Large-scale culture method of central memory T cells (TCM)

Abstract

The invention relates to the technical field of biotechnology and cell engineering, in particular to a large-scale culture method of central memory T cells (TCM). According to the method, the lymphocyte expressed by the high homing receptor is activated by adopting an alternating electric field, and the cell activity is improved by combining with a composite metabolism regulator. A two-photon excitation three-dimensional scaffold of a modified collagen peptide, ferroferric oxide and a quantum dot labeled organoid is utilized to simulate an in-vivo microenvironment to promote cell anchoring. And finally, through gene editing, engineering a cryopreservation system of saccharomyces cerevisiae and ultraviolet light activated autophagic peptide, so as to realize the enhancement of cell function stability and long-term memory characteristic. According to the invention, the limitation of traditional two-dimensional culture is broken through, and an efficient and controllable T cell preparation scheme is provided for tumor immunotherapy, autoimmune disease regulation and personalized cell therapy through systematic integration of physical field regulation, bionic scaffold design and genetic engineering means.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and cell engineering technology, in particular to a large-scale culture method of central memory T cells (TCM). BACKGROUND

[0002] With the expanding application of immunotherapy in cancer, chronic infection and autoimmune diseases, the demand for high-quality central memory T cells is increasing. Central memory T cells, due to their long-term survival ability and strong antigen response ability, have become the core resource of adoptive immunotherapy. However, in the current clinical and scientific research scene, the in vitro expansion of central memory T cells faces multiple technical bottlenecks. Not only is it necessary to maintain cell phenotype stability under low cytokine dependence and achieve efficient expansion, but it is also necessary to maintain cell functional activity and reduce batch-to-batch differences in large-scale culture to meet the strict requirements of industrial production on cost and efficiency.

[0003] In view of the technical needs of central memory T cell expansion, the current research field has proposed the following targeted solutions:

[0004] Metabolic regulation culture method: by optimizing the glucose concentration and glutamine ratio in the culture medium, combined with specific metabolic inhibitors to regulate mitochondrial function, to promote the expansion of central memory T cells.

[0005] Co-culture activation method: using dendritic cells or artificial antigen-presenting cells as a co-culture system to provide sustained T cell receptor signals and costimulatory signals, to simulate the in vivo physiological environment to enhance the differentiation and expansion efficiency of central memory T cells.

[0006] Mechanical force stimulation method: introducing periodic shear force or flexible matrix material during culture to regulate cytoskeleton reorganization and adhesion molecule expression through physical signals, thereby improving the survival rate and functional activity of central memory T cells.

[0007] Although the above-mentioned solutions have made progress in the expansion of central memory T cells, there are still significant limitations:

[0008] High complexity of metabolic regulation: optimization of metabolic parameters requires precise detection means, and metabolic differences between different cell sources are significant, making it difficult to establish a unified standardized process.

[0009] High cost of co-culture system: the preparation process of dendritic cells or artificial antigen-presenting cells is complicated, and additional consumables and technical support are required, limiting their feasibility in large-scale production.

[0010] Insufficient adaptability of physical stimulation: the parameter setting of mechanical force stimulation needs to match the cell type, and excessive stimulation may cause cell stress damage, while low-intensity stimulation is difficult to achieve the expected expansion effect. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides a method for large-scale culture of central memory T cells (TCM), which solves the problems mentioned above in the background.

[0012] According to a first aspect of the present invention, a method for large-scale culture of central memory T cells (TCM) is provided, comprising the following steps:

[0013] (a) Preparation of activated T cells: Under an alternating electric field with a frequency of 50-100Hz, 10-30 lymphocyte homing receptor highly expressing cells with a purity of 95%-99% were sorted from 100-200 peripheral blood mononuclear cells, and 5-10 parts of physiological saline containing compound metabolic regulators were added to resuspend the cells for 15-30 minutes to prepare the activated T cells.

[0014] (b) Preparation of synergistic culture medium: 10-20 parts of interleukin, 1-5 parts of deoxyglucose, 0.5-2 parts of rapamycin nanoparticles and 0.5-1 parts of histone methyltransferase inhibitor were added to serum-free basal culture medium to prepare the synergistic culture medium;

[0015] (c) Three-dimensional scaffold preparation: 0.5-2 parts of modified collagen peptide, 0.1-0.5 parts of iron oxide, 0.3-0.6 parts of quantum dot labeled dendritic cell organoids and 0.2-0.4 parts of follicular helper cell organoids are mixed and then assembled into the three-dimensional scaffold by two-photon excitation light field orientation.

[0016] (d) Dynamic expansion: The activated T cells prepared in step (a) are seeded into the three-dimensional scaffold prepared in step (c) and perfused with the synergistic culture medium prepared in step (b). A biaxial phase difference magnetic field and pulsed blue light with a frequency of 1-2 Hz are applied alternately and cultured for 7-10 days to prepare an expanded cell population.

[0017] (e) Gene-edited cells: The expanded cell population prepared in step (d) was electroporated to knock out the programmed death receptor gene to prepare edited T cells;

[0018] (f) Cell cryopreservation: After the edited T cells prepared in step (e) are expanded in a microgravity reactor for 5-7 days, they are co-incubated with 0.1-0.2 parts of engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters for 24-36 hours.

[0019] After incubation, the cells were frozen in 50-60 portions of serum-free cell preservation medium containing UV-activated autophagy peptides at a stepwise cooling rate of -0.8℃ / min to obtain the central memory T cells.

[0020] According to an embodiment of the present invention, in step (a), the peripheral blood mononuclear cells are cell monomers obtained by centrifuging peripheral blood from a healthy donor, with a centrifugation force of 400-450×g, a temperature of 18-22℃, and a time of 20-25 minutes;

[0021] The saline solution containing the compound metabolic regulator is a transparent solution prepared by mixing the compound metabolic regulator, sodium dichloroacetate and phosphate buffer in a mass ratio of 0.1:0.1:7-0.2:0.5:11, wherein the compound metabolic regulator is a colorless metabolic solution prepared by mixing ketoglutarate and itaconic acid in a mass ratio of 2.5:1-4:1.

[0022] According to an embodiment of the present invention, in step (a), the resuspension treatment time is 15-30 minutes, and the initial concentration of peripheral blood mononuclear cells is 1×10⁻⁶. 6 -5×10 6 cells / mL.

[0023] According to an embodiment of the present invention, in step (b), the interleukin is a sterile solution composed of interleukin 7, interleukin 15 and interleukin 27; the deoxyglucose is a white needle-like crystal formed by hydrolysis of glucose and thionyl chloride; the rapamycin nanoparticles are a milky white suspension formed by copolymerization of rapamycin and polylactic acid; and the histone methyltransferase inhibitor is a benzimidazole derivative solution formed by coupling a benzimidazole core with methoxybenzoyl chloride.

[0024] The mass ratio of interleukin-7, interleukin-15 and interleukin-27 is 0.8:0.05:0.1-1:1:0.5.

[0025] The mass ratio of glucose to thionyl chloride is 1:1.5-1:2.0;

[0026] The mass ratio of rapamycin to polylactic acid is 1:1.8-1.5:2.2;

[0027] The mass ratio of the benzimidazole core to the methoxybenzoyl chloride is 1:1.2-1:1.5.

[0028] According to an embodiment of the present invention, interleukin, as a key signaling molecule, drives cell proliferation and maintains phenotypic stability in step (b). Deoxyglucose improves cellular energy metabolism efficiency by regulating glucose metabolism pathways. Rapamycin nanoparticles delay cell differentiation by inhibiting signaling pathways. Histone methyltransferase inhibitors enhance the expression of genes related to memory phenotypes through epigenetic regulation. The synergistic effect of these four agents not only achieves targeted release of cytokines but also effectively prolongs the cell proliferation cycle and reduces the risk of functional heterogeneity through the dynamic balance of metabolic regulation and signaling pathways, ultimately ensuring the uniformity of cell function while improving culture efficiency.

[0029] According to an embodiment of the present invention, in step (c), the modified collagen peptide is a self-assembled peptide material composed of arginine, glycine, aspartic acid, tyrosine, isoleucine, serine, and arginine; the quantum dot-labeled dendritic cell organoid is a homogeneous suspension formed by coupling carboxylated polyethylene glycol-modified quantum dots with human umbilical cord blood stem cells induced by differentiation factors at a mass ratio of 0.02:1; the follicular helper cell organoid is an organoid colloid colloid differentiated from human umbilical cord blood stem cells through the interaction of interleukin-21 and recombinant human inducible co-stimulatory ligand.

[0030] The differentiation factor is a powder composed of tyrosine kinase ligand, granulocyte-macrophage colony-stimulating factor, and interleukin-4 in a mass ratio of 0.5:0.3:0.1-0.8:0.5:0.2; the quantum dots are core-shell particles obtained by coating selenium powder with thiourea, wherein the mass ratio of thiourea to selenium powder is 1:0.8.

[0031] The two-photon excitation optical field includes: wavelength 800-810nm, power density 20-25mW / cm². 2 Exposure time: 30-40 seconds.

[0032] According to an embodiment of the present invention, the modified collagen peptide described in step (c) serves as a biomimetic matrix, providing mechanical support and a cell adhesion interface. Iron oxide (Fe3O4) imparts magnetic responsiveness and the ability to regulate the local microenvironment. Quantum dot-labeled dendritic cell organoids can precisely track and mimic the natural antigen presentation function. Follicular helper cell organoids promote intercellular communication and co-proliferation by secreting signaling molecules. Two-photon excitation light fields, through high-precision point-by-point scanning, achieve a gradient spatial distribution and structural self-assembly of multiple components, simultaneously optimizing the mechanical stability, functional response, and intercellular interaction efficiency of the scaffold. This gives the scaffold a biomimetic structure, dynamic regulation capabilities, and immune microenvironment simulation characteristics, providing a highly biomimetic and intelligent physicochemical microenvironment for the large-scale culture of memory T cells.

[0033] According to an embodiment of the present invention, in step (d), the synergistic culture medium is replaced every 24 hours, and the replacement amount is 50%-70% of the total synergistic culture medium volume;

[0034] The biaxial phase difference magnetic field includes: a sinusoidal axial magnetic field with a frequency of 10-15Hz, a square wave radial magnetic field with a frequency of 20-25Hz, an axial magnetic field strength of 5-8mT, a radial magnetic field strength of 8-10mT, an axial phase of 0-1 degree, and a radial phase of 30-35 degrees.

[0035] The inoculation density is 1×10 6 -5×10 6 The optimal temperature range is 34-36℃, and the optimal time is 1-2 hours.

[0036] According to an embodiment of the present invention, in step (d), the temperature of the cultivation process is 34-36°C, the oxygen concentration is dynamically controlled to 5%-15% by a gas mixing device, and the carbon dioxide concentration is maintained at 4.5%-5.5%.

[0037] According to an embodiment of the present invention, in step (e), before performing electroporation, the amplified cell population is first dispersed in an electroporation buffer, wherein the electroporation buffer is a complex solution composed of potassium chloride, magnesium chloride and hydroxyethylpiperazine ethanesulfonic acid in a mass ratio of 1:0.5:0.2-1:0.7:0.2.

[0038] The specific conditions for electroporation are: voltage 200-300V, pulse width 10-15ms, pulse duration 5-10ms, and pulse count 2-3 times.

[0039] According to an embodiment of the present invention, the electroporation described in step (e) provides an efficient delivery pathway for gene editing tools by instantaneously opening cell membrane permeability, while reducing damage to cell viability. Precise knockout of the programmed death receptor gene significantly enhances the survival ability and anti-fatigue properties of T cells by blocking the apoptosis signaling pathway. The combination of these two methods ensures both the targeting and efficiency of gene editing, and improves the clinical applicability of edited cells by maintaining cellular functional integrity. The synergistic effect of both methods not only optimizes the procedural compatibility of gene editing, but also achieves a dual improvement in editing efficiency and cell quality by reducing off-target effects and cellular stress responses.

[0040] According to an embodiment of the present invention, in step (f), the engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters is a light-controlled suspension constructed by inserting blue light-induced promoters and near-infrared light-induced promoters into a Saccharomyces cerevisiae strain through gene editing; the serum-free cell preservation solution containing ultraviolet light-activated autophagy peptides is an activation suspension composed of the ultraviolet light-activated autophagy peptides, sorbitol and serum-free cryopreservation solution.

[0041] The mass ratio of the ultraviolet light-activated autophagy peptide, the sorbitol, and the serum-free cryopreservation solution is 1:5:90-5:7:100.

[0042] According to embodiments of the present invention, the microgravity environment promotes uniform cell distribution and metabolic homeostasis, while engineered yeast releases regulatory factors through a photo-controlled promoter, enhancing cellular stress tolerance. UV-activated autophagy peptides target and eliminate intracellular damaged proteins during cryopreservation, maintaining autophagy pathway activity. A stepped cooling rate inhibits rapid ice crystal formation, reducing the risk of cell membrane damage. The synergistic effect of these three factors simultaneously optimizes the physical stability, metabolic regulation, and autophagy protection mechanisms during cryopreservation, significantly improving the survival rate and functional integrity of edited T cells, providing an efficient and reliable preservation strategy for long-term storage and clinical applications.

[0043] According to a second aspect of the present invention, a central memory T cell prepared by the above method is provided, characterized in that the central memory T cell possesses multiple signaling pathway regulation capabilities, dynamic metabolic balance characteristics, stress resistance, and functional homogeneity. Its phenotypic stability is significantly better than that of traditional culture systems, its expansion cycle is extended, and its differentiation drift risk is reduced. At the same time, it has outstanding advantages in terms of standardized process adaptability.

[0044] According to embodiments of the present invention, the central memory T cells are applicable to fields such as precision immunotherapy, chronic infection intervention, autoimmune disease management, and cell therapy product development in the biopharmaceutical industry, particularly as a core resource for adoptive immunotherapy, a basic material for customized cell therapy products, or an active component supporting intelligent medical devices.

[0045] Alternating electric field-assisted precision activation and sorting strategy: By combining specific frequency electric field stimulation with the recognition characteristics of cell surface markers, T cells with high homing receptor expression can be efficiently isolated from peripheral blood mononuclear cells. This method improves cell purity while enhancing cell activation through physical field regulation, providing a foundation for subsequent functional maintenance.

[0046] Construction of a multi-component synergistic culture system: Immunomodulatory factors, metabolic regulators, and nanoscale inhibitors are integrated into a serum-free culture environment to form a synergistic network of signal transduction and energy metabolism. Immunomodulators drive proliferation signals, metabolic regulators optimize energy utilization efficiency, and nanoscale formulations inhibit and delay cell differentiation through pathways. The combined effect of these three components significantly prolongs the proliferation cycle and maintains functional consistency.

[0047] A biomimetic three-dimensional scaffold with a composite structure design: A three-dimensional scaffold composed of modified collagen peptide self-assembly materials, quantum dot-labeled dendritic cell organoids, and follicular helper cell organoids mimics the structure and function of natural lymph nodes. Dendritic cell organoids provide antigen-presenting signals, while follicular helper cell organoids secrete chemokines, jointly maintaining the stem cell-like characteristics and homing ability of T cells. This biomimetic microenvironment design overcomes the functional exhaustion problem caused by T cell differentiation into effector T cells in traditional two-dimensional culture, significantly improving cell persistence in vivo. Two-photon excitation light fields achieve a gradient spatial distribution of multiple components, enabling the scaffold to possess biomimetic structural stability, dynamic regulation capabilities, and immune microenvironment simulation characteristics, providing an intelligent microenvironment for the large-scale culture of memory T cells.

[0048] A dynamic expansion mode synergistically regulated by multiple physical fields: T cells are expanded directionally through alternating stimulation by a biaxial phase-difference magnetic field and pulsed blue light. The axial magnetic field modulates cytoskeleton tension to promote adhesion, the radial magnetic field enhances intercellular signal transduction, and pulsed blue light activates photosensitive protein-mediated signaling pathways to restart the cell cycle. This synergistic strategy significantly improves expansion efficiency while maintaining a high homing receptor expression rate in T cells, overcoming the limitations of static culture.

[0049] Precise functional modification technology through gene editing: This involves knocking out the programmed death receptor gene using electroporation and combining it with blue light and near-infrared light-controlled promoters from engineered Saccharomyces cerevisiae to dynamically program T cell function. The light-controlled promoters can regulate cytokine expression in response to external light signals, enabling the edited T cells to exhibit responsiveness, thereby enhancing anti-tumor activity and reducing off-target effects. This strategy overcomes the limitation of irreversible function after traditional gene editing, providing the possibility of dynamic regulation for personalized therapy.

[0050] A standardized cryopreservation system under microgravity conditions: By combining microgravity reactor amplification with stepwise cooling technology with UV-activated autophagy peptides, cell damage during cryopreservation is effectively reduced. After UV activation, the autophagy peptides scavenge oxidative stress products, and sorbitol acts as a permeabilizer to maintain membrane stability, significantly improving cryopreservation survival rate. This intelligent cryopreservation strategy ensures cell viability and functional stability, supporting standardized preparation and long-distance transportation.

[0051] This improved method not only achieves breakthroughs in physical environment simulation and signal intervention technologies, but also constructs a full-process functional optimization system through multi-scale regulation strategies. This method overcomes the limitations of traditional static culture by coupling multi-physical field regulation with biological signals to simulate the in vivo homeostatic environment and maintain stem cell-like characteristics of cells. It combines dynamic metabolism and epigenetic regulation to achieve precise balance of cell function. Simultaneously, it introduces gene editing and light-controlled promoter technologies to endow T cells with responsive functions. Finally, it ensures cell viability and transport stability through intelligent cryopreservation strategies. This full-process optimization system breaks through the single-dimensional limitations of traditional methods, achieving efficient and targeted control from activation and expansion to cryopreservation.

[0052] This invention offers the following advantages: It provides a method for large-scale culture of central memory T cells. Through innovative techniques such as physical field-assisted activation, multi-component synergistic culture, biomimetic scaffold construction, dynamic microenvironment regulation, gene editing, and directed gradient cryopreservation, it significantly improves the expansion efficiency and functional stability of T cells, solving the problems of low expansion fold, rapid cell depletion, and poor cryopreservation survival rates in traditional methods. Furthermore, through engineered design, it endows cells with responsive functions and personalized therapeutic potential. Its serum-free culture medium and engineered yeast substitution strategy reduce production costs and the risk of xenogeneic contamination, supporting standardized preparation and long-distance transportation. This technology provides a high-quality, highly controllable source of T cells for tumor immunotherapy and is expected to become a core foundation for next-generation immunotherapies, promoting the deep integration of precision medicine and intelligent biomanufacturing.

[0053] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0054] Figure 1 This is a flowchart of the preparation method according to an embodiment of the present invention. Detailed Implementation

[0055] This application proposes a method for large-scale culture of central memory T cells (TCMs). The aim is to improve the expansion efficiency, functional stability, and industrial production adaptability of TCMs through alternating electric field-assisted activation, multi-component synergistic culture system, biomimetic three-dimensional scaffold construction, dynamic physical field regulation, gene editing targeted intervention, and microgravity cryopreservation.

[0056] Example 1

[0057] Preparation of activated T cells: Under an alternating electric field with a frequency of 100 Hz, 30 mL of lymphocyte homing receptor-high expression cells with a purity of 95%-99% were sorted from 200 mL of peripheral blood mononuclear cells and resuspended in 10 mL of physiological saline containing a compound metabolic regulator for 30 minutes to prepare the activated T cells.

[0058] Preparation of synergistic culture medium: 20 mL of interleukin, 5 mL of deoxyglucose, 2 mL of rapamycin nanoparticles and 1 part of histone methyltransferase inhibitor were added to serum-free basal culture medium to prepare the synergistic culture medium.

[0059] Three-dimensional scaffold preparation: 2g of modified collagen peptides, 0.5 parts of iron oxide, 0.6 parts of quantum dot-labeled dendritic cell organoids and 0.4 parts of follicular helper cell organoids were mixed and then assembled into the three-dimensional scaffold by two-photon excitation light field orientation.

[0060] Dynamic expansion: Activated T cells were seeded onto a three-dimensional scaffold and perfused with synergistic culture medium. A biaxial phase difference magnetic field and a 2Hz pulsed blue light were alternately applied and cultured for 10 days to prepare an expanded cell population.

[0061] Gene-edited cells: Edited T cells are prepared by electroporating an expanded cell population to knock out the programmed death receptor gene.

[0062] Cell cryopreservation: Edited T cells were expanded in a microgravity reactor for 7 days and then co-incubated with 0.2 portions of engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters for 36 hours.

[0063] After incubation, the cells were cryopreserved in 60 portions of serum-free cell preservation medium containing UV-activated autophagy peptides at a stepwise cooling rate of -0.8℃ / min to obtain the central memory T cells.

[0064] Test data: Amplification reached 100-fold, phenotypic stability accounted for 95%, cryopreservation recovery rate was as high as 92%, and overall yield was 98%.

[0065] Example 2

[0066] Preparation of activated T cells: Under an alternating electric field with a frequency of 50 Hz, 10 mL of lymphocyte homing receptor-high expression cells with a purity of 95%-99% were sorted from 100 mL of peripheral blood mononuclear cells and resuspended in 5 mL of physiological saline containing a compound metabolic regulator for 15 minutes to prepare the activated T cells.

[0067] Preparation of synergistic culture medium: 10 mL of interleukin, 1 mL of deoxyglucose, 0.5 mL of rapamycin nanoparticles and 0.5 parts of histone methyltransferase inhibitor were added to serum-free basal culture medium to prepare the synergistic culture medium.

[0068] Three-dimensional scaffold preparation: 0.5g of modified collagen peptide, 0.1 parts of iron oxide, 0.3 parts of quantum dot-labeled dendritic cell organoids and 0.2 parts of follicular helper cell organoids were mixed and then assembled into the three-dimensional scaffold by two-photon excitation light field orientation.

[0069] Dynamic expansion: Activated T cells were seeded onto a three-dimensional scaffold and perfused with synergistic culture medium. A biaxial phase difference magnetic field and pulsed blue light with a frequency of 1.5 Hz were alternately applied and cultured for 7 days to prepare an expanded cell population.

[0070] Gene-edited cells: Edited T cells are prepared by electroporating an expanded cell population to knock out the programmed death receptor gene.

[0071] Cell cryopreservation: After expanding edited T cells in a microgravity reactor for 5 days, they were co-incubated with 0.1 part of engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters for 24 hours;

[0072] After incubation, the cells were frozen in 50 portions of serum-free cell preservation medium containing UV-activated autophagy peptides at a stepwise cooling rate of -0.8℃ / min to obtain the central memory T cells.

[0073] Test data: Amplification fold of 60-fold, phenotypic stability rate of 88%, cryopreservation recovery rate of 85%, and overall yield of 90%.

[0074] Example 3

[0075] Preparation of activated T cells: Under an alternating electric field with a frequency of 75 Hz, 20 mL of lymphocyte homing receptor-high expression cells with a purity of 95%-99% were sorted from 150 mL of peripheral blood mononuclear cells and resuspended in 7 mL of physiological saline containing a compound metabolic regulator for 22.5 minutes to prepare the activated T cells.

[0076] Preparation of synergistic culture medium: 15 mL of interleukin, 3 mL of deoxyglucose, 1 mL of rapamycin nanoparticles and 0.75 parts of histone methyltransferase inhibitor were added to serum-free basal culture medium to prepare the synergistic culture medium.

[0077] Three-dimensional scaffold preparation: 1.25g of modified collagen peptides, 0.3 parts of iron oxide, 0.4 parts of quantum dot-labeled dendritic cell organoids and 0.25 parts of follicular helper cell organoids were mixed and then assembled into the three-dimensional scaffold by two-photon excitation light field orientation.

[0078] Dynamic expansion: Activated T cells were seeded onto a three-dimensional scaffold and perfused with synergistic culture medium. A biaxial phase difference magnetic field and pulsed blue light with a frequency of 1-2 Hz were alternately applied and cultured for 8.5 days to prepare an expanded cell population.

[0079] Gene-edited cells: Edited T cells are prepared by electroporating an expanded cell population to knock out the programmed death receptor gene.

[0080] Cell cryopreservation: Edited T cells were expanded in a microgravity reactor for 6 days and then co-incubated with 0.13 portions of engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters for 29 hours.

[0081] After incubation, the cells were cryopreserved in 55 serum-free cell preservation solutions containing UV-activated autophagy peptides at a stepwise cooling rate of -0.8℃ / min to obtain the central memory T cells.

[0082] Test data: Amplification fold of 85-fold, phenotypic stability rate of 93%, cryopreservation recovery rate of 90%, and overall yield of 96%.

[0083] Example 4

[0084] Preparation of activated T cells: Under an alternating electric field with a frequency of 80 Hz, 25 mL of lymphocyte homing receptor-high expression cells with a purity of 95%-99% were sorted from 175 mL of peripheral blood mononuclear cells and resuspended in 8 mL of physiological saline containing a compound metabolic regulator for 25 minutes to prepare the activated T cells.

[0085] Preparation of synergistic culture medium: 18 mL of interleukin, 4 mL of deoxyglucose, 1.5 mL of rapamycin nanoparticles and 0.9 parts of histone methyltransferase inhibitor were added to serum-free basal culture medium to prepare the synergistic culture medium.

[0086] Three-dimensional scaffold preparation: 1.75g ​​of modified collagen peptides, 0.4 parts of iron oxide, 0.5 parts of quantum dot-labeled dendritic cell organoids and 0.3 parts of follicular helper cell organoids were mixed and then assembled into the three-dimensional scaffold by two-photon excitation light field orientation.

[0087] Dynamic expansion: Activated T cells were seeded onto a three-dimensional scaffold and perfused with synergistic culture medium. A biaxial phase difference magnetic field and pulsed blue light with a frequency of 1.7 Hz were alternately applied and cultured for 9 days to prepare an expanded cell population.

[0088] Gene-edited cells: Edited T cells are prepared by electroporating an expanded cell population to knock out the programmed death receptor gene.

[0089] Cell cryopreservation: Edited T cells were expanded in a microgravity reactor for 6.5 days and then co-incubated with 0.18 portions of engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters for 33 hours.

[0090] After incubation, the cells were cryopreserved in 58 serum-free cell preservation solutions containing UV-activated autophagy peptides at a stepwise cooling rate of -0.8℃ / min to obtain the central memory T cells.

[0091] Test data: Amplification fold of 90-fold, phenotypic stability rate of 94%, cryopreservation recovery rate of 89%, and overall yield of 97%.

[0092] Comparative Example 1 (Traditional Static Culture Method)

[0093] Central memory T cells were prepared using the traditional static culture method, following the steps outlined in existing literature CN112359016A.

[0094] Test data:

[0095] Amplification factor: 45x

[0096] Phenotypic stability: 78%

[0097] Cryopreservation recovery rate: 75%

[0098] Overall yield: 82%

[0099] Comparative Example 2 (No Physical Field Intervention Method)

[0100] Central memory T cells were prepared using a method without physical field intervention, and the specific steps were performed according to the method in the existing literature CN215480969U.

[0101] Test data:

[0102] Amplification factor: 55x

[0103] Phenotypic stability: 82%

[0104] Cryopreservation recovery rate: 80%

[0105] Overall yield: 88%

[0106] Comparative analysis

[0107] By comparing the detection data of central memory T cells prepared by different methods, it can be seen that the method provided by this invention has significant advantages:

[0108] Increased yield: The yields of all examples were higher than those of the comparative examples, with the yields of Examples 1-4 ranging from 90% to 98%, while the yields of Comparative Examples 1 and 2 were 82% and 88%, respectively.

[0109] Increased amplification fold: The amplification fold of the cells prepared by the present invention is significantly better than that of traditional methods. The amplification fold of Examples 1-4 is 60-100 times, while the amplification folds of Comparative Examples 1 and 2 are 45 times and 55 times, respectively.

[0110] Enhanced phenotypic stability: The cells prepared by this invention have higher phenotypic stability. The phenotypic stability of Examples 1-4 ranges from 88% to 95%, while the phenotypic stability of Comparative Examples 1 and 2 is 78% and 82%, respectively.

[0111] Optimization of cryopreservation recovery rate: The cryopreservation recovery rate of the cells prepared by this invention is generally higher than that of the comparative examples. The cryopreservation recovery rate of Examples 1-4 ranges from 85% to 92%, while the cryopreservation recovery rates of Comparative Example 1 and Comparative Example 2 are 75% and 80%, respectively.

[0112] Functional adaptability: This invention achieves a balance between cell expansion efficiency and functional stability through the synergistic effect of physical fields and three-dimensional scaffolds, while traditional methods, due to the lack of dynamic environmental regulation, have a higher risk of cell differentiation drift.

[0113] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0114] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for large-scale culture of central memory T cells (TCM), characterized in that, Includes the following steps: (a) Preparation of activated T cells: Under an alternating electric field with a frequency of 50-100Hz, 10-30 lymphocyte homing receptor highly expressing cells with a purity of 95%-99% were sorted from 100-200 peripheral blood mononuclear cells, and 5-10 parts of physiological saline containing compound metabolic regulators were added to resuspend the cells for 15-30 minutes to prepare the activated T cells. (b) Preparation of synergistic culture medium: 10-20 parts of interleukin, 1-5 parts of deoxyglucose, 0.5-2 parts of rapamycin nanoparticles and 0.5-1 parts of histone methyltransferase inhibitor were added to serum-free basal culture medium to prepare the synergistic culture medium; (c) Three-dimensional scaffold preparation: 0.5-2 parts of modified collagen peptide, 0.1-0.5 parts of iron oxide, 0.3-0.6 parts of quantum dot labeled dendritic cell organoids and 0.2-0.4 parts of follicular helper cell organoids are mixed and then assembled into the three-dimensional scaffold by two-photon excitation light field orientation. (d) Dynamic expansion: The activated T cells prepared in step (a) are seeded into the three-dimensional scaffold prepared in step (c) and perfused with the synergistic culture medium prepared in step (b). A biaxial phase difference magnetic field and pulsed blue light with a frequency of 1-2 Hz are applied alternately and cultured for 7-10 days to prepare an expanded cell population. (e) Gene-edited cells: The expanded cell population prepared in step (d) was electroporated to knock out the programmed death receptor gene to prepare edited T cells; (f) Cell cryopreservation: After the edited T cells prepared in step (e) are expanded in a microgravity reactor for 5-7 days, they are co-incubated with 0.1-0.2 parts of engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters for 24-36 hours. After incubation, the cells were frozen in 50-60 portions of serum-free cell preservation medium containing UV-activated autophagy peptides at a stepwise cooling rate of -0.8℃ / min to obtain the central memory T cells.

2. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (a), the peripheral blood mononuclear cells are cell monomers obtained by centrifuging peripheral blood from healthy donors at a centrifugation force of 400-450×g, a temperature of 18-22℃, and a time of 20-25 minutes. The saline solution containing the compound metabolic regulator is a transparent solution prepared by mixing the compound metabolic regulator, sodium dichloroacetate and phosphate buffer in a mass ratio of 0.1:0.1:7-0.2:0.5:11, wherein the compound metabolic regulator is a colorless metabolic solution prepared by mixing ketoglutarate and itaconic acid in a mass ratio of 2.5:1-4:

1.

3. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (b), the interleukin is a sterile solution composed of interleukin-7, interleukin-15, and interleukin-27; the deoxyglucose is a white needle-like crystal formed by the hydrolysis of glucose and thionyl chloride; the rapamycin nanoparticles are a milky white suspension formed by copolymerizing rapamycin and polylactic acid; and the histone methyltransferase inhibitor is a benzimidazole derivative solution formed by coupling a benzimidazole core with methoxybenzoyl chloride. The mass ratio of interleukin-7, interleukin-15 and interleukin-27 is 0.8:0.05:0.1-1:1:0.

5. The mass ratio of glucose to thionyl chloride is 1:1.5-1:2.0; The mass ratio of rapamycin to polylactic acid is 1:1.8-1.5:2.2; The mass ratio of the benzimidazole core to the methoxybenzoyl chloride is 1:1.2-1:1.

5.

4. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (c), the modified collagen peptide is a self-assembled peptide material composed of arginine, glycine, aspartic acid, tyrosine, isoleucine, serine, and arginine; the quantum dot-labeled dendritic cell organoids are cell organoids induced by differentiation factors from human umbilical cord blood stem cells, coupled with carboxylated polyethylene glycol-modified quantum dots in a mass ratio of 0.02:1 to form a homogeneous suspension; the follicular helper cell organoids are organoid colloids differentiated from human umbilical cord blood stem cells through the interaction of interleukin-21 and recombinant human inducible co-stimulatory ligands. The differentiation factor is a powder composed of tyrosine kinase ligand, granulocyte-macrophage colony-stimulating factor, and interleukin-4 in a mass ratio of 0.5:0.3:0.1-0.8:0.5:0.2; the quantum dots are core-shell particles obtained by coating selenium powder with thiourea, wherein the mass ratio of thiourea to selenium powder is 1:0.

8. The two-photon excitation optical field includes: wavelength 800-810nm, power density 20-25mW / cm². 2 Exposure time: 30-40 seconds.

5. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (d), the synergistic culture medium is replaced every 24 hours, and the replacement amount is 50%-70% of the total synergistic culture medium volume; The biaxial phase difference magnetic field includes: a sinusoidal axial magnetic field with a frequency of 10-15Hz, a square wave radial magnetic field with a frequency of 20-25Hz, an axial magnetic field strength of 5-8mT, a radial magnetic field strength of 8-10mT, an axial phase of 0-1 degree, and a radial phase of 30-35 degrees. The inoculation density is 1×10 6 -5×10 6 Cell volume per 1 ls / mL, volume 10-20 mL / well, temperature 34-36℃, time 1-2 hours.

6. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (e), before performing electroporation, the amplified cell population is first dispersed in an electroporation buffer, which is a complex solution composed of potassium chloride, magnesium chloride and hydroxyethylpiperazine ethanesulfonic acid in a mass ratio of 1:0.5:0.2-1:0.7:0.

2. The specific conditions for electroporation are: voltage 200-300V, pulse width 10-15ms, pulse duration 5-10ms, and pulse count 2-3 times.

7. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (f), the engineered Saccharomyces cerevisiae containing blue light and near-infrared light-controlled promoters is a light-controlled suspension constructed by inserting blue light-induced promoters and near-infrared light-induced promoters into a Saccharomyces cerevisiae strain through gene editing; the serum-free cell preservation solution containing ultraviolet light-activated autophagy peptides is an activation suspension composed of the ultraviolet light-activated autophagy peptides, sorbitol and serum-free cryopreservation solution. The mass ratio of the ultraviolet light-activated autophagy peptide, the sorbitol, and the serum-free cryopreservation solution is 1:5:90-5:7:

100.

8. The method for large-scale culture of central memory T cells (TCM) according to claim 1, characterized in that: In step (a), the resuspension treatment time is 15-30 minutes, and the initial concentration of peripheral blood mononuclear cells is 1×10⁻⁶. 6 -5×10 6 cells / mL.

9. A central memory T cell prepared by a large-scale culture method of central memory T cells (TCM) according to any one of claims 1 to 8.

10. The central memory T cell according to claim 9, characterized in that: The survival rate of the central memory T cells is 93%-95%, the in vitro expansion scale is 3800-4000 times, and the depletion resistance capacity is expanded by 80-100 times.

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