µSiM Cell Culturing: Bottom Channel Culturing

Protocol for Culturing Cells in the Bottom Channel

1. Assemble device in hood (see Modular Assembly page), 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. Add sterile water to a small Petri dish or 50 ml conical cap to maintain a humidified environment.
3. Place devices in clamps (Cole-Parmer Cat# 06832-10, made of acetal copolymer; or VWR Cat# 89030-208, made of nylon) to avoid handling directly with gloves during cell culture. Video of Technique for Adding Clamp
   1. Due to concerns about autoclaving and UV exposure causing clamps to emitting a volatile substance, we thoroughly ethanol sterilize clamps prior to use.
4. Add 20 μL of coating solution to the bottom channel of the device if cells require a coating. Please refer to Device Geometry page for surface area. Use the full surface area. 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.
   • Note: some solutions may require a precoat of the chip, prior to device assembly. Reach out for details on how to do this.
   • We prefer to pipet holding the device at an angle, pipetting into the bottom port, as shown in this video and image below, to help avoid bubble formation in the bottom channel. You can stabilize the device in the clamp with your finger gently pressed on the back of the device. Be sure not to apply too much pressure, as this will block the channel.
   • Be sure there is no air at the end of pipet tip or in the port prior to inserting into the port. Only push to the pipet’s first resistance to avoid adding air into the channel.
   • If a bubble does form in the channel, see this page for tips to remove.
     

     Pipetting media into bottom channel.

5. Let the coating solution adhere for 1 hr. Keep device facing up (normal orientation).
6. Remove coating solution by rinsing with 20 µl fresh cell culture media.
7. Add 50-80 µl media to the top well, just enough to coat the surface. The cells need this to adhere and survive.
8. Seed cells at 40,000 cells/cm^2 into bottom channel (or at optimized seeding density for your particular cell type). Please refer to Device Geometry page for surface area and channel volume. Use the top or bottom 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.
   • We calculate seeding density using the channel capacity of 10 µl. However, we pipet 20 µl into the channel to avoid bubble introduction.
   • Be sure not to spill into the top well. Rinse well thoroughly if spilling occurs to remove any cells.
   • For culturing cells on the top surface of the channel, flip the device over. We set the device upside-down in their clamps (see this video and image below). This raises the device off the petri dish and allows gas exchange while cells adhere to the membrane.
     
9. Cover with lid and incubate at 37°C, 5% CO2 for two hours.
10. After 2 hours, replace media in the channel. For culturing cells on the top surface of the channel, flip device back to its normal orientation at this point, as the cells should be adhered to the top surface. Rinse the top well and fill with 100 µl of media. For co-culture, cells can be added to the top well at this point or later, depending on growth kinetics.
    • Be sure not to spill into the top well when pipetting into the channel. Rinse well thoroughly if spilling occurs to remove cells.