Optimized method for expansion and large-scale production of regulatory t cells (Tregs)

An optimized method for expansion of regulatory T cells (Tregs), comprising the following steps:

• (1) preparing a cord blood-derived Treg-containing suspension;
• (2) transferring the cord blood-derived Treg-containing suspension into a sterile tube, adding CD25 Positive Selection Cocktail, and incubating for 5 min;
• vortexing releasable streptavidin-coated magnetic particles for 30 seconds or more until magnetic bead aggregates disappear;
• adding the releasable streptavidin-coated magnetic particles and a CD4+T cell enrichment antibody mixture to the transferred cord blood-derived Treg-containing suspension, and incubating for 5 min;
• placing the sterile tube on a magnetic pole, and incubating for 10 min;
• pouring a first liquid in the sterile tube into a first centrifuge tube;
• placing the sterile tube on the magnetic pole, and incubating for 5 min;
• adding a magnetic bead-removing buffer to the incubated sterile tube;
• then adding a CD127high-removing antibody mixture to the incubated sterile tube, and incubating for 5 min;
• placing the sterile tube on the magnetic pole, and incubating for 5 min;
• pouring the incubated liquid in the sterile tube into a second centrifuge tube to obtain CD4+CD25+CD127− Tregs;
• and
• (3) adding an expansion culture medium to Tregs, conducting a primary culture for 1 d to 2 d, and conducting a subculture once every 1 d to 3 d, wherein a total culture time is 40 d to 50 d,
• wherein in the step (3), in volume fractions, the expansion culture medium comprises 70.54% to 85.27% of a serum-free medium, 2.5% to 5% of a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 1% to 2% of a penicillin-streptomycin solution, 1% to 2% of L-glutamine, 75 μmol/L to 100 μmol/L of 2-mercaptoethanol, 0 U/mL to 1,000 U/mL of recombinant interleukin-2, 50 nmol/L to 200 nmol/L of rapamycin, and 15% to 20% of AB serum.