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

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 5×107 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 127high-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 2×105 to 8×105/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% CO2, 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• (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 2×105 to 8×105/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% CO2 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% CO2, 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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% CO2;
• 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).