ anabolism; i.e. breaking down, combining, removing or releasing molecules), feedback loops, signaling pathways, and also the transport of molecules across a semipermeable barrier, e.g. between microfluidic compartments or molecular uptake/ release across a cell membrane.
This protocol describes a microfluidic cell perfusion setup with an inline fraction collector for automated sample collection from the cell culture medium during perfusion over 20 h. Sequential fractions can be collected at user-defined intervals and volumes. Analysis of samples for a molecule of interest can be performed separately using any appropriate analysis technique (e.g. HPLC, mass spectrometry, ELISA, colorimetric assay).
Applications
Time-resolved fraction collection is highly beneficial for many applications of microfluidic cell perfusion that study changes in molecule concentration or composition, e.g., often related to metabolism, transport or uptake/ release:
- Cell metabolism (break down or appearance of molecules/ drugs with time)
- Chemical/ drug metabolism
- Molecular transport across a membrane (semipermeable support separating 2 compartments; e.g., organ-on-a-chip studies)
- Cell uptake or release of molecules/ drugs (active or passive transport across cell membranes)
- Medium (buffer) exchange (molecule dilution, concentration)
Setup
Materials
Hardware:
- Pressure-driven flow controller (e.g. OB1 MK4 with two 0-2000 mbar channels; Elveflow)
- Flow sensor (e.g. LG16-0431D Sensirion; waterproof, if used in the CO2 incubator)
- 3/2 valve (2-position, 3-way valve)
- Valve controller (e.g. MUX-wire, Elveflow)
- Fraction collector unit, 20 Eppendorf collection tubes
- Recirculation bridge for unidirectional flow through the chip (4 x T-junctions, 4 x check valves)
- Tubings (PTFE, 1/16” outer diameter; OD), fittings and reservoirs
- Microfluidic resistance, 40 cm of 175 µm inner diameter (ID)
- Microfluidic chip (e.g. µ-Slide I Luer, ibidi)
Reagents:
- U-251 MG cells (0.8 x 106 cells/mL)
- Medium (DMEM [+] 4.5g/L D-Glucose; with FBS 10%, penicillin, 100 U/mL / streptomycin, 100 µg/mL)
Design of the chip
This experiment used a straight channel chip (µ-Slide I Luer 0.4; ibidi ®). The simple design and relatively 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 (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
1. Connect a pressure-driven flow controller to an external pressure supply with pneumatic tubing, and to a computer.
2. Turn on the pressure controller and open the software.
3. Install and calibrate the pressure controller following the manufacturer’s instructions.
4. Connect the flow sensors to the pressure controller and software as directed by the manufacturer.
Chip preparation and seeding
1. Prepare the microfluidic chip surface, if needed.
2. Prepare cells as per standard protocol. Disperse all clumps. Count carefully and use immediately.
3. Seed the chip by filling the cell culture chamber with cell suspension. Pay attention to fill only the chamber and not the reservoirs on each side.
4. Wait 5-10 min, then gently fill the reservoirs with fresh medium (containing no cells), taking care not to disturb the cells in the chip.
5. Cover the inlet and outlet and transfer the chip to the CO2 incubator in a sterile petri dish.
6. Leave 6-18h for cells to attach, or as required for your cell type and application.
Recirculation bridge assembly
1. Autoclave the check valves and T-junctions. Similarly, cut all lengths of tubing needed to connect the bridge to the fluidic circuit, attach connectors to the ends, and autoclave.
2. Perform steps 2 and 3 in a biosafety cabinet. Connect the check valves between the T-junctions following the orientation shown in the inset of the figure below (see the full bridge assembly video tutorial or the perfusion cell culture application note for more details).
3. Tighten the structure by firmly pressing along all 4 axes. Repeat tightening, if necessary.
Fraction collector assembly
1. Connect tubing from the 3/2 valve (position “NC”; normally closed) to the center port of rotary valve A on the fraction collector.
2. Connect ports 1-10 of rotary valve A to 10 collection ports. Use a short (5 mm) piece of silicon tubing sleeve and male Luer fluidic connectors (see figure below).
3. Connect port 11 of rotary valve A to the center port of rotary valve B on the fraction collector.
4. Connect ports 1-10 of rotary valve B to the remaining 10 collection ports, using connectors as above.
5. Add 20 Eppendorf tubes to the sample rack. Fold back the lids into the slot.
Fluidic circuit connection
2. Connect reservoir 1 to corner 4 of the recirculation bridge using 1/16” OD tubing.
3. Connect corner 1 of the recirculation bridge to the flow sensor inlet.
4. Connect a piece of resistance tubing to the outlet of the flow sensor. Use a union connector to then add a length of tubing. This will eventually be connected to the inlet of the microfluidic chip, but for now, add another union connector to the free end.
5. Connect tubing from the union connector (used in place of the chip until the circuit is purged of air) to the 3/2 valve that is connected to the fraction collector (use the side of the valve with only one hole). Placing the fraction collector after the chip ensures constant perfusion over cells regardless of whether a sample is being collected or not.
6. Connect the 3/2 valve (position “NO”; normally open) to corner 3 of the recirculation bridge.
7. Connect corner 2 of the recirculation bridge to reservoir 2.
Fill the bridge / Purge the circuit
1. Pressurize reservoir 1 to start flow. Hold the bridge with corners 1 and 2 upwards to aid removal of air (see Bridge Filling video tutorial for more details).
2. When the liquid front has reached reservoir 2, stop pressurizing reservoir 1.
3. Ensure reservoir 2 contains enough liquid, then pressurize reservoir 2 to start flow. Hold the bridge with corners 1 and 4 upwards until the check valve between corners 1 and 2 is filled with liquid.
4. Then tilt the bridge so that corner 4 is pointing upward to remove the remaining air from the circuit.
5. Pressurize alternately reservoir 1 then reservoir 2 a few more times to ensure complete purging of air.
Chip connection to fluidic circuit
1. Once the entire circuit is filled with liquid it is time to attach the seeded chip. Ensure the inlet and outlet of the chip are completely filled with medium.
2. Disconnect the union where the chip will be added. Add a ¼”-28 threaded to male Luer connector (red) to the ¼”-28 threaded connector (blue) and start the flow from reservoir 1 at a low flow rate (e.g. 10 µl/min).
3. Wait until a small droplet of liquid is visible from the end of the tubing, then connect it to the chip inlet, touching meniscus to meniscus to avoid trapping any air.
4. Add the free end of tubing to the chip outlet. Carefully wipe up any split liquid.
5. Add modules to a basket for transfer inside a CO2 incubator
Experiment
2. Schedule sample collection with the dedicated fraction collector software (MIC).
3. Flow at desired flow rate for desired time (e.g., 10 µl/min for 20 h).
4. Analyze (e.g., image cells in the chip or downstream analysis of collected fractions etc).
Results
Cells were cultured with perfusion for 20 h. Samples (150 µl) were collected by the fraction collector module once per hour. Volumes were determined by weighing the tubes before and after collecting fractions, shown below (Figure 1).
More tips included in the Application Note PDF!
Acknowledgements
This application note was written under funding from the European Union programs:
Horizon 2020 research and innovation program, grant agreement no. 101036702 (LIFESAVER, project page),
and H2020-LC-GD-2020-3, grant agreement no. 101037090 (ALTERNATIVE, project page).
Written by MIC in collaboration with LIFESAVER project partner, Institute for Research and Innovation in Health (I3S).
This application note was written by Lisa Muiznieks, PhD.
Published in October 2024.
Contact: Partnership[at]microfluidic.fr
