HUVEC Culturing Protocol
- 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).
- 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.
- Add 100 μL of 5 µg/cm^2 fibronectin to top well. Please refer to ALine Modular Device Geometry for surface area. Be careful to avoid bubble formation over the trench for trench-up devices. Fibronectin Addition
- Let the fibronectin adhere to the membrane for 1 hr at RT (Leave lid off the petri dish to allow EtOH evaporation).
- Remove fibronectin, rinse with fresh cell culture media.
- Add media to the bottom channel. For V1 devices, pipet 20 μl to flush without adding bubbles. Channel capacity is ~10 µl. For V2 devices, pipet 30 μl into channel. Please refer to ALine Modular Device Geometry for a full list of device surface areas and volumes.
- We prefer to pipet holding the device at an angle, pipetting into the bottom port, as shown in image and video link below, to help avoid bubble formation in the bottom channel.
- Be sure there is no air at the end of pipet tip 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, 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
Pipetting media into bottom channel.
- Seed cells at 40,000 cells/cm^2. Please note, available cell seeding area is 37 mm^2 (see ALine Modular Device Geometry) and volume is 100 μl. This leads to a seeding density of ~150,000 cells/ml. Cell Addition
- Cover with lid and incubate at 37°C, 5% CO2 for 2 hrs.
Final device set up.
- After 2 hours, exchange media in the top well and bottom channel.
- Cells are usually ready for assays after 24-48 hours of growth in devices.
- For optimal phase imaging, add media to form an excess layer on the top of the device. Carefully drop a coverslip atop the media to form a flat interface.
HUVECs grown for 48 hours in modular devices. Cells were imaged via brightfield microscopy (using a glass coverslip) and a LIVE/DEAD stain was performed, indicating minimal cell death (green = live, red = dead).