Hi everyone, I'm currently trying to embed endothelial cells in 3D and have them form capillaries (vasculogenesis). However, the cells often look like they're dead after embedding! Has anyone encountered this issue before?
When I detach my cells for a 3D experiment I put some back into 2D for future elements, and those cells seem fine (maybe some limited amount of dead cells floating)! But in 3D they remain as small balls. I've tried different scaffolds (fibrin, collagen, matrigel etc), I've tried different endothelial media compositions, I try to pipette gently etc, but I've been having no luck with getting blood vessels to form!
I've also been doing experiments with muscle cells and some samples are mysteriously... dead (cells stay rounded), but some are alright!
Suggestions: Use a viability stain such as trypan blue. I am not sure why cells in a 3D culture would express linear rather than spherical forms due to contact inhibition. However, the following website
https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=110533206
did mention nanofiber scaffolds. When I cut and paste link name into a browser (Firefox) window it worked for me.
Michael
many cell types change shape in 3D and its hard to see them because they look different to how you used to see them. Use Calcein AM and propidium iodide to get a readout for cell viability/cell death 24-48h after embedding https://www.thermofisher.com/order/catalog/product/C1430#/C1430 . You can do it in 3D. See our paper where we used similar technique to evaluate cell viability in organoids (also 3D) Article Development of a protocol for maintaining viability while sh...
. Basically, my suggestion is similar to Michael's except that trypan will kill you 3D-embedded cells while the fluorescent reagents will give you immediate readout without destroying your 3D gel. Also, use 10 microMolar ROCK inhibitor when embedding and keep ROCK inhibitor for 1-2 days if needed, to increase viability. Let me know the outcome and also -which cell are you using for embedding, are they HuVECs?
Hi Michael Theobald and Igor Nasonkin,
Thank you for the viability testing advice! I think it's true that I should actually check for cell viability rather than go off of them being balled up. The ROCK inhibitor might be a good idea too!! I've been embedding HUVECs or HMEC-1s with C3H10T1/2 cells :)
Hi,
I don't quite understand your experiment.
Do you seed cells on the scaffold (cross-linked biomaterial) or embedding cells in the Matrigel (like in the case of organoids)?
Justyna
Hello everybody,
Igor Nasonkin what ist the function of the ROCK-Inhibitor? Is the mechanism specific for certain cell types?
I am having similar problems as John Yn but with intestinal smooth muscle cells, which i try to embed in collagen type I (0,8 - 3 mg/mL) or "ECM" at concentrations of 5 - 25 x 10⁵ cells/mL. In 2D on normal plastic they look fine and proliferate, but in the gels they seem to just die.
Jörg
Hi Jörg Panzer ,
ROCK inhibitors are usually added to enable cell survival when seeding pluripotent stem cells in a single cell suspension.
For me what ended up working was switching to a low glucose basal media (I found a lot of literature on high glucose conditions harming HUVEC cell viability), sticking to lower gel concentrations (
ROCk inhibitor(s) are used to increase the survival of many cell types after plating (your goal), in addition to, of course, hESCs, the original application Article A ROCK inhibitor permits survival of dissociated human embry...
. I am aware of +impact on neuroepithelial cells. It is recommended to withdraw ROCK a day or 2 after plating, as it may have additional impact on cells. Here is one interesting outcome related to your experimentArticle Inhibition of Rho-dependent kinases ROCK I/II activates VEGF...
In short, this is one of the factors to test to address cell death after plating. Low calcium medium may promote the survival as well
Thank you for your answers, I will consider using ROCK-inhibitors and/or low calcium if the seeding in hydrogel keeps failing.
Best regards
Jörg
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