Cross flow membrane for molecular transport

Introduction

The cross flow membrane chip (Fluidic 480) from microfluidic ChipShop enables to study the dynamic of molecular transport between two chambers and its impact on cell cultures. The microfluidic cell is made of two chambers separated by a permeable membrane. Cells can be seeded on one or both sides of the membrane and exposed to flowing growth medium. The experiment consists of adding a chemical or a drug to one of the chambers. The permeable cross flow membrane will allow molecular transport to the other side of the membrane. In this application note we describe a suitable experimental set-up to use the cross flow membrane microfluidic chip.

Applications of the cross flow membrane chip

• Dilution processes
• Buffer exchange
• Liquid-liquid interface
• In-line filtration
• Organ-on-chip barrier models of molecular transport (cell interaction and signaling)
  • Lung-on-chip: gas exchange, air-liquid interface
  • Intestinal gut-on-chip, placenta-on-chip (mother-fetal barrier), brain-on-chip (blood-brain barrier): molecular transport and uptake (nutrients, toxins, pharmaceuticals, …)
  • Kidney-on-chip: filtration

Cross flow membrane microfluidic setup

Flow controller OB1 Mk3+

Flow sensor

Tubings, fittings and reservoirs

Fluidic 480 chip from microfluidic ChipShop Gmbh

Figure 1: Cross flow membrane experimental set-up

Hardware

1. Flow controller OB1 MK3+with at least 2 channels 0/2000 mbar
2. 2x flow sensors MFS3 2-80 µl/min
3. Microfluidic 3-way valve and 1x MUX Wire valve controller
4. Cross-flow membrane microfluidic chip from microfluidic ChipShop, fluidic design 480
5. 2×12 cm of 125 µm diameter resistance
6. ∼ 75 cm of 1/16” OD tubing
7. ∼ 100 cm of 760 µm OD tubing
8. 4x 15 ml falcon tubes
9. 3x pressure holding caps for 15 ml falcon tubes
10. 20x Eppendorf 1.5 ml test tubes
11. Micropipette
12. Kit fittings start pack luer from Elveflow
13. One pack of Male Mini Luer tube tuck connector from microfluidic ChipShop (Fluidic 997)
14. Computer to control flow controller, flow sensor and MUX Wire

Chemicals

1. Filtered water
2. Tracer (e.g food dye) or chemical of interest

Design of the cross flow microfluidic chip

Figure 2: microfluidic ChipShop Fluidic design 480 cross flow membrane microfluidic chip for molecular transport

The cross flow membrane microfluidic chip consists of two stacked chambers, each with one inlet and one outlet (ports 1 & 2 feed chamber A, ports 3 & 4 feed chamber B), separated by a porous membrane. Molecular transport can be monitored from one chamber to the other across the membrane by diffusive or active flux. The slide itself offers two independent microfluidic chips, where two independent experiments can be run. The chamber’s Mini Luer interfaces fit microfluidic ChipShop‘s Mini Luer connectors and plugs. Tubing 760 µm OD can be directly pushed into the Mini Luer tube tuck connectors.

Figure 3: Mini Luer interfaces fit microfluidic ChipShop‘s Mini Luer connectors and plugs. Tubing 760 µm OD can be attached to Mini Luer tube tuck connectors.

Cross flow membrane quick start guide

Instrument connection

• Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using a USB cable. For detailed instructions on OB1 pressure controller setup, please read the “OB1 user guide”.
• Connect the flow sensors to the OB1. For details refer to “MFS user guide”.
• Turn on the OB1 by pressing the power switch.
• Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the “ESI user guide”. Please refer to the guide for a detailed description.
• Press Add instrument \ choose OB1 \ set as MK3+, set pressure channels if needed, give a name to the instrument and press OK to save changes. Your OB1 should now be on the list of recognized devices.
• OB1 calibration is required for the first use. Please refer to the “OB1 user guide”.
• Add the flow sensors: press Add sensor \ select flow sensor \ analog or digital (choose the working range of flow rate for the sensor if you have an analog one), give a name to the sensor, select to which device and channel the sensor is connected and press OK to save the changes. Your flow sensor should be on the list of recognized devices. For details refer to “MFS user guide”.
• Open the OB1 Window.

SET-UP PREPARATION FOR MOLECULAR TRANSPORT EXPERIMENT

• Fill two reservoirs with water, and one with tracer solution.
• Connect the cap of Reservoir A directly to the OB1 with pneumatic tubing. Branch Reservoir B1 and B2 caps with a T-junction as shown in the schematic and connect to the OB1.
• Connect Reservoirs B1 and B2 to the 3/2 valve with 1/16”OD tubing: water to position N.O. (normally open), tracer to position N.C. (normally closed). Connect the outlet of the 3/2 valve to the flow sensor with 1/16” OD tubing.
• Connect Reservoir A to the flow sensor with 1/16” tubing.
• Add 12 cm of 125 µm ID resistance tubing to the outlet of each flow sensor, then a threaded union, and a piece of 760 µm OD tubing, ready to be reconnected to the appropriate chip inlet after purging (see step below).

TIP:  For more details on the use of resistance to obtain the best performances in terms of flow rate control please refer to the “Flow control tuning” document.

SYSTEM PURGING PROCEDURE

It is recommended to purge the tubing of air while the chip remains disconnected.

• Secure all inlets and outlets of the cross flow membrane microfluidic channel with Mini Luer connectors.
• Set the OB1 in pressure control mode, enter a relatively high pressure value e.g 200 mbar, and start to pressurize the inlet reservoir, once a droplet appears at the tip of the tubing plug it into the MiniLuer connector. Proceed similarly for the second inlet tubing. Once a droplet appears at the channel outlets connect the outlet tubings and place the open end of the tubing in their respective reservoirs (25 ml falcon tube). For the Chamber B line, first purge reservoir B2 (tracer) until a droplet appears at the end of the tubing, switch to reservoir B1, once the droplet appears at the end of the tubing connect to the chip as described above.
• Switch the inlet reservoirs to sensor control mode and set the desired flow rate, e.g 30 µl/min. Adjust the flow control parameters (PID) in ESI to fine tune the sensitivity and responsivity of the flow feedback loop. For more details on this topic please refer to the “User guide MFS flow sensor” document.

MOLECULAR TRANSPORT EXPERIMENT

The principle of the experiment is first to saturate the tubing and the chip with a blank solution, e.g water or culture medium if using cells, then allow a tracer or chemical (compound of interest) to flow in Chamber B and collect samples at regular intervals at the outlet of Chamber A. The collected samples can then be analyzed for molecular transport across the membrane, e.g using a spectrophotometer. The results below show the arrival time of the tracer (a coloured dye) and the steady state concentration in Chamber A. Protocol:

• Prepare 20 x 1.5 ml Eppendorf test tubes, labeled and aligned on a rack as well as a timer within easy reach.
• Define a sampling sequence, e.g for an experimental flow rate of 36 µl/min and tubing length as described in Fig.1, take the first sample after 5 min, the second after 10 min and then one every min. The total experiment duration is 28 min.
• Once the flow value reading is stable in both chambers, e.g at 30 µl/min, switch the valve to allow the dye to flow in Chamber B, place the end of Chamber A outlet tubing in the first Eppendorf tube and start the timer. After 5 min, place the tubing in the second Eppendorf test tube and so on until the end of the sampling sequence
• Stop the flow.
• Using a micropipette add 400 µl of filtered water to each sample to have enough volume for the spectrophotometer measurement.
• Scan each sample through the spectrophotometer and build the tracer breakthrough curve

CHIP SEEDING WITH CELLS

For applications involving cells, the chip needs to be seeded first. Here is a standard protocol to perform cell seeding in the cross flow membrane microfluidic chip. We recommend to prefill the chip with medium to condition the chip and prevent air bubble formation when seeding the cells.

Trypsinize cells from culture flask. Spin down and reconstitute to remove trypsin. Count carefully and prepare cell suspension with cell concentration optimized for your experimental setting. Fill the upper chamber A with cell type A by gently pipetting and fill the lower chamber B with medium. Place the chip in the CO₂ incubator for 6 to 24h for the cells to attach under static conditions (incubation time depends on attachment time of different cell types). To prevent the channels from drying out, close all ports with Mini Luer Plugs. Then fill chamber B with cell type B, plug all inlets tightly and turn the chip over. Leave the chip in the CO2 incubator as above for the cells to attach to the membrane. Once the cells are well attached, follow the system purging procedure and connect the chip to flow system. The cross flow membrane chip seeded with cells will allow for example to monitor how cells impact the transport across the membrane or built an organ on chip experimental set-up.

Breakthrough curve in the cross flow membrane chip from spectrophotometer measurement

Figure 4: Absorbance measured at the outlet of chamber A, with tracer arrival time around 10 min and the steady state plateau.

The graph shows molecular transport measured as the absorbance of samples collected at the outlet of the chamber A. We observe the first arrival time after 10 min and a plateau is reached after ~18 min indicating that the concentration is equilibrated between the two chambers. This gives an indication on the dynamics of the whole microfluidic system in terms of solute transport. A proper and specific (depending on the type of tracer used) calibration is required to obtain concentration values.

Acknowledgements

This work was done with the support from the European Union under H2020-LC-GD-2020-3, grant agreement No. 101036702 (LIFESAVER).

Application note written by Mayumi HAMADA