Hey Guys,
Im working on the HUVEC cells for the first time. Usually, we grow them using on 0.1%, gelatin coated dish and Bullet Kit medium from Lonza. Can you please give some tips for the better culturing of these cells.
Thanks,
Hello Smith,
To have good HUVEC monolayer a critical component is ECM. Coating cell culture dishes (both flasks for expansion and/or experimental dishes/plates/wells) with 0.1% gelatin is fine. If you can afford, fibronectin works better than gelatin. Also, despite which cell culture medium you will use, make sure that ascorbic acid (vitamin C) is included (prolongs expression of eNOS, delays cell senescence etc). For reliable results do not use HUVEC over passage 5-6. They may look nice, but functionally (particularly with respect to permeability) they are very different from passage 1-3. Hope it will help with your experiments.
HI Smith
in my experiment
I used to isolate MSC from wharton jelley .
But basic principles are same , you will got Umbical cord after placental separation under aseptic condition.
In sterile container with PBS added to it 2% pen. strept.
in Laminar flow you will dissect the umbilical vessels from the Whartobn jelley and Amniotic memvbranes., and cutt them to samll pieces.
then washing 3 times with PBS or complete medium with antibiotic.
either you will cutrure these small fragments directly in Your plate or you would digest them using collagenase and then , filter the dislodged cells by cell strainer , then wash and cuture your pellet.
Hello Smith,
To have good HUVEC monolayer a critical component is ECM. Coating cell culture dishes (both flasks for expansion and/or experimental dishes/plates/wells) with 0.1% gelatin is fine. If you can afford, fibronectin works better than gelatin. Also, despite which cell culture medium you will use, make sure that ascorbic acid (vitamin C) is included (prolongs expression of eNOS, delays cell senescence etc). For reliable results do not use HUVEC over passage 5-6. They may look nice, but functionally (particularly with respect to permeability) they are very different from passage 1-3. Hope it will help with your experiments.
I find keeping the cell density a bit higher helps their growth. I seed 1x10^6 in a T175 and grow for 1 week with media replacement every 2-3 days. I would retrieve maybe 11-12x10^6. They do slown down by passage 7-8.
Hello Gediminas,
We got some fibronectin. How much I have to use for T-25 flask ?
Hi Smith,
We use fibronectin (FN) at concentration of 3ug/ml in PBS. Use minimal volume of FN solution to coat any cell culture dish (i.e. just rinse the surface of the flask). You can also "reuse" FN solution for a couple of times. Leave FN-coated cell culture dishes for 2+ hrs in the cell culture hood before seeding the cells. Best of luck!
Update: Thank you guys for your help. Lonza people told me that they will take more time to ship my medium. I tried R&D systems endothelial cell growth media and its going great with HUVEC cells.
Coating is not necessary. Maybe you can follow a protocol, such as published here:
Circ Res. 2009;104(5):589-99
or in:
Biol Chem. 2008;389(10):1333-8
Media were purchased by PromoCell, but other brands should also work (e.g. LONZA).
Standard T175 flasks (from Saarstedt) were used (Falcon, TPP etc. should also work), no coating was done!
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