Efficient expansion culture method of umbilical cord blood gamma delta T cells and application thereof

Abstract

The application provides a high-efficiency expansion culture method of umbilical cord blood gamma delta T cells and application thereof, and belongs to the technical field of biological medicine. Through a specific two-stage strategy of "initial induction culture + expansion culture", the application is simple in operation, stable in system, and high in success rate, effectively overcomes the defects of limited autologous blood source, unstable expansion and low cell activity in the prior art, and lays a foundation for large-scale preparation and allogeneic application of gamma delta T cells. The umbilical cord blood gamma delta T cells obtained by the method of the application are excellent in quantity, purity, activity and anti-tumor function, and have stronger tumor cell killing ability, and are especially suitable for adoptive immunotherapy. The application breaks through the technical bottleneck of the traditional culture system, provides a high-efficiency, stable and clinically convertible gamma delta T cell expansion scheme, and has important application prospect and market value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a highly efficient method for expanding and culturing umbilical cord blood γδT cells and its application. Background Technology

[0002] γδT cells are an important subset of immune cells in the human body. Their surface T cell receptors are composed of γ and δ chains, distinguishing them from the dominant αβT cells. γδT cells are mainly distributed in mucous membranes, skin, and peripheral blood. They are characterized by rapid antigen recognition and do not require major histocompatibility complex (MHC)-restricted presentation, playing a crucial role in anti-infection, anti-tumor activity, and immune regulation. Studies have shown that γδT cells can directly kill virus- or bacterial-infected cells by secreting perforin and granzymes, and can also induce tumor cell apoptosis by recognizing overexpressed stress antigens (such as MICA / MICB) on the surface of tumor cells. Furthermore, some γδT cell subsets are involved in tissue repair and inflammation regulation, showing broad clinical application prospects.

[0003] However, γδT cells constitute a very small percentage of peripheral blood lymphocytes, typically only 1%–5% of total lymphocytes, severely limiting their availability and application in adoptive immunotherapy. To address this issue, researchers have explored various in vitro expansion methods, primarily including the use of bisphosphonates (such as zoledronic acid) combined with interleukin-2 (IL-2) to stimulate peripheral blood mononuclear cells for selective expansion of γδT cells. Bisphosphonates can activate the proliferation of Vγ9Vδ2 subtype γδT cells by inhibiting the mevalonate metabolic pathway, leading to the accumulation of intracellular phosphate antigens. In addition, studies have explored the addition of cytokines such as IL-15 and IL-7 to optimize expansion, or the use of magnetic bead sorting technology to enrich γδT cells before culture.

[0004] Current expansion methods still have many limitations: on the one hand, the expansion efficiency of autologous blood-derived γδT cells is greatly affected by factors such as donor age and physical condition, making it difficult to consistently obtain a sufficient number of cells; on the other hand, existing culture systems often focus on single cytokines or simple combinations, resulting in limited cell expansion efficiency, and the biological activity and cytotoxic function of the expanded cells are often insufficient to meet clinical treatment needs. Furthermore, autologous cells cannot be universally applied, limiting their large-scale application. Therefore, developing a stable, efficient, and highly effective in vitro expansion method for obtaining high quantities and high activity of γδT cells is of great significance for promoting the clinical application of γδT cells in immunotherapy. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient method for expanding and culturing umbilical cord blood γδT cells and its application, which solves the technical problems of low expansion efficiency, insufficient cell activity, and difficulty in obtaining a sufficient number of highly bioactive cells for adoptive immunotherapy in existing in vitro γδT cell expansion methods.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for culturing umbilical cord blood γδT cells, comprising the following steps: Umbilical cord blood γδT cells were seeded into the initial system and subjected to initial induction culture to obtain cells after initial induction culture; The cells after initial induction culture were transferred into an amplification culture system for amplification culture. The initial system comprises 1640 culture medium, a first bisphosphonate, a combination of cytokines, and umbilical cord plasma; The amplification culture system includes 581 medium, second bisphosphonate, rapamycin, a combination of cytokines, and umbilical cord plasma; The combination of cytokines includes IL-2 and IL-15.

[0008] Preferably, the first bisphosphonate is zoledronic acid, and its final concentration in the initial system is 5~10 μmol / L; the second bisphosphonate is sodium ibandronate, and its final concentration in the amplification culture system is 5~10 μmol / L.

[0009] Preferably, in the cytokine combination, the concentration of IL-2 in the initial system is 500~1000 IU / mL, the concentration of IL-15 in the initial system is 500~1000 IU / mL, the concentration of IL-2 in the amplification culture system is 500~1000 IU / mL, and the concentration of IL-15 in the amplification culture system is 500~1000 IU / mL.

[0010] Preferably, the concentration of rapamycin in the amplification culture system is 50~200 nmol / L.

[0011] Preferably, the umbilical cord plasma is inactivated umbilical cord plasma, and its volume percentage concentration in both the initial system and the amplification culture system is 5-10%.

[0012] Preferably, the number of cells after the initial induction culture reaches 4 × 10⁻⁶. 5 When the number of wells is reached, proceed to the amplification culture step; The amplification and culture step includes adjusting the cell density to 2 × 10⁻⁶. 5 ~5×10 5 / mL.

[0013] Preferably, it also includes a umbilical cord blood mononuclear cell separation step and a γδT cell sorting step prior to the initial induction culture step.

[0014] Preferably, the procedure also includes a umbilical cord plasma treatment step prior to the initial induction culture step: after the separated umbilical cord plasma is inactivated by water bath, the upper layer of inactivated umbilical cord plasma is collected by centrifugation.

[0015] The present invention also provides umbilical cord blood γδT cells obtained by the above-described culture method.

[0016] The present invention also provides the application of the above-mentioned umbilical cord blood γδT cells in the preparation of drugs for adoptive immunotherapy.

[0017] The beneficial effects of this invention are: This invention provides a method for culturing umbilical cord blood γδT cells. Through a pioneering two-stage strategy of "initial induction culture + expansion culture," using specific combinations of bisphosphonates and cytokines in 1640 and 581 media respectively, the in vitro expansion efficiency and cell quality of umbilical cord blood-derived γδT cells are significantly improved. This method is not only simple to operate and has a stable system, but also boasts a high success rate, effectively overcoming the shortcomings of existing technologies such as limited autologous blood sources, unstable expansion, and low cell viability. It lays the foundation for the large-scale preparation and allogeneic application of γδT cells.

[0018] The umbilical cord blood γδT cells cultured using the method of this invention exhibit excellent performance in terms of quantity, purity, viability, and anti-tumor function, possessing stronger tumor cell killing ability, and are particularly suitable for adoptive immunotherapy. This invention breaks through the technical bottlenecks of traditional culture systems, providing a highly efficient, stable, and clinically applicable γδT cell expansion protocol, with significant application prospects and market value. Attached Figure Description

[0019] Figure 1 This is a photograph of the cells cultured according to the present invention; Figure 2 This is a flow cytometry analysis diagram of the cells cultured in this invention. Detailed Implementation

[0020] This invention provides a method for culturing umbilical cord blood γδT cells. Using umbilical cord blood as the starting material, this method achieves efficient expansion of umbilical cord blood-derived γδT cells through a specific culture medium combination and culture strategy. The method includes two core culture stages: an initial induction culture stage and a further expansion culture stage. Through the synergistic effect of these two stages, the expansion fold, purity, and biological activity of γδT cells can be significantly improved. This method is simple to operate, has good reproducibility, and is suitable for clinical-grade cell preparation, providing a reliable cell source for subsequent adoptive immunotherapy. Umbilical cord blood, as a source of immune cells, has advantages such as convenient collection, low immunogenicity, and is not affected by the donor's age and health condition; therefore, the method of this invention has good application prospects.

[0021] In the culture method of this invention, umbilical cord blood γδT cells obtained after separation and sorting are first seeded into an initial induction culture system. The purpose of this initial system is to activate the γδT cells and initiate their proliferation program. The initial system comprises 1640 medium, a first bisphosphonate, a cytokine combination, and umbilical cord blood plasma. 1640 medium is a widely used basal medium for lymphocyte culture, containing various amino acids, vitamins, and inorganic salts, which can meet the basic nutritional needs of cells in the early stages of culture. In this invention, the 1640 medium is preferably commercially available RPMI 1640 medium, which can be purchased from well-known brands such as Gibco and HyClone. Initial induction culture is usually carried out in 48-well or 24-well plates under the conditions of 37°C and 5% CO2, with a culture time generally of 3-7 days, until the cell number reaches a certain density. The cell seeding density at this stage can be adjusted according to actual conditions, preferably 1×10⁻⁶ cells / well. 5 ~5×10 5 / well. Through initial induction culture, γδT cells were effectively activated, laying the foundation for subsequent large-scale expansion.

[0022] Once the initial cell number reaches a certain level after induction culture, the cells are transferred to an expansion culture system for further expansion. This expansion culture system contains 581 medium, a second bisphosphonate, rapamycin, a cytokine combination, and umbilical cord plasma. 581 medium is an optimized lymphocyte expansion medium that, compared to 1640 medium, contains higher concentrations of glucose, amino acids, and buffer systems, making it more suitable for high-density cell culture. 581 medium is also commercially available. During the expansion culture phase, cells are typically transferred to T25 or T75 suspension flasks or cultured on a large scale using cell culture bags. During culture, the medium needs to be replenished or changed periodically according to cell growth to maintain a suitable cell density, preferably 2 × 10⁶ cells / year. 5 ~5×10 5 / mL. The expansion culture typically continues until day 14-18, during which time samples can be taken multiple times to assess cell number, viability, and phenotype. Through this stage of culture, the number of γδT cells can be expanded several hundredfold, with cell viability maintained above 98%.

[0023] The initial induction culture system is a key component of this invention. This system is based on 1640 medium, supplemented with a first bisphosphonate, a cytokine combination, and umbilical cord plasma. The first bisphosphonate is preferably zoledronic acid, whose mechanism of action is to inhibit farnesyl pyrophosphate synthase, leading to the accumulation of intracellular pyrophosphate intermediates, thereby mimicking phosphate antigens and stimulating γδT cell activation. The final concentration of zoledronic acid in the initial system is preferably 5–10 μmol / L, more preferably 6–8 μmol / L, and most preferably 7.5 μmol / L. The cytokine combination includes IL-2 and IL-15, where IL-2 is a T cell growth factor that promotes cell proliferation; and IL-15 has anti-apoptotic and cell viability-maintaining effects. The concentration of IL-2 in the initial system is preferably 500-1000 IU / mL, more preferably 600-800 IU / mL, and most preferably 800 IU / mL; the concentration of IL-15 is also preferably 500-1000 IU / mL, more preferably 600-800 IU / mL, and most preferably 800 IU / mL. Umbilical cord plasma is preferably inactivated umbilical cord plasma, and its volume percentage concentration in the initial system is preferably 5-10%, more preferably 6-8%, and most preferably 5%. Umbilical cord plasma contains various growth factors and nutrients, which can promote cell growth. Furthermore, because it is derived from autologous umbilical cord blood, it avoids the risk of immune reactions and pathogen contamination that may arise from xenogeneic serum.

[0024] The amplification culture system is another core component of this invention. This system is based on 581 medium, supplemented with a second bisphosphonate, rapamycin, a combination of cytokines, and umbilical cord plasma. The second bisphosphonate is preferably ibandronate sodium, which, similar to zoledronic acid, also stimulates γδT cell activation, but its potency and kinetic characteristics differ slightly, making it suitable for use in the amplification phase. The final concentration of ibandronate sodium in the amplification culture system is preferably 5–10 μmol / L, more preferably 6–8 μmol / L, and most preferably 7.5 μmol / L. Rapamycin is an mTOR inhibitor that regulates cell metabolism and proliferation; in this invention, it has been found to synergistically enhance the amplification efficiency and functional activity of γδT cells. The concentration of rapamycin in the amplification culture system is preferably 50–200 nmol / L, more preferably 80–120 nmol / L, and most preferably 100 nmol / L. The cytokine combination also includes IL-2 and IL-15, with the same concentration range as the initial system, but which can be fine-tuned according to the cell growth status. Preferably, the concentrations are IL-2 800 IU / mL and IL-15 500 IU / mL. The concentration of umbilical cord plasma added is consistent with the initial system, preferably 5-10%, and more preferably 5%.

[0025] The cytokine combination used in this invention is IL-2 and IL-15. IL-2 (interleukin-2) is a cytokine mainly produced by activated T cells, which can promote T cell proliferation, differentiation, and survival. In the culture system of this invention, the role of IL-2 is to maintain the continuous proliferation and activation of γδT cells. IL-15 (interleukin-15) is a cytokine with similar function to IL-2, but its signaling pathway is slightly different, and it is more inclined to promote the generation and maintenance of memory T cells. In this invention, the role of IL-15 is to enhance the survival and functional activity of γδT cells and reduce apoptosis. The combined use of the two can produce a synergistic effect, both promoting rapid cell proliferation and maintaining high cell activity and function. Both IL-2 and IL-15 can be produced using recombinant DNA technology, and commercial products are available in various forms, such as recombinant human IL-2 and IL-15 lyophilized powder or solution, which can be purchased from companies such as PeproTech and R&D Systems.

[0026] In a preferred embodiment of the present invention, the first bisphosphonate in the initial induction culture system is zoledronic acid, and its final concentration in the system is preferably 5-10 μmol / L. Zoledronic acid is a nitrogen-containing bisphosphonate drug, mainly used clinically to treat osteoporosis and hypercalcemia associated with malignant tumors. In the present invention, its function is to inhibit farnesyl pyrophosphate synthase, leading to the accumulation of intracellular pyrophosphate intermediates (such as IPP), which can be recognized by the TCR of γδT cells, thereby activating the cells. The concentration of zoledronic acid is crucial; too low a concentration will not effectively activate the cells, while too high a concentration may produce cytotoxicity. A concentration range of 5-10 μmol / L can achieve the best activation effect while ensuring cell viability. Further preferred concentrations are 6-8 μmol / L, such as 6.5 μmol / L, 7.2 μmol / L, etc., with 7.5 μmol / L being the most preferred. The second bisphosphonate in the amplification culture system is preferably ibandronate sodium, and its final concentration is also preferably 5-10 μmol / L. Ibandronate sodium is also a nitrogen-containing bisphosphonate, but its pharmacokinetic characteristics differ slightly from zoledronic acid, making it more suitable for use in the amplification phase. A further preferred concentration is 6–8 μmol / L, with an optimal concentration of 7.5 μmol / L. The phased use of these two bisphosphonates allows for a smooth transition from activation to amplification, maximizing cell expansion efficiency.

[0027] In a preferred embodiment of the present invention, the concentrations of IL-2 and IL-15 in both the initial induction culture system and the expansion culture system are preferably 500-1000 IU / mL. This concentration range was determined based on extensive experiments, effectively stimulating cell proliferation without causing excessive cell activation or depletion due to excessive concentration. During the initial induction culture stage, the concentrations of IL-2 and IL-15 are preferably kept consistent at 500-1000 IU / mL, more preferably 600-800 IU / mL, such as 600 IU / mL, 700 IU / mL, 800 IU / mL, etc., with 800 IU / mL being the most preferred. During the expansion culture stage, the concentrations of IL-2 and IL-15 can be fine-tuned according to the cell growth status, preferably still maintained within the range of 500-1000 IU / mL, but IL-2 can be appropriately increased to 800-1000 IU / mL, and IL-15 can be maintained at 500-800 IU / mL. For example, a preferred combination is IL-2 800 IU / mL and IL-15 500 IU / mL. The combined use of IL-2 and IL-15 is one of the key innovations of this invention, and their synergistic effect enables γδT cells to maintain a high activity and high function throughout the expansion process.

[0028] In a preferred embodiment of the present invention, rapamycin is added to the amplification culture system, preferably at a concentration of 50-200 nmol / L. Rapamycin, also known as sirolimus, is a macrolide antibiotic with immunosuppressive and antiproliferative effects. Its mechanism of action is to bind to FKBP12, inhibiting the activity of mTORC1, thereby affecting cell metabolism, proliferation, and differentiation. In this invention, the effect of rapamycin is not to inhibit cell proliferation, but to optimize the metabolic state of cells and enhance cell viability and functional activity by regulating the mTOR signaling pathway. A concentration range of 50-200 nmol / L can significantly improve the cytotoxic activity and cytokine secretion capacity of cells without affecting cell amplification efficiency. Further preferred concentrations are 80-120 nmol / L, such as 90 nmol / L, 100 nmol / L, 110 nmol / L, etc., with 100 nmol / L being the most preferred. The timing and concentration selection of rapamycin addition is another innovation of the present invention; its combined use with bisphosphonates and cytokines constitutes a unique amplification system.

[0029] In a preferred embodiment of the present invention, inactivated umbilical cord plasma is added to both the initial induction culture system and the expansion culture system, with a volume percentage concentration preferably of 5-10%. The umbilical cord plasma is derived from umbilical cord blood and is used after inactivation treatment (56°C water bath for 30 minutes) to eliminate complement activity and inactivate any potential pathogens. Umbilical cord plasma is rich in growth factors, cytokines, hormones, and nutrients, effectively supporting cell growth and proliferation. Compared to fetal bovine serum, umbilical cord plasma has advantages such as being autologous, having low immunogenicity, and high safety. In this invention, the concentration of added umbilical cord plasma needs to be precisely controlled; too low a concentration will not provide sufficient nutritional support, while too high a concentration may introduce uncertainties. A concentration range of 5-10% is optimal, further preferably 6-8%, such as 6.5%, 7%, 7.5%, etc., and most preferably 5%. In practice, umbilical cord plasma can be prepared simultaneously during umbilical cord blood separation, and the supernatant is collected after inactivation and centrifugation and stored at 4°C for later use. Sterility must be ensured before use, and quality testing must be performed.

[0030] In a preferred embodiment of the present invention, when the number of cells after initial induction culture reaches 4 × 10⁻⁶ 5 Once the cells are in the specified number of wells, they can be transferred to the expansion culture step. This cell density is optimized to ensure that the cells are fully activated and expanded while avoiding nutrient competition and metabolic waste accumulation caused by overcrowding. When transferring to the expansion culture system, the cell density should be adjusted to 2 × 10⁹ cells / well. 5 ~5×10 5 / mL, more preferably 2.5×10 5 ~4×10 5 / mL, the optimal value is 3×105 / mL. This density range is beneficial for continuous cell proliferation during the expansion culture phase, while also facilitating subsequent fluid replenishment and replacement. In practice, cell density can be adjusted by centrifugation for concentration or dilution, followed by seeding in T25 or T75 suspension culture flasks, or large-scale culture using cell culture bags. During culture, regular sampling and counting are necessary, and fluid replenishment or replacement should be performed as needed based on cell growth to maintain the cell density within the appropriate range. Through this optimized density control strategy, cells can expand several hundredfold within 14–18 days, reaching the clinically required cell count.

[0031] In a preferred embodiment of the invention, prior to the initial induction culture step, a umbilical cord blood mononuclear cell (PBMC) isolation step and a γδT cell sorting step are included. PBMC isolation is typically performed using density gradient centrifugation with a lymphocyte separation medium (such as Ficoll-Paque). Specifically, umbilical cord blood is diluted with physiological saline and slowly added to the surface of the lymphocyte separation medium. After centrifugation, the white membrane layer is aspirated to obtain mononuclear cells. The key to this step is maintaining cell viability and sterility; centrifugation force, time, and temperature must be strictly controlled. The isolated mononuclear cells contain approximately 1-5% γδT cells. Subsequently, γδT cells are enriched using magnetic bead sorting technology. The present invention preferably uses CD25 MicroBeads II for sorting, at a ratio of 1 × 10⁻⁶ cells per cell. 7 Cells were added to a ratio of 90 μL MACS buffer and 10 μL magnetic beads, incubated at 4°C for 5 minutes, and then separated by column chromatography. The purity of the sorted γδT cells reached over 90%, providing high-quality starting cells for subsequent culture. MACS buffer typically contains PBS, 0.5% BSA, and 2 mmol / L EDTA to maintain cell viability and prevent aggregation.

[0032] In a preferred embodiment of the present invention, a umbilical cord blood plasma treatment step is included before the initial induction culture step. The purpose of this step is to obtain inactivated umbilical cord blood plasma for subsequent culture systems. Specifically, the umbilical cord blood is centrifuged, and the supernatant plasma is aspirated and transferred to a new centrifuge tube. The plasma is then placed in a 56°C water bath for 30 minutes for inactivation. Inactivation aims to destroy complement components in the plasma, preventing non-specific cell-killing effects during culture, and also inactivating any potential pathogens, thus improving safety. After inactivation, the plasma is centrifuged at 2500 rpm for 15 minutes to remove any precipitate generated during inactivation. The clear supernatant inactivated umbilical cord blood plasma is carefully aspirated after centrifugation and transferred to a new sterile centrifuge tube, then stored at 4°C for later use. The preferred concentration of the inactivated umbilical cord blood plasma is 5-10%. Its addition not only provides rich nutritional support but also avoids the risk of immune reactions and pathogen contamination that may arise from xenobiotic serum, making it one of the preferred embodiments of the present invention.

[0033] This invention further provides umbilical cord blood γδT cells obtained by the above-described culture method. These cells have the following characteristics: (1) Sufficient cell quantity: Through the two-stage culture method of this invention, the γδT cell expansion can stably reach more than 700-fold, from the initial 1×10⁻⁶ cells / year. 6 The number of cells increased to 7 × 10⁶. 7 More than 100 cells, meeting the dosage requirements for clinical reinfusion; (2) High cell purity: Flow cytometry showed that the purity of the expanded γδT cells reached more than 97%, significantly higher than that of traditional methods; (3) High cell viability: Throughout the culture process, the cell viability remained above 98%, indicating that the cells were in good condition; (4) Strong cell function: Killing experiment results showed that the cells had significant killing activity against tumor cells (such as HCT-116), and the killing rate reached more than 40% at an effector-to-target ratio of 5:1, which was much higher than that of the control ratio. The cells can be further cryopreserved or directly used for subsequent functional studies and clinical applications. The umbilical cord blood γδT cells provided by this invention have the advantages of stable quality, strong function, and safe source, and are ideal effector cells for adoptive immunotherapy.

[0034] This invention further provides the application of the aforementioned umbilical cord blood γδT cells in the preparation of drugs for adoptive immunotherapy. Adoptive immunotherapy refers to a treatment method in which activated and expanded immune cells are reinfused into a patient to enhance their anti-tumor and anti-infection capabilities. γδT cells, due to their unique MHC non-restrictive recognition ability and strong killing activity, have become a research hotspot in adoptive immunotherapy. The umbilical cord blood γδT cells cultured in this invention have advantages such as sufficient quantity, high purity, high viability, and strong function, making them suitable for the preparation of adoptive immunotherapy drugs. The drugs can be prepared in the form of injections, lyophilized powder injections, or cell suspensions, preferably cell suspensions for intravenous infusion. During the preparation process, the expanded γδT cells need to be washed, concentrated, and resuspended in an infusion medium (such as physiological saline, human serum albumin solution, etc.), and subjected to quality tests such as sterility, endotoxin, and mycoplasma. After passing the tests, they are aliquoted into infusion bags or cryopreservation bags and stored under cold or frozen conditions. In use, the cell suspension is reinfused into the patient via intravenous infusion to treat various malignant tumors (such as colorectal cancer, lung cancer, liver cancer, breast cancer, etc.) or viral infections. This invention provides a stable, efficient, and safe cell source for adoptive immunotherapy, and has significant clinical application value and industrialization prospects.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1) Umbilical cord blood sampling: Use a disposable pipette to transfer the umbilical cord blood from the collection bag to a 50 mL centrifuge tube, and use a Pasteur pipette to extract 2 mL of peripheral blood and aliquot it into two EP tubes. One tube is used for bacterial and fungal testing, and the other is retained as a sample.

[0037] 2) Preliminary separation of umbilical cord blood and plasma processing: Transfer the 50mL centrifuge tube containing umbilical cord blood to a centrifuge and centrifuge at 650g for 15min; use a Pasteur pipette to aspirate the upper plasma layer and transfer it to a new 50mL centrifuge tube; place the plasma in a 56℃ water bath for 30min; after the water bath, centrifuge at 2500rpm for 15min; transfer the upper inactivated plasma layer to a new 50mL centrifuge tube and store at 4℃ for later use.

[0038] 3) Umbilical cord blood separation: In step 2), dilute the lower blood cell layer with an equal volume of physiological saline using a disposable pipette (umbilical cord blood: physiological saline = 1:1) and mix thoroughly. Take a new 50 mL centrifuge tube and add 12.5 mL of lymphocyte separation medium. Slowly transfer the diluted blood to the surface of the lymphocyte separation medium, creating a clear interface between the two (blood diluent: lymphocyte separation medium = 2:1). Transfer the centrifuge tube to a centrifuge and centrifuge at 800g for 15 min. After centrifugation, aspirate the upper layer containing umbilical cord blood mononuclear cells (PBMCs) and transfer it to another 50 mL centrifuge tube, being careful not to aspirate the red blood cell layer. Resuspend the umbilical cord blood mononuclear cells in RPMI 1640 medium using a disposable pipette to 40 mL and centrifuge at 500g for 5 min. (Maintain cell viability.) Discard the supernatant after centrifugation.

[0039] 4) Magnetic bead sorting: according to 1×10 7 Add 90 μL MACS buffer (PBS, 0.5% BSA, and 2 mmol / L EDTA) and 10 μL CD25 MicroBeads II to each cell, mix thoroughly, and incubate at 4 °C in the dark for 5 min. Inject the cell suspension into a pre-washed LS column, wash the column three times with 3 mL MACS buffer, and finally wash the cells off with 5 mL MACS buffer. Centrifuge, discard the supernatant, and count the cells.

[0040] 5) Initial induction culture of umbilical cord blood γδ T cells: Resuspend umbilical cord blood γδ T cells in the initial culture medium and seed them in 48-well round-bottom plates. Incubate at 37 °C and 5% CO2. When the cell number reaches 4 × 10⁶ cells / well... 5 / well, to further expand the cultured cells.

[0041] Initial system: Zoledronic acid (final concentration 7.5 μmol / L), 5% inactivated umbilical cord plasma, IL-2 (concentration 700 IU / mL), and IL-15 (concentration 700 IU / mL) were added to 1640 medium.

[0042] 6) Further expansion and culture of umbilical cord blood γδT cells: Transfer γδT cells into T25 suspension culture flasks, add expansion culture system, and adjust the cell density to 3×10⁻⁶ cells / year. 5 / mL.

[0043] Amplification culture system: Ibandronate sodium at a final concentration of 7.5 μmol / L, 5% umbilical cord blood plasma, IL-2 (concentration of 800 IU / mL), IL-15 (concentration of 500 IU / mL), and rapamycin (concentration of 100 nmol / L) were added to 581 medium.

[0044] 7) Based on cell proliferation, add expansion culture system and adjust the density of γδT cells to 3×10⁻⁶. 5 / mL. Transfer to a cell culture bag and add fluid, culture for 17 days, then perform functional testing. (Repeat the following experiments for 3 batches of cells).

[0045] 8) Cell count and viability detection: Take 10 μL of γδ T cell suspension, add 10 μL of trypan blue dye, mix well and add to a cell counting chamber, and detect cell viability using Cellometer Auto 2000.

[0046] 9) Flow cytometry: Umbilical cord blood mononuclear cells cultured on day 0 and γδ T cells expanded in vitro on day 14 were collected, washed twice with PBS and counted, and the cell concentration was adjusted to 1×10⁻⁶. 6 Each sample was placed in a U-shaped test tube, and 5 μL of anti-human TCR-γδ-PE mAb and 5 μL of anti-human CD3-APC mAb were added respectively. The mixture was then incubated at 4 °C in the dark for 30 min. The sample was washed twice with 1 mL of PBS, and then 500 μL of PBS was added. The percentage of γδ T cells was detected using flow cytometry, and the fold increase was calculated using the following formula: Fold increase = (Total number of cells after expansion × Percentage of γδ T cells after expansion) / (Total number of cells before expansion × Percentage of γδ T cells before expansion).

[0047] 10) Stimulation Experiment: CytoTox96 Non-Radioactive Cytotoxicity Kit Assay: γδT cells cultured for 14 days were selected for the killing experiment. Following the instructions for the lactate dehydrogenase release assay, HCT-116 cells were used as target cells, with effector-to-target cell ratios of 5:1, 10:1, and 30:1. Effector cell control, maximum target cell release, spontaneous target cell release, volume correction, and background control wells were also set up. The 96-well plates were incubated at 37 ℃ in a 50 mL / L CO2 incubator for 4 h, centrifuged, and 50 μL of supernatant was aspirated from each well. The absorbance (D) value was measured at 490 nm using an automated microplate reader. The cell killing rate was calculated using the following formula: Cell killing rate = (Dexperimental group - Deffector cell control group - Dtarget cell control group) / (Dtarget cell maximum release group - Dtarget cell control group) × 100%.

[0048] Cell count and viability (N=3) are shown in Table 1: Table 1. Results of cell number and viability detection

[0049] Photographs of the cultured cells are as follows Figure 1 As shown.

[0050] The results show that γδ T cells, when cultured in vitro, have a latency period of 3 days, and enter the exponential proliferation phase from day 5, with the cell count increasing from the initial 1×10⁶ cells / year. 6 Cells begin to proliferate, reaching a plateau phase on day 15, with the cell number increasing to 7.45 × 10⁻⁶. 7 The cells expanded stably at a fold increase of 745-fold. Cell viability remained above 98% throughout the entire culture process.

[0051] Flow cytometry The control group was prepared according to the existing technology (Su Dan, Zhang Chunhui, Wang Dongliang, et al. High-efficiency expansion of γδT cells and their in vitro killing effect on gastrointestinal tumor cells. Journal of Harbin Medical University [J]; 2018, 52(5): 430-433. Similar existing culture systems are all for the expansion culture system of peripheral blood-derived γδT, and the expansion culture effect of umbilical cord blood-derived γδT is limited). Three samples of umbilical cord blood γδT cells obtained from the culture were taken and recorded as control sample 1, control sample 2 and control sample 3 (three samples of cells obtained by the method of the present invention were also taken and recorded as experimental sample 1, experimental sample 2 and experimental sample 3) (the flow cytometry analysis of the cells obtained by the method of the present invention is shown in the figure). Figure 2 (As shown).

[0052] The results are shown in Table 2 below: Table 2 Results of cell purity and fold expansion detection

[0053] The results showed that, after magnetic bead sorting, the purity of γδ T cells in the three umbilical cord blood samples from the control group ranged from 89% to 91%, and the amplification fold ranged from 225 to 272 times. However, after amplification using the culture system of this invention, the expression of γδ T cells changed to varying degrees: cell purity increased to over 97%, while the amplification fold stabilized between 736 and 743 times. This increased expression of cell activating factors and functional molecules suggests that the amplified γδ T cells possess stronger function.

[0054] The sample acquisition procedure was the same as above, and the killing experiment was conducted (HCT116 target cells: γδ T cell effector cells). The results of the lethality test are shown in Table 3 below: Table 3 Results of Cell Killing Assay 1

[0055] A p-value < 0.05 indicates a statistically significant difference compared to the corresponding experimental sample group. As shown in the table above, the cytotoxicity of umbilical cord blood γδ T cells in the three control groups was 12.73%–15.36% at a target-effect ratio (TAR) of 5:1; 4.26%–5.58% at a TAR of 10:1; and 0.18%–0.42% at a TAR of 30:1. In this embodiment, the TARs at 5:1, 10:1, and 30:1 were 42.45%–43.73%, 12.58%–15.26%, and 6.23%–9.18%, respectively; all higher than the control group's cytotoxicity. This demonstrates that the γδ T cells of this invention have a higher cytotoxicity than existing technologies.

[0056] Based on the solution of this embodiment, the following comparative examples are further provided: Comparative Example 1 (rapamycin in the amplification system was replaced with other mTOR inhibitors): Induction system (unchanged): 1640 basal medium + zoledronic acid (7.5 μmol / L) + IL-2 (700 IU / mL) + IL-15 (700 IU / mL) Amplification system: 581 basal medium + IL-2 + IL-15 + rapalik Comparative Example 2 (the bisphosphonates in the induction and amplification stages were interchanged): Induction system: 1640 basal medium + sodium ibandronate (7.5 μmol / L) + IL-2 (700 IU / mL) + IL-15 (700 IU / mL) Amplification system: 581 basal medium + zoledronic acid (7.5 μmol / L) + IL-2 (700 IU / mL) + IL-15 (700 IU / mL) + rapamycin (100 nmol / L) Comparative Example 3 (Referencing Chinese Patent CN102994448A: Using IL-2+IL-7+IL-15+zoledronic acid, without rapamycin): Patented solution: IL-2 + IL-7 + IL-15 + zoledronic acid The lethality tests were conducted separately, and the results are shown in Table 4 below: Table 4 Results of Cell Killing Assay 2

[0057] The value P < 0.05 indicates a significant difference compared to the present invention.

[0058] As shown in the table above, the killing efficiencies of the three comparative umbilical cord blood γδ T cells at a target-effect ratio of 5:1 were 32.62%, 18.45%, and 15.36%, respectively; all of which showed significant differences compared to the present invention. At a target-effect ratio of 10:1, the killing efficiencies were 9.36%, 7.45%, and 5.58%, respectively; all of which showed significant differences compared to the present invention. At a target-effect ratio of 30:1, the killing efficiencies were 5.52%, 2.42%, and 1.23%, respectively; all of which showed significant differences compared to the present invention. This demonstrates that the umbilical cord blood γδ T cell culture system of the present invention is superior.

[0059] As shown in the above embodiments, this invention provides a method for culturing umbilical cord blood γδT cells. This method employs a two-step strategy of initiation and expansion culture, using 1640 medium containing zoledronic acid and 581 medium containing ibandronate sodium and rapamycin, respectively, and combining cytokines such as IL-2 and IL-15 to expand umbilical cord blood-derived γδT cells in vitro. Experimental results show that γδT cells cultured using this method maintain good cell viability throughout the entire culture period, exhibiting a typical exponential growth trend in cell number, and a significantly expanded cell population can be obtained by day 17. Flow cytometry results show that the purity of γδT cells is significantly improved after culture, and the expansion fold reaches a stable high level, significantly superior to the existing culture method used in the comparative example. Functional experiments show that the γδT cells expanded by the method of this invention have strong killing activity against HCT-116 tumor cells, and the killing efficiency at different effector-to-target ratios is significantly higher than that of the comparative example. In summary, the culture method provided by this invention can stably and efficiently obtain high-purity, high-quantity, and highly active γδT cells from umbilical cord blood, and the expanded cells have good anti-tumor function and can be used for adoptive immunotherapy.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing umbilical cord blood γδT cells, characterized in that, Includes the following steps: Umbilical cord blood γδT cells were seeded into the initial system and subjected to initial induction culture to obtain cells after initial induction culture; The cells after initial induction culture were transferred into an amplification culture system for amplification culture. The initial system comprises 1640 culture medium, a first bisphosphonate, a combination of cytokines, and umbilical cord plasma; The amplification culture system includes 581 medium, second bisphosphonate, rapamycin, a combination of cytokines, and umbilical cord plasma; The combination of cytokines includes IL-2 and IL-15.

2. The cultivation method according to claim 1, characterized in that, The first bisphosphonate is zoledronic acid, and its final concentration in the initial system is 5~10 μmol / L; the second bisphosphonate is sodium ibandronate, and its final concentration in the amplification culture system is 5~10 μmol / L.

3. The cultivation method according to claim 1, characterized in that, In the cytokine combination, the concentration of IL-2 in the initial system is 500~1000 IU / mL, the concentration of IL-15 in the initial system is 500~1000 IU / mL, the concentration of IL-2 in the amplification culture system is 500~1000 IU / mL, and the concentration of IL-15 in the amplification culture system is 500~1000 IU / mL.

4. The cultivation method according to claim 1, characterized in that, The concentration of rapamycin in the amplification culture system is 50~200 nmol / L.

5. The cultivation method according to claim 1, characterized in that, The umbilical cord plasma is inactivated umbilical cord plasma, and its volume percentage concentration in the initial system and the amplification culture system is 5-10%.

6. The cultivation method according to claim 1, characterized in that, The cell number after the initial induction culture reached 4 × 10⁶ cells. 5 When the number of wells is reached, proceed to the amplification culture step; The amplification and culture step includes adjusting the cell density to 2 × 10⁻⁶. 5 ~5×10 5 / mL.

7. The cultivation method according to claim 1, characterized in that, It also includes the umbilical cord blood mononuclear cell isolation step and the γδT cell sorting step before the initial induction culture step.

8. The cultivation method according to claim 1, characterized in that, It also includes a umbilical cord plasma treatment step before the initial induction culture step: after the separated umbilical cord plasma is inactivated by water bath, the upper layer of inactivated umbilical cord plasma is collected by centrifugation.

9. Umbilical cord blood γδT cells obtained by the culture method according to any one of claims 1 to 8.

10. The use of the umbilical cord blood γδT cells of claim 9 in the preparation of a medicament for adoptive immunotherapy.

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