Optimized method for expansion and large-scale production of regulatory t cells (Tregs)
An optimized method for expansion and large-scale production of regulatory T cells (Tregs) includes the following steps: preparing a cord blood-derived Treg-containing suspension; transferring the cord blood-derived Treg-containing suspension into a sterile tube matching with a magnetic pole, and magnetically sorting CD4 + CD25 + CD127 − Tregs out; adding an expansion culture medium to Tregs carrying magnetic beads, conducting a primary culture for 1 d to 2 d, and conducting a subculture once every 1 d to 3 d, where a total culture time is 40 d to 50 d. Through one-step sorting, the method achieves very prominent cell expansion effects (shortened time and high cell viability), and increases an effective expansion time to 40 d to 50 d from 18 d. In addition, an expansion effect will not be gradually deteriorated. Thus, the method greatly increases the expansion generation number and the quantity of Tregs, and can meet the needs of clinical patients.
1 . 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 CD127 high -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.
2 . The optimized method for the expansion of the Tregs according to claim 1 , wherein in the step (1), the cord blood-derived Treg-containing suspension is prepared through the following steps: isolating a peripheral blood mononuclear cell (PBMC) layer from cord blood, removing red blood cells with a red blood cell lysis buffer, washing, and counting; and conducting centrifugation, removing a supernatant, and resuspending.
3 . The optimized method for the expansion of the Tregs according to claim 2 , wherein the resuspending comprises the following steps: after the centrifugation is conducted and the supernatant is removed, resuspending cells to produce 0.5 mL to 6 mL of a suspension with a cell concentration of 5×10 7 cells/mL.
4 . The optimized method for the expansion of the Tregs according to claim 1 , wherein 50 μL of the CD25 Positive Selection Cocktail is added per each 1 mL of a resuspension volume, an amount of the Releasable streptavidin-coated magnetic particles added is the resuspension volume×30 μL, an amount of the CD4 + T cell enrichment antibody mixture added is the resuspension volume×50 μL, an amount of the magnetic bead-removing buffer added is the resuspension volume×100 μL, and an amount of the CD127 high -removing antibody mixture added is the resuspension volume×50 μL.
5 . The optimized method for the expansion of the Tregs according to claim 1 , wherein in the step (3), the Tregs carrying the magnetic beads are added to a 48-well plate at a concentration of 2×10 5 to 8×10 5 /mL per well, and 0.5 mL of the expansion culture medium is added to each well for culture.
6 . The optimized method for the expansion of the Tregs according to claim 1 , wherein in the step (3), both the primary culture and the subculture are conducted at 36° C. to 38° C. and 4% to 6% CO 2 .
7 . The optimized method for the expansion of the Tregs according to claim 6 , wherein on day 1 to day 2 after inoculation, 0.5 mL to 1 mL of the expansion culture medium and 100 U/mL to 400 U/mL of rhIL-2 are added to cells in each well, and then the primary culture is conducted.
8 . The optimized method for the expansion of the Tregs according to claim 7 , wherein during the subculture, 0.5 mL of a medium in each well is removed, a remaining medium is thoroughly mixed with cells, the cells in each well are passaged to 2 wells, and the medium is supplemented to 1 mL per well; and when the subculture is conducted a third time, anti-CD3CD28 magnetic beads are removed, and then fresh anti-CD3CD28 magnetic beads are added with a number ratio of the magnetic beads to the Tregs being 1:1.
9 . The optimized method for the expansion of the Tregs according to claim 6 , wherein during the subculture, 0.5 mL of a medium in each well is removed, a remaining medium is thoroughly mixed with cells, the cells in each well are passaged to 2 wells, and the medium is supplemented to 1 mL per well; and when the subculture is conducted a third time, anti-CD3CD28 magnetic beads are removed, and then fresh anti-CD3CD28 magnetic beads are added with a number ratio of the magnetic beads to the Tregs being 1:1.