Primary AML cell engraftment

[Karolin Stumpf]

Hi everyone,

has anyone optimized media conditions for primary AML cell engraftment (bulk or sorted CD34+ or GPR56+ cell fractions) into the BMOs so far? If so, which medium and cytokine conditions have worked best for you and how patient sample-dependent was this?

Best

Karo

[Abdullah Khan]

Hey Karo, 

Yuqi has done de novo AML in Phase IV medium in the OG model with good success. 

With Aimee we did a couple of formulations with transformed AML (from MF/MPNs I believe) using:

• AML-specific media: StemPro-34, 0.2% P/S, 10ng/mL SCF, FLT3-L, TPO, IL6, G-CSF, GM-CSF, 5ng/mL IL3
• Organoid-standard media: StemPro-34, 0.2% P/S, 10ng/mL SCF, FLT3-L, IL7, 5ng/mL IL3, IL6, mCSF, EPO, 1ng/mL TPO  

Both formulations worked well though my preference is the 'organoid-standard media'. We haven't fully phenotyped the cells, but there is marginally less differentiation in the organoid media (so cells are viable but differentiating/expanding less vigorously). 

BW
Abs

[Salim Atakhanov]

Hi all,

for PBMC-derived CD34+ cell engraftment, do you sort CD34+ and then freeze for later use (what cryopreserved medium do you use? how good is the viability after thawing?) or you freeze PBMCs and from the thawed vial you then sort CD34+ cells? 

I will appreciate if you could share your experience in obtaining enough CD34+ cells from blood.

BW

Salim

[Abdullah Khan]

From standard peripheral blood? HDs? Your numbers will always be small - better from apherisis HDs (or MF samples have loads of circulating CD34s). 

To my knowledge we have some vials which have been pre-enriched for CD34s and frozen, and some that we store and then sort for CD34s after the fact. They are comparable in terms of yield. We use Cryopreserve at the moment but have had good results from other freezing. medium, including sometimes CDMSO/FBS. 

Last comment if you're struggling for numbers, something we haven't quite tried yet but I suspect will work well is using Adam Wilkinson's expansion protocol! 

Hope this helps

Abs