Protocol: Microfluidic Device (Shear and Viability) Protocol

Preparing Raw Devices for Experiments

Inserting Tubing

• Devices arrive with PDMS bonded to glass and ports cored out of the PDMS at either end of the channel.
• For each channel cut two lengths of tubing (BD [company] – Intramedic PE Tubing [PE190] – ID: 1.19mm, OD: 1.70mm) at 15 cm and 75 cm. These will act as the output and input tubing respectively.
• Cut one end of each length of tubing at an oblique angle (30-45°) and insert this end of the tubing into the respective port on the PDMS device. The tubing is cut on an oblique angle to prevent a flat seal with the glass bottom of the channel.

Bonding Tubing with Epoxy

• To bond all the tubing to the device use LePage Speed Set Epoxy™.  Product works best when mixed by hand rather than with twinject system. Eject 3-4mL of product on to some aluminum foil and mix with a craft stick (tongue depressor) before transferring to the barrel of a 10mL syringe. Attach a pipette tip to the end of the syringe and run a small bead of epoxy around all the ports at each end. The epoxy expands as it dries so apply accordingly.
• Let epoxy cures for 2 hours.

Adding Syringe Ports

• Fluids are injected into the ports by syringe. To make changing fluids simple, add an 18 GA 1” needle with a blunted tip into each of the free tubing ends.

Using Microfluidic Devices for Experiments

Cleaning and Wetting Devices

• Prepare the following syringes for cleaning one channel:
  • 1 mL of 100% ethanol in a 1 mL syringe
  • 1 mL of 70% ethanol in a 1 mL syringe
  • 30 mL of D-PBS in a 50 mL syringe
• Inject 100% ethanol through IN-port to wet all surfaces of the device and remove air bubbles. Let stand 1 minute.
• Connect 70% ethanol syringe to OUT-port without introducing any air bubbles (See Attaching Syringes w/o bubbles), remove 100% ethanol syringe and inject 70% ethanol through OUT-port. Let stand 10 minutes.
• Connect D-PBS syringe to IN-port, remove 70% ethanol syringe and flush channel with ~ 15 mL of D-PBS.

Coating Microfluidic Channels with ECM Protein

• Prepare 300 μL of an appropriate concentration of matrix protein (20 μg/mL for Fibronectin) in a 1 mL syringe.
• Connect coating syringe to OUT-port, remove D-PBS syringe and inject 250 μL of solution in to the channel. Allow coating to adhere for 1 hour at room temperature.
• Connect D-PBS syringe to IN-port, remove coating syringe and flush channel with ~10 mL of D-PBS. (Fibronectin coating must remain wet so do not allow channel to dry out before injecting cells)

Growing Cells in Microfluidic Channels

• Prepare the following syringes for seeding cells in one channel:
  • 500 μL of cell suspension with approximately 10⁶ cells/mL in a 1 mL syringe
  • 1 mL of D-PBS in a 1 mL syringe
  • Necessary volume of complete medium for experiment in an appropriate syringe
• Connect 1 mL D-PBS syringe to OUT-port, remove 50 mL D-PBS syringe.
• Connect complete medium syringe to IN-port, remove D-PBS syringe and flush with 5 mL of medium to prepare channel for cells.
• Connect cell suspension syringe to OUT-port, remove medium syringe and inject 400 μL of cell suspension in to the channel.
• Reconnect complete medium syringe to IN-port , remove cell suspension syringe and transfer the microfluidic device to the incubator.
• Place OUT-port in a waste beaker within the incubator, and tape down the device so it remains flat.
• Run the IN-port tubing out of the incubator and secure it in place on the syringe pump (be careful not to move the plunger on the syringe).
• Give the cells 2-6 hours to adhere to the channel.
• For long-term incubation set the flow rate to 1 μL/min in order to replenish medium without applying significant shear to cells.

Shearing and Staining Cells

• Use the formula below to calculate the flow rate required to achieve the necessary shear stress:

τ_w = 6 μ Q / b h²

Where:

τ_w = Wall Shear Stress (Pa)

μ = Fluid Viscosity (Pa*s)

Q = Flow rate (L s^-1)

b = Chamber Width (m)

h = Chamber Height (m)

• Prepare 1 mL of DAPI/FDA stain according to protocol in 1 mL syringe.
• Pause syringe pump, program appropriate shear rate.
• Run pump for predetermined time depending on experiment (be sure to leave some media in the syringe).
• Connect stain syringe to OUT-port, remove medium syringe and inject stain. Let stand for 3 minutes.
• Reconnect medium syringe to IN-port, remove stain syringe and flush with 1 mL of media gently to remove excess fluorescent dyes.
• Image cells on microscope.

Attaching Syringes w/o Bubbles

• It is important that air bubbles are not introduced into the microfluidic channels at any time since they are very destructive to cells.
• The most likely time for introducing bubbles is during the attachment of a syringe to any port on the device.
• In order to reduce the likelihood of introducing bubbles it is important to maintain at least one syringe on either port of the device at all times.
• When attaching a syringe it is best to use a modified form of the ‘droplet merging’ method (http://www.rsc.org/publishing/journals/lc/bubble_injection.asp)

Droplet Merging

• Use the OLD syringe to push fluid through the device so that a bead of fluid is generated at the opposite port.
• Generate a droplet of fluid at the tip of the NEW syringe by applying light pressure to the plunger.
• Carefully merge these two droplets and secure the syringe to the port.
• Detach OLD syringe and inject fluid from NEW syringe. If done properly, no bubbles will be introduced.