M-ATOs were generated as previously described (27 (link)). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free M-ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in 1X PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (of note SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x105 MS5-mDLL4 cells were combined with purified murine HSPCs (100-4000 cells/ATO). M-ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12 and cytokines.
Free full text: Click here
