Method for in-vitro amplification of CD8+T cell and cell subset of CD8+T cell
A cell subset and cell technology, applied in the field of cell biology, can solve the problems of lack of rapid expansion of PD1+CD8+ T cells, difficulty in expansion, and difficulty in meeting anti-tumor immunity and anti-infection immunity, and achieves improved expansion. Efficiency, improve the effect of functional repair
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Embodiment 1
[0033] Example 1: Isolation of Peripheral Blood Mononuclear Cells
[0034] 1. Take 20ml of heparin-anticoagulated human peripheral blood in a centrifuge tube, dilute the peripheral blood with normal saline 1:1, and mix well;
[0035] 2. Take another new 50mL centrifuge tube, add 15ml of lymphocyte separation solution (ficoll), and then according to the volume ratio of ficoll: blood dilution solution 1:2, slowly add the mixed blood dilution solution along the tube wall to the upper layer of lymphocyte separation solution , so that the two form a clear layer, centrifuge at 3000rpm for 30min;
[0036] 3. After centrifugation, absorb the mononuclear cell layer into a new 50ml centrifuge tube, add 30ml X-VIVO-15 medium to wash once, centrifuge at 800g for 5min, and discard the supernatant.
[0037] 4. Add 20ml of X-VIVO-15 medium, blow and aspirate to mix, centrifuge at 200g for 10 minutes at room temperature, and discard the supernatant. Add 10ml X-VIVO-15 medium to resuspend an...
Embodiment 2
[0038] Example 2: Toll-like receptor agonists effectively stimulate the proliferation of CD8+ T cells.
[0039] 1. According to Example 1, healthy human peripheral blood PBMCs were isolated.
[0040] 2. Use EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (Stemcell: 19053) to sort CD8+ T cells.
[0041] 3. Use Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90) to stain the sorted CD8 cells.
[0042] 4. Place the cells in a 96-well U-bottom plate, 1×10 per well 5 cell.
[0043] 5. Add the following stimulators and Toll-like receptor agonists (invivogen: tlr-kit1hw, humanTLR1-9 Agonist KIT) in groups
[0044] (1) Blank;
[0045] (2) Anti-human CD3 antibody;
[0046] (3) Anti-human CD3 antibody + anti-human CD28 antibody;
[0047] (4) Anti-human CD3 antibody + TLR1 / 2 agonist-Pam3CSK4 (0.1-1μg / ml);
[0048] (5) Anti-human CD3 antibody + TLR2 agonist-HKLM (10 8 cells / ml);
[0049] (6) Anti-human CD3 antibody + TLR3 agonist-Poly(I:C) (10ng-10ug / ml);
...
Embodiment 3
[0060] Example 3: Toll-like receptor agonists effectively stimulate the proliferation of CD8+ T cell subsets.
[0061] Toll-like receptor agonists can effectively stimulate CD8+ T cell subset central memory T cells (T CM :Centralmemory T cell and effector memory T cells (T EM :effector memory T cell) proliferation.
[0062] 1. Isolation of healthy human peripheral blood PBMC
[0063] 2. Use EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (stemcell: 19053) to sort CD8+ T cells.
[0064] 3. Use Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90) to stain the sorted CD8 cells.
[0065] 4. Place the cells in a 96-well U-bottom plate, 1×10 per well 5 cells.
[0066] 5. Add the following stimulators and Toll-like receptor agonists in groups (invivogen: tlr-kit1hw, humanTLR1-9 Agonist KIT.)
[0067] (1) Anti-human CD3 antibody;
[0068] (2) Anti-human CD3 antibody + TLR1 / 2 agonist-Pam3CSK4 (0.1-1μg / ml);
[0069] (3) Anti-human CD3 antibody + TLR6 / 2 agonis...
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