Hi all! 2 weeks ago I started culturing a vial of frozen HCMEC/D3 cells from Merck. Even when the process has been long and full of uncertainty, the cells are alive, proliferating and somehow attached to the collagen coating in the flask. NEVERTHELESS they are still in a round shape and not in the characteristic spindle shape... I am not sure what to do or what is going wrong. I have read some people here having the same problem but nobody has provided an answer so far...
Important things to know:
They were thawed from the commercial vial from MERCK following the instructions
They are growing in ENdoGRO-MV medium supplemented with FBS: normally is prepared with 5% FBS BUT I put 10% of FBS after 3 or 4 days of thawing, and have kept them like this. I can see this helps them to proliferate.
I add to the flask FGF-2 growth factor (1ng/mL)
Can someone with more experience with this cells help me? I am getting out of ideas :(
Hello Paulina Fuentes-Zacarías
I have not worked with HCMEC/D3 cells but can always help you.
Some points you need to consider.
1. Cell’s microenvironment has been shown to exert influence on cell behavior. Matrix-cell interactions strongly impact cell morphology and function. So, coating the flask with collagen (type I or IV) would be important. I have described the protocol below.
Thaw Collagen Type I, Rat Tail at room temperature. Dilute 1mL of Collagen Type I with 19mL 1X PBS. Mix gently. Scale up according to the volumes required. Coat flask with 1:20 diluted Collagen Type I solution. Use 5-10mL for T75 flasks. Incubate in 37°C incubator for at least one hour before use. These cells show spindle-shaped, elongated morphology and exhibit contact inhibition at confluence when cultured on collagen type I or IV.
2. I hope you are using cells of low passage. HCMEC/D3 cells maintain their characteristics until passage 33-36.
3. The growth media besides being supplemented with 5% FBS and FGF-2 growth factor (1ng/mL), must contain hydrocortisone (1.4µM) and ascorbic Acid (5µg/mL).
Hope these suggestions help!
Best.
I am facing the same problem with this cell line from Merck. Did you figure out what is the suitable conditions for growing these cells ??
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