Perfusion cell culture with recirculation

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Introduction

Cell culture models are a powerful tool in biomedical research and pharmaceutical development. The ability to mimic physiologically relevant microenvironments is key to improving the clinical relevance of such models and assays. One feature of the in vivo cell environment that is often overlooked in traditional static cultures is the presence of flow and other mechanical stimuli experienced by cells. Microfluidics is a technique that shrinks the cell culture zone to the microscale and provides continuous perfusion of medium (including nutrients exchange and waste removal).

Perfusion cell culture can be achieved by simply flowing medium over cells from a reservoir to a waste collection vessel. However, by connecting the flow path in a loop from the reservoir to the chip and back, referred to as “recirculation”, the experiment won’t stop when the main reservoir is empty, i.e. the experiment length and flow rate are far less dependent on the starting volume of liquid than if a looped circuit is not used. The total volume of the medium in the system is usually many fold larger than the volume of the microfluidic cell culture chamber, meaning that the medium can stay fresh for days to weeks. Recirculation also conditions the medium with molecules secreted by the cells.

Recirculation using a pressure-driven flow controller is based on having two liquid reservoirs that are alternately pressurized so that one reservoir empties while the other collects. This requires a valve system between the reservoirs and the chip to ensure unidirectional flow over the cells. Valves can be active such as a rotary valve or pair of 3/2 valves, or passive such as using a set of check valves (for a detailed overview, check our review comparing different recirculation systems).

In this application note we demonstrate the continuous perfusion over cells in microfluidic culture using a recirculation circuit made from a passive check valve bridge.

Applications

Microfluidic perfusion cell culture using a recirculating flow path has broad applications in:

Microphysiological systems, organ-on-a-chip
Molecular transport assays, barrier models
Cell rolling and adhesion assays (e.g. tumor metastasis)
Drug screening

Specific advantages of recirculating the medium include the ability to:

Conserve medium for long experiments or fast flow rates
Reduce volume of expensive reagents
Circulate non-adherent cells
Mimic physiological multi-organ effects upon connecting multi-organ chips
Mimic physiological residence times of drugs
Condition medium (enrich for secretions)

Setup

The perfusion cell culture setup presented here uses a bridge made from 4 check valves and 4 T-junctions to enable the flow of medium through the microfluidic chip in a single direction (solid pink arrows), regardless of whether reservoir 1 or reservoir 2 is being pressurized.

Check valves allow liquid to pass in one direction only, in this case, out from the apex of the triangle. When two flow directions are possible at one T-junction, e.g. when liquid enters corner 3, the flow will circulate through the check valve with the lowest counter-pressure (see schematic below).

Liquid flow paths

This bridge configuration accommodates two flow paths inside the bridge depending on which reservoir is pressurized (indicated with “mbar”), but only one flow direction through the chip. A. When reservoir 1 is pressurized, liquid is pushed out of the bridge at corner 1 and eventually collects in reservoir 2. B. When reservoir 2 is pressurized, the liquid still exits the bridge at corner 1, maintaining unidirectional flow through the chip, before collecting in reservoir 1.

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 to be used in the CO2 incubator)
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 GFP cells (human glioblastoma astrocytoma transfected with GFP; 7.5 ╳ 105 cells/mL)
Medium (DMEM [+] 4.5g/L D-Glucose; with FBS 10%, penicillin, 100 U/mL / streptomycin, 100 µg/mL)
Cell stains (propidium iodide, 75 µM)

Design of the chip

The perfusion cell culture 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

1. Connect a pressure-driven flow controller to an external pressure supply using 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

The recirculation bridge is essential for the perfusion cell culture setup. The bridge is assembled as a square unit from 4 X check valves and 4 X T-junctions. One T-junction is placed at each corner to split the flow and one check valve is placed on each side. The orientation of each valve is crucial for the correct behavior of the system: each pair of parallel valves should be oriented in the same direction.

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 for improved sterility. Connect the check valves between the T-junctions following the orientation shown in the inset of the figure above (see Bridge assembly video- part 1).

3. Firmly press along all 4 axes to tighten the structure. Repeat tightening, if necessary.

Fluidic circuit assembly

1. Connect the reservoir caps to the pressure controller with pneumatic tubing.

2. Connect reservoir 1 to corner 4 of the recirculation bridge using 1/16” OD tubing (see Bridge assembly video- part 2).

3. Connect corner 1 of the recirculation bridge to the flow sensor inlet (see Bridge assembly video- part 2).

4. Connect 40 cm of 175 µm ID 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 corner 3 of the recirculation bridge (see Bridge assembly video- part 3).

6. Connect corner 2 of the recirculation bridge to reservoir 2 (see Bridge assembly video- part 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

1. Consider performing steps 1-4 in a Biosafety hood to maintain sterility if possible, or to use aseptic conditions on the bench, e.g., bunsen burner. 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. Transfer the setup to the CO2 incubator.

Experiment

1. Schedule liquid recirculation between reservoirs 1 and 2 using the software of the pressure-driven flow controller.

2. Flow at desired flow rate for desired time (e.g., 10 µl/min for 20 h).

3. Analyze (e.g., image cells in the chip).

Results

Cells were cultured under constant flow. Medium was recirculated back and forth between 2 reservoirs, while maintaining a single flow direction over cells in the microfluidic chip. Results of the perfusion cell culture experiment are shown in Figures 1 and 2.