µSiM Cell Culturing: hCMEC/D3

hCMEC/D3 Culturing Protocol

Culture medium: EGM-2 BulletKit (Lonza, Cat Number: CC-3162)
Abbrevations:

1. EGM: Endothelial Growth Medium
2. EBM-2: Endothelial Basal Medium-2
3. hEGF: human epidermal growth factor
4. 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)
5. VEGF: vascular endothelial growth factor
6. hFGF: heparin-binding growth factor 2, basic fibroblast growth factor
7. HC: hydrocortisone
8. AA: ascorbic acid
9. GA: gentamicin sulfate – amphotericin B
10. 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.

1. EBM-2: 100 ml
2. hEGF: 25 µl
3. IGF: 25 µl
4. VEGF: 25 µl
5. hFGF: 100 µl
6. HC: 40 µl
7. AA: 100 µl
8. GA: 100 µl
9. 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).

Assay medium:
Use it for experiments/growing cells for experiments. Cells are added into devices in assay medium.

1. EBM-2: 100 ml
2. hFGF: 400 µl
3. HC: 40 µl
4. GA: 100 µl
5. FBS (2%): 2 ml

Collagen/Fibronectin coating: Due to properties of our silicon nitride 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.

Device Culturing

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. Wash membrane twice with 100 µl sterile water.
5. Add 100 μL of 25 µg/cm^2 collagen type 1 + 5 µg/cm^2 human fibronectin in PBS to top well. Please refer to Device Geometry page for surface area. Be careful to avoid bubble formation over the trench for trench-up devices.
   
   • If a bubble forms over the trench, you should see a bend in the meniscus as shown in video links
   • Meniscus, No Bubble
   • Meniscus, With Bubble
     

     Pipetting coating solution into top well.
6. Let the collagen type 1/fibronectin adhere to the membrane for 1 hr in incubator.
7. 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.
8. 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 Device Geometry page 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 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.

9. Seed cells at 40,000 cells/cm^2. Please note, available cell seeding area is 37 mm^2 (see Device Geometry page) and volume is 100 μl. This leads to a seeding density of ~150,000 cells/ml. Video of Cell Addition Technique
10. Cover with lid and incubate at 37°C, 5% CO2 for 2 hrs.
    

    Left: Final device set up. Right: hCMEC/D3 2 hours post seeding.

11. After 2 hours, exchange media in the top well and bottom channel. Switch media every 2-3 days thereafter.
12. In my experience, cells needed about 2 weeks of growth in devices for optimal barrier function. Alternatively, 10 mM lithium chloride can be added when cells are at ~70-80% confluence to tighten barrier properties.
13. 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.

hCMEC/D3 grown for 5 days in modular devices. Cells were imaged via phase contrast microscopy (using a glass coverslip) at 10X (left) and 4X (right) magnification. A LIVE/DEAD stain was performed, indicating minimal cell death (green = live, red = dead).