hCMEC/D3 Culturing Protocol
Culture medium: EGM-2 BulletKit (Lonza, Cat Number: CC-3162)
- EGM: Endothelial Growth Medium
- EBM-2: Endothelial Basal Medium-2
- hEGF: human epidermal growth factor
- IGF: insulin-like growth factor-1 (recombinant analog of insulin-like growth factor containing the complete human IGF-I amino acid sequence with substitution of Arg for Glu3)
- VEGF: vascular endothelial growth factor
- hFGF: heparin-binding growth factor 2, basic fibroblast growth factor
- HC: hydrocortisone
- AA: ascorbic acid
- GA: gentamicin sulfate – amphotericin B
- FBS: fetal bovine serum
There is also Heparin in the BulletKit, but we DO NOT use it.
Growth medium (EGM-2):
Use it for routine cell culturing.
- EBM-2: 100 ml
- hEGF: 25 µl
- IGF: 25 µl
- VEGF: 25 µl
- hFGF: 100 µl
- HC: 40 µl
- AA: 100 µl
- GA: 100 µl
- FBS (2.5%): 2.5 ml
Collagen coating: All cell culture ware must be coated with collagen prior to cell seeding. We use Collagen, Type 1 solution from rat (Sigma, Cat Number: C3867-1VL). The vial comes as 4-5 mg/ml. Our working solution is ~100 µg/ml Type I Collagen in PBS (can be kept in the fridge for 2 weeks). Coat flasks for 1-2 hr @ 37 °C (tissue culture incubator).
Use it for experiments/growing cells for experiments.
- EBM-2: 100 ml
- hFGF: 400 µl
- HC: 40 µl
- GA: 100 µl
- FBS (2%): 2 ml
Collagen/Fibronectin coating: Due to properties of NPN membranes, devices must be coated with a collagen/fibronectin mix. If there are concerns with consistency, we recommend using this mix for Transwells and other assays as well. We use Collagen, Type 1 solution from rat (Sigma, Cat Number: C3867-1VL) and Fibronectin, Human (Corning, Cat Number: 354008 or VWR, Cat Number: 47743-728). We resuspend our stock solution of fibronectin to 1 mg/ml and aliquot. Our working solution is ~25 µg/cm^2 Type I Collagen + 5 µg/cm^2 Fibronectin in PBS. Coat devices/Transwells for 1-2 hr @ 37 °C (tissue culture incubator). The device protocol is detailed below.
- 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 ~25 µg/cm^2 collagen type 1/5 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. Collagen/Fibronectin Addition
- Let the collagen type 1/fibronectin adhere to the membrane for 1 hr in incubator.
- Remove collagen type 1/fibronectin, rinse with fresh cell culture media. We use assay media, which is growth factor depleted, compared to the growth media used for maintaining cells.
- 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
- 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.
- After 2 hours, exchange media in the top well and bottom channel.
- Cells are usually ready for assays after 4 days to 1 week of growth in devices. 10 mM lithium chloride can be added when cells are at ~70-80% confluence to tighten barrier properties.
- 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.