Method for in vitro expansion of cryopreserved cord blood-derived regulatory t cells (Tregs) with high recovery rate

1 . A method for in vitro expansion of cord blood-derived regulatory T cells (Tregs), comprising the following steps:

(1) preparing Tregs, adding an expansion culture medium to Tregs carrying magnetic beads, and conducting a first expansion culture, wherein a primary culture is conducted for 1 day to 2 days, then a subculture is conducted once every 1 day to 3 days, and a total culture time is 18 days or more; recovering the cord blood-derived Tregs after the first expansion culture, and resuspending cord blood-derived Tregs with a serum-free medium to produce a suspension; and cryopreserving the Tregs produced after the first expansion culture; wherein

a process for the preparing Tregs comprises the following steps:

isolating peripheral blood mononuclear cells (PBMC) from cord blood, removing red blood cells with a red blood cell lysis buffer, washing, and counting the isolated PBMC; conducting centrifugation on the washed and counted PBMC, removing a first resulting supernatant from the centrifuged PBMC, and cryopreserving the centrifuged PBMC after removing the first resulting supernatant; and recovering the cryopreserved PBMC by thawing and counting the recovered PBMC;

conducting centrifugation on the recovered PBMC, removing a second resulting supernatant from the centrifuged PBMC, and resuspending the PBMCs with a magnetic bead to produce 0.5 mL to 6 mL of a cord blood-derived Treg-containing suspension with a cell concentration of 5×10 7 cells/mL; and

transferring the cord blood-derived Treg-containing suspension into a sterile tube contacting a magnetic pole, adding a CD25 selection cocktail to the Treg-containing suspension, and incubating for 5 min; vortexing releasable magnetic bead solution for 30 seconds or more; adding a CD4 + T cell enrichment antibody mixture to the releasable magnetic bead solution, and incubating for 5 min; adding the magnetic bead solution, and mixing 2 times to 3 times; placing the sterile tube on the magnetic pole, and incubating for 10 min; preparing a first centrifuge tube to collect CD25 − cells, and pouring a first liquid in the sterile tube into the first centrifuge tube; removing the sterile tube from the magnetic pole, adding 10 mL of the magnetic bead solution to the sterile tube, mixing 2 times to 3 times, placing the sterile tube on the magnetic pole, and incubating for 5 min; adding the magnetic bead solution to an initial resuspension volume with all cells including cells attached on a tube wall; adding a magnetic bead-removing buffer to the initial resuspension volume that contains the magnetic beads; adding a CD 127 high -removing antibody mixture to the initial resuspension volume that contains the magnetic beads, and incubating for 5 min; adding the magnetic bead solution to the initial resuspension volume that contains the magnetic beads, and mixing 2 times to 3 times; placing the sterile tube on the magnetic pole, and incubating for 5 min; and preparing a second centrifuge tube to collect the Tregs present in the resuspension volume, and pouring a second liquid in the sterile tube into the second centrifuge tube under the magnetic pole to obtain the Tregs; wherein

the first expansion culture comprises the following steps:

inoculating the Tregs into a 48-well plate at 2×10 5 to 8×10 5 /mL, adding 0.5 mL of an expansion culture medium comprising magnetic beads per well on day 0, and culturing at 37° C. and 5% CO 2 , wherein a number of the magnetic beads: a number of the cells=1:1;

on day 1 to day 2 after the inoculation, adding 0.5 mL to 1 mL of the expansion culture medium and 100 U/mL to 400 U/mL of recombinant human interleukin-2 (rhIL-2) to Tregs in each well, and culturing at 37° C. and 5% CO 2 ;

on day 3 to day 4 after the inoculation, a first passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, an expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ; after the addition, 1 mL of the expansion culture medium is presented in each well, and an amount of rhIL-2 added is based on 1 mL of the expansion culture medium, amounts of rhIL-2 added in other steps are equal to the amount in this step;

on day 5 to day 6 after the inoculation, a second passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ;

on day 7 to day 10 after the inoculation, a third passage is conducted as follows: anti-CD3CD28 magnetic beads in a Petri dish are removed, and then washed fresh anti-CD3CD28 magnetic beads are added to Tregs according to a number of the magnetic beads: a number of the Tregs=1:1, and cells are cultured at 37° C. and 5% CO 2 ;

on day 10 to day 11 after the inoculation, a fourth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ;

on day 11 to day 12 after the inoculation, a fifth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ;

on day 12 to day 13 after the inoculation, a sixth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ;

on day 13 to day 14 after the inoculation, a seventh passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ;

on day 14 to day 16 after the inoculation, an eighth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ; and

on day 16 to day 18 after the inoculation, a ninth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with cells, and cells in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the cells are cultured at 37° C. and 5% CO 2 ;

(2) thawing and recovering the ninth passage cryopreserved Tregs from the first expansion culture and adding the recovered Tregs to the expansion culture medium and conducting a second expansion culture; wherein the recovered Tregs exhibit (i) a viability of at least 80% as determined by trypan blue exclusion and (ii) a phenotype of at least 90% CD4+CD25+CD127 − by volume cells as determined by flow cytometry, and wherein a primary culture is conducted for 48 hours, then a subculture is conducted once every 1 day to 3 days, and a total culture time is 13 days or more; wherein

the second expansion culture comprises the following steps:

inoculating the recovered Tregs into a 48-well plate at 2×10 5 to 8×10 5 /mL, adding 0.5 mL of an expansion culture medium comprising 200 U/mL to 600 U/mL of rhIL-2 per well on day 0, and culturing at 37° C. and 5% CO 2 for 48 hours;

on day 2 to day 3 after the inoculation of the Tregs, detecting a viability and number of Tregs with trypan blue, adding 0.5 mL to 1 mL of the expansion culture medium, the magnetic beads, and 100 U/mL to 400 U/mL of rhIL-2 to Tregs in each well, and culturing at 37° C. and 5% CO 2 , wherein a number of the magnetic beads: a number of the Tregs=1:1;

on day 3 to day 4 after the inoculation, a first passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with Tregs, and Tregs in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the Tregs are cultured at 37° C. and 5% CO 2 ; after the addition, 1 mL of the expansion culture medium is presented in each well, and an amount of rhIL-2 added is based on 1 mL of the expansion culture medium, amounts of rhIL-2 added in other steps are equal to the amount in this step;

on day 5 to day 6 after the inoculation, a second passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with Tregs, and Tregs in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 400 U/mL of rhIL-2 is added to 1 mL, and the Tregs are cultured at 37° C. and 5% CO 2 ;

on day 7 to day 10 after the inoculation, a third passage is conducted as follows: anti-CD3CD28 magnetic beads in a Petri dish are removed, and then washed fresh anti-CD3CD28 magnetic beads are added according to a number of the magnetic beads: a number of the Tregs=1:1, and Tregs are cultured at 37° C. and 5% CO 2 ;

on day 10 to day 11 after the inoculation, a fourth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with Tregs, and Tregs in each well are passaged to 2 wells, an expansion culture medium comprising 100 U/mL to 1000 U/mL of rhIL-2 is added to 1 mL, and the Tregs are cultured at 37° C. and 5% CO 2 ;

on day 11 to day 12 after the inoculation, a fifth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with Tregs, and Tregs in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 1000 U/mL of rhIL-2 is added to 1 mL, and the Tregs are cultured at 37° C. and 5% CO 2 ;

on day 12 to day 13 after the inoculation, a sixth passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with Tregs, and Tregs in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 1000 U/mL of rhIL-2 is added to 1 mL, and the Tregs are cultured at 37° C. and 5% CO 2 ; and

on day 13 to day 14 after the inoculation, a seventh passage is conducted as follows: 0.5 mL of the expansion culture medium is removed, a remaining expansion culture medium is thoroughly mixed with Tregs, and Tregs in each well are passaged to 2 wells, the expansion culture medium comprising 100 U/mL to 1000 U/mL of rhIL-2 is added to 1 mL, and the Tregs are cultured at 37° C. and 5% CO 2 ;

wherein, according to a final concentration, the expansion culture medium comprises 70.54% to 85.27% volume fraction of the serum-free medium, 2.5% to 5% volume fraction of a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 1% to 2% volume fraction of a penicillin-streptomycin solution, 1% to 2% volume fraction of L-glutamine, 50 μmol/L to 100 μmol/L of 2-mercaptoethanol, 50 nmol/L to 200 nmol/L of rapamycin, and 10% to 20% volume fraction of AB serum, in addition to the rhIL-2;

the magnetic beads used in the first expansion culture and the second expansion culture are both the anti-CD3CD28 magnetic beads; and

a cryopreservation solution used for cryopreserving the Tregs produced after the first expansion culture is 90% by volume of cord blood-derived plasma and 10% by volume of dimethyl sulfoxide (DMSO).

2 . The method for the in vitro expansion of the cord blood-derived Tregs according to claim 1 , wherein 50 μL of the cocktail added is a resuspension volume, 30 μL of the releasable magnetic bead solution added is the resuspension volume, 50 μL of the CD4 + T cell enrichment antibody mixture added is the resuspension volume, 100 μL of the magnetic bead-removing buffer added is the resuspension volume, and 50 μL of the CD127 high -removing antibody mixture added is the resuspension volume.