Method of treating biological cell culture

Abstract

The invention relates to a method for treating a biological cell culture, which is characterized in that a closed-loop intelligent control system is established, and the physiological status of cells is dynamically deduced by using a pre-trained machine learning model by noninvasively monitoring the concentration change of glucose, lactic acid and dissolved oxygen in a culture environment in real time. Nutrition supplement and environmental parameters are intelligently regulated and controlled, the cells are always in the optimal growth microenvironment, when culture is ended, the system automatically judges and starts a mild harvesting program, and the cells are assisted in mild shedding through a low-frequency weak ultrasonic field, so that mechanical and chemical damage is avoided. When or after harvesting, the cells are mixed and incubated with a treating fluid containing a protective agent and an activating factor in situ, so that the subsequent survival rate and function of the cells are effectively improved. According to the invention, automation, intelligence and standardization of the whole process from culture to harvest post-treatment are realized, and the density, activity, product yield and batch-to-batch consistency of cell culture are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biological cell culture technology, specifically to a method for processing biological cell cultures. Background Technology

[0002] In existing biological cell culture techniques, whether for adherent or suspension cells, static or semi-static culture modes are commonly used. This involves pre-setting parameters such as culture temperature, pH, dissolved oxygen levels, and nutrient addition plans, with limited monitoring and intervention during the culture process. This method has several significant drawbacks:

[0003] 1. Parameter rigidity: Preset parameters cannot respond in real time to the dynamic changes in cellular metabolic activities. For example, after cells enter the logarithmic growth phase, the rate of nutrient consumption and metabolic waste production increases sharply. Fixed feeding and media replacement strategies may lead to nutrient deficiency or accumulation of toxic substances, affecting cell growth and product expression.

[0004] 2. Inhomogeneous cell state: Since the culture environment is not always in the optimal state, the cell population in the culture will undergo state differentiation, resulting in large fluctuations in key indicators such as cell activity and product expression levels, and poor batch-to-batch reproducibility.

[0005] 3. Rough terminal processing: After cell culture is completed, traditional harvesting methods (such as trypsin digestion and centrifugation) may cause mechanical or chemical damage to the cells, affecting the effect of downstream applications. Summary of the Invention

[0006] (I) Technical Issues

[0007] This invention provides an intelligent cell culture method that can sense cell status in real time, automatically optimize the culture environment, and perform gentle and standardized treatment on cells, aiming to solve the problems in the background art.

[0008] (II) Technical Content

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is: a method for processing biological cell cultures, comprising the following steps:

[0010] S1: Cell seeding and initialization: Seed the target cells into a bioreactor containing the initial culture medium and set the initial physicochemical environmental parameters;

[0011] S2: Real-time multi-parameter monitoring: During the culture process, glucose concentration, lactate concentration and dissolved oxygen concentration in the culture environment are monitored in real time and non-invasively;

[0012] S3: Dynamic Feedback and Intelligent Regulation: The data monitored in step S2 is input into a pre-trained intelligent control model. Based on the changing trend of metabolite concentration, the model calculates and outputs control commands in real time, dynamically adjusting the nutrient supplementation strategy, acid-base regulation rate and gas supply ratio.

[0013] S4: Culture Termination Detection and Gentle Harvesting: When the intelligent control model determines that cell growth has entered the late plateau phase or reached the preset target product concentration, the harvesting program is initiated; by applying a weak ultrasonic field or microfluidic field of a specific frequency, the cells are gently detached from the carrier and collected.

[0014] S5: In-situ cell activation treatment: During or after harvesting, the cell suspension is mixed in situ with a treatment solution containing cell protectants and activating factors to incubate the cells for a short period of time, thereby improving the subsequent cell survival rate and function.

[0015] Furthermore, the intelligent control model mentioned in step S3 is a machine learning model, which is trained using historical culture data. The historical culture data includes time-series metabolite concentration data, corresponding regulatory operation data, and final cell growth and product output data. The model has the ability to predict cell metabolic needs in the future and make regulatory decisions in advance.

[0016] Furthermore, the dynamic adjustment nutrient supplementation strategy specifically involves: when the rate of decrease in glucose concentration exceeds a first threshold and the rate of increase in lactic acid concentration is lower than a second threshold, a pulsed supplementation of high-concentration nutrient concentrate is initiated; otherwise, a continuous flow of low-concentration nutrient concentrate is performed.

[0017] Furthermore, in step S4, a weak ultrasonic field of a specific frequency is applied, with a frequency range of 25 kHz to 100 kHz and a power density of less than 5 W / cm². 2 The action time is intermittent, 10-30 seconds, to avoid damage to the cell membrane structure.

[0018] Furthermore, the treatment liquid in step S5 comprises the following components:

[0019] Trehalose, as a cell protectant, is used at a concentration of 10-50 mM;

[0020] Albumin, as a carrier protein, has a concentration of 0.5%-2%;

[0021] At least one cytokine, selected from one or more of interleukin-7, interleukin-15, or transforming growth factor-β, is used to maintain cell viability or guide its differentiation to a specific functional state.

[0022] Furthermore, the method is performed in a closed, integrated bioreactor system that integrates online sensors, actuators, ultrasonic generators, and fluid handling modules. All steps are completed automatically under aseptic conditions without the need to transfer the culture to external equipment.

[0023] Furthermore, the target cells are immune cells used for cell therapy, including chimeric antigen receptor T cells, tumor-infiltrating lymphocytes, or natural killer cells.

[0024] (III) Technical Effects

[0025] The advantages of this invention compared to the prior art are:

[0026] 1. Achieve true dynamic optimization of culture: By monitoring key metabolites in real time, the physiological state of cells can be inferred in reverse, and nutrient supply and environmental parameters can be dynamically adjusted to ensure that cells grow in the optimal microenvironment, which significantly improves cell density, activity and yield of target products.

[0027] 2. Improved stability and repeatability of cultivation: Intelligent control algorithms reduce the uncertainty and reliance on experience in human operation, ensuring a high degree of consistency in cultivation results between different batches, and providing a reliable guarantee for industrial production.

[0028] 3. Achieve gentle, integrated cell harvesting and activation: The post-culture processing steps are integrated into the system, and the cell functional integrity is maintained to the greatest extent through gentle digestion and in-situ activation treatment assisted by physical fields. It is especially suitable for downstream applications with extremely high requirements for cell viability. Attached Figure Description

[0029] Figure 1 This is a schematic flowchart of the method for processing biological cell cultures according to the present invention. Detailed Implementation

[0030] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "inner", "outer", "center", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation structure and operation. Therefore, they should not be construed as limitations on this invention.

[0031] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "provided with," "installed," "connected," "linked," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0032] The present invention will now be described in further detail with reference to the accompanying drawings.

[0033] Combined with appendix Figure 1 A method for processing biological cell cultures includes the following steps:

[0034] S1: Cell seeding and initialization: The target cells are seeded into a bioreactor containing the initial culture medium, and the initial physicochemical environmental parameters are set.

[0035] S2: Real-time multi-parameter monitoring: During the culture process, glucose concentration, lactate concentration and dissolved oxygen concentration in the culture environment are monitored in real time and non-invasively.

[0036] S3: Dynamic Feedback and Intelligent Regulation: The data monitored in step S2 is input into a pre-trained intelligent control model. This model calculates and outputs control commands in real time based on the changing trends of metabolite concentrations, dynamically adjusting the nutrient supplementation strategy, acid-base regulation rate, and gas supply ratio. The intelligent control model is a machine learning model, trained using historical culture data, which includes time-series metabolite concentration data, corresponding regulatory operation data, and final cell growth and product output data. The model has the ability to predict cell metabolic needs in the future and make regulatory decisions in advance. The dynamic adjustment of the nutrient supplementation strategy is as follows: when the rate of decrease in glucose concentration exceeds a first threshold and the rate of increase in lactate concentration is lower than a second threshold, a pulsed supplementation of high-concentration nutrient concentrate is initiated; otherwise, a continuous flow of low-concentration nutrient concentrate is performed.

[0037] S4: Culture Termination Detection and Gentle Harvesting: When the intelligent control model determines that cell growth has entered the late plateau phase or reached the preset target product concentration, the harvesting procedure is initiated; by applying a weak ultrasonic field or microfluidic field of a specific frequency, the cells are gently detached from the carrier and collected. The applied weak ultrasonic field has a frequency range of 25kHz to 100kHz and a power density of less than 5W / cm². 2 The action time is intermittent, 10-30 seconds, to avoid damage to the cell membrane structure.

[0038] S5: In-situ cell activation treatment: During or after harvesting, the cell suspension is mixed in situ with a treatment solution containing cell protectants and activating factors to incubate the cells for a short period of time, thereby improving the subsequent cell survival rate and function. The treatment solution contains the following components:

[0039] Trehalose, as a cell protectant, is used at a concentration of 10-50 mM;

[0040] Albumin, as a carrier protein, has a concentration of 0.5%-2%;

[0041] At least one cytokine, selected from one or more of interleukin-7, interleukin-15, or transforming growth factor-β, is used to maintain cell viability or guide its differentiation to a specific functional state.

[0042] The method is performed in a closed, integrated bioreactor system that integrates online sensors (such as bioimpedance sensors, optical dissolved oxygen / pH probes, and microdialysis sampling-biochemical analyzer systems), actuators, an ultrasonic generator, and a fluid handling module. All steps are completed automatically under sterile conditions without the need to transfer the culture to external equipment. The target cells are immune cells used for cell therapy, including chimeric antigen receptor T cells, tumor-infiltrating lymphocytes, or natural killer cells.

[0043] The working principle of the key steps of this invention is described in detail below:

[0044] S2: Real-time multi-parameter monitoring

[0045] Glucose and lactate monitoring: Traditional offline sampling introduces contamination risks and is subject to delays. This invention preferably employs microdialysis sampling combined with an online biochemical analyzer. The microdialysis probe has a semi-permeable membrane, allowing continuous collection of small molecules (glucose, lactate) from the culture medium, which are then carried along with the perfusion fluid into the online analyzer for concentration determination. This process is completely aseptic, continuous, and automated.

[0046] Dissolved oxygen monitoring: An optical dissolved oxygen sensor based on the fluorescence quenching principle is used. This sensor is consumable-free, has a fast response, high stability, and can reflect the rate of cellular oxygen consumption in real time.

[0047] Working principle: Glucose is the primary carbon source, and lactate is its metabolic product. The glucose consumption rate (GCR) and lactate production rate (LPR) are direct indicators of cellular metabolic activity. The dissolved oxygen rate also reflects the intensity of cellular respiration. By observing the combined changes of these three parameters, the cell's growth stage (lag phase, logarithmic phase, plateau phase) and physiological state can be accurately determined.

[0048] S3: Dynamic Feedback and Intelligent Control

[0049] Construction of intelligent control model:

[0050] Training data: Collect a large amount of historical culture data, including: time series data (glucose, lactate, dissolved oxygen), corresponding operational data (feeding amount, alkali pump start time, aeration adjustment), and final measured results (cell density, cell viability, target product concentration).

[0051] Model Selection and Training: A machine learning model suitable for time series forecasting, such as a Long Short-Term Memory (LSTM) network, is employed. Through learning, the model establishes a complex nonlinear mapping relationship from "historical environment sequence + historical operation sequence" to "future environment state" and "final training result." The training objective is to find a series of operations that optimally predicts the "final training result."

[0052] The logic of real-time control:

[0053] Scenario 1 (Early Logarithmic Growth Phase): A slight increase in GCR and a synchronous rise in LPR are observed, along with a faster rate of dissolved oxygen decline. The model predicts that the cells are about to enter a rapid proliferation phase. At this point, the model may decide to initiate "preventative feeding," continuously adding balanced nutrient solution at a low flow rate to avoid growth stagnation due to sudden nutrient depletion.

[0054] Scenario 2 (Logarithmic growth phase): A sharp increase in GCR is observed, exceeding the first threshold (e.g., a decrease of >1.0 g / L per hour), but the rate of increase in LPR is below the second threshold (indicating high metabolic efficiency and low lactate conversion). The model predicts that the current metabolic load is extremely high. At this point, the model may decide to: initiate "pulsed intensive feeding," injecting a high concentration of nutrient concentrate in a short period of time to quickly meet the cell's needs, while simultaneously fine-tuning the oxygen ratio in the ventilation to prevent excessively low dissolved oxygen.

[0055] Scenario 3 (Plateau / Metabolic Stress): A slowdown in GCR is detected, but LPR remains high or even increases (indicating reduced metabolic efficiency, possibly indicating a plateau or metabolic stress). The model predicts that continued heavy feeding will lead to excessive lactate accumulation. At this point, the model may decide to either stop feeding or significantly reduce the feeding rate, potentially triggering a partial liquid exchange procedure to dilute metabolic waste. Simultaneously, the model will begin preparing for the S4 harvest phase.

[0056] S4: Cultivation Termination Determination and Gentle Harvesting

[0057] Termination Decision Logic: The model does not simply determine termination based on a fixed time or a single cell density. Instead, it considers multiple indicators: the cell growth rate has dropped to near zero; the lactate / glucose ratio remains high, indicating a deteriorating metabolic environment; and the predicted target product concentration growth rate is leveling off. When these indicators simultaneously meet the preset conditions, the model determines that the benefits of continuing culture are less than the risks of declining cell state, and thus issues a harvest instruction.

[0058] Gentle detachment principle (taking adherent cells as an example): Traditional pancreatic digestion involves chemical and enzymatic damage, resulting in uneven action. This invention employs low-frequency, weak ultrasound-assisted physical detachment: applying 25-100kHz ultrasound generates a microfluidic field and stable cavitation effect (non-destructive inertial cavitation) in the liquid. These minute fluid shear forces and bubble oscillation energy primarily act on the junction between the cell and the carrier and the extracellular matrix, physically "loosening" the cells while avoiding tearing damage to the cell membrane itself. Power density is strictly controlled at <5W / cm². 2 The process was conducted in an intermittent mode of "working for 10 seconds and pausing for 20 seconds" to allow cells time to recover and to confirm through microscopic observation that the cells were gradually becoming round and detaching, rather than being broken up.

[0059] S5: In-situ cell activation treatment

[0060] In-situ mixing: Through the multi-channel fluid module integrated within the system, pre-stored, temperature-equilibrium treatment solution is precisely injected into the cell collection flow path in proportion, and mixed online with the cell suspension without the need for additional transfer.

[0061] Functional principles of each component:

[0062] Trehalose is a known osmoregulator and stabilizer. It forms a hydration layer on the outer side of the cell membrane, stabilizing the phospholipid bilayer structure of the cell membrane and reducing apoptosis when cells experience osmotic pressure changes and physical stress.

[0063] Albumin: As a carrier protein, it can adsorb onto the cell surface and the inner wall of the container, reducing cell adhesion and aggregation, and providing a protective coating similar to the in vivo environment.

[0064] Cytokines (such as IL-7 / IL-15): These are key signals for maintaining the survival and function of immune cells such as T cells. During a brief incubation after harvest (e.g., 30-60 minutes), these factors bind to cell surface receptors, activating anti-apoptotic pathways (such as Bcl-2 upregulation) and signaling pathways that maintain metabolic activity, ensuring that cells remain "ready for battle" even after leaving the optimal culture environment.

[0065] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for processing biological cell cultures, characterized in that, Includes the following steps: S1: Cell seeding and initialization: The target cells are seeded into a bioreactor containing the initial culture medium, and the initial physicochemical environmental parameters are set. S2: Real-time multi-parameter monitoring: During the culture process, glucose concentration, lactate concentration and dissolved oxygen concentration in the culture environment are monitored in real time and non-invasively; S3: Dynamic Feedback and Intelligent Regulation: The data monitored in step S2 is input into a pre-trained intelligent control model. Based on the changing trend of metabolite concentration, the model calculates and outputs control commands in real time, dynamically adjusting the nutrient supplementation strategy, acid-base regulation rate and gas supply ratio. S4: Culture Termination Detection and Gentle Harvesting: When the intelligent control model determines that cell growth has entered the late plateau phase or reached the preset target product concentration, the harvesting program is initiated; by applying a weak ultrasonic field or microfluidic field of a specific frequency, the cells are gently detached from the carrier and collected. S5: In-situ cell activation treatment: During or after harvesting, the cell suspension is mixed in situ with a treatment solution containing cell protectants and activating factors to incubate the cells for a short period of time, thereby improving the subsequent cell survival rate and function.

2. The method for processing biological cell cultures according to claim 1, characterized in that, The intelligent control model mentioned in step S3 is a machine learning model, which is trained using historical culture data. The historical culture data includes time-series metabolite concentration data, corresponding regulatory operation data, and final cell growth and product output data. The model has the ability to predict cell metabolic needs in the future and make regulatory decisions in advance.

3. The method for processing biological cell cultures according to claim 2, characterized in that, The dynamic adjustment nutrient supplementation strategy is as follows: when the rate of decrease in glucose concentration exceeds the first threshold and the rate of increase in lactic acid concentration is lower than the second threshold, a pulsed supplementation of high-concentration nutrient concentrate is initiated; otherwise, a continuous flow of low-concentration nutrient concentrate is performed.

4. The method for processing biological cell cultures according to claim 1, characterized in that, The application of a weak ultrasonic field at a specific frequency in step S4, with a frequency range of 25 kHz to 100 kHz and a power density of less than 5 W / cm², is described in step S4. 2 The action time is intermittent, 10-30 seconds, to avoid damage to the cell membrane structure.

5. The method for processing biological cell cultures according to claim 1, characterized in that, The treatment solution in step S5 comprises the following components: Trehalose, as a cell protectant, is used at a concentration of 10-50 mM; Albumin, as a carrier protein, has a concentration of 0.5%-2%; At least one cytokine, selected from one or more of interleukin-7, interleukin-15, or transforming growth factor-β, is used to maintain cell viability or guide its differentiation to a specific functional state.

6. The method for treating biological cell cultures according to any one of claims 1 to 5, characterized in that, The method is performed in a closed, integrated bioreactor system that integrates online sensors, actuators, an ultrasonic generator, and a fluid handling module. All steps are completed automatically under aseptic conditions without the need to transfer the culture to external equipment.

7. The method for processing biological cell cultures according to claim 1, characterized in that, The target cells are immune cells used for cell therapy, including chimeric antigen receptor T cells, tumor-infiltrating lymphocytes, or natural killer cells.

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