Hi I've been trying to find a way to recover cells from matrigel for further culturing. So far the protocols I found are mainly for qPCR and use the cell recovery solution which needs to be kept at 4C for 1 hr to resolve the matrigel. However, I am trying to recover the cells and keep culturing them, so putting them at 4C for 1 hr would damage the cells. Is there a better way to do this? Can I just mix the matrigel with culture medium and pipet up and down to "dissolve" the matrigel? Should I then centrifuge the mixture and will the cells be at the bottom of the tube? Thanks!
Hi Shirley, I have used ice cold media to "dissolve" small patties of matrigel 5-10ul but it is not entirely effective. The most proper way would be to use Corning's "Cell Recovery Solution" formerly called "Matrisperse" when BD was marketing it.
http://catalog2.corning.com/Lifesciences/en-US/Shopping/ProductDetails.aspx?productid=354253%28Lifesciences%29&categoryname=
Thanks, Richard! So that cell recovery solution needs to be used at 2C - 8C (typically at 4C), and I am afraid this temperature would harm the cells. That's why I was hoping to find other ways to dissolve the matrigel...
Hey Shirley,
How are you growing your cells? Is it a 3D system or a monolayer? We grow cardiomyocytes in monolayers on matrigel and often harvest and re-plate for further culturing with high efficiency.
We dissociate with Collagenase II (Worthington) at 37 C for 20 min - 1 hour and gently scrape the cells. Wash with DMEM and spin down. Then we resuspend the pellet in Tryple E for about 5 minutes in the 37 C water bath with light vortexing. Then we wash and spin again in DMEM + DNase. Finally we resuspend in culture media + THIA, filter through cell strainer cap tubes and re-plate on fresh matrigel at the desired density.
The cells never have to be below room temp so there definitely no issue of them being damaged by the cold. The only real damage would come from the Tryple E, but you just need to watch for good dissociation and don't let it go to long. We do this all the time and always have a really high survival rate.
Hope that helps!
Hi Francis, thanks a lot for the answer! I am actually mixing the cells with the matrigel, so 3D culture it is. I will try collagenase then. Can I use trypsin instead of Tryple E?
Trypsin does the same job, but TrypLE is much more gentle and doesn't require inactivation. I can't remember off the top of my head why exactly it's more gentle, but the cells just tend to be happier with TrypLE than with trypsin.
I use dispase solution (BD Biosciences) 1U/ml or 1mg/ml final concentration in culture medium . Aspirate cell culture medium and add dispase solution, incubate plates for 1hour at 37C incubator. Collect everything (gently dissociated Matrigel with gently dissociated organoids, if this is what you culture) into conical tube, spin down, resuspend cells pellet in trypsin/EDTA, incubate 2-5min at 37C, wash cells with culture medium and cells are ready to be plated back to Matrigel again.
Have you ever frozen any cells? If you have, then you should understand that virtually any type of cell can tolerate 4C for an hour just fine.
Accutase or TrypLE are more gentle than trypsin /EDTA and keep surface cadherins intact.
BME? I am assuming, or hoping, that this is not Beta Mercaptoethanol? A 33% solution would seem to be a bad idea.
I still stand by my comment from 2 years ago. Virtually any type of cell can tolerate an hour at 4C, which is sufficient to dissolve matrigel. I would like to know what sort of "damage" can happen to cells in an hour at 4C.
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