Umbilical cord blood-derived regulatory T cell amplification culture medium and use method thereof

Abstract

The invention discloses a cord blood-derived regulatory T cell amplification culture medium and a use method thereof, and relates to the field of animal cell culture and immune cell therapy, the culture medium is a serum-free culture medium, the culture medium is composed of a basic culture medium, a serum substitute combination, a combination of four cell factors, an immunosuppressor, an apparent stabilizer and an antioxidant, and all the components play a role synergistically; according to the use method, cell amplification is realized through a collaborative process of staged environmental regulation and targeted activation. According to the method, the problems of exogenous pollution and batch difference caused by dependence of a serum-containing system on cord blood-derived regulatory T cell amplification in the prior art are solved, the defect that a serum-free scheme is difficult to consider the amplification efficiency and the function stability at the same time is overcome, the cell amplification quality and the clinical application safety are guaranteed, and the method is suitable for large-scale clinical transformation requirements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal cell culture and immune cell therapy, and particularly relates to a culture medium for expanding umbilical cord blood-derived regulatory T cells and a use method thereof. BACKGROUND

[0002] Umbilical cord blood-derived regulatory T cells have important application value in the treatment of immune-related diseases, and the key prerequisite for their clinical transformation is to achieve efficient and stable expansion in vitro. During in vitro expansion, the culture medium as a core element directly affects the proliferation efficiency, functional stability and clinical use safety of cells, so developing a suitable expansion system has become a research focus in the field. Currently, umbilical cord blood-derived regulatory T cell in vitro expansion technology has become a research hotspot in the field of cell culture, and related technologies mainly focus on culture medium component optimization and culture process improvement to meet the strict requirements of clinical cell quantity and quality.

[0003] Existing umbilical cord blood-derived regulatory T cell expansion technology generally relies on serum-containing culture systems. Serum components are complex and heterologous in origin, which not only easily introduces exogenous contamination risk, but also has significant batch differences, resulting in large fluctuations in cell expansion effect, seriously affecting the consistency and safety of clinical application. At the same time, although some serum-free expansion schemes try to avoid serum dependence, they mostly use single serum replacement components, which have the defect of unbalanced nutrient supply, making it difficult to balance cell expansion efficiency and long-term functional stability, and unable to meet the needs of large-scale clinical application.

[0004] In addition, in the existing expansion process, the activation signal regulation and the adaptability of the culture environment are insufficient, further exacerbating the imbalance between expansion efficiency and functional stability. Conventional activation schemes are prone to activate non-target cells, and a single culture environment is difficult to match the growth needs of cells at different stages, limiting the improvement of cell expansion quality. Therefore, developing a serum-free, component-specific umbilical cord blood-derived regulatory T cell expansion culture medium that can synergistically ensure expansion efficiency and functional stability, and an appropriate use method, has become a technical problem to be solved in the field. SUMMARY

[0005] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present application provide a culture medium for expanding umbilical cord blood-derived regulatory T cells and a use method thereof, which solves the problems of exogenous contamination and batch difference caused by the dependence of umbilical cord blood-derived regulatory T cell expansion on serum-containing systems in the prior art, and the problem that serum-free schemes are difficult to balance expansion efficiency and functional stability.

[0006] To solve the above problems, the present application provides the following technical solutions: The umbilical cord blood-derived regulatory T cell expansion culture medium is a serum-free culture medium, which is composed of a basic culture medium, a serum substitute combination, a combination of four cytokines, an immunosuppressant, an epigenetic stabilizer and an antioxidant. The serum substitute combination is a complex of recombinant human albumin and humanized lipoprotein, and plant-derived enzymatic peptides of chickpea protein, and both of them provide nutritional support for umbilical cord blood-derived regulatory T cells. The combination of the four cytokines is interleukin 2, interleukin 33, interleukin 27 and transforming growth factor beta 3, and the four cytokines synergistically regulate the proliferation and function stability of umbilical cord blood-derived regulatory T cells. The immunosuppressant is a sirolimus derivative containing a 42-O-alkyl substituted structure. The epigenetic stabilizer is 4-phenylbutyric acid propyl ester. The antioxidant is a complex of ergothioneine and vitamin E succinate.

[0007] Further, the basic culture medium is X-VIVO15 or RPMI1640. Here, the culture medium is a basic culture medium specially for immune cells, which has clear ingredients and strong adaptability, and can provide a basic nutritional environment for umbilical cord blood-derived regulatory T cells.

[0008] Further, the concentration of each component in the serum substitute combination is 8-12 g / L of the complex of recombinant human albumin and humanized lipoprotein, and 35-45 ng / mL of plant-derived enzymatic peptides of chickpea protein. Here, the optimal interval is screened based on the nutritional requirements of cells, which can not only ensure that the two components synergistically provide comprehensive nutrition (such as protein, lipid, amino acid, etc.), but also avoid the problems of osmotic pressure imbalance caused by too high concentration, component waste and nutritional deficiency caused by too low concentration, further strengthen the nutritional adaptability of the serum-free system, and support efficient cell proliferation.

[0009] Further, in the combination of the four cytokines, the concentration of interleukin 2 is 880-920 U / mL, the concentration of interleukin 33 is 20-30 ng / mL, the concentration of interleukin 27 is 15-20 ng / mL, and the concentration of transforming growth factor beta 3 is 3-8 ng / mL. Here, the concentration combination can synergistically activate cell proliferation signals while maintaining stable expression of FOXP3 molecules (guaranteeing cell function), solving the imbalance problem of "low proliferation efficiency" or "unstable function" caused by single cytokine regulation in the prior art, and ensuring that the expanded cells meet the quantity demand and have normal immune regulation function.

[0010] Further, the concentration of the sirolimus derivative containing a 42-O-alkyl substituted structure is 90-110 nmol / L, and the concentration of the apparent stabilizer 4-phenylbutyric acid propyl ester is 1.0-2.0 mmol / L. The concentration range of the sirolimus derivative containing a 42-O-alkyl substituted structure herein can specifically promote the proliferation of regulatory T cells, inhibit the activation of non-target cells such as effector T cells, and improve cell purity; and the concentration of 4-phenylbutyric acid propyl ester can optimize the epigenetic regulation effect, further stabilize the FOXP3 expression, and avoid cell function exhaustion. The precise matching of the concentrations of the two can synergistically ensure the quality of the expanded cells.

[0011] Further, the concentration of the complex of the antioxidant ergothioneine and vitamin E succinate is 70-90 μmol / L. The concentration range of the complex of ergothioneine and vitamin E succinate herein can effectively scavenge reactive oxygen species generated during cell expansion, reduce the damage of oxidative stress to cells, and maintain cell viability and functional integrity.

[0012] The umbilical cord blood-derived regulatory T cell expansion culture medium of the present application aims to construct a clear composition, safe and controllable, and synergistically efficient expansion system as the core goal in view of the defects of the existing umbilical cord blood-derived regulatory T cell expansion technology, such as exogenous contamination and batch difference in the serum-containing system, unbalanced nutrition supply in the serum-free scheme, and difficulty in balancing the expansion efficiency and functional stability. The umbilical cord blood-derived regulatory T cell expansion culture medium of the present application is designed to have a basic culture medium as a stable carrier, a serum substitute combination to synergistically provide comprehensive nutrition, a combination of four kinds of cytokines to precisely regulate cell proliferation and functional stability, a sirolimus derivative containing a 42-O-alkyl substituted structure to specifically inhibit the activation of non-target cells, 4-phenylbutyric acid propyl ester to strengthen the epigenetic stability, and a complex of ergothioneine and vitamin E succinate to resist oxidative damage. Through the functional adaptation and synergistic effect of each component, the umbilical cord blood-derived regulatory T cell expansion culture medium realizes the efficient and stable expansion of umbilical cord blood-derived regulatory T cells from multiple dimensions of nutrition supply, signal regulation, purity improvement, functional stability, and damage protection, and meets the core needs of cell quality and safety for clinical transformation.

[0013] As a general inventive concept, the present application provides a method for using the umbilical cord blood-derived regulatory T cell expansion culture medium as described above, comprising the following steps, S1. Umbilical cord blood processing and cell sorting, obtaining mononuclear cells by density gradient centrifugation of umbilical cord blood samples, and obtaining CD4+CD25+CD127lo / - umbilical cord blood-derived regulatory T cells by immunomagnetic bead sorting; S2. Hypoxic pretreatment of sorted cells, inoculating the sorted umbilical cord blood-derived regulatory T cells into the expansion culture medium, and pretreating under a hypoxic culture condition of 37℃, 5% CO2, and 5% O2; S3. Activation culture, soluble CD3 / CD28 / OX40L triple activator is added to the pretreated cell system, and the culture is continued under the low-oxygen culture condition of 37°C, 5% CO2, and 5% O2; S4. Stage expansion culture, after the activation culture is completed, expansion medium is supplemented to the system, the combined concentration of four cytokines is adjusted, and the culture is continued under the normoxic culture condition of 37°C, 5% CO2, and 21% O2; S5. Fed-batch culture, the expansion medium is supplemented every 3 days during the expansion culture, and the concentration of each component in the system is maintained consistent with the expansion medium after the supplementation; S6. Cell harvesting, the cord blood-derived regulatory T cells are collected on the 14th day of culture.

[0014] Further, the low-oxygen pretreatment culture time in step S2 is 24-36 h, the inoculated cell density is 1.5×10 6 -2.5×10 6 The cell viability is ≥90% at the end of the pretreatment. The time range and cell density range of the low-oxygen pretreatment are optimized based on the initial activity characteristics of the cord blood-derived regulatory T cells, which can induce the initial stable expression of FOXP3 molecules through the low-oxygen environment, lay a foundation for the subsequent function maintenance, avoid the problems of insufficient nutrient competition caused by too high density and low-efficiency cell signal transmission caused by too low density, and ensure that the cells entering the activation stage have good activity, avoid the influence of excessive or insufficient pretreatment on the subsequent expansion efficiency, and further improve the controllability of the entire culture process.

[0015] Further, the addition concentration of the soluble CD3 / CD28 / OX40L triple activator in step S3 is 30-40 μL / mL, and the activation culture time is 48-72 h. The concentration range of the soluble CD3 / CD28 / OX40L triple activator can specifically bind to the surface receptors of the cord blood-derived regulatory T cells, efficiently start the proliferation signal, and reduce the activation of non-target cells such as effector T cells, solving the problem of non-target cell contamination in the conventional activation scheme. The limitation of the activation culture time is to enable the cells to fully receive the activation signal and complete the initial proliferation preparation, avoid the problems of insufficient proliferation start caused by too short activation time and cell overactivation or functional exhaustion caused by too long activation time, and ensure the balance between the efficiency and cell quality in the activation stage.

[0016] Further, when the step S4 is transferred to the normoxic culture condition for the phase-type expansion culture, the concentration of the combination of the four cytokines is adjusted synchronously, interleukin 2 is 1100-1300 U / mL, interleukin 33 is 10-14 ng / mL, interleukin 27 is 8-12 ng / mL, and transforming growth factor beta 3 is 2-5 ng / mL; the volume of the expansion culture medium supplemented in the step S5 is 50%-70% of the volume of the original culture medium. Here, when the normoxic culture condition is transferred, the concentration of the four cytokines is adjusted synchronously, which can adapt to the change of the growth requirement of the cells from the activation period to the expansion period, the high concentration of interleukin 2 ensures efficient proliferation, and the adjusted concentration of the other cytokines maintains the stability of the function, avoiding the imbalance between proliferation and function caused by asynchronous concentration adjustment. The limitation of the volume of the feed, combined with the requirement of maintaining the consistency of the component concentration after the feed, can ensure that the nutrition supply is sufficient and the environment is stable during the expansion process, avoiding the imbalance of the osmotic pressure caused by too much feed and the lack of nutrition caused by too little feed, and at the same time, it adapts to the large-scale expansion scene, and ensures the uniformity of the quality of batch cell expansion.

[0017] The use method of the umbilical cord blood-derived regulatory T cell expansion culture medium of the present application, through the precise matching of the phase-type oxygen environment regulation and the targeted activation signal, combined with the dynamic adjustment of the cytokine concentration and the standardization of the feeding strategy, adapts to the whole cycle growth requirement of the cells from the initial pretreatment to the large-scale expansion, reduces the activation of non-target cells to improve the cell purity, and at the same time, through the synergistic stability of the environment and the nutrition, the cell viability and the functional integrity are guaranteed, finally realizes the efficient and uniform expansion of the clinical level cells, and meets the strict requirements of the process controllability and the cell quality for the large-scale clinical transformation.

[0018] Compared with the prior art, the present application has the following advantages: (1) The present application eliminates the hidden danger of external pollution by designing a serum-free culture medium, and at the same time avoids the batch difference caused by the heterogeneity of serum source, effectively improves the safety and consistency of cell expansion; (2) The present application guarantees efficient proliferation and maintains the stability of the core function of regulatory T cells through the synergistic effect of the defined components, which makes up for the defects of traditional serum-free schemes that cannot balance efficiency and function; (3) The present application reduces the activation of non-target cells such as effector T cells through the synergistic effect of targeted activation signal and specific immunosuppressants, and guarantees the specificity of the immune regulation function of the cells; (4) The present application adapts to the whole cycle growth requirement of the cells through the synergistic strategy of phase-type oxygen environment regulation and dynamic nutrition supply, improves the stability and repeatability of the expansion process, and meets the requirements of large-scale production scene; (5) The present application effectively resists oxidative stress damage through antioxidant components, and maintains the cell viability and functional integrity through the regulation of epigenetic stabilizers. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a flow chart of an umbilical cord blood derived regulatory T cell expansion medium and a method of using the same according to the present application. DETAILED DESCRIPTION

[0020] For the purpose of facilitating the understanding of the present application, the present application will be described in more detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present application.

[0022] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0023] Example 1 An umbilical cord blood derived regulatory T cell expansion medium according to the present application is a serum-free medium, which is composed of a basal medium, a serum substitute combination, a combination of four cytokines, an immunosuppressant, an epigenetic stabilizer and an antioxidant. The basal medium is X-VIVO 15. The serum substitute combination is a complex of recombinant human albumin and humanized lipoprotein and plant-derived enzymatic peptides of chickpea protein, which synergistically provide nutritional support for umbilical cord blood derived regulatory T cells. The concentration of the complex of recombinant human albumin and humanized lipoprotein is 10 g / L, and the concentration of the plant-derived enzymatic peptides of chickpea protein is 40 ng / mL. The combination of four cytokines is interleukin 2, interleukin 33, interleukin 27 and transforming growth factor β3, which synergistically regulate the proliferation and functional stability of umbilical cord blood derived regulatory T cells. The concentration of interleukin 2 is 900 U / mL, the concentration of interleukin 33 is 25 ng / mL, the concentration of interleukin 27 is 17 ng / mL, and the concentration of transforming growth factor β3 is 5 ng / mL. The immunosuppressant is a sirolimus derivative containing a 42-O-alkyl-substituted structure, and the concentration is 100 nmol / L. The epigenetic stabilizer is 4-phenylbutyric acid propyl ester, and the concentration is 1.5 mmol / L. The antioxidant is a complex of ergothioneine and vitamin E succinate, and the concentration is 80 μmol / L.

[0024] Reference Figure 1 The method of using the umbilical cord blood derived regulatory T cell expansion medium according to the present embodiment includes the following steps, S1. Umbilical cord blood processing and cell sorting, taking the umbilical cord blood sample, separating the mononuclear cells by density gradient centrifugation, and obtaining CD4+CD25+CD127lo / - umbilical cord blood-derived regulatory T cells by immunomagnetic bead sorting, the purity of the sorted cells is not less than 95%; S2. Hypoxic pretreatment of sorted cells, inoculating the sorted umbilical cord blood-derived regulatory T cells into the above-mentioned expansion medium, the inoculation cell density is 2×10 6 S2. Hypoxic pretreatment of sorted cells, inoculating the sorted umbilical cord blood-derived regulatory T cells into the above-mentioned expansion medium, the inoculation cell density is 2×10 6 S2. Hypoxic pretreatment of sorted cells, inoculating the sorted umbilical cord blood-derived regulatory T cells into the above-mentioned expansion medium, the inoculation cell density is 2×10 S3. Activation culture, adding soluble CD3 / CD28 / OX40L triple activator to the pretreated cell system, the concentration is 35 μL / mL, and the activation culture is continued under the condition of 37℃, 5% CO2, and 5% O2 hypoxia for 60h; S4. Stage expansion culture, after the activation culture is completed, the above-mentioned expansion medium is supplemented to the system, and the combined concentration of the four cytokines is adjusted synchronously, after the adjustment, interleukin 2 is 1200 U / mL, interleukin 33 is 12 ng / mL, interleukin 27 is 10 ng / mL, and transforming growth factor β3 is 3 ng / mL, and then the system is transferred to the condition of 37℃, 5% CO2, and 21% O2 normal oxygen culture for continuous culture; S5. Feed culture, the expansion culture is carried out once every 3 days, the above-mentioned expansion medium is supplemented, the supplemented volume is 60% of the original culture medium volume, and after the feeding, the concentration of each component in the system is maintained consistent with the expansion medium; S6. Cell harvest, umbilical cord blood-derived regulatory T cells are collected after 14 days of culture.

[0025] Example 2 The difference between this example and Example 1 is that the basic culture medium of the expansion medium is RPMI1640, and the rest of the culture medium components and the use method are completely consistent with Example 1.

[0026] Comparative Example 1 The difference between this comparative example and Example 1 is that the expansion medium is replaced by X-VIVO15 medium containing 10% fetal bovine serum, and the serum substitute combination and antioxidant are removed, and the rest of the culture medium components and the use method are completely consistent with Example 1.

[0027] Comparative Example 2 The difference between this comparative example and Example 1 is that the serum substitute combination is replaced by single recombinant human albumin with a concentration of 10 g / L, and the rest of the culture medium components and the use method are completely consistent with Example 1.

[0028] Comparative Example 3 The difference between this comparative example and Example 1 is that interleukin-27 was removed from the combination of the four cytokines, while the concentrations of the other three cytokines, interleukin-2, interleukin-33, and transforming growth factor β3, remained at 900 U / mL, 25 ng / mL, and 5 ng / mL, respectively. All other culture medium components and process parameters of the usage method were completely consistent with those of Example 1.

[0029] Comparative Example 4 The difference between this comparative example and Example 1 is that the sirolimus derivative containing the 42-O-alkyl-substituted structure was removed from the amplification culture medium, while the remaining culture medium components and all process parameters of the usage method are completely consistent with Example 1.

[0030] Comparative Example 5 The difference between this comparative example and Example 1 is that the apparent stabilizer propyl 4-phenylbutyrate was removed from the amplification culture medium, while all other culture medium components and process parameters of the usage method are completely consistent with Example 1.

[0031] Comparative Example 6 The difference between this comparative example and Example 1 is that the complex of the antioxidant ergothioneine and vitamin E succinate was removed from the amplification culture medium. All other culture medium components and process parameters of the usage method are completely consistent with Example 1.

[0032] Comparative Example 7 The difference between this comparative example and Example 1 is that the hypoxia pretreatment stage was removed, and the culture environment was maintained at 37°C, 5% CO2, and 21% O2 throughout the entire process. All other culture medium components and process parameters of the usage method were completely consistent with those of Example 1.

[0033] Comparative Example 8 The difference between this comparative example and Example 1 is that the soluble CD3 / CD28 / OX40L triple activator used in the activation culture stage was replaced with a soluble CD3 / CD28 activator at a concentration of 35 μL / mL. All other culture medium components and process parameters of the usage method are completely consistent with Example 1.

[0034] Comparative Example 9 The difference between this comparative example and Example 1 is that the combined concentration of the four cytokines was not adjusted during the staged amplification culture. The concentrations of the four cytokines in the amplification culture medium added after activation remained at their initial values: interleukin-2 at 900 U / mL, interleukin-33 at 25 ng / mL, interleukin-27 at 17 ng / mL, and transforming growth factor β3 at 5 ng / mL. All other culture medium components and process parameters of the usage method were completely consistent with those of Example 1.

[0035] Comparative Example 10 The difference between this comparative example and Example 1 is that the feeding strategy is changed to non-fixed periodic feeding. When the culture medium turns yellow and pH < 7.2, 10%-40% of the volume of amplification culture medium is randomly added. The component concentration is not deliberately maintained to be consistent. All other culture medium components and process parameters of the usage method are completely consistent with Example 1.

[0036] The testing method is as follows: Fold of expansion: The number of cells initially inoculated and the number of cells harvested after 14 days of culture were counted using a fully automated cell counter. The fold of expansion was calculated as the number of harvested cells / the number of initial cells. Cell purity (%): After the harvested cells were fixed and perforated, they were incubated with CD4, CD25 fluorescent antibodies and FOXP3 specific fluorescent antibodies. The proportion of CD4+CD25+FOXP3+ cells in the total number of cells was detected and analyzed by flow cytometry. FOXP3 positivity rate (%): After the harvested cells were fixed and perforated, they were incubated with FOXP3-specific fluorescent antibody, and the proportion of FOXP3-positive cells to CD4+CD25+ cells was detected and calculated by flow cytometry. Cell viability (%): Harvested cells were stained with 0.4% trypan blue and counted under an optical microscope to determine the percentage of unstained cells (live cells) out of the total cells. Batch variation coefficient (%): Set up 3 parallel batches and culture them according to the same protocol. Calculate the standard deviation and mean with the amplification fold as the core indicator. The batch variation coefficient is obtained by (standard deviation / mean) × 100%. IL-10 secretion (pg / mL): Cell supernatant was collected after 14 days of culture, and the IL-10 concentration was detected using a human IL-10 enzyme-linked immunosorbent assay (ELISA) kit, following the instructions of the kit.

[0037] Table 1: Experimental Results of Examples 1-2 and Comparative Examples 1-10

[0038] In summary, referring to Table 1, the amplification fold, cell purity, FOXP3 positivity rate, cell viability, and IL-10 secretion of the embodiment were significantly higher than those of the comparative example, and the batch-to-batch difference coefficient was significantly lower than that of the comparative example, demonstrating the technical advantages of the combination of serum-free components and staged process of the present invention.

[0039] Comparative Example 1 used a serum-containing culture medium. The heterogeneity of serum sources led to a significant increase in the batch-to-batch variation coefficient, and the lack of precise nutritional support from a combination of serum substitutes resulted in a decrease in cell proliferation rate and cell function-related indicators. Comparative Example 2 used a single serum substitute, which failed to achieve synergistic nutrient supply, resulting in insufficient cell proliferation efficiency and viability. Comparative Example 3 lacked interleukin-27, disrupting the synergistic regulatory effect of the four cytokines, and reducing cell proliferation efficiency and FOXP3 expression stability. Comparative Example 4 removed immunosuppressants, which could not specifically inhibit the activation of non-target cells, resulting in a significant decrease in cell purity. Comparative Example 5 removed epigenetic stabilizers, which could not maintain stable FOXP3 molecule expression, and caused a significant decline in core cell function indicators. Comparative Example 6: Removal of antioxidants resulted in oxidative stress damage to cells, leading to a significant decrease in cell viability. Comparative Example 7: Full-process normoxic culture lacked the promoting effect of hypoxic pretreatment on FOXP3 expression, resulting in insufficient cell functional stability. Comparative Example 8: Use of only soluble CD3 / CD28 activators, lacking the OX40L targeted activation signal, resulted in poor specific activation of target cells, leading to a decrease in cell purity and functional indicators. Comparative Example 9: Failure to adjust cytokine concentrations during the expansion phase prevented adaptation to the changing growth requirements of cells from the activation phase to the expansion phase, disrupting the balance between proliferation and function. Comparative Example 10: Use of non-standardized feeding resulted in fluctuations in nutrient concentration and osmotic pressure in the culture system, poor batch consistency, and impact on cell function-related indicators.

[0040] Example 1: The synergistic effect of each component in the serum-free culture medium, combined with staged oxygen environment regulation, targeted triple activation, dynamic cytokine adjustment and standardized feeding process, comprehensively ensures cell proliferation efficiency, purity, functional stability and batch consistency, thus presenting optimal data.

[0041] Example 2 only replaced the basal culture medium with RPMI 1640. All other serum-free components and processes remained the same as in Example 1. The basal culture medium was well compatible with other components and processes, so the data were close to those in Example 1.

Claims

1. A culture medium for expanding regulatory T cells derived from umbilical cord blood, characterized in that: The serum-free culture medium consists of a basal medium, a serum substitute combination, a combination of four cytokines, an immunosuppressant, an epigenetic stabilizer, and an antioxidant. The serum substitute combination comprises a complex of recombinant human albumin and humanized lipoprotein, and plant-derived chickpea protease-digested peptides, which synergistically provide nutritional support for umbilical cord blood-derived regulatory T cells. The combination of four cytokines comprises interleukin-2, interleukin-33, interleukin-27, and transforming growth factor β3, which synergistically regulate the proliferation and functional stability of umbilical cord blood-derived regulatory T cells. The immunosuppressant is a sirolimus derivative containing a 42-O-alkyl-substituted structure. The epigenetic stabilizer is propyl 4-phenylbutyrate. The antioxidant is a complex of ergothioneine and vitamin E succinate.

2. The umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 1, characterized in that: The basal culture medium is X-VIVO15 or RPMI1640.

3. The umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 1, characterized in that: The concentrations of each component in the serum substitute combination are 8-12 g / L for the complex of recombinant human albumin and humanized lipoprotein, and 35-45 ng / mL for plant-derived chickpea protease hydrolysate peptides.

4. The umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 1, characterized in that: In the combination of the four cytokines, the concentration of interleukin-2 is 880-920 U / mL, the concentration of interleukin-33 is 20-30 ng / mL, the concentration of interleukin-27 is 15-20 ng / mL, and the concentration of transforming growth factor β3 is 3-8 ng / mL.

5. The umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 1, characterized in that: The concentration of the sirolimus derivative containing the 42-O-alkyl substituted structure is 90-110 nmol / L, and the concentration of the apparent stabilizer propyl 4-phenylbutyrate is 1.0-2.0 mmol / L.

6. The umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 1, characterized in that: The concentration of the complex of the antioxidant ergothioneine and vitamin E succinate is 70-90 μmol / L.

7. A method of using a culture medium for expanding umbilical cord blood-derived regulatory T cells, applied to the culture medium according to any one of claims 1-6, characterized in that: Includes the following steps, S1. Umbilical cord blood processing and cell sorting: Umbilical cord blood samples were collected and separated into mononuclear cells by density gradient centrifugation. CD4+CD25+CD127lo / - umbilical cord blood-derived regulatory T cells were obtained by immunomagnetic bead sorting. S2. Sorted cells were pretreated with hypoxia. The sorted umbilical cord blood-derived regulatory T cells were seeded into the expansion medium and pretreated under hypoxia culture conditions of 37°C, 5% CO2, and 5% O2. S3. Activation culture: Add soluble CD3 / CD28 / OX40L triple activator to the pretreated cell system and continue to culture under hypoxic conditions of 37℃, 5% CO2, and 5% O2. S4. Staged amplification culture: After activation culture is completed, amplification culture medium is added to the system, the combined concentration of the four cytokines is adjusted, and the system is transferred to normoxic culture conditions of 37℃, 5% CO2, and 21% O2 for continued culture. S5. Feeding culture: During the amplification culture, feeding is performed every 3 days to add amplification medium. After feeding, the concentration of each component in the system is maintained at the same level as that of the amplification medium. S6. Cell harvesting: Cultured until day 14, collect umbilical cord blood-derived regulatory T cells.

8. The method of using the umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 7, characterized in that: In step S2, the hypoxia pretreatment culture time is 24-36 hours, and the inoculated cell density is 1.5 × 10⁻⁶ cells / year. 6 -2.5×10 6 Cells / mL, and the pretreatment was terminated when cell viability was ≥90%.

9. The method of using the umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 7, characterized in that: In step S3, the concentration of the soluble CD3 / CD28 / OX40L triple activator is 30-40 μL / mL, and the activation culture time is 48-72 h.

10. The method of using the umbilical cord blood-derived regulatory T cell expansion culture medium according to claim 7, characterized in that: In step S4, when transferring to normoxic culture conditions for staged amplification culture, the combined concentration of the four cytokines is simultaneously adjusted: interleukin-2 at 1100-1300 U / mL, interleukin-33 at 10-14 ng / mL, interleukin-27 at 8-12 ng / mL, and transforming growth factor β3 at 2-5 ng / mL. In step S5, the volume of amplification culture medium added is 50%-70% of the original culture medium volume.

Images