Artificial antigen presenting cell and preparation method and application thereof
An artificial antigen and cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of complicated DC cell operation, difficulty in forming commercial products, and long culture cycle, etc., to achieve significant T cell expansion, Good biocompatibility, effect of increasing specific surface area
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Embodiment 1
[0080] Example 1. Preparation of PLLA rough microspheres
[0081] 1) Contains Fe 3 O 4 Preparation of PLLA Rough Microspheres
[0082] A. Centrifuge at 8,000 rpm for 5 min, collect smooth microspheres prepared with different molecular weights of PLLA (Mw: 5000, 10000, 50000), and fully remove the water.
[0083] B. Add 5mL of tetrahydrofuran into the centrifuge tube, disperse evenly with a pipette, suspend at 25°C for 30min; centrifuge at 8,000rpm for 5min;
[0084] C. Repeat the above dissolving process twice; centrifuge at 8,000 rpm for 5 min, wash three times with deionized water, and characterize the morphology of the prepared microspheres.
[0085] The PLLA microspheres with molecular weights of 5,000 and 10,000 were completely dissolved by tetrahydrofuran, and the PLLA microspheres with molecular weights of 50,000 were partially dissolved and formed rough morphology with an average particle size of 10 μm. Therefore, the preparation of rough microspheres uses PLLA wit...
Embodiment 2
[0086] Example 2. Preparation of artificial antigen presenting cells
[0087] 1) Contains Fe 3 O 4 Preparation of PLLA Rough Microspheres:
[0088] A. Centrifuge at 8,000 rpm for 5 min to collect Fe-containing 3 O 4 PLLA smooth microspheres, which fully remove moisture.
[0089] B. Add 5mL of tetrahydrofuran into the centrifuge tube, disperse evenly with a pipette, suspend at 25°C for 30min; centrifuge at 8,000rpm for 5min;
[0090] C. Repeat the above dissolving process 2 times; centrifuge at 8,000 rpm for 5 min, wash 3 times with deionized water, and obtain Fe-containing 3 O 4 PLLA rough microspheres (c-MS) with an average particle size of 10 μm.
[0091] A smooth microsphere (s-MS) with a particle size of 2 μm internally distributed with ferric oxide was selected. Others were the same as in this example, and PLLA rough microspheres with holes on the surface, deeper surface roughness and an average particle size of 2 μm were prepared. .
[0092] 2) Physical adsorpti...
Embodiment 3
[0101] Example 3. aAPCs prepared from rough PLLA microspheres for T cell activation and expansion
[0102] 1) Use CD3 negative kit to separate T cells in the spleen of C57 / B6 mice;
[0103] 2) Mix aAPCs with mouse primary T cells according to the number ratio of 1:2, 1:10 and 1:20, in a mixture containing 10% FBS, 1% double antibody, 1% non-essential amino acids and IL2 (300U / mL) in RPMI1640 medium, 37°C, 5% CO 2 After culturing in the incubator for 5 days, the proliferation of T cells was detected by CCK-8 kit, and the level of IFN-γ secreted by T cell culture supernatant was detected by CBA method.
[0104] The example proves that the activation effect is the best when the ratio of aAPCs to mouse primary T cells is 1:2, and the final antibody concentration of aAPC and T cells is 200ng / mL when the final concentration of aAPC and T cells is co-cultured to stimulate the expansion and activation of T cells. Better than 40ng / mL and 1000ng / mL.
[0105] Rough microsphere morpho...
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