ALine Modular Device Cell Culturing: Bottom Channel Culturing

Protocol for Culturing Cells in the Bottom Channel

  1. Assemble device in hood (see ALine Modular Device Assembly), place in sterilized petri dish, and let sit under UV light for 20 minutes (Leave lid off the petri dish to allow sufficient UV exposure).
  2. Roll a Kimwipe and generously wet with DI water, spray with ethanol to sterilize or use sterile water, and place around the device(s) inside the petri dish for humidity control. If you spray with EtOH, be sure to let EtOH evaporate before adding cells.
  3. Add 20 μL of coating solution to the bottom channel of the device. Please refer to ALine Modular Device Geometry for surface area. Use the full surface area, not including ports. For collagen type 1, coat at 25 µg/cm^2 and for fibronectin, coat at 5 µg/cm^2. Be careful to avoid bubble formation.
    • We prefer to pipet from the bottom up, as shown in image below and video link below, to help avoid bubble formation in the bottom channel. This is most useful for trench-down devices.
    • Only push to the pipet’s first resistance to avoid adding air to the channel.
    • If a bubble does form in the channel, hold the device at an angle and pipet 100 µl through the channel more aggressively to push it out. Pipetting too aggressively can burst the membrane, however. Finding the right amount of force will come with experience.
    • Bottom Channel Pipetting (Version 2)

      Pipetting into bottom channel.

  4. Let the coating solution adhere for 1 hr. Keep device facing up (normal orientation).
  5. Remove coating solution, rinse twice with 20 µl fresh cell culture media.
  6. Add 80 µl media to the top well, just enough to coat the surface. The cells need this to adhere.
  7. Seed cells at 40,000 cells/cm^2 into bottom channel. Please refer to ALine Modular Device Geometry for surface area and channel volume. Use the top surface area only. Note the effective area is less than for the coating solution. While the coating solution coats all surfaces, the cells settle on the bottom surface only.
    • For culturing cells on the top surface of the channel, flip the device over. We set the device upside-down in a slide holder (see image below). This raises the device off the petri dish and allows gas exchange while cells adhere to the membrane.
  8. Cover with lid and incubate at 37°C, 5% CO2 for two hours.
  9. After 2 hours, flip device back to its normal orientation. Exchange media in the top well and bottom channel. The top well can be filled to 100 µl.