I've been trying to activate and expand naive T cells that have previously been isolated from PBMCs with a pan-T cell isolation kit from Miltenyi and cryopreserved in 90% FBS 10% DMSO. I have decent viability post thaw >85% with ~10% loss in viability from the pre-freeze viability. However, over the 10 days that i'm expanding the T cells, I initially see good activation, but then the T cells start dying off and I end up with abysmal viability by day 10.

Here's the protocol i'm using:

I rapidly thaw the vial of T cells in the water bath and when the vial is almost completely thawed, add 1mL T cell media dropwise. I transfer the full volume to a 15mL tube with more T cell media dropwise, then spin at 300xg for 5 minutes. I activate the T cells in Miltenyi TexMACS medium supplemented with 300 IU/mL IL2 and CD3/CD38 Dynabeads at a 1:1 T cell:bead ratio. I plate the cells at 2 million/mL in a 24 well plate for the initial activation. I expand the T cells for 10 days, checking the viability every 2 days and replenishing media/cytokine if the media starts looking yellow, keeping T cells between 1.5-2 million/mL.

This protocol has worked very well for me from T cells isolated from freshly prepared PBMCs, but hasn't been working well with the cryopreserved T cells. I initially see very good clumps forming that get larger, but towards the second half of the 10 days the clumps don't get very large and the T cells start dying. Over 10 days I had less than a 2-fold expansion, whereas with fresh T cells I could get a 4-5 fold expansion over 10 days.

Does anyone have experience activating and expanding thawed naive T cells or have thoughts on why my T cells may be dying? Whats weird is that I've used the exact same vial of frozen naive T cells to generate CART cells using a 3 day Dynabead activation with IL7 & IL15 followed by viral spinoculation, and the viability of these CART cells is fine after a few days of expansion after spinoculation.