Hey, all!
I've recently started working with human aortic endothelial cells. I acquired a vial from Lonza, and I feed these cells with their EGM-2 medium which comes with the many growth factors that these cells require. Up to the first 3 passages, everything was going well. However, the 4th and 5th passages look terrible: there are hardly any adherent cells, and there is a lot of dead cell material in there such that the medium looks pale pink and chunky/turbid. I have a few freezer stocks of these cells, but I am reluctant to thaw these until I know what the problem could be.
When I split these cells, I use accutase (instead of EDTA or the harsher trypsin). I haven't changed cell culture flask brands either (Corning T-75s throughout).
I'd appreciate any input/ideas. Thanks!
A.
try growing the cells in MCDB-131 or McCoy's supplemented with 10% FBS instead of the Lonza media. I had a similar problem with an epithelial cell line, and when I tried media and FBS instead of all the growth factors the cells were much happier
Akshat, can you suggest what assays you would use these cells for?
Dr. Jarajapu,
I'm co-culturing HAECs with iNKT cells under different cytokine milieux. It's a minimalist system to evaluate how CD1d-restricted cells may behave in an inflammatory situation like atherosclerosis.
We have had bad luck with keeping multiple passaged-endothelial cells alive as well. They need endothelial growth factor if that is not in the media already...pre-warm all media before passing the cells back into fresh media.
You may have tried all this, but just in case...
Oh, yes, Dr. Gibbs! The medium has VEGF, EGF, insulin-like growth factor, hydrocortisone...I don't even remember what else. Currently, I've gathered the live "happy" cells from the T75 flask, and moved them to a T25. Maybe being in closer proximity to each other might help? Homotypic interactions have generally kept T-cells and NK cells alive and activated, so I'm hoping that maybe that helps here, too!
OK, so you aren't doing any angiogenesis-relevant assays, so you can switch to a simple medium as per Chris's suggestion, which MAY help. I am pretty sure technically you are not doing anything wrong, basing on your extensive experience. I can only suggest trying commonly used adhesion factors for enhancing the attachment.
BTW, what medium you are using for co-culturing?
Nice to see your updated picture.
Are you going to be at EB-meeting in two weeks?
Endothelial cells want to be crowded...and we would pass 5% spent media back to the new cells. I would try maybe seeding more cells after a couple of passages?
Dr. Jarajapu, that sounds like a great idea! The more I read, the more I find that folks use gelatin-coated flasks to keep their HUVECs or HAECs happy. I think I'll try that.
The co-culturing medium is RPMI spiked with 10% BCS and 3% human AB serum. I need to have the latter in there because the iNKT cells are sluggish (or, worse, just die, if there's no human AB serum around!).
And thank you! I rather like that picture, too! :-) I'd love to go to EB, but I think I may end up going to the AAAI meeting instead either this May or the next. I have some interesting data right now, but it's a little all over the place. I'm not sure what this story is going to turn out to be, but I can't wait to find out!
Ooh, Dr. Gibbs! The spent media idea is an excellent one! I remember the good Tom Gustad extolling the virtues of "bathwater media!" Yes! That may really help! Thanks!
... since I am not aware of mechanisms by which endos support iNKTs, the following concern may or may not be applicable for your case!
Endos secrete factors in a different profile depending on their proliferative or functional phenotype. In your test system they are mostly in a proliferative phenotype. for my functional assays I generally derive them of serum/fbs for at least 12 hours in advance... again not sure if its a concern with your assay system or not...
Good luck with your studies...
This is, again, something worth trying in a side-by-side format to see how the responses are skewed with or without serum starvation. The cells are doing better in a smaller flask, but there is still a lot of debris around. I'm going to try gelatin-coated surfaces now. I *will* figure this out if it's the last thing I do!
I coat my dishes with fibronectin (costs a bit more but not too bad) or you can also use 0.1% gelatin (more common) to help cells adhere. Also how thinly are you splitting them? I wouldn't go below 30 or 40% confluence when splitting into new flasks or plates (try 1:3 or if you see a survival problem try 1:2). There WILL be a decrease in viability and doubling time for these cells after passage 5 in my experience, but a 1:2 split usually still works well after this passage. Also isyour media staying fresh? Was it being warmed in a water bath or stored at room temp which deteriorates the quality of EGM2? let it come to room temp in the dark before using. Best of luck
Thanks, Natalie! These are all excellent points. I store the media at 4C, and I did pre-warm it prior to splitting. I'd split the cells when they were around 70-ish% confluent. But, we are past the fifth passage, for sure. I did freeze down some happy cells from the 1st and 2nd split. Do these thaw up well?
Thank you so much!
A.
They should thaw well. In my experience, spinning the thaw down to remove DMSO stresses the cells more than the DMSO itself, so I let the vial thaw fast in water bath, add a few drops of 100% FBS to quench thaw shock, and plate into a high volume of media to dilute the DMSO to a negligible amount (.01% just like you would for a vehicle control in an experiment). I also leave them be for an extra day to attach and get paracrine signaling and change media about 36 hours after thaw instead of the next morning. If there are a lot of dead cells the next morning then change the media though. You can also always call Lonza to ask about the lot of cells and any pointers they could have as well.
I realize my first answer sounds a bit misleading, when i wrote 30-40% I meant the newly plated passage shouldn't be lower than 30-40% for seeding. The original plate of cells you are splitting into a new set of plates should be at LEAST 80% and 90-100% still works well for splitting (like if you forget to split over the weekend). I like 70% density of EC's for performing actual experiments, it suggests they are in a linear phase growth and are not likely to be under any type of contact inhibition quiescence or senescence which could possibly result in a sub par activation of kinase pathways ( if they're metabolically happy enough to be dividing then they will probably have a better response to any stimuli).
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