A method for establishing an in vitro culture model of oral squamous cell carcinoma

Abstract

The invention relates to a method for establishing an in vitro culture model of oral squamous cell carcinoma; the method comprises the following steps: collecting patient data, and analyzing oral squamous cell carcinoma in a patient and its living environment; periodically obtaining tissue samples and separating cells of oral squamous cell carcinoma, and establishing an in vitro culture model, and placing the oral squamous cell carcinoma into the in vitro culture model for culture; collecting oral squamous cell carcinoma in a patient at different cycles and oral squamous cell carcinoma cultured in vitro; analyzing the obtained data to adjust parameters in the model to be closer to in vivo generation characteristics, so that the oral squamous cell carcinoma generated in vitro is consistent with the oral squamous cell carcinoma grown in vivo, and by integrating the patient's specific nutrition, metabolism and gene data, the model can more realistically reflect the growth characteristics and microenvironment of individual tumors, and compared with the traditional 2D culture method, the three-dimensional culture adopted in this scheme can better simulate the arrangement and interaction of cells in vivo.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro culture of cell carcinoma, and in particular to a method for establishing an in vitro culture model of oral squamous cell carcinoma. Background Art

[0002] Oral squamous cell carcinoma is one of the most common malignant tumors in the head and neck, and its common sites are the tongue, gums, cheeks, palate, lips, and floor of mouth. Squamous cell carcinoma is characterized by the formation of keratin beads and the appearance of cell bridges. The tumor originates from the surface oral mucosal epithelium, and the neoplastic epithelial masses or epithelial islands infiltrate the underlying connective tissue. The cell characteristics can be manifested as abundant eosinophilic cytoplasm, large nuclear-cytoplasmic ratio, different degrees of cell and nuclear polymorphism, keratin beads formed by squamous skin, and single cell keratinization. OSCC accounts for more than 80% of oral, neck and facial cancers, and the 5-year survival rate of patients is about 50%. For patients with advanced and recurrent tumors, the survival rate is even lower. Therefore, it is very important to find an effective treatment method.

[0003] Tumor cell culture is an important means to study the mechanism of carcinogenesis, the sensitivity of anticancer drugs, and the biological characteristics of tumor cells and cancer molecules. Cancer cells are the most common cell lines currently established. The application of in vitro cell culture technology for tumor research has many advantages: it can be free from the influence of environmental factors in the body, avoids individual differences, and makes it easy to explore the influence of various physical, chemical and biological factors on the life activities of tumor cells. It is convenient to study the structure and function of tumor cells at the cellular level, and to study the mechanism of carcinogenesis at the genetic and molecular levels; it can be preserved for a long time, which is convenient for observing the changes in the biological characteristics and genetic behavior of tumor cells; it can be used to quickly screen anticancer drugs and study drug resistance mechanisms; the research cycle is short and relatively economical.

[0004] However, due to the different personal conditions of different patients, there are obvious differences in the growth rate of oral squamous cell carcinoma, etc. It is difficult to reflect the situation in the patient's body based on the in vitro culture of oral squamous cell carcinoma. Therefore, the present invention proposes a method for establishing an in vitro culture model of oral squamous cell carcinoma. Summary of the invention

[0005] The present invention aims at the technical problems existing in the prior art and provides a method for establishing an in vitro culture model of oral squamous cell carcinoma.

[0006] The technical solution of the present invention to solve the above technical problem is as follows: a method for establishing an in vitro culture model of oral squamous cell carcinoma; comprising the following steps:

[0007] S1: Collect patient data and analyze oral squamous cell carcinoma and its living environment in patients;

[0008] S2: Periodically obtain tissue samples and separate cells from oral squamous cell carcinoma, establish an in vitro culture model, and culture oral squamous cell carcinoma in the in vitro culture model;

[0009] S3: Collect oral squamous cell carcinoma in patients at different cycles and oral squamous cell carcinoma cultured in vitro;

[0010] S4: Analyze the obtained data to adjust the parameters in the model to be closer to the in vivo generation characteristics, so that the oral squamous cell carcinoma generated in vitro is consistent with the oral squamous cell carcinoma grown in vivo.

[0011] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma comprises in S1:

[0012] The collection of patient data includes the collection of nutritional intake analysis and metabolic analysis of the patient:

[0013] The food consumed by the patient is input through the nutrition analysis software, and the macronutrients and micronutrients are output; the macronutrients include: total carbohydrates, total protein and total fat; the micronutrients include the average daily intake of major vitamins and minerals;

[0014] The metabolic analysis includes: estimating metabolic characteristics by Monte Carlo simulation or linear regression based on the expression levels of transport proteins and enzymes;

[0015] Assume that the gene expression level data E and the metabolic activity M of multiple gene pathways P are expressed as:

[0016]

[0017] where w i is the weight of each path, E i is the gene expression of the ith pathway.

[0018] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma, the analysis of oral squamous cell carcinoma in a patient and its living environment comprises:

[0019] The patient's tumor microenvironment is analyzed based on the patient's clinical data, including oxygen concentration, pH value, nutrient levels (such as glucose, amino acids), and the activation status of key signaling pathways.

[0020] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma comprises in S2:

[0021] Sample collection: A small piece of tumor tissue is obtained through a routine biopsy procedure;

[0022] Prepare enzyme solution: Prepare enzyme solution such as 0.1% collagenase and 0.25% trypsin according to tissue type and needs;

[0023] Tissue digestion: Place the tissue block in the enzyme solution and shake slowly in a 37°C warm water bath for 1-2 hours to allow the enzyme to fully degrade the tissue structure;

[0024] Centrifugation and washing; Centrifugation: Use low-speed centrifugation (such as 300g, 5 minutes) to separate cells in the cell suspension; Washing: Wash the cell pellet with sterile PBS (phosphate buffered saline), repeat two to three times to remove enzymes and cell debris.

[0025] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma further comprises in step S2:

[0026] Media optimization: Based on the patient data collected in step S1, adjust the media composition, including appropriate sugar, amino acid, vitamin, and mineral levels;

[0027] Cell seeding: isolated OSCC cells were resuspended in optimized culture medium and seeded in culture dishes or 3D scaffolds;

[0028] Biocompatible materials such as sodium alginate, gelatin or polylactic acid are used as scaffolds, which are suitable for the three-dimensional arrangement of cells.

[0029] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma comprises in S3:

[0030] Regular sampling: set multiple observation time points (e.g., days 0, 3, 7, and 14 of in vitro culture) for data collection; for in vivo samples, set an appropriate sampling frequency based on the course of treatment or disease;

[0031] Light microscopy and phase contrast microscopy were used to observe cell morphology, division pattern, and colony morphology;

[0032] The cell proliferation rate was measured using BrdU labeling combined with flow cytometry;

[0033] Measure oxygen consumption rate (OCR) and glycolytic rate using specific biochemical assays or software (e.g., Seahorse XF assay);

[0034] qPCR was used to quantify specific gene expression levels, and RNA-seq was used for global gene expression profiling;

[0035] By detecting specific proteins and their modified forms, we can understand the activation status of cell signaling pathways;

[0036] Detect metabolites such as ATP and lactate in cells to reflect metabolic activity;

[0037] The data of oral squamous cell carcinoma in vivo and oral squamous cell carcinoma in vitro were statistically analyzed through the above-mentioned tests.

[0038] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma comprises:

[0039] Input the data collected in S3 into statistical software:

[0040] The differences in proliferation rate, metabolic indexes, gene expression, etc. between in vivo and in vitro samples were analyzed using t-test or ANOVA;

[0041] Use principal component analysis (PCA) or cluster analysis to see the overall differences between different samples;

[0042] Fit the relationship between in vivo and in vitro growth characteristics using linear or nonlinear regression;

[0043] Regression Model:

[0044] Y=β0+β1X1+β2X2+...+β n X n +ε;

[0045] Where Y is the output variable, X i is the independent variable, β i is the regression coefficient and ε is the error term.

[0046] Furthermore, the method for establishing an in vitro culture model of oral squamous cell carcinoma comprises in S4:

[0047] Modify parameters based on statistical analysis results:

[0048] Nutrient ratio: For nutrients with significant effects (such as glucose and amino acids), adjust their concentration in the culture medium;

[0049] Adjust the oxygen level in the incubator to simulate a low oxygen environment if analysis shows that low oxygen better mimics in vivo tumors.

[0050] The beneficial effects of the present invention are:

[0051] By integrating the patient's specific nutritional, metabolic and genetic data, the model can more realistically reflect the growth characteristics and microenvironment of individual tumors; compared with traditional 2D culture methods, the three-dimensional culture used in this scheme can better simulate the arrangement and interaction of cells in the body.

[0052] Because the model is close to the actual physiological state of patients, drug screening results are more clinically relevant, improving the effectiveness of treatment plan design, and providing an experimental basis for treatment optimization based on individual tumor characteristics, such as targeted nutritional intervention and customized chemotherapy plans;

[0053] An iterative feedback mechanism is used to continuously adjust in vitro model parameters to ensure the accuracy and effectiveness of the research process; by using statistical analysis and machine learning methods, the model can quickly respond to new research data and significantly improve decision-making efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A schematic diagram of a process for establishing an in vitro culture model of oral squamous cell carcinoma according to the present invention; DETAILED DESCRIPTION

[0055] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0056] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, "plurality" means two or more, unless otherwise clearly and specifically defined.

[0057] In the description of the present application, the term "for example" is used to mean "used as an example, illustration or description". Any embodiment described as "for example" in the present application is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is given to enable any technician in the field to implement and use the present invention. In the following description, details are listed for the purpose of explanation. It should be understood that a person of ordinary skill in the art can recognize that the present invention can be implemented without using these specific details. In other examples, well-known structures and processes will not be elaborated in detail to avoid unnecessary details to obscure the description of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown, but is consistent with the widest scope consistent with the principles and features disclosed in the present application.

[0058] like Figure 1 As shown: In one embodiment, a method for establishing an in vitro culture model of oral squamous cell carcinoma comprises the following steps:

[0059] S1: Collect patient data and analyze oral squamous cell carcinoma and its living environment in patients. Specifically: obtain clinical information of patients, such as age, gender, medical history, tumor stage, and treatment history;

[0060] S2: Periodically obtain tissue samples and separate cells from oral squamous cell carcinoma, and establish an in vitro culture model. Oral squamous cell carcinoma is cultured in the in vitro culture model. Specifically: Tumor samples obtained through surgery are processed into single-cell suspensions and then inoculated in a culture medium containing high concentrations of glucose. At the same time, gelatin scaffolds are used to support three-dimensional growth to reflect the real tumor environment in the patient's body;

[0061] S3: Collect oral squamous cell carcinoma samples from patients at different cycles and oral squamous cell carcinoma samples cultured in vitro. Specifically: The test showed that the tumor cells cultured in vitro showed a proliferation rate similar to that of the in vivo samples, but the metabolic activity of glucose was low, indicating the possible need for nutritional adjustment;

[0062] S4: Analyze the obtained data to adjust the parameters in the model to be closer to the in vivo generation characteristics, so that the oral squamous cell carcinoma generated in vitro is consistent with the oral squamous cell carcinoma grown in vivo. Specifically: Through analysis, it was found that the regulation of lactic acid accumulation by cells is different from that in vivo, so the pH and neutralization dose of the culture medium are adjusted to improve the metabolic matching. After the subsequent test confirms the effect of the adjustment, the model can be established to more accurately reflect the in vivo situation.

[0063] In this example, a dynamic adjustment and optimization framework is provided, which enables the in vitro model to more accurately represent the real tumor growth characteristics of patients through precise data collection, analysis and iterative optimization. This framework is not only applicable to basic research, but also provides a solid experimental foundation for the development of personalized cancer treatment strategies.

[0064] In one embodiment, S1 includes:

[0065] The collection of patient data includes the collection of nutritional intake analysis and metabolic analysis of the patient:

[0066] The food consumed by the patient is input through the nutrition analysis software, and the macronutrients and micronutrients are output; the macronutrients include: total carbohydrates, total protein and total fat; the micronutrients include the average daily intake of major vitamins and minerals;

[0067] The metabolic analysis includes: estimating metabolic characteristics by Monte Carlo simulation or linear regression based on the expression levels of transport proteins and enzymes;

[0068] Assume that the gene expression level data E and the metabolic activity M of multiple gene pathways P are expressed as:

[0069]

[0070] where w i is the weight of each path, E i is the gene expression of the ith pathway.

[0071] In this embodiment, a deep learning network is used to process the patient's metabolic data:

[0072] S11: Collect gene expression data and metabolism-related gene sets of patients;

[0073] S12: Construct metabolic activity calculation formula:

[0074]

[0075] S13: Predicting metabolic network output using neural network models;

[0076] S131: Network structure: Input layer: gene expression data and environmental data;

[0077] Hidden layer: multi-layer fully connected layer, ReLU is used as activation function;

[0078] Output layer: metabolic indicators (such as lactate production, oxygen consumption);

[0079] S132: The loss function uses mean square error MSE to optimize the prediction:

[0080]

[0081] where y j is the true metabolic output, is the predicted value.

[0082] S14: Optimize network parameters through gradient descent algorithm (such as Adam), use patient historical data for cross-validation, and evaluate the prediction accuracy of the model.

[0083] At the same time, based on conventional clinical data, single-cell transcriptomics data is introduced to capture the heterogeneity of tumor cells and further personalize culture conditions:

[0084] Single cells from tumor samples were isolated using flow cytometry.

[0085] Single-cell RNA expression profiles were obtained using high-throughput sequencing technology.

[0086] Data normalization: Gene expression data were log-transformed and Z-score normalized.

[0087] Dimensionality reduction analysis: Use t-SNE or UMAP method to reduce the dimension of high-dimensional single-cell data:

[0088]

[0089] Where P i,j Represents the similarity distribution in high-dimensional space, Q i,jRepresents the similarity distribution in low-dimensional space.

[0090] Use K-means or graph-based clustering (such as Louvain algorithm) to cluster cells:

[0091]

[0092] Among them C i represents a cluster, u i is the center of the ith cluster, x j Indicates the cells to be grouped.

[0093] The clustering results were annotated according to known cell marker genes (such as immune cells and fibroblasts); transcriptomics data were integrated with metabolic data to establish a multidimensional feature matrix for the personalized design of culture medium components.

[0094] In a specific embodiment: Clinical data collection:

[0095] Patient: A 55-year-old male with mid-stage oral squamous cell carcinoma.

[0096] Clinical background: Hyperglycemia, tumors are significantly dependent on glycolysis.

[0097] Diet analysis: high daily sugar intake, insufficient amino acids and minerals.

[0098] Metabolic analysis: Using RNA-seq and gene expression databases, we confirmed that the glycolysis pathway of tumor cells was significantly activated; lactate metabolism was enhanced under hypoxic conditions.

[0099] Sample processing and cell culture: Biopsy to obtain tumor tissue blocks (3-5 mm 3 ).

[0100] Single cell suspension was prepared by enzymatic digestion (0.1% collagenase + 0.25% trypsin, 37°C, 2 hours).

[0101] Medium Optimization:

[0102] According to the metabolic characteristics of the patients, high glucose (25 mM glucose) and low oxygen (1% oxygen concentration) conditions were added.

[0103] Increased amino acids (glutamine 4mM) and minerals (calcium 2mM, magnesium 1.5mM).

[0104] Three-dimensional culture matrix:

[0105] Scaffold material: Sodium alginate and gelatin (1:1) were mixed and cross-linked into a 3D scaffold.

[0106] Seeding density: 2x105 cells per well, cultured in 24-well plates.

[0107] Periodic data collection: samples were collected on days 0, 7, and 14 of culture.

[0108] Metabolic analysis: Oxygen consumption rate (OCR) and lactate secretion were measured using a Seahorse XF analyzer.

[0109] Gene expression: qPCR was used to detect the expression of key genes in glycolysis (such as LDHA).

[0110] Cell proliferation: After BrdU labeling, the cell proliferation rate was measured using flow cytometry.

[0111] Model optimization and validation: Comparison of in vivo and in vitro metabolic data revealed that lactate levels were too high in in vitro culture and cell proliferation was slow.

[0112] Adjust the medium pH (add HEPES buffer to 7.4) to reduce lactate accumulation.

[0113] Re-testing: Sampling was performed again (day 14), and it was found that the metabolic match was improved and the in vitro model was closer to the in vivo situation.

[0114] Experimental results:

[0115] Metabolic profile matching:

[0116] OCR and lactate secretion were close to the average values ​​of tumors in patients.

[0117] Increased proliferation rate:

[0118] After adjustment, the cell proliferation rate in the in vitro model increased by approximately 25%.

[0119] The analysis of oral squamous cell carcinoma in the patient and its living environment includes:

[0120] The patient's tumor microenvironment is analyzed based on the patient's clinical data, including oxygen concentration, pH value, nutrient levels (such as glucose, amino acids), and the activation status of key signaling pathways.

[0121] Included in S2:

[0122] Sample collection: A small piece of tumor tissue is obtained through a routine biopsy procedure;

[0123] Prepare enzyme solution: Prepare enzyme solution such as 0.1% collagenase and 0.25% trypsin according to tissue type and needs;

[0124] Tissue digestion: Place the tissue block in the enzyme solution and shake slowly in a 37°C warm water bath for 1-2 hours to allow the enzyme to fully degrade the tissue structure;

[0125] Centrifugation and washing; Centrifugation: Use low-speed centrifugation (such as 300g, 5 minutes) to separate cells in the cell suspension; Washing: Wash the cell pellet with sterile PBS (phosphate buffered saline), repeat two to three times to remove enzymes and cell debris.

[0126] The S2 step also includes:

[0127] Media optimization: Based on the patient data collected in step S1, adjust the media composition, including appropriate sugar, amino acid, vitamin, and mineral levels;

[0128] Cell seeding: isolated OSCC cells were resuspended in optimized culture medium and seeded in culture dishes or 3D scaffolds;

[0129] Biocompatible materials such as sodium alginate, gelatin or polylactic acid are used as scaffolds, which are suitable for the three-dimensional arrangement of cells.

[0130] Included in S3:

[0131] Regular sampling: set multiple observation time points (e.g., days 0, 3, 7, and 14 of in vitro culture) for data collection; for in vivo samples, set an appropriate sampling frequency based on the course of treatment or disease;

[0132] Light microscopy and phase contrast microscopy were used to observe cell morphology, division pattern, and colony morphology;

[0133] The cell proliferation rate was measured using BrdU labeling combined with flow cytometry;

[0134] Measure oxygen consumption rate (OCR) and glycolytic rate using specific biochemical assays or software (e.g., Seahorse XF assay);

[0135] qPCR was used to quantify specific gene expression levels, and RNA-seq was used for global gene expression profiling;

[0136] By detecting specific proteins and their modified forms, we can understand the activation status of cell signaling pathways;

[0137] Detect metabolites such as ATP and lactate in cells to reflect metabolic activity;

[0138] The data of oral squamous cell carcinoma in vivo and oral squamous cell carcinoma in vitro were statistically analyzed through the above-mentioned tests.

[0139] Input the data collected in S3 into statistical software:

[0140] The differences in proliferation rate, metabolic indexes, gene expression, etc. between in vivo and in vitro samples were analyzed using t-test or ANOVA;

[0141] Use principal component analysis (PCA) or cluster analysis to see the overall differences between different samples;

[0142] Fit the relationship between in vivo and in vitro growth characteristics using linear or nonlinear regression;

[0143] Regression Model:

[0144] Y=β0+β1X1+β2X2+...+β n X n +ε;

[0145] Where Y is the output variable, X i is the independent variable, β i is the regression coefficient and ε is the error term.

[0146] Included in S4:

[0147] Modify parameters based on statistical analysis results:

[0148] Nutrient ratio: For nutrients with significant effects (such as glucose and amino acids), adjust their concentration in the culture medium;

[0149] Adjust the oxygen level in the incubator to simulate a low oxygen environment if analysis shows that low oxygen better mimics in vivo tumors.

[0150] Specific examples:

[0151] Patient Background:

[0152] Age: 55

[0153] Gender: Male

[0154] Diagnosis: Intermediate stage oral squamous cell carcinoma

[0155] Medical history: Hyperglycemia, long-term dietary preference for high-sugar and high-fat foods

[0156] Traditional cultivation methods:

[0157] Standard oral squamous cell carcinoma cell lines (such as SCC-25) were used for 2D plate culture in a universal culture medium (such as RPMI-1640 or DMEM plus 10% fetal bovine serum) without personalized condition settings.

[0158] S1: Collect and analyze patient data

[0159] Nutritional analysis: Using nutritional analysis software, it was confirmed that the patient's daily sugar intake was significantly higher than normal levels.

[0160] Metabolic analysis: Genetic testing showed that the glycolysis pathway of the patient's cancer cells was enhanced. Oxygen concentration testing showed that the patient's tumor was mainly grown in a low-oxygen environment.

[0161] S2: Sample acquisition and in vitro culture model establishment

[0162] Tissue Collection and Processing: Biopsy samples were obtained from patient tumors and digested with collagenase and trypsin to prepare single cells.

[0163] Optimization of culture conditions: Design a three-dimensional scaffold culture environment with high sugar concentration and low oxygen, and mix scaffold materials such as gelatin and sodium alginate to simulate the intercellular spaces in the body.

[0164] S3: Collect periodic data

[0165] Regular observation: Microscopic observation was performed on days 0, 7, and 14, and cell proliferation was detected using flow cytometry.

[0166] Metabolic measurements: Lactate and ATP metabolites were measured using the Seahorse XF system, and glycolytic gene expression was analyzed using qPCR.

[0167] S4: Analysis and Optimization

[0168] Data comparison: Compared with the in vivo data, it was found that the cells in the in vitro environment responded consistently to high sugar, but lactate production was higher.

[0169] Model adjustments: Add a pH adjustment pathway to optimize lactate metabolism, and continue to optimize through iterations until the performance of the two converges.

[0170] Traditional methods: lack of personalized physiological conditions and cannot accurately reflect the patient's specific metabolic abnormalities, such as high glucose tolerance and hypoxia adaptability. At the same time, the in vitro cell proliferation rate and metabolic characteristics are inconsistent with those in vivo, affecting the effectiveness of drug screening.

[0171] This program: By accurately simulating the nutritional and metabolic characteristics of patients, the model is made more representative of individuals. Through periodic data collection and feedback adjustment, the proliferation and metabolic performance of the in vitro model is closer to the in vivo situation, improving the credibility of the experimental results.

[0172] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0173] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for establishing an in vitro culture model of oral squamous cell carcinoma, characterized in that: The following steps are involved: S1: Collect patient data and analyze oral squamous cell carcinoma and its living environment in patients; S2: Periodically obtain tissue samples and separate cells from oral squamous cell carcinoma, establish an in vitro culture model, and culture oral squamous cell carcinoma in the in vitro culture model; S3: Collect oral squamous cell carcinoma in patients at different cycles and oral squamous cell carcinoma cultured in vitro; S4: Analyze the obtained data to adjust the parameters in the model to be closer to the in vivo generation characteristics, so that the oral squamous cell carcinoma generated in vitro is consistent with the oral squamous cell carcinoma grown in vivo.

2. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 1, characterized in that: Included in S1: The collection of patient data includes the collection of nutritional intake analysis and metabolic analysis of the patient: The food consumed by the patient is input through the nutrition analysis software, and the macronutrients and micronutrients are output; the macronutrients include: total carbohydrates, total protein and total fat; the micronutrients include the average daily intake of major vitamins and minerals; The metabolic analysis includes: estimating metabolic characteristics by Monte Carlo simulation or linear regression based on the expression levels of transport proteins and enzymes; Assume that the gene expression level data E and the metabolic activity M of multiple gene pathways P are expressed as: where w i is the weight of each path, E i is the gene expression of the ith pathway.

3. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 1, characterized in that: The analysis of oral squamous cell carcinoma in the patient and its living environment includes: The patient's tumor microenvironment is analyzed based on the patient's clinical data, including oxygen concentration, pH value, nutrient levels, and activation status of key signaling pathways.

4. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 1, characterized in that: Included in S2: Sample collection: A small piece of tumor tissue is obtained through a routine biopsy procedure; Prepare enzyme solution: Prepare enzyme solution such as 0.1% collagenase and 0.25% trypsin according to tissue type and needs; Tissue digestion: Place the tissue block in the enzyme solution and shake slowly in a 37°C warm water bath for 1-2 hours to allow the enzyme to fully degrade the tissue structure; Centrifugation and washing; Centrifugation: Use low-speed centrifugation to separate cells from the cell suspension; Washing: Wash the cell pellet with sterile PBS, repeat two to three times to remove enzymes and cell debris.

5. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 1, characterized in that: The S2 step also includes: Media optimization: Based on the patient data collected in step S1, adjust the media composition, including appropriate sugar, amino acid, vitamin, and mineral levels; Cell seeding: isolated OSCC cells were resuspended in optimized culture medium and seeded in culture dishes or 3D scaffolds; Biocompatible materials such as sodium alginate, gelatin or polylactic acid are used as scaffolds, which are suitable for the three-dimensional arrangement of cells.

6. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 1, characterized in that: Included in S3: Regular sampling: set multiple observation time points for data collection; for in vivo samples, set an appropriate sampling frequency based on the course of treatment or disease; Light microscopy and phase contrast microscopy were used to observe cell morphology, division pattern, and colony morphology; The cell proliferation rate was measured using BrdU labeling combined with flow cytometry; Measure oxygen consumption rate and glycolytic rate using specific biochemical assays or software; qPCR was used to quantify specific gene expression levels, and RNA-seq was used for global gene expression profiling; By detecting specific proteins and their modified forms, we can understand the activation status of cell signaling pathways; Detect metabolites such as ATP and lactate in cells to reflect metabolic activity; The data of oral squamous cell carcinoma in vivo and oral squamous cell carcinoma in vitro were statistically analyzed through the above-mentioned tests.

7. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 6, characterized in that: include: Input the data collected in S3 into statistical software: The differences in proliferation rate, metabolic indexes, gene expression, etc. between in vivo and in vitro samples were analyzed using t-test or ANOVA; Principal component analysis was used to look at the overall differences between different samples; Fit the relationship between in vivo and in vitro growth characteristics using linear or nonlinear regression; Regression Model: Y=β0+β1X1+β2X2+...+β n X n +e; Where Y is the output variable, X i is the independent variable, β i is the regression coefficient and ε is the error term.

8. The method for establishing an in vitro culture model of oral squamous cell carcinoma according to claim 1, characterized in that: Included in S4: Modify parameters based on statistical analysis results: Nutrient ratio: For nutrients with significant effects, adjust their concentration in the culture medium; Adjust the oxygen level in the incubator to simulate a low oxygen environment if analysis shows that low oxygen better mimics in vivo tumors.

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