Feb 11, 2026
hCMEC/D3 growth, subculture and seeding on device
This protocol is a draft, published without a DOI.
- yunran Feng1
- 1Duke University
Protocol Citation: yunran Feng 2026. hCMEC/D3 growth, subculture and seeding on device. protocols.io https://protocols.io/view/hcmec-d3-growth-subculture-and-seeding-on-device-hrk7b54zp
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 11, 2026
Last Modified: February 11, 2026
Protocol Integer ID: 243071
Keywords: device hcmec, d3 growth, hcmec
Abstract
hCMEC/D3 growth, subculture and seeding on device
Guidelines
**hCMEC/D3 Coating on Device**
Coat the top chambers with 25 µg/cm2 of collagen I and 5 µg/cm2 of human fibronectin mixed in phosphate-buffered saline (PBS). Let it sit for 1–2 h at 37 °C or overnight at 4 °C.
*consider the top chamber as 1cm2 surface area instead of 0.3 cm2 because the wall of the top chamber also contact with the coating solution.
**hCMEC/D3 Seeding on Device**
1. Rinse the device top and bottom channels with warm assay medium and keep them in the incubator.
2. Follow the same protocol as the Subculture.
3. Resuspend cells in 1ml.
4. Count the cell concentration.
5. Calculate the cells needed for all the devices. 40000-60000 cells/ cm2 (I personally keep it around 45000), 0.3cm2 per device.
6. Dilute to 1.5x10^5^ cells/ml and seed 100 ul per device.
Materials
Culture medium: EBM-2 media with EGM-2 BulletKit (Lonza, Cat Number: CC-3162) (composed of CC-3156 CC-4176)
**hCMEC/D3 growth medium**
- EBM-2**
- 500 ml
- hEGF 125 µl
- IGF 125 µl
- VEGF 125 µl
- hFGF 500 µl
- Hydrocortisone 200 µl
- Ascorbic Acid 500 µl
- Gentamycin (GA) 500 µl
- FBS (2.5%) 12.5 ml
- 100 ml
- hEGF 25 µl
- IGF 25 µl
- VEGF 25 µl
- hFGF 100 µl
- Hydrocortisone 40 µl
- Ascorbic Acid 100 µl
- Gentamycin (GA) 100 µl
- FBS (2.5%) 2.5 ml
**Assay medium**
- EBM-2**
- 500 ml
- hFGF 2 ml
- Hydrocortisone 200 µl
- Gentamycin (GA) 500 µl
- FBS (2%) 10 ml
- 100 ml
- hFGF 400 µl
- Hydrocortisone 40 µl
- Gentamycin (GA) 100 µl
- FBS (2%) 2 ml
**Normal Culture Collagen coating**
- COLLAGEN, TYPE I SOLUTION FROM FROM RAT (from Sigma; Cat Number: C3867-1VL)
- Collagen working solution: ~100 µg/ml Type I Collagen in PBS
3 ml / T25 flask, 9 ml/ T75 flask, 500 µl/12-well plate, 200 µl/Transwell insert of 12-well format etc.
**Additional materials for subculture and cryopreservation**
- DPBS**
- Trypsin (3-5 ml of Accumax™ or trypsin-EDTA solution/ TrypLE)
- Cryotubes**
- DMSO**
- Cold FBS**
Troubleshooting
Safety warnings
There is also Heparin in the BulletKit, but we DO NOT use it.
Before start
- 1-2 hr @ 37 °C (tissue culture incubator/ dry incubator next to high content computer- in this case cover all dish with parafilm) or 2hr @ RT or Overnight @ 4 °C
- Wash coating two times with 1x PBS and keep all culture ware in PBS until using it (3^^rd PBS)
- No drying is necessary. In case it is required, use sterile and filtered MilliQ water to avoid salt crystal formation
hCMEC/D3 growth medium
EBM-2 media with EGM-2 BulletKit (Lonza, Cat Number: CC-3162) (composed of CC-3156 & CC-4176)
Abbreviations:
EGM Endothelial Growth Medium
EBM-2 Endothelial Basal Medium-2
FBS fetal bovine serum
HC Hydrocortisone
rhFGF-B heparin-binding growth factor 2, basic fibroblast growth factor
VEGF vascular endothelial growth factor
R3-IGF-1 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)
AA absorbic acid
rhEGF humanepidermal growth factor
GA-1000 Gentamicin sulfate - Amphotericin B
There is also Heparin in the BulletKit,but we DO NOT use it.
hCMEC/D3 growth medium
EBM-2 500ml 100ml
hEGF 125 μl 25 µl
IGF 125μl 25 µl
VEGF 125 μl 25 µl
hFGF 500 μl 100 µl
Hydrocortisone 200 μl 40 µl
Ascorbic Acid 500 μl 100 µl
Gentamycin (GA) 500 μl 100 µl
FBS (2.5%) 12.5 ml 2.5 ml
Assay medium
EBM-2 500ml 100ml
hFGF 2 ml 400 µl
Hydrocortisone 200 μl 40 µl
Gentamycin (GA) 500μl 100µl
FBS (2%) 10 ml 2 ml
Collagen Coating for culture in cell culture flasks
1. Prepare a collagen working solution of ~100 µg/ml Type I Collagen in PBS.
2. Incubate the solution for 1-2 hr at 37 °C or overnight at 4 °C.
3. Wash the coating two times with 1x PBS and keep all culture ware in PBS until using it (3rd PBS).
Note: No drying is necessary. If required, use sterile and filtered MilliQ water to avoid salt crystal formation.
hCMEC/D3 recovery from liquid nitrogen
1. Have the pre-warmed growth media (37°C) and coated flasks ready.
2. Place the frozen vial in a 37°C water bath. Hold and rotate the vial gently until the contents 80% thaw.
3. Promptly remove the vial from the water bath, wipe it down with 70% ethanol, and transfer it to the sterile field.
4. Transfer cells to a sterile 15 mL conical tube (do not introduce any bubbles during the transfer process) and slowly add dropwise add 3 ml (9 for Millipore vial) growth medium.
IMPORTANT: Do not add the whole volume of media at once to the cells. This may result in decreased cell viability due to osmotic shock.
5. Pipet up and down twice slowly to mix.
6. Centrifuge pellet (to remove DMSO) before seeding the flask (3 min 300xg). Remove supernatant and resuspend in 4 ml (10ml for Millipore vial) media.
7. Plate cells in a collagen-coated T25 flask. When thawing the vial purchased, follow company instructions for this step (add vial to collagen-coated T75 flask/ 10 cm dish or 3 T25 flasks, if use 3 T25, add the volume to 12 and distribute 4 ml each flask).
8. Refresh growth medium after 2-4h to remove residual DMSO and unattached cells.
9. Change the medium every other day, and grow cells to be ~90% confluent and passage (take 4-5 days, depending on the defrosted batch).
hCMEC/D3 maintenance culture
Change media every 2 days, until the cell reaches 70%, then change media every day.
hCMEC/D3 Subculture
Note: Amounts are for cells culturing in a T25 flask. For T75 flasks, the volume is generally 3 times that for T25 because of the surface area.
1. Have the pre-warmed complete media (37°C), DPBS (37°C), TrypLE (We use TrypLE ad it's fine, from previous protocols, Accumax™ and trypsin-EDTA solution are also fine) (37°C), 15 ml conical tube and coated flasks ready.
2. Remove old medium from the flask and wash the cells with 3 ml of pre-warmed DPBS.
3. Trypsinize cells with 1 ml of TrypLE for 3 min (incubator: 37°C, 5 % CO2/95 % air and saturated humidity).
4. Check under the microscope. Once cells are completely rounded up, transfer the solution from the flask to the 15 ml centrifuge tube that has 0.5ml of media in it.
5. Continue to incubate the flask at 37°C for another minute (no solution in the flask at this time, no more than one minute in case the residual gets dried).
6. Add total 3.5 ml of growth medium to stop the trypsin and wash down all the cells from the wall by gently pipetting up and down.
7. Collect cell suspension in the 15 ml conical tube.
8. Pellet cells at 300 x g for 5 min at RT.
9. Re-suspend cells in 1 ml growth medium (or in assay medium in case the cells are for an experiment.)
10. Count the cells with the hemocytometer (1:2 dilution before adding Trypan blue).
11. Seed at 10000 cells/cm2, and the cell will be ready in 4-5 days.
12. Keep the cells in the incubator (37 °C, 5 % CO2/95 % air and saturated humidity).
hCMEC/D3 Cryopreserve
1. Prepare ice bucket, cryotubes, DMSO, cold FBS, and all reagent need for subculturing the cells.
2. Rinse the cells with DPBS.
3. Add 3 ml of TrypLE to cells in a T75 flask or a 10cm petri dish (1ml for T25 flask) for 3 min. Move the cell back into the incubator.
4. Add 3ml of media to a 15 ml centrifuge tube.
5. Check under the microscope. Once cells are completely rounded up, transfer the solution from the flask to the 15 ml centrifuge tube with media and continue to incubate the flask at 37°C for another minute (no solution in the flask at this time, no more than one minute in case the residual gets dried).
6. Gently tap the side of the flask to dislodge cells from the surface.
7. Check under a microscope to make sure that all cells detach.
8. Add 5 ml of growth media to the flask and transfer detached cells to the centrifuge tube. Rinse the flask with another 4 ml of growth media to collect the residual cells.
9. Examine the flask under a microscope for a successful cell harvest by looking at the number of cells being left behind; there should be fewer than 5%.
10. Top off to 15ml.
11. Pellet the cells at 300xg for 5 minutes.
12. Gently resuspend cells in 1ml growth medium.
13. Count (1:5 dilution before adding Trypan blue).
14. Mix the cell suspension, cold media, cold FBS, and DMSO together. The aim is to freeze down no less than 3x10^5^ cells per vial for future use in T25 flasks, in a concentration no less than 0.5x10^6^ cells/ml. The final freezing should contain 10%DMSO, 40% FBS. Add DMSO dropwise to the media first. Then add FBS, and then add the cell suspension.
15. Aliquot the cell suspension as needed.
16. Freeze in a slow freeze cryopreservation box in -80 and transfer to liquid nitrogen after 24h.
hCMEC/D3 Coating on Device
Coat the top chambers with 25 µg/cm2 of collagen I and 5 µg/cm2 of human fibronectin mixed in phosphate-buffered saline (PBS). Let it sit for 1–2 h at 37 °C or overnight at 4 °C.
Note: At first, I did the calculation based on 0.3 cm2 surface area and so I used 92.5ug/ml collagen I and 18.5 ug/ml fibronectin. However, I found the previous paper by Hudzec, and in that paper, they used 50ug/ml fibronectin, and their cells have a better expression. I think it's because the surface area is considered as 1cm2 surface area instead of 0.3 cm2 because the wall of the top chamber also contact with the coating solution.
hCMEC/D3 Seeding on Device
1. Rinse the device top and bottom channels with warm assay medium and keep them in the incubator.
2. Follow the same protocol as the Subculture.
3. Resuspend the cells in 1ml.
4. Count the cell.
5. Calculate the cells needed for all the devices. 40,000-60,000 cells/ cm2 (I keep it around 50,000), 0.3cm2 per device.
6. Dilute to 1.5x105 cells/ml (for 50,000 cells/ cm2)and seed 100 ul per device.