Please share the protocols which are tried for the same and explain the critical points to take care.e.g.
How to get rid of other cell population apart from blast cells?
What should be the culture medium and how does culture behave?
How long can we maintain such cultures?
Can we transfect such cells with lipofectamine for transient expression of a gene?
When my colleague worked with primary AML cells, he was immunomagnetically enriching CD34+ cells from mononuclear cells after density centrifugation of blood / bone marrow. For immunomagnetical enrichment he was using MACS system (Miltenyi). Then, he cultured them in serum-free HPGM (hemato-poietic progenitor growth medium; Lonza) supplemented with IL-3, IL-6 (both 10 ng/ml), and SCF (25 ng/ml; all from Peprotech). He could maintain them for up to 1 week in culture. Transfection was done in our lab with Amaxa system, so I don't know if Lipofectamine will work.
Look at the protocol in Klco et al. Blood February 28, 2013 vol. 121 no. 9 1633-1643. They use an Hs27 co-culture approach that maintains most AMLs for 1-2 weeks and many cases can be cultured for 5-6 weeks. This maintains the subclonal mutation architecture as well.
From the methods: Primary AML cells (from either peripheral blood or bone marrow) cryopreserved in 10% DMSO were quickly thawed, resuspended in 40 mL of PBS with 20% FBS and centrifuged at 600g for 5 minutes. Cells were resuspended in DMEM supplemented with 15% FBS, 50μM β-mercaptoethanol, penicillin/streptomycin, and human cytokines (Peprotech) including SCF (100 ng/mL), IL3 (10 ng/mL), IL-6 (20 ng/mL), TPO (10 ng/mL), and FLT3L (10 ng/mL), and then plated on confluent irradiated stromal cells. Most experiments were performed in a 6-well plate with 500 000 stromal cells and 350 000 human AML cells added per well. Fresh growth media was added weekly; hemi-depopulation was performed when growth was excessive. Using this approach, approximately 70% of tested cases expanded > 2-fold during 1 week of culture.
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