A factor combination composition for treg cell expansion and its dynamic regulation method

Abstract

This invention belongs to the field of in vitro cell expansion technology, specifically relating to a factor composition for cell expansion and its dynamic regulation method. The factor composition includes: membrane-bound co-stimulatory factors, soluble growth factors, and nutritional support factors; it can simulate the continuous and efficient binding of ligands to cell surface receptors under physiological conditions, precisely initiating core proliferation signaling pathways. The method involves: developing the factor composition; establishing a detection system integrating metabolic characteristics and molecular markers to accurately capture key nodes in the cell initiation, logarithmic, and stationary phases; and finally, dynamically regulating the type and concentration of factors based on these phase nodes. This invention can significantly improve cell proliferation initiation efficiency, expansion rate, and functional stability, providing sufficient and functionally stable cells for the treatment of autoimmune diseases, prevention of organ transplant rejection, and regulation of the tumor immune microenvironment, demonstrating significant clinical application value.

Description

Technical Field

[0001] This invention relates to In the field of in vitro cell expansion technology, specifically, a method for... Compositions of factors for cell expansion and their dynamic regulation methods. Background Technology

[0002] Regulation Cells are a core cellular subset in the immune system that maintains immune homeostasis. Their core function lies in inhibiting excessive immune responses and regulating the activation and proliferation of immune cells, demonstrating key clinical application value in areas such as the treatment of autoimmune diseases, prevention of organ transplant rejection, and regulation of the tumor immune microenvironment. The clinical application of cells requires a sufficient quantity of functionally stable cells, and in vitro expansion is the way to obtain cells that meet therapeutic needs. The core technology of cells.

[0003] However, in the existing technology used Key factors for cell proliferation are easily overlooked in practical applications. Cellular signal activation is specifically dependent on the structural state of factors, resulting in low efficiency of proliferation signal transduction. At the same time, existing dynamic regulation relies on preset time nodes or rough cell concentration indicators, does not take into account the deviation of molecular markers of cell proliferation stages caused by differences in culture environment, and lacks the means to accurately capture key nodes of stage transition, causing factor adjustment to lag behind the actual needs of cells.

[0004] Therefore, the present invention proposes a method for... Compositions of factors for cell expansion and their dynamic regulation methods. Summary of the Invention

[0005] In order to solve the problems mentioned in the background technology The modes of action of cell expansion factors and Mismatch between cell signaling activation needs and The present invention addresses the technical problem of determining the cell proliferation stage and the synergistic lag of factor adjustment. The objective of this invention is to provide a method for... Compositions of factors for cell expansion and their dynamic regulation methods.

[0006] The present invention adopts the following technical solution.

[0007] The first aspect of the present invention discloses a method for Treg A cell expansion factor composition, the factor composition comprising: membrane-bound co-stimulatory factors, soluble growth factors, and nutritional cofactors; The membrane-bound costimulatory factor is membrane-bound. The target of action is cell surface Specific receptors used to initiate Intracellular Proliferation signaling pathway; The soluble growth factor is recombinant human. The target of action is cell surface Specific receptors are used to maintain proliferation signals, inhibit cell differentiation, and promote metabolism; The nutritional cofactors are glutamine and vitamins. Glutamine acts on intracellular glutaminase, vitamins It acts on methyltransferase and methionine synthase to supplement metabolic substrates, alleviate lactic acid toxicity, and stabilize microenvironment parameters.

[0008] The second aspect of the present invention discloses a method for Treg A method for dynamic regulation of cell expansion, based on the factor composition described in the first aspect, includes the following steps: S1: Preparation for A concentrated composition of factors for cell expansion, and the concentrated composition of factors is prepared according to... The initial ratio is set according to the synergistic requirements of cell proliferation signaling pathways to form a factor concentration system for direct dilution and use; S2: Based on the aforementioned factor concentration system, inoculate at the appropriate concentration. Cells and modulated recombinant human After setting the initial glutamine concentration and culture environment parameters, the process was monitored. Cell density and survival rate are dynamically replenished with depleting factors, and in When the cell density reaches a threshold, the cells are passaged for use. Cells steadily and continuously expand; S3: Using the cell density and viability as core parameters, and adding accumulated lactate concentration as a metabolic auxiliary parameter, a comprehensive judgment is made. According to the preset stage-factor ratio, the factor concentrated composition is diluted and dynamically added according to the stage of cell proliferation to achieve a match between factor supply and cell stage requirements.

[0009] Preferably, S1 includes: S101: The factor concentration composition includes: membrane-bound co-stimulatory factor, soluble growth factor and nutritional cofactor; S102: The initial proportions of the factor-concentrated composition are calculated as follows: The concentration of the membrane-bound co-stimulatory factor is calculated using the following formula: ; in, For membrane-bound type in factor concentration systems The final concentration; for The minimum signal intensity threshold for initiating cell proliferation; This is the signal amplification factor; For signal transmission efficiency; This refers to the subsequent dilution factor of the factor concentration system; The formula for calculating the concentration of the soluble growth factor is as follows: ; in, Recombinant human in the factor concentration system The final concentration; These are the signal matching coefficients; Bioavailability; This is the signal maintenance coefficient; The formula for calculating the concentration of the nutritional cofactor is as follows: ; in, This represents the final concentration of glutamine in the factor concentration system. for Glutamine consumption rate during cell initiation; for Duration of cell initiation phase; Glutamine utilization efficiency; This refers to the volume of a single culture system.

[0010] Preferably, S2 includes: S201: After completing the preparation of the factor concentration system, perform inoculation, initial concentration adjustment and culture environment parameter setting in sequence; S202: In After the basic environment for cell expansion is constructed, Cells continuously consume recombinant human Both glutamine and other factors proliferate, and on Cell status is monitored to dynamically adjust factor concentrations; S203: When monitoring detects that the cell density has reached the aforementioned threshold density At the critical value, the proliferation is ensured by expanding the culture scale through the aforementioned subculturing operation. Adjusting cell volume and density; in, Cell density after passage; This refers to the volume of the cell expansion culture system after passage. In order to cultivate Cell density after hours.

[0011] Preferably, in S201, the inoculation and initial concentration adjustment are performed by adjusting the logarithmic growth phase... Cells with cell / mL High-density vaccination, recombinant human Initial and final concentrations The calculation formula is Initial and final glutamine concentrations The calculation formula is ;in, , All are the final concentrations of the factor concentration system in step S102; This refers to the volume of a single culture system. for Initial culture volume for cell expansion.

[0012] Preferably, S202 includes: S2021: Take the volume as Culture medium, cell density was measured. ; S2022: Cell density based on monitoring Data calculation of recombinant human Based on the consumption of glutamine factor, targeted supplementation can be provided to maintain proliferation signals and nutrient supply; S203: When monitoring detects that the cell density has reached the aforementioned threshold density When the critical value is reached, the proliferation is ensured by expanding the culture scale through subculturing.

[0013] Preferably, the cell density and the volume of the cell expansion culture system after the passage operation need to satisfy the formula, ; in, Cell density after passage; This refers to the volume of the cell expansion culture system after passage. In order to cultivate Cell density after hours.

[0014] Preferably, S3 includes: S301: Yes Cell status was monitored, and a monitoring data table was established to simultaneously record the detection time and cell density. The survival rate The lactic acid concentration and the cumulative amount of lactic acid ; S302: Based on the cell density The survival rate The lactic acid concentration and the cumulative amount of lactic acid Establish criteria for determining the proliferation stage to classify the proliferation stages; S303: Based on the factor concentration composition, the proportioning coefficient is set in stages, and the factor concentration of the fed-batch system in each stage is calculated by the dilution formula; S304: Dynamically regulates membrane-bound costimulatory factors through a stepwise increase in cell density. The fed-batch volume is adjusted to ensure that the intensity of the co-stimulation signal matches the needs of the cellular stage. S305: Based on lactate accumulation Dynamic regulation of soluble growth factors in recombinant human The concentration, and based on that concentration, the recombinant human... Dynamic fed-batch volume to balance proliferation signals and metabolic stress; S306: Combines the metabolic demand coefficient of each stage with a fixed molar ratio to dynamically supplement nutritional cofactors in order to maintain cellular metabolic homeostasis. S307: Based on the dynamic fed-batch volume and replenishment logic of membrane-bound co-stimulatory factors, soluble growth factors and nutritional cofactors, the system verifies and adjusts after each operation, re-determines the cell proliferation stage, and repeats steps S303-S306 to achieve a closed loop of dynamic factor regulation.

[0015] Preferably, the proliferation phase described in S302 includes an initiation phase, a logarithmic phase, and a stationary phase; The criteria for determining the start-up period are as follows: (Unit: cells / ) mL ), (unit: mmol / L ) 、 ; The criteria for determining the logarithmic period are as follows: , , ; The criteria for determining the stable period are as follows: , , ; The dilution basis formula described in S303 is: , The final concentration of the target factor in a certain stage of the fed-batch system; The initial concentration of the factor concentration system in S1; The corresponding ratio coefficients for each stage are taken from the ratio coefficient table for each stage of the factor concentration composition.

[0016] Preferably, the calculation of the dynamic supplementation volume of the nutritional support factors in S306 includes: S3061: The formula for glutamine supplementation is: ;in, This refers to a single dose of glutamine. The target concentration of glutamine for the current stage; The remaining concentration of glutamine in the culture system before fed-batch culture; This is the metabolic demand coefficient; The target concentration of glutamine at the current stage ,in, The initial concentration of the glutamine concentration system in S1; The stage ratio coefficient of glutamine; S3062: Vitamin Supplement amount The formula is: ;in, Vitamins The amount of a single replenishment.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least the following: 1. The factor composition of this invention adopts a membrane-bound factor design, accurately simulating the interaction between ligands and... Sustained and efficient binding to cell surface receptors can stably initiate [the process]. The core proliferation signaling pathway significantly enhances the strength and duration of signal transduction, efficiently promoting... Cells from quiescent period The transformation during the preparation period for splitting makes... The efficiency of cell proliferation initiation and the rate of expansion have been substantially improved.

[0018] 2. The dynamic regulation method of this invention integrates multiple dimensions of precise indicators such as cell metabolic characteristics and molecular markers, enabling real-time and accurate capture of... This involves identifying key transition points during the cell initiation, logarithmic, and stationary phases to achieve timely and precise regulation of factor types and concentrations, effectively preventing factor insufficiency or excess and ensuring [the cell's health]. The cell's functional stability at each stage of proliferation is significantly improved, resulting in increased cell viability and fold expansion, fully meeting clinical needs for sufficient quantities of functionally stable cells. The needs of cells.

[0019] 3. The factor composition and dynamic regulation method of the present invention work synergistically to maintain [the function] during in vitro amplification. The immunosuppressive phenotype of cells can also enhance their survival and migration abilities in the in vivo microenvironment, thus providing... Cells provide a high-quality cell source for clinical applications in areas such as autoimmune disease treatment, organ transplant rejection prevention, and tumor immune microenvironment regulation, and have significant clinical value and broad application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the overall system workflow of the present invention; Figure 2 This is a schematic diagram of the preparation and verification process of the factor concentration system of the present invention; Figure 3 This is a schematic diagram of the proliferation stage determination and dynamic factor addition process of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. The embodiments described in this application are merely some embodiments of this invention, and not all embodiments. Based on the spirit of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this invention.

[0023] To achieve the above objectives, the present invention provides the following technical solution: Embodiment 1 of the present invention provides a method for... Treg A cell expansion factor composition, the factor composition comprising: membrane-bound co-stimulatory factors, soluble growth factors, and nutritional cofactors; The membrane-bound costimulatory factor is membrane-bound. The target of action is cell surface Specific receptors, whose core function is to initiate Intracellular Proliferation signaling pathway; The soluble growth factor is recombinant human. The target of action is cell surface Specific receptors, whose core functions are to maintain proliferation signals, inhibit cell differentiation, and promote metabolism; The nutritional cofactors are glutamine and vitamins. Glutamine acts on intracellular glutaminase, vitamins It acts on methyltransferase and methionine synthase, and its core functions are to replenish metabolic substrates, alleviate lactic acid toxicity, and stabilize microenvironment parameters.

[0024] Embodiment 2 of the present invention provides a method for TregA method for dynamic regulation of cell expansion, based on a method described in Example 1 for... Treg Combinations of cell expansion factors, such as Figures 1 - 3 As shown, the method includes the following steps: S1: Preparation for A concentrated composition of factors for cell expansion, and the concentrated composition of factors is prepared according to... The initial ratio is set according to the synergistic requirements of cell proliferation signaling pathways to form a factor concentration system that can be directly diluted and used.

[0025] The concentrated factor composition contains three types of functional components: membrane-bound co-stimulatory factors, soluble growth factors, and nutritional cofactors. The molecular type, target site, and core function of each component must be matched. Cell proliferation signaling pathways and metabolic needs, among which membrane-bound co-stimulatory factors can be matched. Cell surface receptors activate proliferation signals, with specific parameters as follows: S101: The molecular type of the membrane-bound co-stimulatory factor is limited to membrane-bound. The target point is cell surface Specific receptor; its core function is to activate the proliferation signaling pathway, that is, by interacting with... Specific receptors bind specifically, initiating Intracellular The proliferation signaling pathway promotes cell proliferation. quiescent period The transformation during the division preparation period is for Cell proliferation provides the initiation signal; The molecular type of the soluble growth factor is limited to recombinant human. The target point is cell surface Specific receptors; their core functions include three aspects: first, maintaining the persistence of proliferation signals, ensuring that the signals activated by membrane-bound co-stimulatory factors do not decay, and avoiding cell proliferation arrest; second, inhibiting cell differentiation and preventing... Cellular and Cell transformation, maintaining cell proliferation capacity and immunosuppressive properties, and promoting cell metabolism to enhance... Treg The efficiency with which cells take up nutrients such as glucose and amino acids provides energy and material support for cell division. The molecular selection of the nutritional cofactor is limited to glutamine and vitamins. The target site of the glutamine target is... Intracellular glutaminase, the vitamin The target point is Intracellular methyltransferases and methionine synthases are two types of metabolism-related enzymes, and vitamins It can activate methyltransferase and methionine synthase, promote methionine synthesis, and support nucleic acid replication and protein synthesis; the core functions include three aspects: first, supplementing metabolic substrates, for It provides key raw materials for nucleic acid synthesis and energy metabolism during cell proliferation; secondly, it alleviates lactic acid toxicity by promoting the activity of enzymes related to lactic acid metabolism and reducing the damage to cells caused by lactic acid accumulation in the culture system; and thirdly, it stabilizes microenvironment parameters and maintains the stability of the culture system. Values, osmotic pressure, and other indicators are within a suitable range, which is... Cell proliferation creates a stable metabolic environment; S102: To ensure that the factor concentration composition meets the requirements For cell proliferation, the initial formulation must meet three core requirements: signal activation threshold, signal synergy ratio, and metabolic supply-demand balance. The specific concentrations of each component are calculated as follows: S1021: The formula for calculating the concentration of the membrane-bound co-stimulatory factor is as follows: ; in, For membrane-bound type in factor concentration systems Final concentration (unit: ng / mL ); for The minimum signal intensity threshold for initiating cell proliferation; This is the signal amplification factor (unitless); Signal transmission efficiency (unitless); This refers to the subsequent dilution factor of the factor concentration system (unitless). In this embodiment, the minimum signal strength threshold Preliminary experimental determinations indicate that the source of peripheral blood... cell The value is 5.2 × 10³, and the source of umbilical cord blood is... cell The value is 5.0 × 10³, with an error range of ±5%; membrane-bound type The signal amplification factor The value is 1.8, which is due to membrane binding. The molecular properties were determined; experimental verification showed that it is a membrane-bound type. and The efficiency of signal transduction by specific receptor binding The value is 0.62, with an error range of ±0.03; the subsequent dilution factor of the factor concentration system. for The standard operating parameters for cell culture are set at 100 for laboratory-scale culture and 200 for large-scale culture. This embodiment provides an example: the culture scale is at the laboratory level. =100), Pick Substituting into the formula, we get: (unit: ng / mL The actual value is 150. ng / mL To adapt to pipetting equipment 1 ng / mL The accuracy range.

[0026] S1022: The formula for calculating the concentration of the soluble growth factor is as follows: ; in, Recombinant human in the factor concentration system The final concentration, in units of ng / mL, The corresponding activity unit is 1 ng / mL ≈ 12 IU / mL (Based on the recombinant human used in this embodiment) Batch test reports for reagents confirm that specific activities vary between different manufacturers or batches, with a typical range of 10–15. IU / mL (Requires separate labeling); The signal matching coefficient (unitless); Bioavailability (unitless); This is the signal maintenance factor (unitless); In this embodiment, recombinant human was experimentally determined to be... Membrane-bound type The signal matching coefficient The value is 0.35, for recombinant human The bioavailability The value is 0.45, with an error range of ±0.02, for recombinant human. The signal maintenance coefficient It is 1.2; This embodiment provides an example: a membrane-bonded type. ( =150 ng / mL The cultivation scale is at the laboratory level. Substituting 100 into the formula, we get: (unit: ng / mL The actual value is 0.2. ng / mL To adapt to pipetting equipment 0.01 ng / mL The accuracy range.

[0027] S1023: The formula for calculating the concentration of the nutritional cofactor is as follows: ; in, The final concentration of glutamine in the factor concentration system (unit: mmol / L This provides initial substrates to maintain metabolic stability in the culture system; for Glutamine consumption rate during cell initiation (unit: mmol / ( cell· h )); for Cell initiation phase duration (unit: h ); Glutamine utilization efficiency (unit not specified); Volume of a single culture system (unit: L This is to build the system for the subsequent S2 step. Volume reference standard for the initial system of cell expansion; In this embodiment, the glutamine consumption rate was experimentally determined. The experimentally determined value was 0.08, with an error range of ±0.01; preliminary experiments verified that the... Cell initiation period duration 24 h The utilization efficiency of glutamine was determined experimentally. The value is 0.75, with an error range of ±0.04; the volume of the single culture system is... for The standard setting in cell culture, for laboratory-scale culture, is 0.5. L Large-scale cultivation is set at 10 L ; This embodiment provides an example: the culture scale is at the laboratory level. =0.5 L , Substituting 100 into the formula, we get: (0.08×24×100) / (0.75×0.5)=192 / 0.375=512 (unit: mmol / L), actual value is 500 mmol / L To be compatible with commercially available glutamine reagent 500 mmol / L Specifications.

[0028] S103: To ensure that the factor concentrate composition meets the requirements for sterility, stability, and dilution compatibility, three validations must be performed sequentially: sterility validation, stability validation, and dilution compatibility validation. Specifically: The sterility validation procedure is as follows: the factor concentration system prepared by mixing the component concentrations determined in S102 is passed through a 0.22mm pore size filter.µm The polyethersulfone filter membrane is used for filtration and sterilization (the filter membrane needs to be autoclaved at 121°C for 30 minutes). min Take 100 ml of the filtered concentrate. µL Evenly coated on The solid culture medium surface was placed in an incubator at 37°C and inverted for 48 days. h If no bacterial or fungal colonies grow on the surface of the culture medium, it is considered sterile and qualified. The stability verification procedure is as follows: The sterile and qualified factor concentration system is dispensed into 1... ml / Each batch of cryovials should be divided into portions of no less than 9 portions and stored at -80°C. Three cryovials should be removed on the day of freezing (when freshly prepared), on the 7th day of freezing, and on the 14th day of freezing. 100 ml of each cryovial should be taken from each cryovial. µL Concentrate ， Thaw rapidly in a 37°C water bath; Method for detecting membrane binding at various time points and reorganized persons The actual concentration; if the concentration on the 14th day of freezing has an error of ≤10% compared with the concentration on the day of freezing and the relative standard deviation of the parallel samples is ≤5%, it is judged to be of acceptable stability. The dilution compatibility verification process is as follows: Dilute to a preset dilution factor (e.g., ...). =100), the factor concentration system was treated with serum-free... Cell culture medium was diluted to prepare a simulated culture system, and precise methods were employed. The system after dilution was tested. The osmotic pressure should be maintained between 7.2 and 7.4, and the osmotic pressure of the diluted system should be maintained between 280 and 320, measured using an osmometer. mOsm / kg When both parameters meet the requirements, the dilution compatibility is deemed acceptable.

[0029] S2: Based on the aforementioned factor concentration system, inoculate at the appropriate inoculation density. Cells and modulated recombinant human After setting the initial glutamine concentration and culture environment parameters, the process was monitored. Cell density and survival rate are dynamically replenished with depleting factors, and in When the cell density reaches a threshold, passage is performed to achieve [the desired result]. Cells steadily and continuously proliferate.

[0030] S201: After completing the preparation of the aforementioned factor concentration system, the first step is to complete... Cell seeding procedures, and the recombinant human cells in the factor concentration system Adjust the initial concentration of glutamine to a suitable level. The level of cell proliferation, in order to build The basic environment for cell expansion is as follows: S2011: First, those in the logarithmic growth phase... Cells with cell / mL The recombinant human cells were inoculated at a density into a culture system containing serum-free medium, and then the recombinant human cells were calculated based on the parameters of the factor concentration system. The initial addition of glutamine is diluted to match the initial cell proliferation requirements, resulting in a culture system after dilution of the factor concentration system, which is the system used for cell culture. Calculate recombinant human The formula is:

[0031] in, For reorganization Initial and final concentrations (unit: ng / mL ); Recombinant human for the factor concentration system in step S1 Final concentration; This refers to the volume of a single culture cycle in step S1. for Initial culture volume for cell expansion; The formula for calculating glutamine is:

[0032] in, Initial and final glutamine concentrations (unit: mmol / L ); The final concentration of glutamine in the factor concentration system during step S1; This refers to the volume of a single culture cycle in step S1. for Initial culture volume for cell expansion; Dilution should be done with serum-free solution. The cell culture medium is a diluted matrix, prepared according to the initial and final concentrations of recombinant human IL-2 as described. and the initial and initial concentrations of glutamine Take the corresponding volume of the factor concentration system (containing recombinant human IL-2 and glutamine) and slowly add it to the inoculated cells. In the basic cell culture system, add the ingredients while gently mixing. This embodiment provides an example: First, those in the logarithmic growth phase Cells at 10 6 Cells are seeded into the culture system at a density of 8 × 10⁶ cells / mL. 6 One Treg cell can determine the initial culture volume. The initial and final concentrations of recombinant human IL-2 were calculated using the formula based on the factor concentration system parameters determined in step S1 (recombinant human IL-2 concentration G = 800 U / mL, single culture volume V = 20 mL, glutamine concentration N = 150 mmol / L). The initial and final concentrations of glutamine were 2000 U / mL. The concentration was 375 mmol / L. Subsequently, using serum-free Treg cell culture medium as the dilution matrix, the corresponding volume of the factor concentration system was measured according to the calculated initial addition amount and slowly added to the basal culture system of the seeded cells while gently mixing.

[0033] S2012: Next, the culture environment parameters are set. Based on the culture system diluted from the factor concentration system obtained in S2011, the various parameters of the culture environment are adjusted to achieve precise matching. Cellular physiological metabolism and proliferation requirements; Preferably, the culture system is placed at 37°C. In a constant temperature incubator, maintain the system 7.2-7.4, dissolved oxygen concentration of 40%-60% saturated dissolved oxygen, to ensure that cells are in a suitable growth microenvironment; S202: In After the basic environment for cell expansion is constructed, The cells will continuously consume the recombinant human Both glutamine and other factors proliferate, requiring periodic monitoring to control their activity. Cell state is dynamically adjusted to adjust factor concentrations, specifically: S2021: To keep abreast of developments Cell proliferation progress and survival status need to be monitored by cell density every 24 hours. Detection: Take the volume as (unit: L The cell density was measured using the culture medium. (unit: cell / mL When the cell density Reaching the threshold density When this happens, it is necessary to initiate fluid replacement or passage procedures to avoid insufficient nutrition due to excessively high cell density. In this embodiment, based on The threshold density is a commonly used industry practice in cell culture. The value is 2.0; S2022: Cell density based on monitoring Further calculations of the data on recombinant human Based on the consumption of both glutamine and other factors, targeted supplementation is provided to maintain proliferation signals and nutrient supply, specifically as follows: Recombinant human Will follow Cell proliferation continuously consumes nutrients; the consumption rate should be determined before calculating the culture time. The remaining concentration after one hour is calculated using the following formula:

[0034] in, In order to cultivate Reconstruction of human after hours Remaining concentration (unit: ng / mL ); For reorganization Initial and final concentrations (unit: ng / mL ); For reorganization Consumption rate (unit: ng / ( cell h )); In order to cultivate Average cell density per hour (unit: cell / mL ); For cultivation time (unit: h );

[0035] In this embodiment, the recombinant human IL-2 consumption rate First, with 10 6 Preliminary experiments were conducted using Treg cells in the logarithmic growth phase (cells / mL). IL-2 concentration was measured periodically under standard culture conditions, and the average value was calculated from multiple parallel experiments as the baseline. Subsequent dynamic calibration was performed based on the proliferation phase determination results. The baseline value was set at 80%-90% for the initiation phase, 100%-120% for the logarithmic phase, and 70%-80% for the stationary phase to match the metabolic and proliferation requirements of cells at different stages. when concentrations below the threshold (unit: ng / mL When this happens, the replenishment amount needs to be calculated. Reorganization The formula for restoring the concentration to the target value is:

[0036] in, For reorganization Supplement amount (unit: ng ); For reorganization Target concentration after supplementation (unit:ng / mL ); It serves as a unit conversion factor, used to unify the units of volume and concentration, thereby obtaining the correct unit for supplementary quantities; In this embodiment, based on Research consensus and industry standards in the field of cell proliferation and cell culture, the threshold concentration mentioned The value is 0.1.

[0037] Glutamine as The core nutrients of cell metabolism, their consumption patterns and Similarly, first calculate the culture The remaining concentration after one hour is calculated using the following formula:

[0038] in, In order to cultivate Remaining glutamine concentration after hours (unit: mmol / L ); Initial and final glutamine concentrations (unit: mmol / L ); Glutamine consumption rate (unit: mmol / ( cell h )); When the remaining concentration of glutamine Below the concentration threshold (unit: mmol / L When ), the supplementary amount needs to be calculated to restore the glutamine concentration to the target value. The formula is:

[0039] in, Glutamine supplementation (unit: mmol ); Target concentration after glutamine supplementation (unit: mmol / L ); In this embodiment, based on Research consensus and industry standards in the field of cell proliferation and cell culture, including the concentration threshold. The value is 0.5.

[0040] S203: When monitoring detects that the cell density has reached the aforementioned threshold density At the critical point, fluid supplementation alone cannot meet the cell growth requirements, and it is necessary to ensure continued proliferation by increasing the culture scale through passages. Specifically: Passaging must adhere to the principle of cell conservation, meaning the total number of cells before and after passage must remain constant, and the cell density and volume of the cell expansion culture system after passage must satisfy the following formula: ; in, Cell density after passage (unit: cell / mL ); Volume of cell expansion culture system after passage (unit: L ); In order to cultivate Cell density after hours (unit: cell / mL ); Finally, based on the new volume of the passaged system... Referring to the initial factor concentration calculation formula in S201, the recombinant human cells were readjusted simultaneously with changes in cell density. and glutamine concentration to target value and This ensures that the cells after passage remain in a suitable growth environment, maintaining the continuity and stability of the expansion process.

[0041] In this embodiment, the threshold density Based on industry practice, the threshold value is set at 2.0 × 10⁻⁶. 6 Cells / mL.

[0042] S3: Using cell density and viability as core parameters, and adding accumulated lactate concentration as a metabolic auxiliary parameter, a comprehensive judgment is made. According to the preset stage-factor ratio, the factor concentrated composition is diluted and dynamically added according to the stage of cell proliferation to achieve a match between factor supply and cell stage requirements.

[0043] It is worth noting that both S2 and S3 revolve around As the cell expansion process unfolds, S3 supplements metabolic parameters and optimizes factor regulation logic based on S2. It is not a sequential process, but rather the two work together to match the needs of the cells.

[0044] S301: First, clarify Cell state monitoring parameters and detection protocols are as follows: The cell density (unit: cell / mL ) and survival rate (Unit:%) The fully automated cell counter was used to detect the cells once every 24 hours, and the data was directly linked to the S2 monitoring results. Increased lactic acid concentration (unit: mmol / L The detection method used was the lactate dehydrogenase method, referring to... GB / T 20574-2006 "Determination of Lactic Acid in Fermentation Broth", take 1mL Culture medium 3000 rpm Centrifuge 5 min The supernatant was collected and analyzed using a biochemical analyzer (lactic acid detection was performed using a Hitachi 7600 fully automated biochemical analyzer, detection wavelength 340 nm). nm (Reaction temperature 37℃), measured every 12 hours, and the cumulative lactic acid amount over 12 hours was calculated. The formula is ,in, The lactate concentration was measured 12 hours prior, reflecting changes in cellular metabolic intensity. Parameter recording requires the establishment of a monitoring data table to synchronously record the detection time and the cell density. The survival rate The lactic acid concentration and the cumulative amount of lactic acid To ensure data traceability; S302: Based on the cell density The survival rate The lactic acid concentration and the cumulative amount of lactic acid Establish criteria for determining the proliferation stage to classify the proliferation stages and avoid misjudgment of the stage, specifically as follows: The proliferation phase includes the initiation phase, the logarithmic phase, and the stationary phase; The phase characteristics of the initiation period are as follows: Cells are in a "dormant-activated" transition phase, with proliferation signals just initiated, low metabolic intensity, and low lactic acid production; the criteria for judgment are... (Unit: cells / ) mL ), (unit: mmol / L ) 、 ; The phase characteristics of the logarithmic period are as follows: The cells exhibit the fastest proliferation rate, vigorous metabolic activity, and moderate lactic acid production, requiring a supply of enhancing factors; the criteria for judgment are... , , ; The phase characteristics of the stable period are as follows: Cell density is approaching the passage threshold, growth rate is slowing, and lactic acid is accumulating significantly, requiring optimization of factor ratios to reduce metabolic stress; the judgment criteria are... , , ; When the cell density Prioritize passage of S203 cells. After passage, re-determine the proliferation stage based on the new cell density (post-passage cell density is...). cell / mIf the cumulative lactic acid content is <0.5 mmol / L, This is considered the initiation phase if 0.5 ≤ lactic acid accumulation < 1.2. mmol / L (and the survival rate is ≥95%, which is determined to be in the logarithmic phase). S303: Based on the factor concentration composition, the proportioning coefficient is set in stages, and the factor concentration of the fed-batch system in each stage is calculated by the dilution formula; The following is a table of proportioning coefficients for the concentrated factor composition at different stages:

[0045] The basic formula for dilution is: ;in, The final concentration of the target factor in a certain stage of the fed-batch system; The initial concentration of the factor concentration system in S1 ( 150 ng / mL; 0.2 ng / mL; glutamine: 500 mmol / L); The corresponding proportioning coefficients for each stage are taken from the proportioning coefficient table of the factor concentrated composition stage; This example provides two stages of dilution concentration calculation examples: Example 1: If S1 Initial concentration of concentrate It was 150 ng / mL, during the logarithmic phase. Stage ratio coefficient In the logarithmic phase addition system Final concentration (Unit: ng / mL); Example 2: If the recombinant person Initial concentration of concentrate It is 0.2 ng / mL During the stable period Stage ratio coefficient If the value is 0.6, then the recombinant human in the steady-state flow system... Final concentration (unit: ng / mL ); S304: Regarding the existing technology The supply of (membrane-bound costimulatory factors) was not dynamically adjusted according to cell density, leading to... This step addresses the mismatch between cell proliferation signals and cell numbers by dynamically regulating cell density using a stepwise gradient rule. The fed-batch volume is adjusted to ensure that the intensity of the co-stimulatory signal matches the needs of the cellular stage. The dynamic flow volume is calculated as follows:

[0046] in, for The single-batch volume (unit: mL); For the current stage The final concentration (calculated from S303); In the pre-culturing system The remaining concentration (unit: ng / mL, by...) (Legal testing); This represents the total volume of the current culture system (unit: mL). For S1 The concentration of the concentrated system; The stepwise increasing rule is: when the cell density... Each increase cell / mL The current stage final concentration The matching factor for the corresponding stage is increased by 10%, but the maximum increase is no more than 1.2 times the matching factor for the logarithmic period. The final maximum concentration is 150 × 1.2 = 180. ng / mL Once this value is exceeded, the increment stops.

[0047] This embodiment provides a calculation example: Currently in the logarithmic period, Target final concentration The concentration was 150 ng / mL in the culture system before feeding. Remaining concentration The concentration is 80 ng / mL, and the total volume of the current culture system is... It is 500mL. Initial concentration of concentrate If the concentration is 150 ng / mL, then the required supplement is... Concentrated liquid volume (Unit: mL) S305: Targeting soluble growth factors (recombinant human) in existing technologies. Fixed concentration leads to This step addresses the issue of excessive lactate accumulation during the cell stabilization phase by dynamically fine-tuning the amount of accumulated lactate in recombinant human cells. Concentration to balance proliferation signals and metabolic stress, recombinant human The dynamic flow volume is calculated as follows: First, calculate the recombinant human... The concentration adjustment factor is calculated using the following formula:

[0048] in, For reorganization Concentration adjustment factor (unitless); This represents the target value for lactic acid accumulation at the current stage. The current 12-hour cumulative lactate content (unit: mmol / L ); In this embodiment, based on Based on research findings on cell metabolic characteristics and industry practices in immune cell culture, the recombinant human... Concentration adjustment coefficient The value ranges from 0.5 to 1.5, and this is the target value for lactic acid accumulation in the current stage. The start-up period is 0.3. mmol / L The logarithmic period is 0.8. 1.0 mmol / L The stable period is 1.2. mmol / L ; Based on the recombinant human Concentration adjustment coefficient The target concentration is calculated using the following formula: ;in, After fine-tuning Target concentration (unit: ng / mL); This is the baseline target concentration for the current stage (calculated from S303); Based on the aforementioned fine-tuned recombinant human Target concentration The formula for calculating the feed volume is:

[0049] in, For reorganization Single-flow volume (unit: µL ); Recombinant human cells in the pre-infusion culture system Remaining concentration (unit: ng / mL, pass (Legal testing); Recombinant human in S1 Specific activity of the concentrated system (unit: IU / mL In this embodiment, 1 ng / mL ≈12 IU / mL ,Right now ); This embodiment provides a calculation example: The current period is a stable period, and the reorganized person initial target final concentration It is 0.12 ng / mL Target value for lactic acid accumulation 1 mmol / L The actual cumulative amount of lactic acid detected so far. It is 1.3 mmol / L Then the recombinant human Concentration adjustment coefficient Recombinant human after fine-tuning Target final concentration (unit: ng / mL ); If recombinant human cells are in the pre-infusion culture system Remaining concentration It is 0.05 ng / mL The total volume of the current cultivation system For 500 mL The additional recombinant person required. Concentrated liquid volume (unit: µL ), that is, 7.08 mL ; S306: Addressing the issues of nutritional cofactors (glutamine and vitamins) in existing technologies. The supply was not precisely matched to the metabolic stage, resulting in To address the issue of cellular metabolic imbalance, this step combines the stage-specific metabolic demand coefficient with a fixed molar ratio (1:1000 molar ratio) to dynamically supplement nutrient cofactors to maintain cellular metabolic homeostasis. The dynamic supplementation volume of nutrient cofactors is calculated as follows: S3061: The formula for glutamine supplementation is: ;in, This refers to a single dose of glutamine (unit: mmol); The target concentration of glutamine for the current stage (calculated from S303) ,in, The initial concentration of the glutamine concentration system in S1; (This refers to the stage ratio coefficient of glutamine, taken from the stage ratio coefficient table of the factor concentrated composition in S303). The remaining concentration of glutamine in the culture system before fed-batch culture (unit: mmol / L, detected by a biochemical analyzer); This is the metabolic demand coefficient, adjusted according to the metabolic intensity of the stage. In this embodiment, based on The metabolic demand coefficient was determined by combining research findings on cell metabolic characteristics with industry practices in immune cell culture. The value is 0.9 during the initiation period, 1.2 during the logarithmic period, and 1.5 during the stationary period. S3062: Vitamin The formula for supplementation is: ;in, Vitamins Single replenishment amount (unit: mmol ), calculated proportionally based on glutamine supplementation; This embodiment provides a calculation example: Currently in the logarithmic phase, the initial concentration of the glutamine concentrate... For 500 mmol / L The stage ratio coefficient of glutamine in the logarithmic phase If the value is 1.0, then the final concentration of glutamine in the logarithmic feed system is... (unit: mmol / L If the residual concentration of glutamine in the culture system before feeding is... 300 mmol / L The total volume of the current cultivation system It is 0.5 L Glutamine metabolic demand coefficient in the logarithmic phase If the value is 1.2, then the single dose of glutamine supplementation is... Vitamins single replenishment amount (unit: mmol ); S307: The dynamic feed volume and replenishment logic based on membrane-bound co-stimulatory factors, soluble growth factors, and nutritional cofactors require verification and adjustment after each operation, as detailed below: Six hours after the factor flow addition operation was completed, the culture system was tested. Value (maintained at 7.2) 7.4), Osmotic pressure (280) 320 mOsm / kg If the range is exceeded, fine-tune using sterile saline or concentrated factor solution. Subsequent monitoring should be performed according to the established cycle (cell density / viability measured every 24 hours, lactate measured every 12 hours) to reassess the cell proliferation stage and repeat step S303. Step S306 achieves a closed loop for dynamic regulation of factors; like <7.0 or osmotic pressure>350 mOsm / kg Immediately stop the subsequent factor addition and add sterile saline at 10% of the system volume. 7.3, osmotic pressure 300 mOsm / kg After 30 minutes, retest and resume flow feeding only after the target is met.

[0050] This embodiment provides a complete example of dynamic regulation of Treg cell in vitro expansion in a logarithmic phase-dominated expansion scenario: Amplification started 0h, with an initial culture volume of 5L and an initial inoculation density of 1.0×10⁻⁶ during the logarithmic phase. 6 cells / mL Cells, with a total cell count of 5 × 10⁻⁶ 6Cells were cultured in a serum-free medium at 37°C and 5% CO2 (initial pH = 7.3, osmotic pressure = 300 mOsm / kg) with initial concentrations of 1.0 ng / mL recombinant human IL-2, 2.0 mmol / L glutamine, and 5.0 μg / mL membrane-bound co-stimulatory factor (adapted to the initial cell density, with the concentrations maintained throughout). The remaining concentrations of the two factors were then calculated using the adjusted formula. The first monitoring was performed 24 hours after culture, at which time the cell density had increased to 1.5 × 10⁻⁶. 6 Cells / mL, viability 95%, lactate accumulation 0.6 mmol / L, meeting the criteria for logarithmic phase; IL-2 consumption rate set at 100% logarithmic phase was 0.04 ng / (10⁻⁶). 6 cell Calculated at h), the cumulative consumption concentration was 0.0144 ng / mL, and the remaining concentration was 0.9856 ng / mL (above the threshold of 0.1 ng / mL). Glutamine was consumed at 0.08 mmol / (10 6 cell The rate of consumption (h) was 0.0288 mmol / L, with a remaining concentration of 1.9712 mmol / L. No additional factor was required, and the culture was continued under the current conditions. When cultured for 48 hours, the cell density was monitored again and increased to 2.8 × 10⁻⁶. 6 Cells / mL (viability 93%), lactate accumulation 1.0 mmol / L (still in logarithmic phase, close to the stationary phase threshold), IL-2 consumption rate increased to the upper limit of logarithmic phase 0.048 ng / (10⁻⁶) 6 cell (h), the cumulative consumption concentration over 48 hours was 0.0645 ng / mL, with a remaining concentration of 0.9355 ng / mL; the cumulative consumption of glutamine was 0.1075 mmol / L, with a remaining concentration of 1.8925 mmol / L; due to cell density exceeding 2.0 × 10⁻⁶ h. 6 The cell / mL passage threshold was set, and the passage operation was initiated. Following the principle of total cell population conservation, the culture volume was adjusted to 14 L to ensure the cell density returned to 1.0 × 10⁻⁶ cells / mL after passage. 6 Cells / mL, and simultaneously replenish the two factors according to the new volume to maintain the initial concentration; The first monitoring was completed at 72 hours after passage, and the cell density recovered to 1.3 × 10⁻⁶. 6 Cells / mL, viability 94%, lactate accumulation 0.7 mmol / L, IL-2 consumption rate reduced to 0.04 ng / (10⁻⁶) 6 cell h), after passage, the cell consumption concentration was 0.0125 ng / mL and glutamine consumption was 0.02496 mmol / L. The remaining concentrations of both factors remained stable, requiring no further action, and the next monitoring cycle began; at the 72-hour mark, the total cell count decreased from the initial 5 × 10⁻⁶. 6 The cell count increased to 18.2 × 10⁻⁶. 6 The cells expanded 3.64 times, and all key indicators throughout the process met the dynamic regulation targets.

[0051] This disclosure can be a system, method, and / or computer program product. A computer program product may include a computer-readable storage medium having computer-readable program instructions loaded thereon for causing a processor to implement various aspects of this disclosure.

[0052] Computer-readable storage media can be tangible devices capable of holding and storing instructions for use by an instruction execution device. Computer-readable storage media can be, for example—but not limited to—electrical storage devices, magnetic storage devices, optical storage devices, electromagnetic storage devices, semiconductor storage devices, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of computer-readable storage media include: portable computer disks, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable compact disc read-only memory (CD-ROM), digital multifunction disc (DVD), memory sticks, floppy disks, mechanical encoding devices, such as punch cards or recessed protrusions storing instructions thereon, and any suitable combination of the foregoing. The computer-readable storage media used herein are not to be construed as transient signals themselves, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through waveguides or other transmission media (e.g., light pulses through fiber optic cables), or electrical signals transmitted through wires.

[0053] The computer-readable program instructions described herein can be downloaded from computer-readable storage media to various computing / processing devices, or downloaded via a network, such as the Internet, local area network, wide area network, and / or wireless network, to an external computer or external storage device. The network may include copper transmission cables, fiber optic transmission, wireless transmission, routers, firewalls, switches, gateway computers, and / or edge servers. A network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards them to the computer-readable storage media in the respective computing / processing device.

[0054] Computer program instructions used to perform the operations of this disclosure may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, status setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Smalltalk, C++, etc., and conventional procedural programming languages ​​such as the "C" language or similar programming languages. The computer-readable program instructions may execute entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In cases involving a remote computer, the remote computer may be connected to the user's computer via any type of network—including a local area network (LAN) or a wide area network (WAN)—or may be connected to an external computer (e.g., via the Internet using an Internet service provider). In some embodiments, electronic circuitry, such as programmable logic circuitry, field-programmable gate arrays (FPGAs), or programmable logic arrays (PLAs), is personalized by utilizing the status information of the computer-readable program instructions to implement various aspects of this disclosure.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A method for Treg A cell proliferation factor composition, characterized in that, The factor composition includes: membrane-bound co-stimulatory factors, soluble growth factors, and nutritional cofactors; The membrane-bound costimulatory factor is membrane-bound. The target of action is cell surface Specific receptors used to initiate Intracellular Proliferation signaling pathway; The soluble growth factor is recombinant human. The target of action is cell surface Specific receptors are used to maintain proliferation signals, inhibit cell differentiation, and promote metabolism; The nutritional cofactors are glutamine and vitamins. Glutamine acts on intracellular glutaminase, vitamins It acts on methyltransferase and methionine synthase to supplement metabolic substrates, alleviate lactic acid toxicity, and stabilize microenvironment parameters.

2. A method for Treg A method for dynamic regulation of cell expansion, based on the factor composition described in claim 1. Its features are, Includes the following steps: S1: Preparation for A concentrated composition of factors for cell expansion, and the concentrated composition of factors is prepared according to... The initial ratio is set according to the synergistic requirements of cell proliferation signaling pathways to form a factor concentration system for direct dilution and use; S2: Based on the aforementioned factor concentration system, inoculate at the appropriate concentration. Cells and modulated recombinant human After setting the initial glutamine concentration and culture environment parameters, the process was monitored. Cell density and survival rate are dynamically replenished with depleting factors, and in When the cell density reaches a threshold, the cells are passaged for use. Cells steadily and continuously expand; S3: Using the cell density and viability as core parameters, and adding accumulated lactate concentration as a metabolic auxiliary parameter, a comprehensive judgment is made. According to the preset stage-factor ratio, the factor concentrated composition is diluted and dynamically added according to the stage of cell proliferation to achieve a match between factor supply and cell stage requirements.

3. A method for use according to claim 2 Treg A method for dynamic regulation of cell expansion, characterized in that, S1 includes: S101: The factor concentration composition includes: membrane-bound co-stimulatory factor, soluble growth factor and nutritional cofactor; S102: The initial proportions of the factor-concentrated composition are calculated as follows: The concentration of the membrane-bound co-stimulatory factor is calculated using the following formula: ; in, For membrane-bound type in factor concentration systems The final concentration; for The minimum signal intensity threshold for initiating cell proliferation; This is the signal amplification factor; For signal transmission efficiency; This refers to the subsequent dilution factor of the factor concentration system; The formula for calculating the concentration of the soluble growth factor is as follows: ; in, Recombinant human in the factor concentration system The final concentration; These are the signal matching coefficients; Bioavailability; This is the signal maintenance coefficient; The formula for calculating the concentration of the nutritional cofactor is as follows: ; in, This represents the final concentration of glutamine in the factor concentration system. for Glutamine consumption rate during cell initiation; for Duration of cell initiation phase; Glutamine utilization efficiency; This refers to the volume of a single culture system.

4. A method for use according to claim 2 Treg A method for dynamic regulation of cell expansion, characterized in that, S2 includes: S201: After completing the preparation of the factor concentration system, perform inoculation, initial concentration adjustment and culture environment parameter setting in sequence; S202: In After the basic environment for cell expansion is constructed, Cells continuously consume recombinant human Both glutamine and other factors proliferate, and on Cell status is monitored to dynamically adjust factor concentrations; S203: When monitoring detects that the cell density has reached the aforementioned threshold density At the critical value, the proliferation is ensured by expanding the culture scale through the aforementioned subculturing operation. Adjusting cell volume and density; in, Cell density after passage; This refers to the volume of the cell expansion culture system after passage. In order to cultivate Cell density after hours.

5. A method for use according to claim 4 Treg A method for dynamic regulation of cell expansion, characterized in that, In S201, the inoculation and initial concentration adjustment are as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] Cells with cell / mL High-density vaccination, recombinant human Initial and final concentrations The calculation formula is Initial and final glutamine concentrations The calculation formula is ;in, , All are the final concentrations of the factor concentration system in step S102; This refers to the volume of a single culture system. for Initial culture volume for cell expansion.

6. A method for use according to claim 4 Treg A method for dynamic regulation of cell expansion, characterized in that, S202 includes: S2021: Take the volume as Culture medium, cell density was measured. ; S2022: Cell density based on monitoring Data calculation of recombinant human Based on the consumption of glutamine factor, targeted supplementation can be provided to maintain proliferation signals and nutrient supply; S203: When monitoring detects that the cell density has reached the aforementioned threshold density When the critical value is reached, the proliferation is ensured by expanding the culture scale through subculturing.

7. A method for use according to claim 6 Treg A method for dynamic regulation of cell expansion, characterized in that, The cell density and cell expansion culture system volume after the passage operation must meet the following formula. ; in, Cell density after passage; This refers to the volume of the cell expansion culture system after passage. In order to cultivate Cell density after hours.

8. A method for use according to claim 2 Treg A method for dynamic regulation of cell expansion, characterized in that, S3 includes: S301: Yes Cell status was monitored, and a monitoring data table was established to simultaneously record the detection time and cell density. The survival rate The lactic acid concentration and the cumulative amount of lactic acid ; S302: Based on the cell density The survival rate The lactic acid concentration and the cumulative amount of lactic acid Establish criteria for determining the proliferation stage to classify the proliferation stages; S303: Based on the factor concentration composition, the proportioning coefficient is set in stages, and the factor concentration of the fed-batch system in each stage is calculated by the dilution formula; S304: Dynamically regulates membrane-bound costimulatory factors through a stepwise increase in cell density. The fed-batch volume is adjusted to ensure that the intensity of the co-stimulation signal matches the needs of the cellular stage. S305: Based on lactate accumulation Dynamic regulation of soluble growth factors in recombinant human The concentration, and based on that concentration, the recombinant human... Dynamic fed-batch volume to balance proliferation signals and metabolic stress; S306: Combines the metabolic demand coefficient of each stage with a fixed molar ratio to dynamically supplement nutritional cofactors in order to maintain cellular metabolic homeostasis. S307: Based on the dynamic fed-batch volume and replenishment logic of membrane-bound co-stimulatory factors, soluble growth factors and nutritional cofactors, the system verifies and adjusts after each operation, re-determines the cell proliferation stage, and repeats steps S303-S306 to achieve a closed loop of dynamic factor regulation.

9. A method for use according to claim 8 Treg A method for dynamic regulation of cell expansion, characterized in that, The proliferation phase described in S302 includes the initiation phase, the log phase, and the stationary phase; The criteria for determining the start-up period are as follows: (Unit: cells / ) mL ), (unit: mmol / L ) 、 ; The criteria for determining the logarithmic period are as follows: , , ; The criteria for determining the stable period are as follows: , , ; The dilution basis formula described in S303 is: , The final concentration of the target factor in a certain stage of the fed-batch system; The initial concentration of the factor concentration system in S1; The corresponding ratio coefficients for each stage are taken from the ratio coefficient table for each stage of the factor concentration composition.

10. A method for use according to claim 8 Treg A method for dynamic regulation of cell expansion, characterized in that, The calculation of the dynamic supplementation volume of nutritional cofactors as described in S306 includes: S3061: The formula for glutamine supplementation is: ;in, This refers to a single dose of glutamine. The target concentration of glutamine for the current stage; The remaining concentration of glutamine in the culture system before fed-batch culture; This is the metabolic demand coefficient; The target concentration of glutamine at the current stage ,in, The initial concentration of the glutamine concentration system in S1; The stage ratio coefficient of glutamine; S3062: Vitamin Supplement amount The formula is: ;in, Vitamins The amount of a single replenishment.

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