A Treg cell expansion and culture process

Abstract

The present invention discloses a Treg cell expansion and culture process, which relates to cell therapy. The process comprises: step 1, separating umbilical cord blood to obtain a peripheral blood mononuclear cell layer; step 2, using flow cytometry to sort CD4 + CD25 + CD127 lo / ‑ Treg cells; step three, Treg cell activation and expansion, cord blood Treg cells cryopreservation; the expansion culture medium includes: AB serum, IL-2 concentration of, rapamycin concentration of, α-tocopherol, iguratimod, and gypenosides; the formula of the present invention, α-tocopherol, iguratimod, and gypenosides are synergistic in increasing the Treg ratio and cell activity; the formula and method of the present invention can prolong the expansion days, and can have a good expansion effect even at 78 days.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to a Treg cell expansion and culture process. Background Art

[0002] Treg is a subpopulation of CD4+T cells. Its most specific label is the nuclear transcription factor Foxp3. In addition, it also highly expresses the IL-2α receptor (IL2Rα) - CD25. Therefore, the classic Treg marker is CD4+CD25+Foxp3+T. Studies have shown that the expression of CD127 on Treg cells is negatively correlated with the expression of Foxp3, so CD4 can also be used. + CD25 + CD127 lo / - Treg is used to define Treg cells.

[0003] Cell culture includes the following steps: separation, activation, and expansion. During the expansion phase of Treg cells, it may be necessary to continuously provide IL-2 and regularly replace the culture medium to maintain the cell concentration. The main disadvantage of the Treg cell expansion methods on the market is low proliferation ability: natural Tregs proliferate slowly in vitro and require repeated stimulation, which can easily lead to cell aging or apoptosis. The expansion period is generally 14 days. Starting from the 18th day, the cell multiplier will drop rapidly, and the CD4 + CD25 + CD127 lo / - The proportion is rapidly decreasing, and the market needs a culture process that can increase the number and generation of regulatory T cells. Summary of the invention

[0004] To address the deficiencies of the prior art, the present invention aims to provide a Treg cell expansion and culture process. The formula of the expansion culture fluid of the present invention can improve cell activity and passage number.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A Treg cell expansion and culture process, comprising:

[0007] Step 1: thawing frozen umbilical cord blood, separating the thawed umbilical cord blood to obtain a peripheral blood mononuclear cell layer, lysing red blood cells using a red blood cell lysing solution, and removing red blood cells in the peripheral blood by density gradient centrifugation using a density gradient centrifuge tube and a lymphocyte separation solution, thereby obtaining a peripheral blood mononuclear cell suspension;

[0008] Step 2: The density of peripheral blood mononuclear cells was adjusted to 0.6×10 6 -1.8×10 6cells / mL; then flow cytometry was used to sort CD4 + CD25 + CD127 lo / - Treg cells;

[0009] Step 3, Treg cell activation and expansion, includes the following steps:

[0010] The sorted CD4 + CD25 + CD127 lo / - Treg cells were adjusted to 0.6×10 6 -1.8×10 6 cells / mL;

[0011] On the first day, regulatory T cells were inoculated, and CD3 / CD28 immunomagnetic beads and expansion culture medium were added;

[0012] On the second and fourth days, expansion medium was added;

[0013] On the sixth day, half of the culture medium in the culture flask was discarded and RPMI culture medium was added;

[0014] On the eighth and tenth days, expansion medium was added;

[0015] On the twelfth day, half of the culture medium in the culture flask was discarded and RPMI culture medium was added;

[0016] Add expansion medium every two days for two consecutive days. After two days, remove half of the medium in the culture bottle and discard it, add RPMI medium, and repeat this cycle. Test the amplification multiple every two cycles.

[0017] After centrifugation, the cord blood Treg cells were collected and counted, and the cord blood Treg cells were frozen after quality testing;

[0018] The expansion culture medium includes: AB serum 0.25-2.0 mg / mL, IL-2 concentration of 100-300 IU / mL, rapamycin concentration of 20-40 nmol / L, α-tocopherol concentration of 0.25-2.0 mg / mL, ilamod concentration of 60-100 μmol / L, and gypenosin concentration of 60-100 μmol / L.

[0019] In the aforementioned Treg cell expansion and culture process, the density gradient centrifuge tube is SepTube™ density gradient centrifuge tube-15 or SepTube™ density gradient centrifuge tube-50; and the lymphocyte separation fluid is Lymphoprep™ / lymphocyte separation fluid.

[0020] In the aforementioned Treg cell expansion culture process, the RPMI culture medium includes, by mass percentage, 79.4% water, 15% FBS, 0.2% β-mercaptoethanol, 0.2% primocin antibiotic, 1.5% non-essential amino acids, 2.5% HEPS balanced solution, and 1.2% sodium pyruvate.

[0021] The aforementioned Treg cell expansion culture process, the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, α-tocopherol concentration of 1.2 mg / mL, elamod concentration of 70 μmol / L, and gypenosin concentration of 80 μmol / L.

[0022] A Treg cell expansion and culture process described above, flow cytometry sorting of CD4 + CD25 + CD127 lo / - The method of Treg cells is:

[0023] 1) Take peripheral blood mononuclear cells and place them in flow cytometry tubes, and prepare blank tubes, CD4 single staining tubes, CD25 single staining tubes, CD127 single staining tubes and full staining tubes for sorting;

[0024] 2) Add 1 mL PBS to each tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 100-200 ul volume for flow cytometry staining;

[0025] 3) FCR blocking: except for the Blank tube, add 1ul anti-mouse CD16 / 32 antibody to each tube to block the non-specific antigen binding sites on the cell surface;

[0026] 4) Add CD4, CD25, and CD127 flow cytometry antibodies to each tube, mix well, and incubate;

[0027] 5) Wash off unbound antibodies with PBS and centrifuge;

[0028] 6) Discard the supernatant, add PBS buffer to each tube, and sort CD4 + CD25 + CD127 lo / - Treg cell population;

[0029] 7) Collect the CD4 + CD25 + Treg cells were expressed at 0.6×10 6 -1.8×10 6 The cells were cultured at a density of 1.5 cells / mL in RPMI medium.

[0030] The aforementioned Treg cell expansion and culture process, the method for freezing cord blood Treg cells: using cell freezing solution at 5×10 6 / mL density to resuspend Treg cells, the freezing volume is 1mL, the cell suspension is transferred into the cryotube and quickly placed in a programmed cooling box in a -80℃ refrigerator, and then transferred to liquid nitrogen for storage after 24 hours.

[0031] The aforementioned Treg cell expansion and culture process has a cycle number of 1-11 times.

[0032] The present invention is beneficial in that:

[0033] The α-tocopherol, iguratimod and gypenosides in the formula of the present invention are synergistic in increasing the Treg ratio and cell activity.

[0034] The formula and method of the present invention are used to expand CD4 in regulatory T cells for 18 days. + CD25 + CD127 lo / - The proportion of Treg was 96.76±3.46%, the proportion of CD3+CD8+ was 3.97±2.38%; + CD25 + CD127 lo / - The proportion of Treg is high.

[0035] The formula and method of the present invention are used to expand the regulatory T cell CD4 to 78 days + CD25 + CD127 lo / - The proportion of Treg was 82.72±1.97%, and the proportion of CD3+ CD8+ was 7.7±3.16%. Even after expansion to 78 days, CD4 + CD25 + CD127 lo / - The proportion of Treg is still high. DETAILED DESCRIPTION

[0036] The present invention is specifically described below through specific embodiments.

[0037] Embodiment 1: A Treg cell expansion and culture process, comprising:

[0038] Step 1, thawing frozen umbilical cord blood, separating the thawed umbilical cord blood to obtain a peripheral blood mononuclear cell layer, lysing red blood cells by using red blood cell lysis solution, removing red blood cells in the peripheral blood by density gradient centrifugation using a density gradient centrifuge tube and a lymphocyte separation solution, thereby obtaining a peripheral blood mononuclear cell suspension; the density gradient centrifuge tube is SepTube™ density gradient centrifuge tube-15 or SepTube™ density gradient centrifuge tube-50; the lymphocyte separation solution is Lymphoprep™ / lymphocyte separation solution.

[0039] Step 2: The density of peripheral blood mononuclear cells was adjusted to 0.6×10 6 -1.8×10 6 cells / mL; then flow cytometry was used to sort CD4 + CD25 + CD127 lo / - Treg cells:

[0040] Flow cytometry CD4 + CD25 + CD127 lo / - The method of Treg cells is:

[0041] 1) Take peripheral blood mononuclear cells and place them in flow cytometry tubes, and prepare blank tubes, CD4 single staining tubes, CD25 single staining tubes, CD127 single staining tubes and full staining tubes for sorting;

[0042] 2) Add 1 mL PBS to each tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 100-200 ul volume for flow cytometry staining;

[0043] 3) FCR blocking: except for the Blank tube, add 1ul anti-mouse CD16 / 32 antibody to each tube to block the non-specific antigen binding sites on the cell surface;

[0044] 4) Add CD4, CD25, and CD127 flow cytometry antibodies to each tube, mix well, and incubate;

[0045] 5) Wash off unbound antibodies with PBS and centrifuge;

[0046] 6) Discard the supernatant, add PBS buffer to each tube, and sort CD4 + CD25 + CD127 lo / - Treg cell population;

[0047] 7) Collect the CD4 + CD25 + Treg cells were expressed at 0.6×106 -1.8×10 6 The cells were cultured at a density of 1.5 cells / mL in RPMI medium.

[0048] Step 3, Treg cell activation and expansion, includes the following steps:

[0049] In a 100 mL culture flask, 50 mL of RPMI medium was used to culture the sorted CD4 + CD25 + CD127 lo / - Treg cells were adjusted to 0.6×10 6 -1.8×10 6 cells / mL, culture conditions were: 37°C, 5% CO 2 Concentration culture; RPMI medium includes, by mass percentage, 79.4% water, 15% FBS, 0.2% β-mercaptoethanol, 0.2% primocin antibiotics, 1.5% non-essential amino acids, 2.5% HEPS balanced solution, and 1.2% sodium pyruvate.

[0050] On the first day, regulatory T cells were inoculated and CD3 / CD28 immunomagnetic beads and 10 mL of expansion culture medium were added;

[0051] On the second and fourth days, 10 mL of expansion medium was added;

[0052] On the sixth day, half of the culture medium (40 mL) in the culture flask was discarded and 20 mL of RPMI medium was added;

[0053] On the eighth and tenth days, 10 mL of expansion medium was added;

[0054] On the twelfth day, half of the culture medium (40 mL) in the culture flask was discarded and 20 mL of RPMI culture medium was added;

[0055] Add expansion medium every two days for two consecutive days. After two days, remove half of the medium in the culture bottle and discard it, add RPMI medium, and repeat this cycle. Test the amplification multiple after each cycle.

[0056] The cord blood Treg cells were collected by centrifugation and counted. After quality testing, the cord blood Treg cells were frozen. The method for freezing cord blood Treg cells was as follows: 5×10 6 / mL density to resuspend Treg cells, the freezing volume is 1mL, the cell suspension is transferred into the cryotube and quickly placed in a programmed cooling box in a -80℃ refrigerator, and then transferred to liquid nitrogen for storage after 24 hours.

[0057] The expansion culture medium includes: AB serum 0.25 mg / mL, IL-2 concentration of 100 IU / mL, rapamycin concentration of 40 nmol / L, α-tocopherol concentration of 2 mg / mL, iguratimod concentration of 100 μmol / L, and gypenosin concentration of 100 μmol / L.

[0058] Embodiment 2:

[0059] The rest is the same as Example 1, except that the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, α-tocopherol concentration of 1.2 mg / mL, iguratimod concentration of 70 μmol / L, and gypenosin concentration of 80 μmol / L.

[0060] Embodiment 3:

[0061] The rest is the same as Example 1, except that the expansion culture medium includes: AB serum 2 mg / mL, IL-2 concentration of 300 IU / mL, rapamycin concentration of 20 nmol / L, α-tocopherol concentration of 0.25 mg / mL, iguratimod concentration of 60 μmol / L, and gypenosin concentration of 60 μmol / L.

[0062] Embodiment 4:

[0063] The rest is the same as in Example 2, except for the number of cycles, which is 11 times and the amplification time is 78 days.

[0064] Comparative Example 1:

[0065] The rest is the same as Example 2, only the formula is different, the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, iguratimod concentration of 70 μmol / L, gypenosin concentration of 80 μmol / L, and lacks α-tocopherol.

[0066] Comparative Example 2:

[0067] The rest is the same as Example 2, only the formula is different, the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, α-tocopherol concentration of 1.2 mg / mL, gypenosin concentration of 80 μmol / L. Iguratimod is missing.

[0068] Comparative Example 3:

[0069] The rest is the same as Example 2, except that the expansion medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, α-tocopherol concentration of 1.2 mg / mL, and iguratimod concentration of 70 μmol / L, but lacks gypenosin.

[0070] Comparative Example 4:

[0071] The rest is the same as Example 2, only the formula is different, the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, α-tocopherol concentration of 1.2 mg / mL, and lacks iguratimod and gypenosides.

[0072] Comparative Example 5:

[0073] The rest is the same as Example 2, only the formula is different, the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, gypenosin concentration of 80 μmol / L, and lacks iguratimod and α-tocopherol.

[0074] Comparative Example 6:

[0075] The rest is the same as Example 2, only the formula is different, the expansion culture medium includes: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, iguratimod concentration of 70 μmol / L, lacking α-tocopherol and gypenosin.

[0076] Experiment 1:

[0077] 1. Experimental process

[0078] Antibody selection and staining;

[0079] CD4-FITC / APC (human: CD4; mouse: CD4), CD25-PE / PerCP-Cy5.5, CD127-APC-Cy7 / PE-Cy7 (low expression of CD127 is a Treg characteristic), CD8+ T cell phenotype: CD3-BV421 (pan-T cell marker), CD8-BV510 / APC;

[0080] Other necessary reagents:

[0081] Live and dead cell dyes (such as 7-AAD or Fixable Viability Dye), Fc receptor blockers (such as FcBlock for humans and anti-CD16 / 32 for mice);

[0082] Staining steps

[0083] Blocking nonspecific binding;

[0084] The cells were incubated with Fc receptor blocker (1:100) on ice for 10 min;

[0085] surface staining;

[0086] Add antibody mixture (CD3, CD4, CD8, CD25, CD127) and incubate on ice for 30 minutes in the dark.

[0087] washing

[0088] Wash twice with PBS + 2% FBS and centrifuge to remove the supernatant;

[0089] Dead and live cell staining;

[0090] Add 7-AAD (5 μL / test) and incubate on ice for 5 minutes in a dark place;

[0091] Fixation (if intracellular staining is required);

[0092] Cells were fixed with 1% paraformaldehyde.

[0093] Total lymphocyte selection

[0094] FSC-A vs SSC-A: Circle the lymphocyte population. Exclude contigs: Remove doublets by FSC-H vs FSC-A.

[0095] Live cell selection

[0096] 7-AAD- (or dead and live dye-): exclude dead cells (if dead and live dye is used);

[0097] T cell subset analysis;

[0098] CD3+ T cells: Circle CD3+ cells from live cells (a pan-T cell marker). CD4+ T cells: Sort CD4+ cells from the CD3+ population. Treg analysis: From the CD4+ population, circle CD25+CD127- (or CD25hiCD127lo) cells (a characteristic surrogate marker for Foxp3+ Tregs). CD8+ T cells: Sort CD8+ cells from the CD3+ population.

[0099] Treg (CD4+CD25+CD127-) ratio:

[0100] formula:

[0101] Treg proportion = (number of CD4+CD25+CD127- cells / total number of CD4+ T cells) × 100%;

[0102] Treg ratio = total number of CD4+ T cells CD4+CD25+CD127-cell number × 100%;

[0103] CD8+ T cell ratio = (number of CD3+CD8+ cells / total number of CD3+ T cells) × 100%.

[0104] Table 1 Results of Treg ratio and CD8+ T cell ratio

[0105]

[0106] It can be seen from the above experiments that the formula of α-tocopherol, iguratimod and gypenosides of the present invention are synergistic in increasing the proportion of Tregs; and the present invention can be expanded to 78 days and still have a high Treg proportion.

[0107] Experiment 2: Cell proliferation and CFSE detection:

[0108] CFSE labeled Teff;

[0109] Marking steps:

[0110] Teff was resuspended in pre-warmed PBS (containing 0.1% BSA) at a concentration of 1×10 6 / mL;

[0111] CFSE was added to a final concentration of 2.5 μM and incubated at 37°C in the dark for 10 min;

[0112] Add 5 volumes of pre-cooled culture medium to terminate the reaction, and wash by centrifugation 3 times;

[0113] Establishment of co-cultivation system;

[0114] Group setting (each group has 3 replicate holes);

[0115] Experimental group: CFSE + Teff + Treg (ratio 1:1);

[0116] Positive control: CFSE + Teff culture alone (without Treg);

[0117] Negative control: CFSE + Teff no stimulation (background proliferation);

[0118] Inhibition control: adding anti-TGF-β / IL-10 antibody or Transwell separation;

[0119] Stimulus conditions:

[0120] Anti-CD3 (1 μg / mL per plate) + soluble anti-CD28 (2 μg / mL);

[0121] or use T cell activation magnetic beads (Dynabeads);

[0122] IL-2 (20 U / mL) was added to support Treg survival;

[0123] Culture conditions:

[0124] Cell density: 1×10 5 Teff / well (96-well plate), 37°C, 5% CO 2 Cultivate for 3-5 days;

[0125] Flow cytometry assay;

[0126] Cell collection;

[0127] The cells were collected and washed with PBS containing EDTA to avoid aggregation;

[0128] Live-dead dye (7-AAD) was added to exclude dead cells;

[0129] Staining protocol:

[0130] Surface staining: anti-CD4-APC, CD25-PE (avoid FoxP3 channel);

[0131] Intracellular staining (after fixation and membrane permeabilization): FoxP3-PE-Cy7 (to confirm the purity of Treg);

[0132] CFSE signal analysis:

[0133] CFSE was detected by flow cytometry (FITC channel) and CD4 + CFSE + Proliferation of Teff.

[0134] Software analysis: Calculate the proliferation index or split peak using FlowJo or ModFit.

[0135] Inhibition rate calculation:

[0136] Inhibition rate = (1-proliferation index of experimental group / proliferation index of positive control group) × 100%;

[0137] Inhibition rate = (1-proliferation index of positive control group / proliferation index of experimental group) × 100%.

[0138] Table 2 T cell expansion fold

[0139]

[0140] Table 3 CFSE inhibition rate results

[0141]

[0142] As can be seen from Tables 2 and 3, the formula and method of the present invention can obtain excellent cell activity and good expansion effect. α-Tocopherol, iguratimod and gypenosides have a synergistic effect in improving cell activity. The expansion days are extended, and there is still a good expansion effect at 78 days.

[0143] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A Treg cell expansion and culture process, characterized in that: include: Step 1: thawing frozen umbilical cord blood, separating the thawed umbilical cord blood to obtain a peripheral blood mononuclear cell layer, lysing red blood cells using a red blood cell lysing solution, and removing red blood cells in the peripheral blood by density gradient centrifugation using a density gradient centrifuge tube and a lymphocyte separation solution, thereby obtaining a peripheral blood mononuclear cell suspension; Step 2: The density of peripheral blood mononuclear cells was adjusted to 0.6×10 6 -1.8×10 6 cells / mL; then flow cytometry was used to sort CD4 + CD25 + CD127 lo / - Treg cells; Step 3, Treg cell activation and expansion, includes the following steps: The sorted CD4 + CD25 + CD127 lo / - Treg cells were adjusted to 0.6×10 6 -1.8×10 6 cells / mL; On the first day, regulatory T cells were inoculated, and CD3 / CD28 immunomagnetic beads and expansion culture medium were added; On the second and fourth days, expansion medium was added; On the sixth day, half of the culture medium in the culture flask was discarded and RPMI culture medium was added; On the eighth and tenth days, the expansion medium was added; On the twelfth day, half of the culture medium in the culture flask was discarded and RPMI culture medium was added; Add expansion medium every two days for two consecutive days. After two days, remove half of the medium in the culture bottle and discard it, add RPMI medium, and repeat this cycle. Test the amplification multiple every two cycles. After centrifugation, the cord blood Treg cells were collected and counted, and the cord blood Treg cells were frozen after quality testing; The expansion culture medium includes: AB serum 0.25-2.0 mg / mL, IL-2 concentration of 100-300 IU / mL, rapamycin concentration of 20-40 nmol / L, α-tocopherol concentration of 0.25-2.0 mg / mL, ilamod concentration of 60-100 μmol / L, and gypenosin concentration of 60-100 μmol / L.

2. A Treg cell expansion and culture process according to claim 1, characterized in that: The RPMI culture medium includes, by mass percentage, 79.4% water, 15% FBS, 0.2% β-mercaptoethanol, 0.2% primocin antibiotics, 1.5% non-essential amino acids, 2.5% HEPS balanced solution, and 1.2% sodium pyruvate.

3. A Treg cell expansion and culture process according to claim 1, characterized in that: The expansion culture medium comprises: AB serum 0.85 mg / mL, IL-2 concentration of 200 IU / mL, rapamycin concentration of 30 nmol / L, α-tocopherol concentration of 1.2 mg / mL, iguratimod concentration of 70 μmol / L, and gypenosin concentration of 80 μmol / L.

4. A Treg cell expansion and culture process according to claim 1, characterized in that: The flow cytometry separation of CD4 + CD25 + CD127 lo / - The method of Treg cells is: 1) Take peripheral blood mononuclear cells and place them in flow cytometry tubes, and prepare blank tubes, CD4 single staining tubes, CD25 single staining tubes, CD127 single staining tubes and full staining tubes for sorting; 2) Add 1 mL PBS to each tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 100-200 ul volume for flow cytometry staining; 3) FCR blocking: except for the Blank tube, add 1ul anti-mouse CD16 / 32 antibody to each tube to block the non-specific antigen binding sites on the cell surface; 4) Add CD4, CD25, and CD127 flow cytometry antibodies to each tube, mix well, and incubate; 5) Wash off unbound antibodies with PBS and centrifuge; 6) Discard the supernatant, add PBS buffer to each tube, and sort CD4 + CD25 + CD127 lo / - Treg cell population; 7) Collect the CD4 + CD25 + Treg cells were expressed at 0.6×10 6 -1.8×10 6 The cells were cultured at a density of 1.5 cells / mL in RPMI medium.

5. A Treg cell expansion and culture process according to claim 1, characterized in that: Method for cryopreservation of cord blood Treg cells: Use cell cryopreservation solution at 5×10 6 / mL density to resuspend Treg cells, the freezing volume is 1mL, the cell suspension is transferred into the cryotube and quickly placed in a programmed cooling box in a -80℃ refrigerator, and then transferred to liquid nitrogen for storage within 24 hours.

6. A Treg cell expansion and culture process according to claim 1, characterized in that: The number of cycles is 1-11 times.

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