T cell activation expansion medium and culture process thereof

Abstract

This invention relates to the field of microorganisms or enzymes, and discloses a T-cell activation and expansion medium and its culture process. The culture process includes: inoculating isolated T cells into a basal medium containing IL-2 and culturing them; on the 4th day of culture, adding IL-7, IL-15, and a defined molar ratio of nicotinamide and N-acetyl-L-cysteine ​​to the system; calculating the metabolic quotient by monitoring changes in lactate and glucose concentrations in the supernatant; and triggering a feeding action based on the metabolic quotient. During the feeding stage, the supplementation ratio of nicotinamide and N-acetyl-L-cysteine ​​is adjusted according to the threshold of the metabolic quotient. This invention achieves a precise coupling between mitochondrial energy drive and oxidative damage quenching, thereby solving the technical contradiction of difficulty in balancing expansion efficiency and phenotypic maintenance, effectively blocking the late-stage metabolic exhaustion pathway and directionally enriching memory subpopulations.

Description

Technical Field

[0001] This invention relates to a T-cell activation and amplification culture medium and its culture process, belonging to the field of microbial or enzyme technology. Background Technology

[0002] The core of current research lies in the effective activation and expansion of patient-derived cell populations in vitro to obtain biologically functional cell products. Existing processes typically employ culture systems containing antibody signaling stimulation and basal culture media, driving cells into the proliferation cycle by adding cytokines such as interleukins. Such approaches focus on maintaining the strength of external stimulus signals to gain a numerical advantage in cell numbers. However, under the pressure of pursuing large-scale production, existing processes face inherent constraints between proliferation rate and differentiation phenotype quality. To achieve high-density expansion, the supply concentration of cytokines often needs to be increased, which often leads to phenotypic depletion in the late logarithmic growth phase of the culture. The physical essence of this phenomenon stems from the transformation of the cellular metabolic network. During the transition of cells from the initial activation phase to the expansion phase, energy uptake pathways change. Although metabolic reprogramming supports the formation of memory phenotypes, high-throughput mitochondrial respiratory activity is accompanied by excessive endogenous oxidative byproducts.

[0003] The approach of mitigating damage by directly introducing antioxidants is logically contradictory. Introducing reducing agents in the early stages of culture can quench the signal transduction required for activation, leading to a decrease in initial activation efficiency. Simultaneously, the conventional static feeding mode, which triggers nutrient replenishment only based on glucose consumption, cannot detect and mitigate the nonlinear metabolic load surges under high-density environments. This mismatch between supply logic and dynamic physiological needs results in the implicit cost of increased expression of depletion markers and a decrease in the proportion of memory T cells in the later stages of expansion. For example, Chinese invention patent CN114990061B discloses a method for inducing the expansion of central memory T cells. The method improves the anti-apoptotic ability and phenotype ratio of T cells by adding a specific concentration of nicotinamide to the culture medium. However, in the context of engineered large-scale culture, the static supply mode faces an essential mismatch in the underlying metabolic logic. Introducing a fixed dose of regulatory components in the early stage of cell activation can easily quench the endogenous reactive oxygen species (ROS) signals required for initial activation, thus limiting proliferation efficiency. During the high-density expansion stage, the mitochondrial metabolic load increases nonlinearly, and the static concentration setting cannot sense and offset the instantaneous ROS shock of the high-load respiratory chain in real time. Due to the lack of closed-loop feedback on the real-time metabolic state of cells, the existing technology is unable to block the cell drift towards the depleted phenotype from the bottom layer of metabolic flux when ensuring high-fold expansion, and the product quality lacks stability when dealing with microenvironment fluctuations.

[0004] Therefore, the technical problem to be solved by this invention is how to block metabolic depletion pathways while ensuring proliferation efficiency by adjusting the timing and ratio of exogenous components based on the evolutionary characteristics of cellular metabolic state. Summary of the Invention

[0005] To address the problems in the background art, the technical solution of the present invention is as follows: A culture process for a T-cell activation and expansion medium, comprising the following steps: Step S1: The isolated T cells were seeded in basal medium containing IL-2 at a concentration of 50 IU / mL to 300 IU / mL and incubated at 37°C with 5% IL-2. Activation culture was performed under specific concentration conditions, and the basal medium did not contain nicotinamide or N-acetyl-L-cysteine. Step S2: Starting from day 4 of culture, add IL-7, IL-15 and a redox regulator composed of nicotinamide and N-acetyl-L-cysteine ​​to the culture system. The final concentration of nicotinamide in the culture system is 1.0 mM to 20.0 mM, and the initial molar ratio of nicotinamide to N-acetyl-L-cysteine ​​is 4.0:1 to 5.0:1. Step S3: Measure the difference in lactate concentration and the difference in glucose concentration in the supernatant of the culture system, and calculate the ratio of the difference in lactate concentration to the difference in glucose concentration to obtain the apparent metabolic quotient. Step S4: Monitor the glucose concentration in the culture system. When it is lower than the preset glucose threshold, add supplementary solution. Specifically, when the apparent metabolite quotient is greater than 1.2, the supplementary solution is a first supplementary solution containing nicotinamide and N-acetyl-L-cysteine ​​in a molar ratio of 4.0:1 to 5.0:1; when the apparent metabolite quotient is less than or equal to 1.2, the supplementary solution is a second supplementary solution containing nicotinamide and N-acetyl-L-cysteine ​​in a molar ratio of 2.0:1 to 2.5:1.

[0006] Preferably, step S2 includes: step S21, controlling the initial concentration of nicotinamide in the culture system to be 2.0 mM to 5.0 mM, and controlling the initial concentration of N-acetyl-L-cysteine ​​in the culture system to be 0.4 mM to 1.0 mM.

[0007] Preferably, step S2 further includes: step S22, controlling the concentration of IL-7 in the culture system to be from 5 ng / mL to 15 ng / mL, and controlling the concentration of IL-15 in the culture system to be from 5 ng / mL to 20 ng / mL.

[0008] Preferably, in step S1, the basal culture medium is selected from one or more of AIMV medium, X-VIVO15 medium, OpTmizer medium or TexMACS medium.

[0009] Preferably, step S4 includes: step S41, maintaining the addition rate of the first supplementary solution when the apparent metabolite quotient is in the range of 1.5 to 1.8; step S42, when the apparent metabolite quotient exceeds... At that time, the addition rate of nicotinamide and N-acetyl-L-cysteine ​​in the first replenishment solution was increased.

[0010] Preferably, the glucose concentration in the replenishment solution in step S4 is higher than the initial glucose concentration in the basal culture medium.

[0011] Preferably, the method further includes the following steps: Step S5, from day 10 to day 14 of culture, the expression ratio of PD-1 and TIM-3 on the surface of T cells in the culture system is detected, and T cells are harvested when the proportion of PD-1 positive cells is less than 20% and the proportion of TIM-3 positive cells is less than 15%.

[0012] Preferably, in step S1, activation signals are provided by anti-CD3 antibody and anti-CD28 antibody, and the concentration ratio of anti-CD3 antibody to anti-CD28 antibody is 1:1.

[0013] Preferably, in the T cell products obtained by controlling the culture process, the proportion of CD45RO+ and CD62L+ double-positive central memory T cell subsets is not less than 60%, and the cell expansion fold is 500 to 1000 times that of the initial inoculum.

[0014] A T-cell activation and expansion medium, wherein the T-cell activation and expansion medium is prepared by a T-cell activation and expansion medium culture process.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. In T cell activation and expansion culture medium, a synergistic mechanism of mitochondrial energy engine driving and associated oxidative damage targeted quenching is constructed to overcome the physiological coupling limitation of high metabolic load and phenotypic depletion. This scheme does not achieve gains through the isolated effects of each component, but uses nicotinamide as an energy metabolism precursor to forcibly drive mitochondrial oxidative phosphorylation, and simultaneously combines it with a specific molar ratio of N-acetyl-L-cysteine ​​to construct a microscopic driving and counteracting system. Under this synergistic effect, nicotinamide is responsible for providing the high-energy substrate required to maintain memory phenotypic differentiation, while N-acetyl-L-cysteine, which is bound to it in proportion, neutralizes the associated mitochondrial reactive oxygen species that are inevitably generated by the high-load respiratory chain. This dynamic balance mechanism based on molecular ratio enables cells to maintain a high metabolic level to support rapid proliferation while avoiding terminal depletion pathways triggered by endogenous oxidative stress, thereby achieving decoupling of expansion efficiency and phenotypic quality in the C12N domain during in vitro culture of live cells.

[0016] 2. Implement a segmented process pathway based on the timing of activation signal transduction and metabolic pathways to ensure the fidelity of initial signal transduction and the orderly connection of subsequent state reprogramming. This process utilizes the essential differences in the nature of T cells' reactive oxygen species (ROS) requirements at different physiological stages. In the initial stage of culture, by limiting the culture environment without adding any antioxidant components, endogenous ROS are retained as second messengers to participate in the transduction of activation signaling pathways. At the critical node when cells complete initial signal transduction and enter metabolic reprogramming, a combination of functional regulatory factors is precisely inserted. This intervention method, which is highly consistent with the physiological rhythm of cells in the time domain, avoids the defects of the existing technology caused by the mixed addition of all stages, such as the suppression of initial activation or the lack of protection in the later stage, and ensures the natural and coherent transition from the resting state to the activated state and to the amplified state.

[0017] 3. A nonlinear feedback feeding logic based on the threshold determination of the apparent metabolic quotient is established. Based on the adaptive adjustment capability of the culture process to the evolutionary state of the cell population, this scheme abandons the static feeding mode of the traditional process that relies on the consumption of a single carbon source. By monitoring the molar ratio of lactate production to glucose consumption in the culture medium, an objective physical index reflecting the mitochondrial load status of the cells is constructed. When this metabolic ratio falls below the preset threshold, the process automatically switches to a nonlinear feeding mode with a higher density of thiol electron donors to offset the extreme oxidative pressure caused by the exponential increase in cell density. This parameter adjustment mechanism driven by the real-time metabolic phenotype of cells enhances the stability of the system in the face of drastic fluctuations in the microenvironment during the later stages of high-density amplification and prevents product quality fluctuations caused by metabolic imbalance. Attached Figure Description

[0018] Figure 1 This is a flowchart of the T-cell segmented activation and expansion process for determining the epigenetic metabolite quotient threshold in this invention. Figure 2 This is a logical architecture diagram of the adaptive feedback feeding system with multi-parameter sensor compensation function of the present invention.

[0019] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0021] A culture process for a T-cell activation and expansion medium includes the following steps: Step S1: The isolated T cells were seeded in basal medium containing IL-2 at a concentration of 50 IU / mL to 300 IU / mL and incubated at 37°C with 5% IL-2. Activation culture was performed under specific concentration conditions, and the basal medium did not contain nicotinamide or N-acetyl-L-cysteine. Step S2: Starting from day 4 of culture, add IL-7, IL-15 and a redox regulator composed of nicotinamide and N-acetyl-L-cysteine ​​to the culture system. The final concentration of nicotinamide in the culture system is 1.0 mM to 20.0 mM, and the initial molar ratio of nicotinamide to N-acetyl-L-cysteine ​​is 4.0:1 to 5.0:1. Step S3: Measure the difference in lactate concentration and the difference in glucose concentration in the supernatant of the culture system, and calculate the ratio of the difference in lactate concentration to the difference in glucose concentration to obtain the apparent metabolic quotient. Step S4: Monitor the glucose concentration in the culture system. When it is lower than the preset glucose threshold, add supplementary solution. Specifically, when the apparent metabolite quotient is greater than 1.2, the supplementary solution is a first supplementary solution containing nicotinamide and N-acetyl-L-cysteine ​​in a molar ratio of 4.0:1 to 5.0:1; when the apparent metabolite quotient is less than or equal to 1.2, the supplementary solution is a second supplementary solution containing nicotinamide and N-acetyl-L-cysteine ​​in a molar ratio of 2.0:1 to 2.5:1.

[0022] Preferably, step S2 includes: step S21, controlling the initial concentration of nicotinamide in the culture system to be 2.0 mM to 5.0 mM, and controlling the initial concentration of N-acetyl-L-cysteine ​​in the culture system to be 0.4 mM to 1.0 mM.

[0023] Preferably, step S2 further includes: step S22, controlling the concentration of IL-7 in the culture system to be from 5 ng / mL to 15 ng / mL, and controlling the concentration of IL-15 in the culture system to be from 5 ng / mL to 20 ng / mL.

[0024] Preferably, in step S1, the basal culture medium is selected from one or more of AIMV medium, X-VIVO15 medium, OpTmizer medium or TexMACS medium.

[0025] Preferably, step S4 includes: step S41, maintaining the addition rate of the first replenishment solution when the apparent metabolite quotient is in the range of 1.5 to 1.8; step S42, increasing the replenishment rate of nicotinamide and N-acetyl-L-cysteine ​​in the first replenishment solution when the apparent metabolite quotient exceeds 1.8.

[0026] Preferably, the glucose concentration in the replenishment solution in step S4 is higher than the initial glucose concentration in the basal culture medium.

[0027] Preferably, the method further includes the following steps: Step S5, from day 10 to day 14 of culture, the expression ratio of PD-1 and TIM-3 on the surface of T cells in the culture system is detected, and T cells are harvested when the proportion of PD-1 positive cells is less than 20% and the proportion of TIM-3 positive cells is less than 15%.

[0028] Preferably, in step S1, activation signals are provided by anti-CD3 antibody and anti-CD28 antibody, and the concentration ratio of anti-CD3 antibody to anti-CD28 antibody is 1:1.

[0029] Preferably, in the T cell products obtained by controlling the culture process, the proportion of CD45RO+ and CD62L+ double-positive central memory T cell subsets is not less than 60%, and the cell expansion fold is 500 to 1000 times that of the initial inoculum.

[0030] A T-cell activation and expansion medium, wherein the T-cell activation and expansion medium is prepared by a T-cell activation and expansion medium culture process.

[0031] Example 1: In the late logarithmic expansion stage, when the cell density reaches or exceeds In a closed bioreactor culture setting with T cells / mL, a non-linear shift in the metabolic flux between glucose consumption and lactate production leads to a burst of endogenous mitochondrial reactive oxygen species (ROS) in T cells under high metabolic load. This associated oxidative stress directly induces an increase in the expression of depletion markers such as PD-1 and TIM-3 on the cell surface, causing the memory subset, which originally had high anti-tumor potential, to rapidly differentiate into a terminally depleted state. This phenomenon is unavoidable and cannot be reversed by simple nutrient supplementation in traditional static processes that rely solely on proportional feeding based on residual glucose concentration. The culture process of this invention, in this high-density culture environment, during the initial activation phase from day 0 to day 3 after inoculation, involves inoculating the isolated T cells into a basal medium containing IL-2 at a concentration of 50 IU / mL to 300 IU / mL, and incubating at 37°C with 5% IL-2. Activation culture was carried out under specific concentration conditions, and the basal medium was kept free of nicotinamide and N-acetyl-L-cysteine ​​during this stage to ensure that endogenous reactive oxygen species, as second messengers, could fully participate in the transduction of the TCR signaling pathway, thus avoiding signal quenching caused by premature introduction of reducing substances in the early stages of activation.

[0032] From day 4 of culture, the process entered the metabolic rebalancing phase. This involved simultaneously introducing IL-7, IL-15, and a redox regulator composed of nicotinamide and N-acetyl-L-cysteine ​​into the system. The final concentration of nicotinamide in the culture system was controlled within the range of 1.0 mM to 20.0 mM, while the initial molar ratio of nicotinamide to N-acetyl-L-cysteine ​​was limited to 4.0:1 to 5.0:1. Nicotinamide was used as an energy precursor to drive mitochondrial oxidative phosphorylation to support memory phenotype differentiation. The N-acetyl-L-cysteine, bound to nicotinamide, neutralized the associated reactive oxygen species in mitochondria inevitably generated by the high-load respiratory chain. During the feed maintenance process, supernatant samples were collected every 24 to 48 hours, and the differences in lactate and glucose concentrations were measured to calculate the epigenetic quotient. The specific calculation formula is as follows: ,in, For the epigenetic metabolic quotient, This represents the difference in lactic acid concentration in the supernatant between the current sampling point and the previous sampling point. The value represents the difference in glucose concentration between the current sampling point and the previous sampling point in the supernatant. When the apparent metabolic quotient is detected... When the level drops to 1.2 or below, the system initiates a non-linear feeding action. This threshold of 1.2 is based on a correlation analysis of the maintenance of the T cell central memory phenotype and the distribution of lactate / glucose metabolic flux. Experimental data show that when... When the reactive oxygen species (ROS) level is between 1.3 and 1.8, cells primarily obtain energy through oxidative phosphorylation, and the ROS level is within the equilibrium threshold of intracellular scavenging capacity; once... A drop below 1.2 indicates that the mitochondrial load has reached saturation and is accompanied by a large-scale overflow of oxygen free radicals. At this point, the CD45RO+ / CD62L+ subset ratio will decrease. Statistical analysis of five different batches of culture samples confirmed that 1.2 is the underlying biochemical characteristic point that triggers the cells to drift towards a depleted state. This was used as the sole logical trigger point for switching to a high-thiol electron donor feeding mode. The feeding medium was changed from the initial first feeding medium to the second feeding medium. The molar ratio of nicotinamide to N-acetyl-L-cysteine ​​in the second feeding medium was compressed to the range of 2.0:1 to 2.5:1. This allows for the construction of a dynamic antioxidant defense barrier by increasing the relative concentration of thiol electron donors when cells enter the mitochondrial load limit window.

[0033] Under this process pathway based on real-time metabolic state feedback, the proportion of CD45RO-positive and CD62L-positive double-positive central memory T cell subsets in the final culture product is stably maintained above 60%, and an expansion efficiency of 500-fold to 1000-fold is achieved throughout the total culture cycle. This effectively solves the physicochemical decoupling problem between expansion fold and maintenance of stem phenotype in the large-scale preparation of immune cells in the C12N field. Under the initial conditions for clinical-scale preparation, the basal culture medium adopts a chemically defined medium that meets the specifications of serum-free and animal-derived components. Its core nutrient components include 4.0 to 6.0 g / L glucose and 2.0 to 4.0 mM L-glutamyl The system contains amines and 10.0 to 20.0 mg / L of recombinant human insulin, which together maintain the basal metabolic homeostasis of cells from day 0 to day 3 during the initial inoculation phase. In this initial phase, to trigger the T cell receptor signaling pathway, the system is pre-filled with magnetic beads coated with anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies. The amount of magnetic beads added is determined according to the ratio of magnetic beads to cells of 1.0:1.0 to 3.0:1.0. Since the basal medium does not contain reducing nicotinamide and N-acetyl-L-cysteine ​​at this stage, the transient reactive oxygen species induced by magnetic bead binding can initiate the transduction process of downstream kinase chains, thereby providing an activated cell seed stream for entering the amplification mode on day 4.

[0034] Example 2: Under this condition, an experimental scenario targeting cell exhaustion induced by high metabolic load was established to verify the efficacy of the culture process in maintaining the proportion of memory phenotypes while increasing cell expansion. The original T cells used in the experiment were extracted from peripheral blood mononuclear cell samples authorized by the donor, and the initial seeding density was set at [missing information]. cells / mL; core monitoring parameter: apparent metabolite quotient The sampling period was set to 24 hours. This period was used to balance the real-time response of biochemical indicators with the physical interference of sampling operations on the sterility barrier of the culture system. When the concentration rise rate of metabolites in the supernatant was in a rapidly changing range, the sampling period tended to the lower limit of 24 hours to capture the critical point of metabolic transition. To simulate the instability of sensor signals in the production environment, random electrical noise with a signal-to-noise ratio of 25dB was actively superimposed on the lactate concentration difference and glucose concentration difference fed back by the biochemical analyzer. The experiment was divided into the present invention sample group, control sample group A, and control sample group B. Control sample group A adopted the conventional fed-batch method based on the residual glucose concentration, and the feed did not contain redox regulators. Control sample group B introduced nicotinamide and N-acetyl-L-cysteine ​​from the 4th day of culture, but the molar ratio of the two was kept constant at 4.5:1 and the ratio was not changed with the fluctuation of the metabolic quotient. In the early stage of fed-batch culture of the present invention sample group, the supernatant monitoring showed It is 8.42mM. The initial epigenetic quotient was calculated to be 6.24 mM. The value was 1.35. Based on this feedback value, the system started a mixed feed path consisting of 90% basal culture medium and 10% first supplement solution. The molar ratio of nicotinamide to N-acetyl-L-cysteine ​​in the first supplement solution was set to 4.8:1. To simulate a non-ideal culture environment, dissolved oxygen fluctuations of ±5% were generated by adjusting the gas supply flow rate during the experiment.

[0035] When the culture period reached day 9, the cell density increased to The mitochondrial respiratory chain load was approaching its physical limit, and the epigenetic quotient of the sample group of this invention was detected. The value decreased to 1.14; at this point, the fluorescence intensity of endogenous reactive oxygen species (ROS) produced by mitochondria, measured by the ROS fluorescence probe, increased from 124.5 RFU at the initial activation stage to 938.2 RFU. This data reflects the nonlinear overflow phenomenon when the metabolic pathway transitions to a high-load state; the apparent metabolic quotient of the sample group in this invention... The triggering condition was to switch the supplementary solution from the first supplementary solution to the second supplementary solution, and reduce the molar ratio of nicotinamide to N-acetyl-L-cysteine ​​in the supplementary stream to 2.2:1. The comparative data showed that the reactive oxygen species fluorescence intensity of control group A at the same sampling point was 2456.7 RFU, and the proportion of CD45RO-positive and CD62L-positive central memory T cells decreased from 72.4% to 28.3%. Due to the lack of antioxidant intensity regulation targeting the metabolic inflection point, the proportion of double-positive cells in control group B eventually remained at 41.6%.

[0036] When the initial molar ratio of nicotinamide to N-acetyl-L-cysteine ​​deviated from the range of 4.0:1 to 5.0:1, and was set as an out-of-range control group of 6.0:1, the high concentration of nicotinamide induced mitochondrial oxidative phosphorylation intensity exceeding the system's reducing equilibrium capacity, resulting in a decrease in cell viability to 78.4% on day 12 of culture. When the molar ratio was set below 3.0:1, the reducing component excessively quenched the reactive oxygen species messengers required for T cell receptor activation in the early stages of inoculation, leading to a prolonged cell doubling time of 42.8 hours in the first 72 hours after inoculation and a deterioration in the expansion rate. Under the dynamic feedback method of the sample group of this invention, the proportion of central memory T cell subsets in the finally harvested T cells remained at 62.4%, and the total expansion fold reached 845.2-fold. The experimental results confirmed that the composition ratio of nicotinamide to N-acetyl-L-cysteine ​​and the results based on the epigenetic metabolic quotient were effective. There is an interaction between the ratio switching actions, through the apparent metabolic quotient Using this as a quantitative characterization parameter of the limits of mitochondrial metabolism and adjusting the electron donor concentration accordingly, the culture system can build a dynamic antioxidant defense layer while maintaining the supply of energy metabolism precursors, thus achieving a balance between amplification efficiency and maintenance of phenotypic stemness.

[0037] Example 3: This example combines Figures 1 to 2 A description of a T-cell activation and expansion culture medium and its culture process, such as... Figure 1 As shown, the process includes four sequentially connected operation nodes. The step numbers of each node are independently placed outside the main flowchart frame and connected to the corresponding flowchart frame by irregular wavy lines. The first step is labeled S1, and its content is objectively described as inoculating T cells in IL-2-containing basal medium for activation. A downward-pointing hollow triangular arrow leads to the node labeled S2, where the specific operation is to add IL-7, IL-15, and redox regulators on day 4. After that, the process cascades to the node labeled S3 via downward hollow triangular arrows. In this node, the operation of measuring the difference between lactate and glucose concentrations and calculating the epigenetic quotient is performed. Finally, the process follows the hollow triangular arrows to the node labeled S4, completing the steps of monitoring glucose concentration and adding supplementary fluid according to the epigenetic quotient.

[0038] like Figure 2 As shown, the top layer of the system houses an online biochemical monitoring module for the supernatant. This module connects to the glucose sensor, lactate sensor, and dissolved oxygen monitoring sensor below via three downward-pointing solid arrows. Each of these sensor nodes extends a downward-pointing solid arrow and transmits its output to the centrally located control unit. To the left of this control unit, two modules—the automatic sensor calibration process and the mass transfer correlation matrix compensation module—are presented in dashed boxes. Both modules provide signal input to the control unit via paths with dashed arrows. The bottom output of the control unit leads to three independent downward control arrows, pointing separately to the first feed pump on the left, the centrally located agitator power control system, and the second feed pump on the right. Furthermore, along the material transport path, the first feed storage tank at the bottom is connected to the first feed pump via an upward-pointing solid arrow, and the second feed storage tank at the lower right is also connected to the second feed pump via an upward-pointing solid arrow. This achieves a system-level closed loop for online monitoring and action execution. All text within the diagram is formatted in 20-point Song typeface.

[0039] Example 4: In a 50L automated stirred bioreactor culture scenario, when T cells enter the high-density expansion phase, due to the cell population sensing effect and the accumulation of metabolic byproducts, the system's nutrient uptake rate and metabolic waste excretion rate exhibit nonlinear fluctuations. This leads to the feeding method being unable to maintain the biochemical microenvironment homeostasis within the system, thereby inducing mitochondrial dysfunction and phenotypic exhaustion. This experimental platform uses a bioreactor system equipped with multiple feed pumps and an online biochemical monitoring module for the supernatant. The implementation process involves setting a glucose threshold... Calibration was performed to balance the inhibitory effect of carbon source limitation on expansion rate and the damage of high glucose environment to T cell stemness. The specific calibration procedure was as follows: During the metabolic stabilization period from day 4 to day 7 of expansion, the residual glucose concentration was maintained at gradient levels of 1.0 mM, 2.0 mM, 3.0 mM, and 4.0 mM by adjusting the feeding rate. Flow cytometry was used to detect the distribution of mitochondrial membrane potential at different concentrations. When the glucose concentration was below 2.0 mM, mitochondria underwent polarization damage, while when the concentration was above 3.0 mM, the proportion of cells differentiating towards effector phenotypes increased. Based on this, the preset glucose threshold was determined. Set to 2.5mM.

[0040] During the culture process, the system acquires the lactate concentration difference in the supernatant every 12 hours using an online biochemical analyzer. Difference from glucose concentration And calculate the epigenetic quotient. Simultaneously monitor the residual glucose concentration in the culture system in real time. When the conditions are met At that time, the feeding path is initiated: the control unit determines the feeding path based on the apparent metabolic quotient. Numerical start path branch determination, if If so, the first feed pump is activated to deliver the first supplemental solution, maintaining the baseline level of mitochondrial energy metabolism using the initial molar ratio of nicotinamide to N-acetyl-L-cysteine; if This indicates that mitochondrial oxidative phosphorylation flux is approaching saturation and is accompanied by a risk of reactive oxygen species overflow. The control unit switches to a second feed pump to deliver the second replenishment solution. By reducing the molar ratio of nicotinamide to N-acetyl-L-cysteine ​​to 2.2:1, the system's reduction buffer capacity is increased in situ. When determining the feed rate, the system uses a prediction method based on the current consumption rate. The specific replenishment solution flow rate... The calculation formula is as follows: ,in, The instantaneous volumetric flow rate of the feed in the replenishing liquid. The glucose consumption rate during the current sampling period The real-time total volume of the current culture system This is the initial concentration of glucose in the replenishment solution. With a preset glucose threshold of 2.5 mM, under this non-linear feeding path based on real-time calibration of metabolic trajectory, the cumulative rate of reactive oxygen species in the system was controlled below 15.2 RFU / h by day 14, lower than the 84.6 RFU / h in the control group using static feeding. Because mitochondrial metabolic stress was compensated for at the performance inflection point, the CD45RO-positive and CD62L-positive double-positive cell subsets in the finally harvested T cells maintained a proportion of 64.8% after 920-fold expansion, achieving dynamic adaptation of the process to cellular metabolic demands. When the process entered the metabolic rebalancing stage from day 4, the supplementary concentrations of interleukin-7 and interleukin-15 were locked within the ranges of 5.0–20.0 ng / mL and 5.0–15.0 ng / mL, respectively. The determination of these concentrations followed the correlation between mitochondrial membrane potential and expansion rate: when the control unit calculated the apparent metabolic quotient... When the interleukin-7 concentration is in the high oxidative phosphorylation range of 1.3 to 1.5, the concentration is kept at the lower limit of this range to maintain the homeostatic signal of the central memory subset; while when the epigenetic metabolic quotient... As the value approaches 1.2 or below, the system upregulates the supplementation ratio of interleukin-15, utilizing the effect of interleukin-15 on increasing the oxidative capacity of the respiratory chain, and synergistically with nicotinamide to regulate energy turnover under high metabolic load, so that cells can maintain proliferation rate after crossing the metabolic transition point.

[0041] Example 5: In an engineering scenario involving large-scale T-cell expansion using a multi-stage stirred bioreactor, the conductivity variations among different batches of synthetic culture medium components cause a baseline shift in the comparison between the raw signal from the biochemical analysis module and the actual solute concentration. During the equilibration period after loading the basal culture medium and before inoculation, the system initiates an automatic calibration process for the glucose and lactate sensors. This process uses a peristaltic pump to introduce a pre-set concentration of standard solution into the detection chamber. By recording the difference in response current at concentrations of 0.0 mM and 5.0 mM, the system calculates and updates the slope compensation coefficient, thus providing a basis for calculating the epigenetic quotient in subsequent expansion stages. The concentration data provides a traceable metrological basis.

[0042] When the system culture scale increases from 3L to 50L, the operating conditions are affected by differences in flow field and changes in mass transfer resistance. To maintain the stability of the nonlinear feeding action, the control unit performs a pre-calibration of respiration intensity based on the dissolved oxygen change rate before switching the feeding mode on the 4th day of culture. The mitochondrial oxygen load at the current cell density is checked by monitoring the slope of dissolved oxygen decrease after gas supply is stopped, and this load data is added to the feed fluid flow rate as a dynamic correction. In the computational model, under this operating condition, since the molar ratio of nicotinamide to N-acetyl-L-cysteine ​​in the second replenishment solution is locked within the range of 2.0:1 to 2.5:1, the flux of thiol electron donors within the system varies with the apparent metabolic quotient. The stepwise increase occurs when the mitochondrial membrane potential is reduced to below 1.2, allowing T cells in the metabolic transition phase to return to the low-oxidative-stress growth range after fluctuations in mitochondrial membrane potential.

[0043] Example 6: In a process scale-up scenario from 50L preparation scale to 500L industrial scale, due to the bioreactor diameter-to-height ratio and the tip speed of the impeller... The change in oxygen mass transfer coefficient within the system The non-uniformity of the flow field generates gradient fluctuations, leading to discrepancies in the dissolved oxygen partial pressure and metabolic rate sensed by T cells at different axial positions, which in turn triggers the epimetabolic quotient. Signal deviation at the sampling level; the procedure first uses the dynamic exhaust method to determine the basic oxygen mass transfer coefficient of the reactor under no-load conditions. The specific calculation formula is as follows: ,in, The oxygen mass transfer coefficient. This represents the saturated dissolved oxygen concentration at the current temperature and pressure. and At the respective time points and The measured actual dissolved oxygen concentration in the liquid phase, and As the sampling time point, the system obtains data at different stirring speeds. Numerical mass transfer correlation matrix is ​​established, and this correlation matrix is ​​used as the underlying input to calculate the apparent metabolic quotient. The differences in lactate and glucose concentrations involved in the process are weighted and compensated. Specifically, the mass transfer correlation matrix is ​​stored in the read-only memory of the control unit, and its data structure is a binary feature map table with stirring speed and aeration rate as independent variables. Its element values ​​are pre-calibrated ideal oxygen mass transfer coefficients. In the actual culture process, the sensor collects the output power signal of the stirring impeller in real time and converts it into the Reynolds number. The control unit extracts the corresponding reference transfer coefficient from the matrix according to the change of the Reynolds number using a lookup table method and transmits it as an input parameter to the aforementioned weight calculation formula. This matrix-based data retrieval and linear interpolation algorithm ensures that the control unit can respond in real time to flow field fluctuations caused by changes in culture medium viscosity or scale-up, and output deterministic compensation commands. Based on the empirical diffusion kinetics model of gas-to-liquid mass transfer, the volumetric oxygen mass transfer of the reaction system is constrained by the mechanical power input of the stirring. To implement weight compensation, it is necessary to collect the actual output power of the cell-free basic culture medium under continuous gradient stirring speed and the corresponding measured reference transfer coefficient, and solidify the data into the matrix after data mapping. As the expansion culture progresses, the high cell density causes an increase in apparent viscosity, which locally weakens the flow field mixing efficiency. This causes cells in the oxygen transfer retardation zone to initiate anaerobic glycolysis by bypassing the flow field, secreting redundant lactic acid that is detached from the global real metabolic load into the culture system. In order to eliminate the physical and biochemical coupling interference caused by the inhomogeneity of the multiphase flow field, the control unit extracts the current stirring output power collected in real time and retrieves the reference transfer coefficient calibrated under the corresponding power in the matrix. Based on the current online measured transfer coefficient of the system Simultaneous calculation of mass transfer compensation weights The data correction algebraic mapping relationship is set as follows: The expression relates to the flow field damping constant. To characterize the physical geometric parameters of shear flow transport attenuation, the measured flow field diameter-to-height ratio of the reactor used in the embodiment was constantly assigned a value of 0.42. In this calculation process, the mass transfer compensation weight... As a mapping operator between microscopic metabolic flow and macroscopic flow field physical quantities, its physical essence is a quantitative characterization of the contribution of oxygen mass transfer efficiency to the glycolytic bypass. Because T cells undergo an instantaneous transition from oxidative phosphorylation to anaerobic glycolysis in oxygen-limited microenvironments, leading to a nonlinear increase in lactate secretion, the actual transfer coefficient under the current stirring power is measured. With ideal transfer coefficient The deviation can be used to calculate the pseudo-incremental weight of lactate due to physical mass transfer resistance; specifically, applying this weighting coefficient to the original lactate concentration difference within a single sampling period can subtract metabolic signal noise caused by local flow field dead zones, thereby correcting the lactate / glucose ratio (after reconstruction). It can accurately pinpoint the actual respiratory load state of mitochondria inside the cell. The control unit manipulates the underlying logic to combine the original lactate concentration difference from a single sampling with the mass transfer compensation weight. Multiplication filters out test noise introduced by physical interference in the flow field. The resulting product of differences is divided by the glucose concentration difference during the same period, outputting a reconstructed apparent metabolic quotient that reflects the true global mitochondrial respiratory load. With a correction mechanism, when a nonlinear shift in the stirring output power is detected due to increased cell density, the control unit automatically adjusts the output power based on the mass transfer correlation matrix. The gain coefficient of the feedback signal enables physical alignment of the feeding action at the 500L scale with the mitochondrial metabolic turnaround point at the 3L scale on the time axis.

[0044] In the preparation of the second replenishment solution for different batches of reducing components, to address the physicochemical instability of N-acetyl-L-cysteine ​​in aqueous solution at room temperature due to oxidative dehydrogenation and disulfide bond formation, a standardized anaerobic preparation procedure was introduced into the process flow. This procedure first heats the water for injection in the preparation container to 90°C to 100°C and introduces nitrogen gas while continuously stirring for 30 minutes to remove dissolved oxygen. Once the water temperature drops to between 4°C and 8°C, the solution is added according to the ratio of nicotinamide to N-acetyl-L-cysteine... With a molar ratio of 2.0:1 to 2.5:1, N-acetyl-L-cysteine ​​was first added and stirred until completely dissolved under nitrogen protection, followed by the introduction of nicotinamide. The liquid phase temperature and oxygen-deficient environment were used to inhibit the premature consumption of thiol electron donors. During actual feeding, to address the oxidative loss of N-acetyl-L-cysteine ​​in the 37°C culture system, the feeding pipeline was jacketed with a 4°C circulating cooling water system to ensure the regulator maintained its physicochemical activity before entering the reactor. The control unit was based on the aforementioned apparent metabolic quotient. The system employs a rapid, small-step, high-frequency pulse feeding method to ensure that the freshly added reducing components neutralize the localized oxidative micro-regions around the cells before complete oxidation. This closed-loop process, consisting of a cold chain and high-frequency pulse injection, offsets the reduction efficiency degradation caused by environmental factors, effectively maintaining the dynamic reduction potential of the extracellular microenvironment. When the second supplementary solution is introduced into the culture system through a sterile feeding line, the control unit compares the apparent metabolic quotient before and after sampling in real time. rate of change ; Calculate using the following formula: ,in, The rate of change over time of the apparent metabolic quotient. It is the epigenetic metabolic quotient; For the cultivation time, if determined If the value is negative and its absolute value exceeds the preset metabolic decay threshold, the system automatically increases the pulse frequency of the feed pump. This metabolic decay threshold is determined based on the critical rate of T-cell mitochondrial membrane potential collapse, and its physical meaning represents the maximum allowable decrease in the apparent metabolic quotient per unit time. In actual process calibration, through extreme pressure testing in a 3L bioreactor, it was observed that when… Two consecutive sampling periods below -0.05 At this point, the expression rate of PD-1, a marker of T cell exhaustion, began to show an inflection point increase; therefore, the threshold was set at 0.05. When the system detects that the real-time rate of change exceeds this upper limit, it determines that the current reduction buffer capacity is insufficient to offset the sudden surge in oxidative pressure, thus triggering a pulse feeding compensation action. This utilizes a specific ratio of reducing components to repair localized oxidative damage caused by mass transfer resistance in situ, stabilizing the proportion of CD45RO-positive and CD62L-positive central memory T cells in the culture product at 65.3%, and ensuring that the total number of viable cells reaches [a certain value] within the 14-day expansion cycle. indivual.

[0045] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A culture process for a T-cell activation and expansion medium, characterized in that, Includes the following steps: Step S1: The isolated T cells were seeded in basal medium containing IL-2 at a concentration of 50 IU / mL to 300 IU / mL and incubated at 37°C with 5% IL-2. Activation culture was performed under specific concentration conditions, and the basal medium did not contain nicotinamide or N-acetyl-L-cysteine. Step S2: Starting from day 4 of culture, add IL-7, IL-15 and a redox regulator composed of nicotinamide and N-acetyl-L-cysteine ​​to the culture system. The final concentration of nicotinamide in the culture system is 1.0 mM to 20.0 mM, and the initial molar ratio of nicotinamide to N-acetyl-L-cysteine ​​is 4.0:1 to 5.0:

1. Step S3: Measure the difference in lactate concentration and the difference in glucose concentration in the supernatant of the culture system, and calculate the ratio of the difference in lactate concentration to the difference in glucose concentration to obtain the apparent metabolic quotient. Step S4: Monitor the glucose concentration in the culture system. When it is lower than the preset glucose threshold, add supplementary solution. Specifically, when the apparent metabolite quotient is greater than 1.2, the supplementary solution is a first supplementary solution containing nicotinamide and N-acetyl-L-cysteine ​​in a molar ratio of 4.0:1 to 5.0:1; when the apparent metabolite quotient is less than or equal to 1.2, the supplementary solution is a second supplementary solution containing nicotinamide and N-acetyl-L-cysteine ​​in a molar ratio of 2.0:1 to 2.5:

1.

2. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, Step S2 includes: Step S21, controlling the initial concentration of nicotinamide in the culture system to be 2.0 mM to 5.0 mM, and controlling the initial concentration of N-acetyl-L-cysteine ​​in the culture system to be 0.4 mM to 1.0 mM.

3. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, Step S2 further includes: step S22, controlling the concentration of IL-7 in the culture system to be from 5 ng / mL to 15 ng / mL, and controlling the concentration of IL-15 in the culture system to be from 5 ng / mL to 20 ng / mL.

4. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, In step S1, the basal culture medium is selected from one or more of AIMV medium, X-VIVO15 medium, OpTmizer medium or TexMACS medium.

5. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, Step S4 includes: Step S41, when the apparent metabolic quotient is in the range of 1.5 to 1.8, maintaining the addition rate of the first replenishment solution; Step S42, when the apparent metabolic quotient exceeds 1.8, increasing the addition rate of nicotinamide and N-acetyl-L-cysteine ​​in the first replenishment solution.

6. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, The glucose concentration in the replenishment solution in step S4 is higher than the initial glucose concentration in the basal medium.

7. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, It also includes the following steps: Step S5: From day 10 to day 14 of culture, detect the expression ratio of PD-1 and TIM-3 on the surface of T cells in the culture system. When the proportion of PD-1 positive cells is less than 20% and the proportion of TIM-3 positive cells is less than 15%, harvest T cells.

8. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, In step S1, activation signals are provided by anti-CD3 antibody and anti-CD28 antibody, with the concentration ratio of anti-CD3 antibody to anti-CD28 antibody being [missing information]. .

9. The culture process of the T cell activation and expansion medium according to claim 1, characterized in that, In the T cell products obtained by controlling the culture process, the proportion of CD45RO+ and CD62L+ double-positive central memory T cell subsets is not less than 60%, and the cell expansion fold is 500 to 1000 times that of the initial seeding amount.

10. A T-cell activation and expansion culture medium, characterized in that, The T-cell activation and expansion medium is prepared by the culture process of the T-cell activation and expansion medium described in claim 1.

Images