A novel tumor cell expansion medium without animal-derived components and a process thereof

Abstract

The application relates to the field of tumor cell amplification and metabolic regulation, and discloses a novel tumor cell amplification culture medium without animal components and a process, which comprises the following steps: inoculating tumor cells with a culture medium without animal components, monitoring residual glucose concentration in the exponential growth phase, stopping the supply of a basic liquid when the glucose concentration decreases to a trigger threshold interval, single-injecting a sugar-free metabolic reverse pulse liquid containing sodium pyruvate and N-acetyl cysteine into the culture system, maintaining the intervention state, utilizing the concentration gradient formed by the exogenous pyruvate to drive the reverse catalysis of lactate dehydrogenase, inducing the tumor cells to uptake and assimilate lactic acid, and restoring the supply of the sugar-containing basic liquid after the intervention is over. The application establishes an endogenous closed-loop regulation mechanism for metabolic flow, realizes self-assimilation of acidic metabolic waste through endogenous lactic acid removal, avoids self-secretion microenvironment damage caused by physical dilution, and ensures the stability of the genetic characteristics of the cell line.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell amplification and metabolic regulation technology, and in particular relates to a novel tumor cell amplification culture medium and process without animal-derived components. Background Technology

[0002] Currently, in vitro expansion of tumor cells uses a basal culture medium system containing fetal bovine serum. However, the uncertainty of serum components and the biological differences between batches lead to poor reproducibility of experimental data. Furthermore, there are potential risks of exogenous viral contamination and interference from xenogeneic immunogenicity. With the increasing demands for process standardization in the biopharmaceutical field, the development of serum-free culture systems with clearly defined components has become an inevitable direction for industry evolution. Tumor cells exhibit aerobic glycolysis, producing lactic acid and causing media acidification. Conventional processes employ a culture medium replacement strategy, which physically dilutes lactic acid to maintain the survival environment, but this creates fundamental limitations. The flushing effect eliminates cell-secreted growth factors, extracellular vesicles, and key signaling molecules, disrupting the local microenvironment established by tumor cells in the early stages of proliferation. This technical cost of maintaining media homeostasis leads to phenotypic drift and downregulation of tumorigenic genes during long-term passage. Drastic fluctuations in metabolic stress induce the production of excessive reactive oxygen species within cells, causing genomic oxidative stress damage and reducing the genetic fidelity of tumor models.

[0003] After the overall physical replacement method revealed irreconcilable contradictions, the industry attempted to seek breakthroughs at the level of surface chemical components. However, such conventional component intervention strategies also face inherent limitations in underlying mechanisms. For example, Chinese invention patent application CN110835622A discloses a culture medium for regulating lactate metabolism in mammalian cells and its application. It inhibits lactate accumulation by adding a specific concentration of ginsenoside tricarboxylic acid or ginsenoside tricarboxylic acid ammonium salt to the culture medium. However, this method of bypassing intervention by simply relying on non-physiological exogenous chemical complexes has a fundamental mismatch between the core presupposition and the actual metabolic law of cells. Its essence remains at the level of passively suppressing acidic metabolic waste, without reshaping the metabolic flow at the bottom layer of the cell. Although the introduction of macromolecular chemical substances can delay the appearance of lactate accumulation, it is powerless to drive the endogenous assimilation of existing lactate, let alone reverse its conversion into energy substrates for the tricarboxylic acid cycle. At the same time, the long-term residence of non-metabolic intermediate components will impose an additional homeostasis maintenance load on the normal signal transduction and oxidative stress defense pathways of cells, and cannot solve the risk of DNA damage induced by metabolic fluctuations.

[0004] Therefore, the technical problem to be solved by this invention is how to construct an animal-free amplification system that synergistically balances endogenous assimilation of metabolites and maintenance of cellular genetic fidelity, and eliminates genomic instability and phenotypic degradation induced by metabolic stress in serum-free culture. Summary of the Invention

[0005] This invention aims to address the problems of loss of genetic stability caused by metabolic stress and tumorigenic phenotypic drift resulting from damage to the autocrine microenvironment during serum-free cell amplification.

[0006] In this technical solution, a novel tumor cell amplification culture medium and process without animal-derived components are disclosed, comprising the following steps: Step 101: Inoculate tumor cells with an animal-free culture medium. The animal-free culture medium includes a basal solution and a core nutrient supplement. The core nutrient supplement contains recombinant human serum albumin at a weight-to-volume ratio of 50 μg / mL to 150 μg / mL, transferrin at a weight-to-volume ratio of 5 μg / mL to 10 μg / mL, insulin at a weight-to-volume ratio of 5 μg / mL to 20 μg / mL, sodium selenite at a weight-to-volume ratio of 5 nM to 50 nM, and sodium pyruvate at a weight-to-volume ratio of 0.51 mM to 1 mM. Step 102: During the time window when tumor cells transition from the adaptation phase to the explosive expansion phase, the residual glucose concentration in the culture system is monitored in real time using a metabolic sensor. Step 103: When the residual glucose concentration is measured to drop to the metabolic trigger threshold range of 1.0 mM to 1.5 mM, stop adding the basal solution and inject a single dose of sugar-free metabolic reversal pulse solution into the culture system. The sugar-free metabolic reversal pulse solution is composed of sodium pyruvate and 1.5 mM to 2.0 mM N-acetylcysteine ​​in the sugar-free basal solution. Step 104: By adjusting the injection volume of sugar-free metabolic reversal pulse solution, the local transient concentration of sodium pyruvate in the culture system is increased to 2.0 mM to 2.5 mM, and this pulse intervention state is maintained for 2 to 4 hours. The concentration gradient formed by exogenous pyruvate inside and outside the cell membrane drives the reverse catalysis of lactate dehydrogenase in tumor cells, inducing tumor cells to re-take up lactate in the culture system and convert it into tricarboxylic acid cycle energy substrate. After the maintenance phase of steps 105 and 104 is completed, the routine replenishment of the glucose-containing basal solution is resumed, and the residual glucose concentration is maintained above 2 mM to stabilize the genetic characteristics of tumor cells by endogenously clearing acidic metabolic waste.

[0007] Preferably, the basal solution in step 101 is composed of RPMI-1640 culture medium and DMEM culture medium mixed in a volume ratio of 2:1 to 1:1; the process also includes a step of segmented delivery of growth factors: within 0 to 24 hours after inoculation, 5 ng / mL to 10 ng / mL of epidermal growth factor and 50 μg / mL to 100 μg / mL of heparin are added to the culture system.

[0008] Preferably, the inoculation density in step 101 is set to 10,000. Up to 20000 This is used to reduce contact inhibition and simulate the in vivo diffusion environment; the basic culture conditions in step 101 are set as follows: 37°C, 5% carbon dioxide concentration and saturated humidity to ensure that the cells are in a controlled growth environment.

[0009] Preferably, the process employs a half-volume medium replacement method, replacing 50% to 70% of the culture medium every 48 hours, utilizing the retained autocrine signaling molecules to maintain intercellular communication; during passage, the process uses a trypsin substitute without EDTA to digest tumor cells, thereby reducing mechanical damage to cell surface receptors.

[0010] Preferably, when the culture reaches the steady-state stage of 48 to 72 hours, 5 μM to 10 μM of apoptosis enzyme inhibitor or 10 μM of ROCK inhibitor is added to the culture system. The apoptosis enzyme inhibitor is used to reduce the proportion of apoptosis under high-density amplification, and the ROCK inhibitor is used to improve the adhesion efficiency under low serum conditions.

[0011] Preferably, the core nutritional supplement also includes 1 μM to 10 μM of ethanolamine; the core nutritional supplement, through the synergistic effect of sodium selenite and N-acetylcysteine ​​concentration in step 103, neutralizes reactive oxygen free radicals generated by tumor cells during lactic acid uptake, thereby blocking DNA oxidative damage induced by metabolic fluctuations.

[0012] Preferably, for specific cell lines at risk of p53 gene mutation, the process involves adding an extremely low dose of p53 pathway regulator during the steady-state phase after 48 hours of culture, and using genomic monitoring pressure to screen cell clones that have not undergone genetic drift, ensuring the genetic fidelity of the amplification products relative to the original standard strain.

[0013] Preferably, when the pH of the culture system drops below 6.8 or the residual glucose concentration is below 2 mM, the feeding step is initiated: glucose solution is added according to metabolic needs; sodium pyruvate solution is added; and the acid-base balance and substrate supply stability in the culture system are maintained through combined feeding.

[0014] Preferably, the tumor cells produced by the process have a coefficient of variation of less than 5% in the sensitivity index of chemotherapy drugs after five consecutive passages, and the short tandem repeat sequence typing of the tumor cells matches the original standard strain 100%, which proves the role of sugar-free metabolism reversal pulse solution in maintaining the genetic stability of cells.

[0015] Compared with existing technologies, the novel tumor cell expansion culture medium and process of this invention, which are free of animal-derived components, have the following advantages: 1. In tumor cell expansion culture, an endogenous closed-loop regulatory mechanism for metabolic flow is established to avoid microenvironmental disruption caused by external physical dilution. This invention utilizes a metabolic regulation system constructed from sodium pyruvate and sodium selenite, combined with a specific feeding logic. When the glucose concentration in the culture system drops to a critical threshold, the chemical potential gradient of external pyruvate is maintained, inducing the reverse catalytic action of lactate dehydrogenase in tumor cells. This mechanism enables tumor cells to actively take up and consume lactate in the microenvironment, converting it into an energy substrate for the tricarboxylic acid cycle, thus achieving endogenous self-clearance of acidic metabolic waste. Compared with the passive mode of relying on frequent medium changes to physically replace lactate in existing technologies, this invention does not require large-scale removal of the culture supernatant, ensuring the continuous enrichment of cell-secreted growth factors, extracellular vesicles, and key signaling molecules in the system, thereby avoiding cell communication interruption and phenotypic drift caused by physical flushing.

[0016] 2. To achieve simultaneous remodeling of oxidative stress defense and energy metabolism flux, ensuring the genetic fidelity of nucleic acid materials, this invention physically binds the dynamic delivery of growth factors with the activation of the antioxidant chain, addressing the risk of genomic damage caused by a sudden increase in metabolic flux during the explosive expansion phase of tumor cells. During the cell cycle initiation and expansion window, sodium selenite and glutathione precursor work synergistically to construct an immediate free radical neutralization shield within the cell during the instantaneous overload of the tricarboxylic acid cycle, targeting and clearing reactive oxygen species generated by the metabolic surge. This synergistic mechanism directly reduces the oxidative stress pressure on tumor cell DNA, ensuring the absolute stability of genomic characteristics during long-term passage of the cell line, and making the amplification products exhibit high consistency in genetic locus comparison.

[0017] 3. Eliminating signal interference caused by heterologous components and constructing a drug response evaluation model with high monotonic determinism: This invention uses a chemical formula with clearly defined components and no animal-derived ingredients, combined with a combination of factors that maintain the dedifferentiation state of tumor cells, to eliminate growth signal fluctuations caused by batch-to-batch differences in traditional serum. Because the culture system provides a stable and predictable metabolic substrate environment, the stress response pathway of tumor cells is in a highly controlled homeostasis, enabling the cell model to exhibit extremely high response consistency in the sensitivity evaluation of chemotherapy drugs or targeted drugs. This technical approach not only avoids the risk of animal-derived viral contamination, but also maintains the coefficient of variation of drug test results at an extremely low level through standardization at the mechanism level, thereby improving the engineering reproducibility value of experimental data. Attached Figure Description

[0018] Figure 1 This is a flowchart of the metabolic control process for tumor cell expansion without animal-derived components according to the present invention; Figure 2 This is a schematic diagram of the logical interaction principle of the tumor cell proliferation and metabolic regulation system of the present invention. Detailed Implementation

[0019] 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.

[0020] It should be noted that all directional and positional terms used in this invention, such as: up, down, left, right, front, back, vertical, horizontal, inner, outer, top, bottom, transverse, longitudinal, center, etc., are only used to explain the relative positional relationship and connection between components in a specific state (as shown in the accompanying drawings). They are only for the convenience of describing this invention and do not require that this invention be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention. In addition, the descriptions of "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms installation, connection, and linking should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to the internal connection of two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0022] In the description of this specification, references to the terms "an embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0023] A novel tumor cell amplification culture medium and process free of animal-derived components, comprising the following steps: Step 101: Inoculate tumor cells with an animal-free culture medium. The animal-free culture medium includes a basal solution and a core nutrient supplement. The core nutrient supplement contains recombinant human serum albumin at a weight-to-volume ratio of 50 μg / mL to 150 μg / mL, transferrin at a weight-to-volume ratio of 5 μg / mL to 10 μg / mL, insulin at a weight-to-volume ratio of 5 μg / mL to 20 μg / mL, sodium selenite at a weight-to-volume ratio of 5 nM to 50 nM, and sodium pyruvate at a weight-to-volume ratio of 0.51 mM to 1 mM. Step 102: During the time window when tumor cells transition from the adaptation phase to the explosive expansion phase, the residual glucose concentration in the culture system is monitored in real time using a metabolic sensor. Step 103: When the residual glucose concentration is measured to drop to the metabolic trigger threshold range of 1.0 mM to 1.5 mM, stop adding the basal solution and inject a single dose of sugar-free metabolic reversal pulse solution into the culture system. The sugar-free metabolic reversal pulse solution is composed of sodium pyruvate and 1.5 mM to 2.0 mM N-acetylcysteine ​​in the sugar-free basal solution. Step 104: By adjusting the injection volume of sugar-free metabolic reversal pulse solution, the local transient concentration of sodium pyruvate in the culture system is increased to 2.0 mM to 2.5 mM, and this pulse intervention state is maintained for 2 to 4 hours. The concentration gradient formed by exogenous pyruvate inside and outside the cell membrane drives the reverse catalysis of lactate dehydrogenase in tumor cells, inducing tumor cells to re-take up lactate in the culture system and convert it into tricarboxylic acid cycle energy substrate. After the maintenance phase of steps 105 and 104 is completed, the routine replenishment of the glucose-containing basal solution is resumed, and the residual glucose concentration is maintained above 2 mM to stabilize the genetic characteristics of tumor cells by endogenously clearing acidic metabolic waste.

[0024] Preferably, the basal solution in step 101 is composed of RPMI-1640 culture medium and DMEM culture medium mixed in a volume ratio of 2:1 to 1:1; the process also includes a step of segmented delivery of growth factors: within 0 to 24 hours after inoculation, 5 ng / mL to 10 ng / mL of epidermal growth factor and 50 μg / mL to 100 μg / mL of heparin are added to the culture system.

[0025] Preferably, the inoculation density in step 101 is set to 10,000. Up to 20000 This is used to reduce contact inhibition and simulate the in vivo diffusion environment; the basic culture conditions in step 101 are set as follows: 37°C, 5% carbon dioxide concentration and saturated humidity to ensure that the cells are in a controlled growth environment.

[0026] Preferably, the process employs a half-volume medium replacement method, replacing 50% to 70% of the culture medium every 48 hours, utilizing the retained autocrine signaling molecules to maintain intercellular communication; during passage, the process uses a trypsin substitute without EDTA to digest tumor cells, thereby reducing mechanical damage to cell surface receptors.

[0027] Preferably, when the culture reaches the steady-state stage of 48 to 72 hours, 5 μM to 10 μM of apoptosis enzyme inhibitor or 10 μM of ROCK inhibitor is added to the culture system. The apoptosis enzyme inhibitor is used to reduce the proportion of apoptosis under high-density amplification, and the ROCK inhibitor is used to improve the adhesion efficiency under low serum conditions.

[0028] Preferably, the core nutritional supplement also includes 1 μM to 10 μM of ethanolamine; the core nutritional supplement, through the synergistic effect of sodium selenite and N-acetylcysteine ​​concentration in step 103, neutralizes reactive oxygen free radicals generated by tumor cells during lactic acid uptake, thereby blocking DNA oxidative damage induced by metabolic fluctuations.

[0029] Preferably, for specific cell lines at risk of p53 gene mutation, the process involves adding an extremely low dose of p53 pathway regulator during the steady-state phase after 48 hours of culture, and using genomic monitoring pressure to screen cell clones that have not undergone genetic drift, ensuring the genetic fidelity of the amplification products relative to the original standard strain.

[0030] Preferably, when the pH of the culture system drops below 6.8 or the residual glucose concentration is below 2 mM, the feeding step is initiated: glucose solution is added according to metabolic needs; sodium pyruvate solution is added; and the acid-base balance and substrate supply stability in the culture system are maintained through combined feeding.

[0031] Preferably, the tumor cells produced by the process have a coefficient of variation of less than 5% in the sensitivity index of chemotherapy drugs after five consecutive passages, and the short tandem repeat sequence typing of the tumor cells matches the original standard strain 100%, which proves the role of sugar-free metabolism reversal pulse solution in maintaining the genetic stability of cells.

[0032] Example 1: A specific operational example in the high-fidelity expansion of the A549 lung cancer cell line. During the large-scale expansion of A549 cells, due to the unique aerobic glycolytic metabolism of tumor cells, i.e., the Warburg effect, a large amount of lactic acid secretion occurs in the culture system, leading to medium acidification and inducing cell phenotypic drift. To address this technical bottleneck, the basal medium used in this example is a 1:1 volume mixture of RPMI-1640 culture medium and DMEM culture medium, with an inoculation density of 15,000 cells / day. During the amplification initiation phase, i.e., from 0 to 24 hours after inoculation, 10 ng / mL of epidermal growth factor and 100 μg / mL of heparin were added to the culture system to activate growth signals. Core nutritional supplements included 100 μg / mL of recombinant human serum albumin, 10 μg / mL of transferrin, 10 μg / mL of insulin, 20 nM sodium selenite, and 0.5 mM sodium pyruvate. This combination was used to replace fetal bovine serum and provide necessary metabolic support. The calibration procedure for determining the metabolic trigger threshold of residual glucose concentration included monitoring the specific oxygen uptake rate of the culture system using an online polarographic dissolved oxygen electrode. The rate of lactic acid production was determined using a biochemical analyzer. Within the time window during which tumor cells transition from the adaptation phase to the explosive expansion phase, supernatant samples are extracted at predetermined time intervals to calculate... and When the ratio calculated over three consecutive sampling periods decreases from a value greater than 1 to a set range of 0.8 to 0.95, the residual glucose concentration value output by the metabolic sensor at the corresponding time is read. The lower quartile of the value range is identified as the lower limit of the residual glucose concentration metabolic trigger threshold range, so that the pulse injection action trigger node corresponds to the physical moment of the transfer of cellular glucose metabolic flow.

[0033] When A549 cells enter the burst expansion phase, the residual glucose concentration in the culture system is monitored in real time using a metabolic sensor. When the residual glucose concentration drops to the metabolic trigger threshold of 1.2 mM, the addition of glucose-containing basal medium is stopped, and a single injection of glucose-free metabolic reversal pulse solution is injected into the culture system. After the injection of the glucose-free metabolic reversal pulse solution, the online monitoring program for redox potential based on Raman spectroscopy coenzyme pool is initiated. The controller receives real-time data from the Raman spectrometer on the oxidation state of nicotinamide adenine dinucleotide in the cytoplasm. With reduced state Characteristic peak intensity signal, based on which calculations are performed and output. and The ratio data serves as a process feedback parameter. Exogenous sodium pyruvate enters the cell via a monocarboxylic acid transporter and interacts with N-acetylcysteine ​​in the culture system. The decision logic unit confirms this. and When the ratio remains above 1.5 for a set time interval, the system is determined to have entered an endogenous lactate clearance state, triggering a timing action for the maintenance pulse intervention state. During the online monitoring process, to eliminate the masking of the weak coenzyme signal by complex amino acids and impurity proteins in the liquid phase background, the controller incorporates a baseline subtraction algorithm module based on adaptive iterative penalized least squares. This module performs second derivative calculations on the real-time acquired spectral data to anchor the baseline. In 1096 and In 1690 By dynamically fitting and stripping non-specific fluorescence background contours at nearby characteristic peak positions, the characteristic Raman scattering photon intensity of the coenzyme pool is converted into accurate concentration ratios in a liquid-phase environment without physical isolation. This pulsed solution is composed of sodium pyruvate and 1.8 mM N-acetylcysteine ​​in a sugar-free basal solution. The injection volume is adjusted to raise the local transient concentration of sodium pyruvate in the culture system to 2.2 mM, and this pulsed intervention state is maintained for 3 hours. During this adjustment of the injection volume, to ensure that the addition of the overall global volume is accurately converted into a local transient concentration on the surface, the system directly guides the injection path of the pulsed solution to the impeller end flow region with the highest shear force inside the bioreactor. Utilizing the hydrodynamic characteristics of this region, where the characteristic mixing time is much shorter than the transmembrane mass transfer time constant of the exogenous substrate,… The high-concentration pulsed droplets are rapidly broken up and uniformly dissipated by turbulent vortices before contacting cells, thereby causing the concentration of components in the cellular microenvironment to instantaneously exceed the threshold for triggering metabolic reversal without causing a surge in global osmotic pressure. During this intervention, the exogenous high-concentration pyruvate forms a chemical potential gradient across the cell membrane, driving the reverse catalysis of lactate dehydrogenase in A549 cells. This induces the cells to re-take up lactate from the culture system and convert it into energy substrates for the tricarboxylic acid cycle, achieving endogenous autophagic clearance of acidic metabolic waste. Specifically, the exogenous high-concentration pyruvate preferentially occupies monocarboxylic acid transporters on the cell membrane surface, blocking the exocytosis of intracellular free lactate through competitive inhibition of transmembrane transport, leading to the instantaneous accumulation of intracellular lactate and disrupting the original thermodynamic equilibrium. Simultaneously, it combines with the system's internal... and A high-oxidation-state environment with a ratio greater than 1.5 acts as an allosteric regulatory signal, inducing a catalytic bias in the isozyme profile of lactate dehydrogenase towards a configuration that consumes lactate. This reverses the original direction of glycolysis in physicochemical kinetics, allowing the intracellularly enriched lactate to be used as a substrate for assimilation and consumption. Simultaneously present N-acetylcysteine ​​neutralizes reactive oxygen species generated during metabolic remodeling, blocking DNA oxidative damage induced by metabolic fluctuations. During the pulsed intervention period, the absolute value of lactate concentration in the culture system is periodically measured using a bypass liquid chromatograph. The trigger condition for restoring the routine replenishment of the glycobase solution is set when the first derivative of the lactate concentration calculated from two consecutive sampling periods approaches zero from a negative value and the current absolute value of lactate concentration drops below 30% of the initial peak value before intervention. At this point, the glycobase solution is injected at the set pump rate to maintain the residual glucose concentration in the culture system above 2 mM within the set time window.

[0034] After the intervention, the basal solution containing glucose was restored, and the residual glucose concentration was maintained above 2 mM. Experimental data showed that the A549 cells produced by this process expanded 18-fold within 72 hours, with a cell viability greater than 95%, and the short tandem repeat sequence typing matched the original standard strain 100%. After five consecutive passages, this cell line showed good resistance to the chemotherapy drug cisplatin. The coefficient of variation was less than 5%, confirming that the metabolic remodeling procedure effectively enriched cell-secreted growth factors and exosomes without requiring a large proportion of physical media replacement, thus maintaining the stability of the genetic characteristics of tumor cell lines and the consistency of drug sensitivity screening.

[0035] Example 2: In the MCF-7 breast cancer cell line fidelity verification scenario, the experimental platform has a temperature control accuracy of 0.1℃ and a carbon dioxide concentration control accuracy of 0.1%. The raw data required for the experiment are acquired by a metabolic sensor integrated in the tubing. The glucose detection resolution of this sensor is 0.02mM, and the sampling period is set to 1h. The sampling period is determined based on the balance between monitoring the dynamic slope of glucose concentration decrease and maintaining the homeostasis of the cell microenvironment. That is, when the glucose consumption rate exceeds the judgment threshold of 0.5mM / h, the sensor maintains the sampling frequency to locate the metabolic trigger threshold range of 1.0mM to 1.5mM. The experimental groups are set as experimental group, control sample group A, and control. Sample group B and control group C were used. The experimental group used a culture medium and process without animal-derived components, in which the local transient concentration of sodium pyruvate was controlled at 2.35 mM and the concentration of N-acetylcysteine ​​was set at 1.75 mM. The pulse intervention state was maintained for 3 h. Control group A did not contain N-acetylcysteine ​​in the sugar-free metabolic reversal pulse solution. Control group B set the local transient concentration of sodium pyruvate in the sugar-free metabolic reversal pulse solution at 3.5 mM. Control group C used a basal culture medium containing 10% fetal bovine serum and a physical medium change process every 48 h. In order to simulate the measurement error and environmental fluctuation in the industrial environment, random noise disturbance with a signal-to-noise ratio of 25 dB was superimposed on the metabolic sensor readings.

[0036] After the experiment was started, the original input data of each group were recorded. In the experimental group, the glucose concentration decreased from the initial 15.2 mM to 1.28 mM 36 h after inoculation. The system injected sugar-free metabolic reversal pulse solution into the culture system. During the intervention period, the lactate concentration in the experimental group showed a decreasing trend, decreasing from 22.4 mM to 12.6 mM. This data reflects that the chemical potential gradient formed by exogenous pyruvate inside and outside the cell membrane drives the reverse catalytic reaction of lactate dehydrogenase. In the control group B, under the condition of sodium pyruvate concentration of 3.5 mM, the lactate assimilation rate did not increase with the increase of concentration, and the osmotic pressure stress caused the cell viability to decrease from 96.2% to 84.5%. The experimental data determined that the concentration range of 2.0 mM to 2.5 mM is the working window that takes into account both metabolic assimilation efficiency and cell viability.

[0037] At the end of the experiment, the final product performance indicators of each group were obtained. The MCF-7 cells produced in the experimental group expanded 16.8 times after 72 hours, and the short tandem repeat sequence typing matched the original standard strain 100%. Due to the scavenging effect of N-acetylcysteine ​​on reactive oxygen species, the concentration of 8-OHdG, the DNA oxidative damage index, in the experimental group was at the level of 1.25 ng / mL. The control group A, lacking an antioxidant synergistic mechanism, had an 8-OHdG concentration of 8.42 ng / mL, and after 5 consecutive passages, the half-maximal inhibitory concentration against cisplatin was lower. The coefficient of variation was 18.6%, and the MCF-7 cells produced in the experimental group were effective against cisplatin. The coefficient of variation was 4.2%, and the data obtained reflect the characteristics of metabolic reversal pulse solution and dynamic monitoring process in maintaining the genetic fidelity of tumor cells and reducing the fluctuation of drug sensitivity screening data.

[0038] Example 3: This example combines Figures 1 to 2 Description of a novel tumor cell amplification culture medium and process without animal-derived components, such as... Figure 1 As shown, the novel tumor cell amplification process without animal-derived components proceeds from step 101, inoculating tumor cells with a culture medium without animal-derived components, to step 102, monitoring the residual glucose concentration in real time using a metabolic sensor, then to step 103, injecting a metabolic reversal pulse solution when glucose drops to a threshold, to step 104, increasing the pyruvate concentration to drive enzyme reverse catalysis to take up lactate, and finally to step 105, restoring and clearing acidic metabolites to stabilize genetic characteristics.

[0039] like Figure 2 As shown, a metabolic sensor is connected to monitor the residual glucose concentration, an online pH electrode is connected to monitor the rate of change in the system's pH, and a controller is connected to pause the replenishment of the basal solution, calculate the injection volume of the pulse solution, inject the sugar-free metabolic reversal pulse solution, and resume the replenishment of the basal solution containing sugar. The injected sugar-free metabolic reversal pulse solution is pointed to the reverse catalytic lactate dehydrogenase via the dotted line, and the reverse catalytic lactate dehydrogenase is pointed to the uptake of lactate in the culture system via the dotted line. Tumor cells are connected to the reverse catalytic lactate dehydrogenase and the uptake of lactate in the culture system and its conversion into tricarboxylic acid cycle substrates.

[0040] Example 4: In the large-scale expansion of the A549 cell line using a 10L stirred bioreactor, to address monitoring deviations caused by sensor drift and dynamic increases in culture volume, the system used glucose standard solutions with concentrations of 1.0 mM, 5.0 mM, and 15.0 mM as inputs to perform slope calibration on the metabolic sensor, establishing the sensor's transfer function as follows: ,in, The original voltage signal output by the sensor. Glucose concentration, coefficient and The seeding density was determined through linear fitting and set at 20,000 cells / h as the concentration conversion baseline for subsequent 72-hour continuous amplification.

[0041] When the amplification enters the burst phase and the glucose concentration sequence collected by the metabolic sensor shows a monotonically decreasing trend, the logic decision unit built into the controller performs a first-order differential operation to calculate the real-time glucose consumption rate. And a moving average window with a step size of 5 is introduced to smooth the noise, when the measured When the sampling rate exceeds 0.5 mM / h for three consecutive sampling cycles and the residual glucose concentration decreases to 1.25 mM, the system calculates the injection volume of the glucose-free metabolic reversal pulse solution based on the volume balance principle. The calculation formula is as follows: ,in, This refers to the pulse injection volume. The target value for the local transient concentration of sodium pyruvate is 2.25 mM. This represents the initial concentration of sodium pyruvate in the system before injection. The total volume of the culture system is expressed in liters (L). The concentration of sodium pyruvate in the sugar-free metabolic reversal pulse solution.

[0042] The system drives the peristaltic pump to calculate the... A pulsed liquid was injected into the reactor, and the rate of change in pH of the system was monitored by an online pH electrode. Within 15 minutes after injection, it was observed that The change from negative to positive values ​​reflects the reverse catalysis of lactate dehydrogenase induced by the chemical potential gradient formed by exogenous pyruvate, enabling cells to re-take lactate and release hydrogen ions. Simultaneously, reactive oxygen free radicals are neutralized by 1.75mMN-acetylcysteine, which works in synergy with sodium selenite. After intervention for 3.5 hours, regular feeding was resumed. The resulting A549 cells were subjected to short tandem repeat sequence typing by capillary electrophoresis. The fluorescence spectra of D3S1358, vWA, and the remaining 13 core sites showed a consistency deviation of 0% with the original standard strain, demonstrating the maintenance of cellular genetic stability by this calibration and feeding procedure in the treatment of large-scale bioreactor environments.

[0043] Example 5: In the adaptive expansion of tumor cell lines using a 5L gas-propelled bioreactor, the system was calibrated to address the fluctuations in cofactor enrichment between different batches of recombinant human serum albumin and recombinant insulin. The initial environment consisted of an animal-free culture medium. A basal solution of RPMI-1640 and DMEM culture medium at a 1:1 volume ratio was prepared as a blank reference before inoculation. A glucose standard solution with a known concentration gradient was used to generate a response voltage sequence at the metabolic sensor built into the bioreactor. The measured voltage signal was then analyzed. Compared with known concentration The zero-point offset of the sensor is determined by a linear regression relationship. and slope coefficient This is used to maintain the monitoring accuracy of residual glucose concentration at 0.05 mM in subsequent amplification.

[0044] When the A549 cells in the bioreactor enter the burst expansion phase and the residual glucose concentration in the system drops to the trigger point of 1.25 mM, the controller, based on the current total volume of the culture system... and dissolved oxygen change rate Calculate the injection volume of sugar-free metabolic reversal pulsed solution Using the formula The amount of sodium pyruvate required for a single pulse intervention was determined, among which, The target value for the local transient concentration was set at 2.3 mM. This represents the initial concentration of sodium pyruvate in the system before injection. The concentration of sodium pyruvate in the sugar-free metabolic reversal pulse solution was determined by adjusting the stirring speed during the 3-hour pulse intervention period to maintain the oxygen mass transfer coefficient within the system. Assimilation rate with lactate By maintaining kinetic matching, the pH of the system was eventually raised back to the neutral range of 7.2 without changing the culture medium. The resulting A549 cell viability remained above 96.5% and no short tandem repeat sequence shifts were observed.

[0045] Example 6: In an industrial deployment scenario of large-scale tumor cell expansion using a 50L stirred bioreactor, the system calibrates the stirring parameters based on the internal geometry of the reactor, and uses the formula... Calculate the tip velocity, where, For the tip velocity, Pi The diameter of the agitator blades, The stirring speed is adjusted by regulating the stirring speed. Increase the speed of the propeller tip The flow rate was maintained at 0.85 m / s to ensure efficient dissolved oxygen mass transfer while avoiding shear stress that could reduce cell viability. Before inoculation, the mass of the animal-free culture medium was obtained using an online electronic balance, and the initial culture volume was calculated. The data is stored in the controller, and glucose concentration sequences are subsequently acquired at an hourly sampling frequency to monitor the metabolic evolution of A549 cells.

[0046] When the amplification enters the burst phase and the residual glucose concentration reaches the trigger point of 1.15 mM, the controller initiates the online electrode calibration program. This program utilizes the differential signals from the primary and backup pH sensors to eliminate zero-point drift, based on the formula... The amount of sugar-free metabolic reversal pulse solution to be added was determined, among which, This refers to the pulse injection volume. To preset the target value for the local transient concentration of sodium pyruvate, This represents the sodium pyruvate concentration in the system prior to injection. The total volume of the current cultivation system, To determine the reserve concentration of sodium pyruvate in the pulsed solution, adjust the pulsed pump flow rate to achieve the calculated concentration. The pulsed solution was injected over 8 minutes, and the rate of pH recovery was monitored during the intervention. When measured When the concentration of 8-OHdG, a marker of cellular oxidative damage, reached 0.08 pH units / h and remained at 1.32 ng / mL, the endogenous lactate assimilation mechanism was determined to be triggered. The tumor cells produced were confirmed by short tandem repeat sequence typing to have no phenotypic drift at their core sites.

[0047] Example 7: In a long-cycle expansion of highly differentiated MCF-7 cell lines exceeding 10 passages, to address contact inhibition and downregulation of tumorigenesis markers caused by high cell density during the later stages of explosive expansion, a stabilization enhancer was injected into a 50L stirred bioreactor via a high-precision peristaltic pump during the 48-hour steady-state phase of culture. The flow rate of the stabilization enhancer was adjusted to maintain the final concentration of the ROCK inhibitor Y-27632 in the culture system at 10 μM, while simultaneously maintaining the final concentration of the p53 pathway regulator Nutlin-3 at 5 nM. This allowed Y-27632 to reduce cell shrinkage caused by myosin light chain phosphorylation. To improve cell adhesion, Nutlin-3 is used to maintain genomic surveillance pressure at the transcriptional level. Here, p53 pathway regulators are not limited to Nutlin-3, but include non-peptide small molecule compounds that can specifically bind to MDM2 protein and create steric hindrance in its p53 binding pocket region. The objective screening criterion is that the half-maximal inhibitory concentration against the interaction between MDM2 and p53 is less than 100 nM in in vitro binding experiments. These regulators competitively block the ubiquitination and degradation pathway of p53 by MDM2, allowing unmutated wild-type p53 protein to accumulate stably in the cell, thereby constructing a concentration threshold-dependent active apoptosis defense against genomic abnormalities.

[0048] Through the synergistic intervention of the above components, cell samples were obtained at the 72-hour culture termination point, and the expression intensity of the markers CD133 and CD44 was detected by flow cytometry. The positive percentage of CD133 was 94.2%, and the positive percentage of CD44 was 91.5%, which was 35.8% higher than that of the control group without the above regulators. Furthermore, the short tandem repeat genotyping sites remained 100% matched with the original standard strain after 10 consecutive passages, indicating a significant increase in expression abundance against cisplatin. The measured value was 2.15 μM with a coefficient of variation of 3.2%. This quantitative result confirms the lock-in characteristics of apoptosis inhibition and gene surveillance mechanisms on the consistency of tumor cell phenotype and drug sensitivity in long-cycle amplification tasks.

[0049] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A novel tumor cell amplification culture medium and process free of animal-derived components, characterized in that, Includes the following steps: Step 101: Inoculate tumor cells with an animal-free culture medium. The animal-free culture medium includes a basal solution and a core nutrient supplement. The core nutrient supplement contains recombinant human serum albumin at a weight-to-volume ratio of 50 μg / mL to 150 μg / mL, transferrin at a weight-to-volume ratio of 5 μg / mL to 10 μg / mL, insulin at a weight-to-volume ratio of 5 μg / mL to 20 μg / mL, sodium selenite at a weight-to-volume ratio of 5 nM to 50 nM, and sodium pyruvate at a weight-to-volume ratio of 0.51 mM to 1 mM. Step 102: During the time window when tumor cells transition from the adaptation phase to the explosive expansion phase, the residual glucose concentration in the culture system is monitored in real time using a metabolic sensor. Step 103: When the residual glucose concentration is measured to drop to the metabolic trigger threshold range of 1.0 mM to 1.5 mM, stop adding the basal solution and inject a single dose of sugar-free metabolic reversal pulse solution into the culture system. The sugar-free metabolic reversal pulse solution is composed of sodium pyruvate and 1.5 mM to 2.0 mM N-acetylcysteine ​​in the sugar-free basal solution. Step 104: By adjusting the injection volume of sugar-free metabolic reversal pulse solution, the local transient concentration of sodium pyruvate in the culture system is increased to 2.0 mM to 2.5 mM, and the pulse intervention state is maintained for 2 to 4 hours. The concentration gradient formed by exogenous pyruvate inside and outside the cell membrane drives the reverse catalysis of lactate dehydrogenase in tumor cells, inducing tumor cells to re-take up lactate in the culture system and convert it into tricarboxylic acid cycle energy substrate. After the maintenance phase of steps 105 and 104 is completed, the routine replenishment of the glucose-containing basal solution is resumed, and the residual glucose concentration is maintained above 2 mM to stabilize the genetic characteristics of tumor cells by endogenously clearing acidic metabolic waste.

2. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, The basal solution in step 101 is composed of RPMI-1640 culture medium and DMEM culture medium mixed in a volume ratio of 2:1 to 1:1; the process also includes a step of segmented delivery of growth factors: within 0 to 24 hours after inoculation, 5 ng / mL to 10 ng / mL of epidermal growth factor and 50 μg / mL to 100 μg / mL of heparin are added to the culture system.

3. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, The inoculation density in step 101 is set to 10000. Up to 20000 This is used to reduce contact inhibition and simulate the in vivo diffusion environment; the basic culture conditions in step 101 are set as follows: 37°C, 5% carbon dioxide concentration and saturated humidity to ensure that the cells are in a controlled growth environment.

4. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, The process employs a half-volume medium replacement method, replacing 50% to 70% of the culture medium every 48 hours, utilizing the retained autocrine signaling molecules to maintain intercellular communication; during passage, the process uses a trypsin substitute without EDTA to digest tumor cells, thereby reducing mechanical damage to cell surface receptors.

5. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, When the culture reaches the steady-state stage of 48 to 72 hours, 5 μM to 10 μM of apoptosis enzyme inhibitor or 10 μM of ROCK inhibitor is added to the culture system. The apoptosis enzyme inhibitor is used to reduce the proportion of anolysmic apoptosis under high-density amplification, and the ROCK inhibitor is used to improve the adhesion efficiency under low serum conditions.

6. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, The core nutritional supplement also includes 1 μM to 10 μM ethanolamine; the core nutritional supplement, through the synergistic effect of sodium selenite and N-acetylcysteine ​​concentration in step 103, neutralizes reactive oxygen free radicals generated by tumor cells during lactate uptake, thereby blocking DNA oxidative damage induced by metabolic fluctuations.

7. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, For specific cell lines at risk of p53 gene mutation, the process involves adding an extremely low dose of p53 pathway regulator during the steady-state phase after 48 hours of culture. This utilizes genomic monitoring pressure to screen cell clones that have not undergone genetic drift, ensuring the genetic fidelity of the amplification products relative to the original standard strain.

8. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, When the pH of the culture system was monitored to drop to When the pH is below 6.8 or the residual glucose concentration is below 2 mM, initiate the feeding procedure: supplement glucose solution according to metabolic needs; supplement sodium pyruvate solution; maintain the acid-base balance and substrate supply stability in the culture system through combined feeding.

9. The novel tumor cell amplification culture medium and process without animal-derived components according to claim 1, characterized in that, The tumor cells produced by the process had a coefficient of variation of less than 5% in the sensitivity index of chemotherapy drugs after five consecutive passages, and the short tandem repeat sequence typing of the tumor cells matched the original standard strain 100%, which proved the role of sugar-free metabolic reversal pulse solution in maintaining the genetic stability of cells.

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