Culture method for amplifying stem cell-like memory T cells
A culture method and memory technology, applied in the field of cell culture, can solve the problems of expanding TSCM subgroups, and achieve the effect of simple operation, high clinical application value and scalable scale
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Embodiment 1
[0035] Example 1 Stem cell-like memory T cells (T cells) in human peripheral blood mononuclear cells (PBMCs) SCM ) Amplification of (1)
[0036] In this example, PBMCs were freshly isolated from human whole blood by density centrifugation for activation and expansion of T cells. Transfer the separated PBMC cell suspension to the prepared 6-well plate culture dish, and the seeding density is 1x10 6 / ml, use 2mL / well X-Vivo-15 (Lonza) medium, and add CD3 antibody at a final concentration of 10μg / mL, CD28 antibody at a final concentration of 5μg / mL, and IL2 at a final concentration of 100IU / mL, and put Continue culturing in a 5% carbon dioxide incubator at 37°C.
[0037] On the third day of cultivation, transfer the above culture to a 125ml shake flask, use X-Vivo-15 (Lonza) medium containing IL2 at a final concentration of 100IU / mL to make up the volume to 15mL, and set the rotation speed of the shaking incubator to 40rpm .
[0038] On the 6th day of culture, collect the cel...
Embodiment 2
[0042] Example 2 Expansion of stem cell-like memory T cells (TSCM) in human peripheral blood mononuclear cells (PBMC) (2)
[0043] In this example, PBMCs were freshly isolated from human whole blood by density centrifugation for activation and expansion of T cells. Transfer the separated PBMC cell suspension to the prepared 6-well plate culture dish, and the seeding density is 1x10 6 / ml, use 2mL / well OpTmizer (ThermoFisher) medium, and add CD3 antibody at a final concentration of 10 μg / mL, CD28 antibody at a final concentration of 5 μg / mL, and IL2 at a final concentration of 100 IU / mL, and put in 37 ° C, 5 Continue culturing in the % carbon dioxide incubator.
[0044] On the third day of culture, the above culture was transferred to a 125 mL shake flask, and the volume was made up to 40 mL using OpTmizer (ThermoFisher) medium containing IL2 at a final concentration of 100 IU / mL, and the rotation speed of the shaking incubator was set at 60 rpm.
[0045] On the 6th day of cu...
Embodiment 3
[0047] Example 3 Stem cell-like memory T cells (T cells) in human peripheral blood mononuclear cells (PBMCs) SCM ) Amplification (3)
[0048] In this example, PBMCs were freshly isolated from human whole blood by density centrifugation for activation and expansion of T cells. Transfer the separated PBMC cell suspension to the prepared 6-well plate culture dish, and the seeding density is 1x10 6 / ml, use 2mL / well X-Vivo-15 (Lonza) medium, and add CD3 antibody at a final concentration of 10μg / mL, CD28 antibody at a final concentration of 5μg / mL, and IL2 at a final concentration of 100IU / mL, and put Continue culturing in a 5% carbon dioxide incubator at 37°C.
[0049] On the third day of cultivation, transfer the above culture to a 125ml shake flask, use X-Vivo-15 (Lonza) medium containing IL2 at a final concentration of 100IU / mL to make up the volume to 30mL, and set the rotation speed of the shaking incubator to 70rpm .
[0050] On the 6th day of culture, collect the cells ...
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