Effects of physioxia on ATO T-cell development Cells cultured in serum and feeder-free medium and rhuDLL4-Fc-coated culture plates were transferred to ATO at week 2. more efficiently when maintained under physioxia, compared to ambient air. Low oxygen tension acts as a positive regulator of HSC commitment and HPC differentiation toward proT cells in the feeder-free culture system and for further maturation into T cells in the ATO. Culturing HSCs/HPCs in physioxia is an enhanced method of effective progenitor T and mature T-cell production ex vivo and may be of future use for HCT and T-cell immunotherapies. for 5 minutes at 4C. Cell pellets were loosened by light vortexing, and the cell slurry (about 5 L) was gently placed on a 0.4-m Costar Transwell insert (Corning, Kennebunk, Maine, Cat. 3450) in six-well plates. One insert with two ATOs was placed in a well containing 2 mL medium. Serum-free medium to maintain ATOs was composed of Roswell Park Memorial Institute (RPMI) 1640 (Gibco), 4% B27 supplement (ThermoFisher Scientific, Grand Island, New York), 1% Glutamax, 1% penicillin/streptomycin, 0.5% amphotericin B (Gibco), 5 ng/mL rhuFLT3L, 5 ng/mL rhuIL-7, ERK5-IN-1 with and without 100 M AA. Medium for ATO was made fresh weekly and completely changed every 3 to 4 4 days. ATOs were maintained up to 6 weeks in an incubator at 37C,5% CO2 and either ~21% O2, or 5% O2. ATOs were maintained for RAB7A up to 10 weeks in some experiments. At the time of ATO cell harvest, ATOs were briefly disaggregated and aspirated using 1000 L pipettes with flow cytometry staining buffer, and passed through a 70 m nylon strainer (Corning, Cat. 431751) to filter MS5-hDLL1 cells out. Two ATOs placed onto a single well were harvested and pooled for cell counts and further analysis. 2.4 |. Cells and organoids cultured under hypoxia To minimize effects of ambient air, cells were cultured in a hypoxia incubator (5% O2, ERK5-IN-1 5% CO2, and N2 balance), and media for feeder-free and organoid cultures changed inside a custom-configured, O2-, and CO2-controlled glove box (Hypoxic Chamber, Coy), maintained at 3% O2, 5% CO2, and N2 balance. For subsequent ATO culture, plates from the hypoxia incubator (5% O2) were quickly moved into a hypoxia chamber ERK5-IN-1 set at 3% O2 within 30 seconds. A 5% O2 was appropriate for cell incubation and the hypoxia chamber was at 3% O2.19 All supplies and media were acclimated for at least 18 hours in the hypoxia chamber. Liquids with less than 1 mL were acclimated in the hypoxia chamber for at least 3 hours, considering the time of equilibrium of a hypoxic environment.20 Transfer of cells under physioxia to ATO was performed in the 3% O2 hypoxia chamber to minimize ambient air O2 (~21%) effects. 2.5 |. Flow cytometry Cells were harvested and stained with antibodies at 4C for 30 minutes, by washing with 1 mL flow cytometry staining buffer, fixed with 1% formalin solution at 4C for 30 minutes, and resuspended with staining buffer (PBS, 0.5% BSA, and 2 mM EDTA) before analysis. For flow cytometry analysis of ATO cell harvests, a Fc receptor blocking solution, Human being TruStain FcX and a fixable viability dye, zombie yellow (BioLegend, San Diego, California) were added. Antibodies ERK5-IN-1 (BD Biosciences, San Jose, California) utilized for cell surface staining were: CD34-APC (581), CD38-PE (HIT2), CD45RA-PE-CF594 (HI100), CD90-BV421 (5E10), ERK5-IN-1 CD10-PE-Cy7 (HI10a), CD7-APC-H7 (M-T701), CD5-BV421 (UCHT2), CD1a-FITC (HI149), CD3-FITC or APC (UCHT1), CD4-PE-CF594 (RPA-T4), CD8-PerCP-Cy5.5 (RPA-T8), TCR-FITC (IP26), TCR-BV421 (B1), CD19-PE (HIB19), CD33-PE-Cy7 (WM53), CD56-FITC (B159). Samples analysis was on an LSR4 circulation cytometer (BD Biosciences), and data analyzed using the FlowJo software (Tree Celebrity, Washington). 2.6 |. ATO T-cell cytokine assays Harvested cells were seeded at 1 105 cells/well with Minimum amount Essential Medium.