In-vitro amplification culture method and culture medium for regulatory T cells

Abstract

The invention provides an in-vitro amplification culture method of regulatory T cells and a culture medium, and belongs to the technical field of medicines. Comprising the following steps: separating and purifying peripheral blood CD4 < + > CD25 < + > regulatory T cells, co-culturing the peripheral blood CD4 < + > CD25 < + > regulatory T cells and dendritic cells, and separating and purifying to obtain stimulated CD4 < + > CD25 < + > regulatory T cells; the preparation method comprises the following steps: adding tris (9-cis retinoic acid) rapamycin ester and IL-2 into an RPMI 1640 complete culture solution to prepare a culture medium; and adding the stimulated CD4 + CD25 + regulatory T cells into a culture medium, carrying out stimulation culture, and separating to obtain the regulatory T cells amplified in vitro. According to the method, a step-by-step stimulation method is adopted, the amplification efficiency of the obtained in-vitro amplified regulatory T cells is high, proliferation of other cells can be inhibited, expression of the obtained amplified regulatory T cells Foxp3 is improved to a certain extent, the inpotency of the regulatory T cells Foxp3 can be kept, good inhibition performance is shown, and good medical value is achieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an in vitro expansion and culture method and culture medium for regulatory T cells. Background Technology

[0002] Immunological diseases are caused by autoimmune disorders leading to damage to multiple organ and tissue functions. They have a wide spectrum, including autoimmune diabetes and systemic lupus erythematosus. Autoimmune diseases are also listed as one of the ten major diseases in my country's Medium- and Long-Term Science and Technology Development Plan. my country has a large population and a large absolute number of patients. Immunological diseases not only impose a heavy economic burden on individuals and families but also seriously impact the country's economic development. Currently, treatment largely relies on traditional methods such as immunosuppressants, but the root cause—immune dysregulation—remains unresolved.

[0003] Regulatory T cells (Tregs) are a special subset of T cells that maintain dynamic immune homeostasis by inhibiting and regulating effector T cells. Since their isolation and identification by Japanese researchers in the late 20th century, regulatory T cells have become a hot topic in immunology research in recent years. Several clinical trials related to regulatory T cells have been initiated abroad, with the representative case of regulatory T cell therapy for graft-versus-host disease demonstrating the safety and efficacy of this therapy in immune diseases. However, regulatory T cell therapy for immune diseases remains a blank area in China. Regulatory T cell therapy can restore balance to the immune system, prevent further damage to organs and tissues from autoimmune responses, improve or even reverse the condition, and fundamentally cure immune diseases.

[0004] Increasing research indicates that umbilical cord blood is rich in regulatory T cells. Because these cells are predominantly T cells that have not been activated by antigens, and the Treg cells expanded in vitro exhibit good function and low immunogenicity, they offer new hope for the treatment of immune diseases. Currently, high-purity CD4+ can be obtained through magnetic bead separation or flow cytometry. + CD25 + Treg cells, but CD4+ cells obtained through simple in vitro isolation techniques due to their extremely low in vivo content. + CD25 + Treg cells are insufficient to meet the needs of experimental research. Meanwhile, CD4... + CD25 + The in vitro expansion of Treg cells is complex and inefficient due to their high insensitivity to in vitro stimuli. Summary of the Invention

[0005] The purpose of this invention is to provide an in vitro expansion and culture method and culture medium for regulatory T cells. The method uses a stepwise stimulation approach, which yields in vitro expanded regulatory T cells with high expansion efficiency and the ability to inhibit the proliferation of other cells. The expression of Foxp3 in the expanded regulatory T cells is enhanced to a certain extent, and the cells are kept incompetent, showing good inhibitory performance and good medical value.

[0006] The technical solution of this invention is implemented as follows: This invention provides a method for in vitro expansion and culture of regulatory T cells, comprising the following steps: S1. Peripheral blood CD4 + CD25 + Isolation and purification of regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells were co-cultured with dendritic cells, then isolated and purified to obtain stimulated CD4+. + CD25 + Regulatory T cells; Foxp3 is a key transcription factor identified in recent years that influences the development of regulatory T cells. The Foxp3 gene encodes the scurfin protein, which affects CD4+. + CD25 + Treg cells play a crucial role in growth, development, and function; therefore, Foxp3 is considered a key component of CD4+. + CD25 + A specific marker of Treg.

[0007] This invention first involves peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were co-cultured. After the first stimulation step, the cells began to proliferate significantly, but the expression of the FOXP3 gene was poor. Therefore, after the proliferation stimulation, the dendritic cells were promptly removed, and other effector T cells were purified to obtain stimulated CD4+ cells. + CD25 + Regulatory T cells were added to a culture medium containing tris-9-cis-retinoic acid rapamycin ester and IL-2 for the second step of stimulation.

[0008] S3. Rapamycin tris-9-cis-retinoic acid was prepared by esterification of 9-cis-retinoic acid with rapamycin. S4. Rapamycin tris-9-cis-retinoic acid and IL-2 (interleukin-2) were added to RPMI 1640 complete culture medium to prepare the culture medium; S5. Stimulated CD4 + CD25+ Regulatory T cells were added to the culture medium and stimulated with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads. After 6-8 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed and cultured again. After 10 days, the medium was replaced with fresh RPMI 1640 complete culture medium, and 9-cis retinoic acid, IL-2, and CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were added for further stimulation. The expanded regulatory T cells were isolated using the MACS immunomagnetic bead method.

[0009] Rapamycin is an immunosuppressant widely used in transplant rejection. It selectively promotes the in vitro expansion of Tregs and inhibits the expansion of a small number of T effector cells mixed in with Tregs. All-trans retinoic acid (ATA) is a vitamin A metabolite. In animal models with selective deletion of the retinoic acid receptor α gene, the expression of Treg Foxp3 is also significantly impaired. Therefore, ATA plays a role in stabilizing Foxp3 expression. ATA combined with rapamycin has a synergistic effect on the in vitro expansion of Tregs. However, since ATA can induce the activation of effector T cells (Teff) and induce Teff differentiation into Th1 and Th17 cells, this invention uses 9-cis-retinoic acid instead of ATA to achieve a better effect on stimulating the in vitro expansion of Tregs. Furthermore, through the esterification reaction conjugate, both drugs can be released simultaneously within Treg cells, ensuring that both drugs enter the same cell and avoiding Teff activation caused by paracrine effects, thereby improving the efficiency of in vitro expansion of regulatory T cells.

[0010] As a further improvement of the present invention, the peripheral blood CD4 in step S1 + CD25 + The method for isolating and purifying regulatory T cells involved separating peripheral blood single cells using the Ficoll density gradient separation method, followed by separation of peripheral blood CD4 cells using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells.

[0011] As a further improvement of the present invention, the co-culture method in step S2 is to use peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded in RPMI 1640 medium and cultured at 36-38°C with 5% CO2 for 3-5 days. Stimulated CD4+ cells were then isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells, peripheral blood CD4 + CD25 +The ratio of regulatory T cells to dendritic cells is 8-12:1.

[0012] As a further improvement of the present invention, the molar ratio of 9-cis-retinoic acid to rapamycin in step S3 is 3-3.3:1, and the reaction time is 2-4 hours.

[0013] As a further improvement of the present invention, the culture medium in step S4 contains 8-12% inactivated fetal bovine serum, penicillin 40-60 U / mL, streptomycin 40-60 U / mL, rapamycin tris-9-cis-retinoic acid ester 25-35 nmol / L, and IL-2 15-25 U / mL.

[0014] As a further improvement of the present invention, when adding the culture medium in step S5, the CD4+ levels after stimulation are adjusted. + CD25 + The number of regulatory T cells is 10 4 -10 5 The CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4 cells / mL + CD25 + The ratio of regulatory T cells is 3-5:1.

[0015] As a further improvement of the present invention, the stimulation culture conditions in step S5 are 36-38℃, 5% CO2, and saturated humidity; the fresh RPMI 1640 complete culture medium is a culture medium containing 8-12% inactivated fetal bovine serum, penicillin 40-60 U / mL, and streptomycin 40-60 U / mL; in the continued stimulation culture, 9-cis-retinoic acid is added to the culture medium to a final concentration of 30-50 nmol / L, and IL-2 is added to the culture medium to a final concentration of 10-20 U / mL; the amount of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads added is 3-5 times the number of cells.

[0016] The present invention further protects an in vitro expanded regulatory T cell prepared by the above-described culture method.

[0017] This invention further protects the use of the above-mentioned in vitro expanded regulatory T cells in the preparation of drugs for treating immune diseases.

[0018] The present invention further protects a culture medium containing 8-12% inactivated fetal bovine serum, penicillin 40-60 U / mL, streptomycin 40-60 U / mL, rapamycin tris-9-cis-retinoic acid ester 25-35 nmol / L, and IL-2 15-25 U / mL.

[0019] The present invention has the following beneficial effects: 1. This invention first involves peripheral blood CD4... + CD25 + Co-culturing regulatory T cells and dendritic cells, after the first step of stimulation, resulted in significant cell proliferation, thereby greatly improving the proliferation efficiency.

[0020] 2. This invention prepares 3-9-cis-retinoic acid rapamycin ester via esterification. The conjugate obtained through esterification can be cleaved within Treg cells to release both drugs simultaneously, ensuring that both drugs enter the same cell. This overcomes the problem of all-trans retinoic acid activating effector T cells (Teff) and inducing Teff differentiation into Th1 and Th17 cells. At the same time, it increases the expression of Treg Foxp3 and improves the performance of the obtained in vitro expanded regulatory T cells.

[0021] 3. The present invention uses a stepwise stimulation method, which results in high expansion efficiency of the in vitro expanded regulatory T cells and can inhibit the proliferation of other cells. The expression of Foxp3 in the expanded regulatory T cells is enhanced to a certain extent, and the cells can maintain their incompetence, showing good inhibitory performance and good medical value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The infrared spectrum of rapamycin tris-9-cis-retinoic acid ester.

[0024] Figure 2 CD4 in Example 1 + CD25 + Image showing the sorting results of T cells.

[0025] Figure 3 This refers to one case of regulatory T cells expanded in vitro from each group acting on autologous CD4. + CD25 - Flow cytometry plot of Teff proliferation inhibition. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Dendritic cells were enriched from peripheral blood single cells using the Dynabeads™ Human Dendritic Cell (DC) Enrichment Kit.

[0028] Example 1 This embodiment provides a method for in vitro expansion and culture of regulatory T cells, including the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded into RPMI 1640 medium (containing IL-2 10 μg / mL + TGFβ1 2 ng / mL + 100 IU / M streptomycin + 1 mmol / L L-glutamine + 0.01 mol / L HEPES buffer + 50 μmol / L 2β-mercaptoethanol + 10% fetal bovine serum) to adjust peripheral blood CD4 levels. + CD25 + The cell concentration of regulatory T cells was 10. 6 CD4+ in peripheral blood + CD25 + The ratio of regulatory T cells to dendritic cells was 8:1. Cells were cultured at 36°C and 5% CO2 for 5 days. Stimulated CD4 cells were isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S3. Add 0.3 mol of 9-cis-retinoic acid, 0.1 mol of rapamycin, and 0.025 mol of DMAP (4-dimethylaminopyridine) to 500 mL of N,N-dimethylformamide. Lower the temperature to 0 °C, then add 0.7 mol of EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). Stir the reaction at room temperature for 2 h. Wash successively with 0.1 mol / L hydrochloric acid, 0.3% sodium bicarbonate solution, and saturated brine. Dry, filter, concentrate, and purify to obtain the trisubstituted esterification product, namely tris-9-cis-retinoic acid rapamycin ester; the infrared spectrum is shown below. Figure 1 As shown.

[0029] S4. Rapamycin tris-9-cis-retinoic acid ester and IL-2 were added to RPMI 1640 complete culture medium to prepare a culture medium containing 8% inactivated fetal bovine serum, 40 U / mL penicillin, 40 U / mL streptomycin, 25 nmol / L rapamycin tris-9-cis-retinoic acid ester, and 15 U / mL IL-2.

[0030] S5. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust CD4 levels after stimulation. + CD25 + The number of regulatory T cells was 5 × 10 4 The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 + Regulatory T cells were cultured at a ratio of 3:1, at 36°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed, and the cells were cultured under the original conditions. On day 10, the medium was replaced with fresh RPMI 1640 complete medium (containing 8% inactivated fetal bovine serum, 40 U / mL penicillin, and 40 U / mL streptomycin). 9-cis-retinoic acid was added to the culture medium to a final concentration of 30 nmol / L, and IL-2 was added to the culture medium to a final concentration of 10 U / mL. DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added to the cell count three times the normal amount. The cells were cultured under the original conditions for another 3 days. The expanded regulatory T cells were then isolated using the MACS immunomagnetic bead method.

[0031] Example 2 This embodiment provides a method for in vitro expansion and culture of regulatory T cells, including the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded into RPMI 1640 medium (containing IL-2 10 μg / mL + TGFβ1 2 ng / mL + 100 IU / M streptomycin + 1 mmol / L L-glutamine + 0.01 mol / L HEPES buffer + 50 μmol / L 2β-mercaptoethanol + 10% fetal bovine serum) to adjust peripheral blood CD4 levels. + CD25 + The cell concentration of regulatory T cells was 10. 7 CD4+ in peripheral blood + CD25 + The ratio of regulatory T cells to dendritic cells was 12:1. Cells were cultured at 38°C and 5% CO2 for 3 days. Stimulated CD4 cells were then isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S3. Add 0.33 mol 9-cis-retinoic acid, 0.1 mol rapamycin, and 0.025 mol DMAP to 500 mL N,N-dimethylformamide, lower the temperature to 0 °C, add 0.7 mol EDC·HCl, stir the reaction at room temperature for 4 h, wash with 0.1 mol / L hydrochloric acid, 0.3% sodium bicarbonate solution and saturated brine in sequence, dry, filter and concentrate, separate and purify to obtain the trisubstituted esterification product, which is tris-9-cis-retinoic acid rapamycin ester; S4. Rapamycin tris-9-cis-retinoic acid ester and IL-2 were added to RPMI 1640 complete culture medium to prepare a culture medium containing 12% inactivated fetal bovine serum, 60 U / mL penicillin, 60 U / mL streptomycin, 35 nmol / L rapamycin tris-9-cis-retinoic acid ester, and 25 U / mL IL-2.

[0032] S5. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust CD4 levels after stimulation. + CD25 + The number of regulatory T cells was 5 × 10 4The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 + Regulatory T cells were cultured at a ratio of 5:1, at 38°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed, and the cells were cultured under the original conditions. On day 10, the medium was replaced with fresh RPMI 1640 complete medium (containing 12% inactivated fetal bovine serum, 60 U / mL penicillin, and 60 U / mL streptomycin). 9-cis-retinoic acid was added to the medium to a final concentration of 50 nmol / L, and IL-2 was added to a final concentration of 20 U / mL. DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added to the medium, and the cells were cultured under the original conditions for another 3 days. The expanded regulatory T cells were then isolated using the MACS immunomagnetic bead method.

[0033] Example 3 This embodiment provides a method for in vitro expansion and culture of regulatory T cells, including the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded into RPMI 1640 medium (containing IL-2 10 μg / mL + TGFβ1 2 ng / mL + 100 IU / M streptomycin + 1 mmol / L L-glutamine + 0.01 mol / L HEPES buffer + 50 μmol / L 2β-mercaptoethanol + 10% fetal bovine serum) to adjust peripheral blood CD4 levels. + CD25 + The cell concentration of regulatory T cells was 5 × 10⁻⁶. 6 CD4+ in peripheral blood + CD25 + The ratio of regulatory T cells to dendritic cells was 10:1. Cells were cultured at 37°C and 5% CO2 for 4 days. Stimulated CD4+ cells were isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S3. Add 0.32 mol 9-cis-retinoic acid, 0.1 mol rapamycin, and 0.025 mol DMAP to 500 mL N,N-dimethylformamide, lower the temperature to 0 °C, add 0.7 mol EDC·HCl, stir the reaction at room temperature for 3 h, wash with 0.1 mol / L hydrochloric acid, 0.3% sodium bicarbonate solution and saturated brine in sequence, dry, filter and concentrate, separate and purify to obtain the trisubstituted esterification product, which is tris-9-cis-retinoic acid rapamycin ester; S4. Rapamycin tris-9-cis-retinoic acid ester and IL-2 were added to RPMI 1640 complete culture medium to prepare a culture medium containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, 50 U / mL streptomycin, 30 nmol / L rapamycin tris-9-cis-retinoic acid ester, and 20 U / mL IL-2.

[0034] S5. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust CD4 levels after stimulation. + CD25 + The number of regulatory T cells was 5 × 10 4 The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 + Regulatory T cells were cultured at a ratio of 4:1, at 37°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed, and the cells were cultured under the original conditions. On the 10th day, the medium was replaced with fresh RPMI 1640 complete medium (containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, and 50 U / mL streptomycin). 9-cis-retinoic acid was added to the culture medium to a final concentration of 40 nmol / L, and IL-2 was added to the culture medium to a final concentration of 15 U / mL. DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added at a cell count of 4 times, and the cells were cultured under the original conditions for another 3 days. The expanded regulatory T cells were then isolated using the MACS immunomagnetic bead method.

[0035] Comparative Example 1 The difference from Example 3 is that step S2 was not performed.

[0036] Includes the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method.+ CD25 + Regulatory T cells; S2. Add 0.32 mol 9-cis-retinoic acid, 0.1 mol rapamycin, and 0.025 mol DMAP to 500 mL N,N-dimethylformamide, lower the temperature to 0 °C, add 0.7 mol EDC·HCl, stir the reaction at room temperature for 3 h, wash with 0.1 mol / L hydrochloric acid, 0.3% sodium bicarbonate solution and saturated brine in sequence, dry, filter and concentrate, separate and purify to obtain the trisubstituted esterification product, which is tris-9-cis-retinoic acid rapamycin ester; S3. Rapamycin tris-9-cis-retinoic acid ester and IL-2 were added to RPMI 1640 complete culture medium to prepare a culture medium containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, 50 U / mL streptomycin, 30 nmol / L rapamycin tris-9-cis-retinoic acid ester, and 20 U / mL IL-2.

[0037] S4. Peripheral blood CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust peripheral blood CD4 levels. + CD25 + The number of regulatory T cells was 5 × 10 4 The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 + Regulatory T cells were cultured at a ratio of 4:1, at 37°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed, and the cells were cultured under the original conditions. On the 10th day, the medium was replaced with fresh RPMI 1640 complete medium (containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, and 50 U / mL streptomycin). 9-cis-retinoic acid was added to the culture medium to a final concentration of 40 nmol / L, and IL-2 was added to the culture medium to a final concentration of 15 U / mL. DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added at a cell count of 4 times, and the cells were cultured under the original conditions for another 3 days. The expanded regulatory T cells were then isolated using the MACS immunomagnetic bead method.

[0038] Comparative Example 2 Compared with Example 3, the difference is that step S3 involves mixing 0.32 mol of 9-cis-retinoic acid and 0.1 mol of rapamycin evenly to obtain a mixture.

[0039] Includes the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded into RPMI 1640 medium (containing IL-2 10 μg / mL + TGFβ1 2 ng / mL + 100 IU / M streptomycin + 1 mmol / L L-glutamine + 0.01 mol / L HEPES buffer + 50 μmol / L 2β-mercaptoethanol + 10% fetal bovine serum) to adjust peripheral blood CD4 levels. + CD25 + The cell concentration of regulatory T cells was 5 × 10⁻⁶. 6 CD4+ in peripheral blood + CD25 + The ratio of regulatory T cells to dendritic cells was 10:1. Cells were cultured at 37°C and 5% CO2 for 4 days. Stimulated CD4+ cells were isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S3. Mix 0.32 mol of 9-cis-retinoic acid and 0.1 mol of rapamycin until homogeneous to obtain a mixture; S4. Add the mixture and IL-2 to RPMI 1640 complete culture medium to prepare a culture medium containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, 50 U / mL streptomycin, 30 nmol / L of the mixture, and 20 U / mL of IL-2.

[0040] S5. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust CD4 levels after stimulation. + CD25 + The number of regulatory T cells was 5 × 10 4 The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 +Regulatory T cells were cultured at a ratio of 4:1, at 37°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed, and the cells were cultured under the original conditions. On the 10th day, the medium was replaced with fresh RPMI 1640 complete medium (containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, and 50 U / mL streptomycin). 9-cis-retinoic acid was added to the culture medium to a final concentration of 40 nmol / L, and IL-2 was added to the culture medium to a final concentration of 15 U / mL. DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added at a cell count of 4 times, and the cells were cultured under the original conditions for another 3 days. The expanded regulatory T cells were then isolated using the MACS immunomagnetic bead method.

[0041] Comparative Example 3 The difference from Example 3 is that rapamycin tris-9-cis-retinoic acid was not added in step S4.

[0042] Includes the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded into RPMI 1640 medium (containing IL-2 10 μg / mL + TGFβ1 2 ng / mL + 100 IU / M streptomycin + 1 mmol / L L-glutamine + 0.01 mol / L HEPES buffer + 50 μmol / L 2β-mercaptoethanol + 10% fetal bovine serum) to adjust peripheral blood CD4 levels. + CD25 + The cell concentration of regulatory T cells was 5 × 10⁻⁶. 6 CD4+ in peripheral blood + CD25 + The ratio of regulatory T cells to dendritic cells was 10:1. Cells were cultured at 37°C and 5% CO2 for 4 days. Stimulated CD4+ cells were isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S3. Add IL-2 to RPMI 1640 complete culture medium to prepare a culture medium containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, 50 U / mL streptomycin, and 20 U / mL IL-2.

[0043] S4. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust CD4 levels after stimulation. + CD25 + The number of regulatory T cells was 5 × 10 4 The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 + Regulatory T cells were cultured at a ratio of 4:1, at 37°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed, and the cells were cultured under the original conditions. On the 10th day, the medium was replaced with fresh RPMI 1640 complete medium (containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, and 50 U / mL streptomycin). 9-cis-retinoic acid was added to the culture medium to a final concentration of 40 nmol / L, and IL-2 was added to the culture medium to a final concentration of 15 U / mL. DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added at a cell count of 4 times, and the cells were cultured under the original conditions for another 3 days. The expanded regulatory T cells were then isolated using the MACS immunomagnetic bead method.

[0044] Comparative Example 4 The difference from Example 3 is that 9-cis-retinoic acid was not added in step S5.

[0045] Includes the following steps: S1. White blood cells were collected from blood samples of healthy volunteers. Peripheral blood single cells were separated using the Ficoll density gradient separation method, and peripheral blood CD4 were obtained using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells and dendritic cells were seeded into RPMI 1640 medium (containing IL-2 10 μg / mL + TGFβ1 2 ng / mL + 100 IU / M streptomycin + 1 mmol / L L-glutamine + 0.01 mol / L HEPES buffer + 50 μmol / L 2β-mercaptoethanol + 10% fetal bovine serum) to adjust peripheral blood CD4 levels. + CD25 + The cell concentration of regulatory T cells was 5 × 10⁻⁶. 6 CD4+ in peripheral blood +CD25 + The ratio of regulatory T cells to dendritic cells was 10:1. Cells were cultured at 37°C and 5% CO2 for 4 days. Stimulated CD4+ cells were isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells; S3. Add 0.32 mol 9-cis-retinoic acid, 0.1 mol rapamycin, and 0.025 mol DMAP to 500 mL N,N-dimethylformamide, lower the temperature to 0 °C, add 0.7 mol EDC·HCl, stir the reaction at room temperature for 3 h, wash with 0.1 mol / L hydrochloric acid, 0.3% sodium bicarbonate solution and saturated brine in sequence, dry, filter and concentrate, separate and purify to obtain the trisubstituted esterification product, which is tris-9-cis-retinoic acid rapamycin ester; S4. Rapamycin tris-9-cis-retinoic acid ester and IL-2 were added to RPMI 1640 complete culture medium to prepare a culture medium containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, 50 U / mL streptomycin, 30 nmol / L rapamycin tris-9-cis-retinoic acid ester, and 20 U / mL IL-2.

[0046] S5. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium to adjust CD4 levels after stimulation. + CD25 + The number of regulatory T cells was 5 × 10 4 The concentration of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads was increased to 1 / mL, along with CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4+. + CD25 + The regulatory T cells were cultured at a ratio of 4:1, at 37°C, with 5% CO2 and saturated humidity. After 7 days, the CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads were removed and cultured under the original conditions. After 10 days, the medium was replaced with fresh RPMI 1640 complete medium (containing 10% inactivated fetal bovine serum, 50 U / mL penicillin, and 50 U / mL streptomycin). IL-2 was added to the culture medium to a final concentration of 15 U / mL, and DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added at a cell count of 4. The cells were then cultured under the original conditions for another 3 days. The expanded regulatory T cells were isolated using the MACS immunomagnetic bead method.

[0047] Test Example 1 The in vitro expanded regulatory T cells obtained from Examples 1-3 and Comparative Examples 1-5 were counted, and the expansion fold was calculated by comparing them with the cells when the culture medium was added.

[0048] Take the in vitro expanded regulatory T cell suspensions from Examples 1-3 and Comparative Examples 1-5, stain them with trypan blue, count the cells under a microscope, and calculate the viability.

[0049] The in vitro expanded regulatory T cell suspensions from Examples 1-3 and Comparative Examples 1-5 were subjected to triple monoclonal antibody staining, including CD4 antibody and CD25 antibody, and intracellular FoxP3 antibody or isotype control staining. The proportion of triple antibody positive cells was detected by flow cytometry.

[0050] The results are shown in Table 1.

[0051] Table 1

[0052] As shown in the table above, the methods described in Examples 1-3 can rapidly expand regulatory T cells with a high viability rate. Among these, CD4+... + CD25 + FoxP3 + The purity of the triple-positive cells is high.

[0053] Figure 2 This is a diagram showing the sorting results of CD4+CD25+ T cells in Example 1. As can be seen from the figure, CD4+CD25+ T cells... + CD25 + FoxP3 + The purity of the triple-positive cells is high.

[0054] Test Example 2: Self- or Allogeneic CD4 + CD25 – T cell proliferation inhibition test Use CFSE to label autologous or allogeneic CD4. + CD25 – T cells. CFSE-labeled effector CD4+ + CD25 – T cells (Teff) and in vitro expanded regulatory T cells (Treg) from Examples 1-3 and Comparative Examples 1-5 were co-cultured. The initial Teff number was 5 × 10⁻⁶. 4 The sample size was 1 / mL. The positive group (with magnetic beads but no Treg) and the mixed culture groups of Examples 1-3 and Comparative Examples 1-3 were set up (Teff: Treg prepared in Examples 1-3 or Comparative Examples 1-4 was 1:1).

[0055] Cells were cultured in 96-well plates using RPMI 1640. The co-culture system contained 10% human AB serum, 2 mmol / L glutamine, 25 mmol / L HEPES buffer, 50 U / mL penicillin, 50 g / mL streptomycin, 50 µmol / L 2-ME, and 400 U / mL IL-2. Except for the negative control group, all other groups were supplemented with CD3 / CD28 monoclonal antibody-coated amplification magnetic beads (Teff: magnetic beads = 1:2).

[0056] After culturing cells in 37℃, 5% CO2 incubator for 5 days, the magnetic beads were removed using a magnetic rack, and the cells were collected. CFSE fluorescence was detected by flow cytometry, and CD4+ was analyzed. + CD25 – The proliferation of Teff. (FACS Canto) TM II. Flow cytometry analysis. All experiments were performed in triplicate, with at least five replicates per well.

[0057] CD4 in the positive group + CD25 – Teff proliferation percentage minus CD4 in the experimental group + CD25 – The percentage of Teff proliferation, divided by the percentage of proliferation in the positive group, yields the Treg-CD4 proliferation rate for each group. + CD25 – The inhibition rate of Teff proliferation.

[0058] The results are shown in Table 2.

[0059] Table 2

[0060] As shown in the table above, the expanded regulatory T cells obtained in Examples 1-3 of this invention exhibit improved resistance to autologous or allogeneic CD4+. + CD25 - Teff proliferation showed good inhibitory effects in all groups, with significant differences among the groups. Furthermore, the inhibitory effects on autologous and allogeneic CD4+ were observed in each group. + CD25 - There was no statistically significant difference in the inhibition of T cell proliferation among the groups. It can be concluded that the expanded regulatory T cells obtained in vitro showed no significant difference in the inhibition rate of autologous and allogeneic Teff proliferation among the groups.

[0061] Figure 3 This refers to one case of regulatory T cells expanded in vitro from each group acting on autologous CD4. + CD25 - Flow cytometry plot of Teff proliferation inhibition. As shown in the figure, the expanded regulatory T cells obtained in Examples 1-3 inhibited the proliferation of autologous CD4+.+ CD25 - Teff proliferation was effectively inhibited.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro expansion and culture of regulatory T cells, characterized in that, Includes the following steps: S1. Peripheral blood CD4 + CD25 + Isolation and purification of regulatory T cells; S2. Peripheral blood CD4 + CD25 + Regulatory T cells were co-cultured with dendritic cells, then isolated and purified to obtain stimulated CD4+. + CD25 + Regulatory T cells; S3. Rapamycin tris-9-cis-retinoic acid was prepared by esterification of 9-cis-retinoic acid with rapamycin. S4. Rapamycin tris-9-cis-retinoic acid ester and IL-2 were added to RPMI 1640 complete culture medium to prepare the culture medium; S5. Stimulated CD4 + CD25 + Regulatory T cells were added to the culture medium and stimulated with DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies. After 6-8 days, the DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were removed and cultured again. After 10 days, the medium was replaced with fresh RPMI 1640 complete culture medium, and 9-cis-retinoic acid and DynaBeads magnetic beads coated with CD3 / CD28 monoclonal antibodies were added and cultured again. The expanded regulatory T cells were isolated using the MACS immunomagnetic bead method.

2. The cultivation method according to claim 1, characterized in that, The peripheral blood CD4 mentioned in step S1 + CD25 + The method for isolating and purifying regulatory T cells involved separating peripheral blood single cells using the Ficoll density gradient separation method, followed by separation of peripheral blood CD4 cells using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells.

3. The cultivation method according to claim 1, characterized in that, The co-culture method described in step S2 involves using peripheral blood CD4+. + CD25 + Regulatory T cells and dendritic cells were seeded in RPMI 1640 medium and cultured at 36-38°C with 5% CO2 for 3-5 days. Stimulated CD4+ cells were then isolated using the MACS immunomagnetic bead method. + CD25 + Regulatory T cells, peripheral blood CD4 + CD25 + The ratio of regulatory T cells to dendritic cells is 8-12:

1.

4. The cultivation method according to claim 1, characterized in that, In step S3, the molar ratio of 9-cis-retinoic acid to rapamycin is 3-3.3:1, and the reaction time is 2-4 hours.

5. The cultivation method according to claim 1, characterized in that, The culture medium described in step S4 contains 8-12% inactivated fetal bovine serum, penicillin 40-60 U / mL, streptomycin 40-60 U / mL, rapamycin tris-9-cis-retinoic acid ester 25-35 nmol / L, and IL-2 15-25 U / mL.

6. The cultivation method according to claim 1, characterized in that, When adding the culture medium in step S5, adjust the CD4 levels after stimulation. + CD25 + The number of regulatory T cells is 10 4 -10 5 The CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads and stimulated CD4 cells / mL + CD25 + The ratio of regulatory T cells is 3-5:

1.

7. The cultivation method according to claim 1, characterized in that, The stimulation culture conditions described in step S5 are 36-38℃, 5% CO2, and saturated humidity; the fresh RPMI 1640 complete culture medium contains 8-12% inactivated fetal bovine serum, 40-60 U / mL penicillin, and 40-60 U / mL streptomycin; in the continued stimulation culture, 9-cis retinoic acid is added to the culture medium to a final concentration of 30-50 nmol / L; the amount of CD3 / CD28 monoclonal antibody-coated DynaBeads magnetic beads added is 3-5 times the number of cells.

8. An in vitro expanded regulatory T cell prepared by the culture method according to any one of claims 1-7.

9. The use of the in vitro expanded regulatory T cells as described in claim 8 in the preparation of a medicament for treating immune diseases.

10. A culture medium, characterized in that, The culture medium contains 8-12% inactivated fetal bovine serum, penicillin 40-60 U / mL, streptomycin 40-60 U / mL, rapamycin tris-9-cis-retinoic acid ester 25-35 nmol / L, and IL-2 15-25 U / mL.

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