Dear colleagues,
I want to establish a in vivo model utilizing primary adult murine ventricular myocytes (AMVMs). I need to culture them for at least 72 hours. However, my cell gradually die with already insufficient amount of viable cells at 48 hours.
Cells are isolated from mouse hearts by collaborators, so I cannot adjust anything at this step. I use MEM-HBSS supplemented with 10% FBS, Pen/Strep, 2 mM L-Glut, 2 mM ATP and 10 mM BDM for palting. After 1 hrs of plating media is switched to MEM-HBSS with Pen/Step, 0,1% BSA, 2 mM L-Glut, 10 mM BDM and ITS ( 5 ug/mL insulin, 5 ug/mL transferrin, and 5 ng/mL selenium). Cells are incubated at 37oC 2% CO2. I use laminin-coated glass coverslips or plastic dishes.
Am I doing something wrong? Or do you know some ways to improve AMVMs survival?
Great thanks!
Hi Stanislovas,
How do you get the AMVMs from your colaborators - cryopreserved?
If so, how are you thawing them at the moment?
Maybe tweaking the thawing protocol a bit to make it less stressful for the cells will improve their survivability. Here's a protocol for human primary cells, but I would suggest to try it with you AMVMs:
https://www.promocell.com/videos/getting-started-with-primary-cells-standard-procedures-in-cell-culture/
You could also try to culture the cells without antibiotics - somewhat risky, if thcells from your collaborators might be contaminated, but if the cells are already struggling, the antibiotics are not helping with that.
Also, are the AMVMs already well attached after 1 hour? What if you wait longer (4-16 hours) before changing the media after plating?
Cheers,
Alex
Dear Alexander Trampe , thanks a lot!
I receive cells immediately after isolation and transfer them to my building.
After 1 hour they a well attached. But 24 hours latter some unattached cells appears.
And at that time you are already using the media for co-cultivation? (i.e. it's not the recommended/optimal media for AMVMs?)
You could try a 50/50 mix of the media recommended for each cell type - unless that's what you are already using?
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