A three-dimensional culture model of HeLa cells, preparation method and use

Abstract

The application discloses a HeLa cell three-dimensional culture model, a preparation method and application. HeLa cells are mixed with a collagen-sodium alginate composite hydrogel precursor solution to obtain a cell-precursor mixture, the cell-precursor mixture is mixed with a CaCl2 solution to obtain a mixed solution, and the mixed solution is placed in an incubator for standing culture for 10-60 min, so that the hydrogel is cross-linked and solidified to form a three-dimensional culture system; complete culture medium is further added for continuous culture, and a HeLa cell three-dimensional culture model is constructed. The HeLa cell three-dimensional culture model is constructed by using the hydrogel, defects existing in simulation of a tumor microenvironment by two-dimensional culture and an animal model are effectively solved, the body microenvironment can be better simulated, cells form regular three-dimensional cell spheroids, a malignant phenotype is maintained, the accuracy and repeatability of drug screening are improved, and a reliable technical platform is provided for cervical cancer mechanism research and anti-tumor drug screening.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cell engineering, and in particular to a three-dimensional culture model of HeLa cells, its preparation method, and its uses. Background Technology

[0002] Cell culture models are an important tool in cervical cancer research and anti-tumor drug development. Traditional two-dimensional culture models are widely used due to their ease of operation. However, in this model, cells grow in a monolayer on a flat surface, which leads to problems such as abnormal cell morphology, limited intercellular interactions, and an inability to simulate the nutrient gradient, oxygen gradient, and drug penetration barrier in the tumor microenvironment in vivo. As a result, the drug response and functional assessment results deviate significantly from the actual in vivo state.

[0003] The emergence of three-dimensional cell culture technology has made it possible to overcome the aforementioned limitations. Currently, commonly used three-dimensional culture methods mainly include ultra-low adsorption culture, hanging drop culture, and scaffold-based three-dimensional culture. While matrix gels can better simulate the in vivo microenvironment, they suffer from inherent limitations such as complex composition, large batch-to-batch variability, and difficulty in independently controlling mechanical properties, leading to poor experimental reproducibility and failing to meet the needs of standardized and large-scale drug screening. Furthermore, existing three-dimensional culture methods often suffer from cumbersome operation, difficulty in maintaining cell viability, and a tendency for central necrosis during long-term culture. Therefore, developing a simple, well-defined, mechanically adjustable, and precisely simulated in vivo tumor microenvironment HeLa cell three-dimensional culture model is of great significance for cervical cancer research and drug screening. Summary of the Invention

[0004] In view of the problems existing in the above-mentioned background technology, the purpose of this invention is to provide a method for constructing and applying a three-dimensional culture model of HeLa cells, so as to solve the problems of complex operation, poor model stability, and difficulty in accurately simulating the tumor microenvironment in vivo in existing three-dimensional culture methods.

[0005] To achieve the above objectives, the present invention provides a method for constructing a three-dimensional culture model of HeLa cells, characterized by comprising the following steps: HeLa cells were mixed with a collagen-sodium alginate composite hydrogel precursor solution to obtain a cell-precursor mixture; the cell-precursor mixture was then mixed with CaCl2 solution to obtain a mixture solution, and the mixture solution was placed in an incubator and statically cultured for 10-60 minutes to allow the hydrogel to cross-link and solidify, forming a three-dimensional culture system. Complete culture medium was added to the three-dimensional culture system, and the cells were cultured continuously to construct a three-dimensional culture model of HeLa cells. The preparation method of the collagen-sodium alginate composite hydrogel precursor solution is as follows: S1. Preparation of aminoated collagen: Collagen was fully dissolved in acetic acid solution, then EDA aqueous solution was added to the solution and mixed well, then EDC solution was added, and the reaction was stirred at room temperature. After the reaction was completed, the reaction solution was purified by dialysis using a dialysis bag, and then freeze-dried under vacuum to obtain aminoated collagen; it was then dissolved in dilute acetic acid to prepare an aminoated collagen solution with a mass fraction of 2±1wt%. S2. Preparation of oxidized sodium alginate: Sodium alginate was completely dissolved in hot distilled water and cooled to room temperature. NaIO4 aqueous solution was added dropwise to the sodium alginate solution and reacted in the dark at room temperature. Ethylene glycol was then added and stirring was continued to terminate the reaction and reduce the residual NaIO4. Dialysis was performed using a dialysis bag to remove unreacted reagents and small molecules. After vacuum freeze-drying, oxidized sodium alginate was obtained. The oxidized sodium alginate was then dissolved in PBS buffer solution with a pH of near neutral to prepare a 10-20 wt% oxidized sodium alginate solution. The order of steps S1 and S2 is not important; they can be performed simultaneously. The amino-collagen solution is mixed with the sodium oxidized alginate solution to obtain a collagen-sodium alginate composite hydrogel precursor solution. Preferably, the amino-collagen solution and the sodium oxidized alginate solution are mixed at a volume ratio of 1:(1±0.2).

[0006] Furthermore, the density of the HeLa cells is 0.5 × 10⁻⁶. 5 ~5×10 5 per mL.

[0007] Furthermore, the volume ratio of HeLa cells to the collagen-sodium alginate composite hydrogel precursor solution was 1:8.

[0008] Furthermore, the Ca in the mixture 2+ The concentration is 5-80 mM.

[0009] Furthermore, the continuous culture refers to replacing the complete culture medium every 48 hours and culturing for 1-14 days.

[0010] Furthermore, in step S1, the acetic acid solution is a 0.02 mol / L acetic acid solution, the EDA aqueous solution is a pH 6.0, 2.5 mol / L EDA aqueous solution, and the EDC solution is a 0.2 mol / L EDC solution; More preferably, the ratio of collagen to 0.02 mol / L acetic acid solution to pH 6.0, 2.5 mol / L EDA aqueous solution to 0.2 mol / L EDC solution is 3 g: 740 mL: 60 mL: 36 mL; Optionally, in step S1, the reaction time at room temperature is 8-20 hours, and the dialysis bag is a 3500 Da dialysis bag.

[0011] Furthermore, in step S2, the ratio of sodium alginate, distilled water, NaIO4, and ethylene glycol is 2 g: 100 mL: 1.5 g / 20 mL: 1 mL; Optionally, in step S2, the reaction time at room temperature in the dark is 24-60 hours, and the dialysis bag is a 3500 Da dialysis bag.

[0012] This invention also protects a three-dimensional culture model of HeLa cells constructed by the aforementioned construction method.

[0013] This invention also protects the application of the HeLa cell three-dimensional culture model in the screening of antitumor drugs.

[0014] Furthermore, the antitumor drug is doxorubicin.

[0015] This invention constructs a three-dimensional culture model of HeLa cells using hydrogels, effectively addressing the shortcomings of two-dimensional culture and animal models in simulating the tumor microenvironment. It can better simulate the body's microenvironment, enabling cells to form regular three-dimensional cell spheres, maintain a malignant phenotype, and improve the accuracy and reproducibility of drug screening. This provides a reliable technical platform for cervical cancer mechanism research and anti-tumor drug screening. Attached Figure Description

[0016] Figure 1 Bright-field images of HeLa cells cultured for 7 days under different calcium ion concentrations.

[0017] Figure 2 The image shows the green fluorescence staining of HeLa cell spheroids under different ion concentrations.

[0018] Figure 3 The image shows the red fluorescence staining of HeLa cell spheroids under different ion concentrations.

[0019] Figure 4 Immunofluorescence staining of HeLa cell spheroids under different ion concentrations.

[0020] Figure 5 Immunofluorescence staining of HeLa cell spheroids with DAPI under different ion concentrations.

[0021] Figure 6 Fluorescent staining images of phalloidin in HeLa cells under different ion concentrations.

[0022] Figure 7 The image shows the viability of HeLa cells cultured in hydrogels with different concentrations of calcium ions.

[0023] Figure 8 The figure shows the average diameter of HeLa cells cultured in composite hydrogels with different concentrations of calcium ions for different culture times.

[0024] Figure 9 The figure shows the effect of doxorubicin on the survival rate of HeLa cells under different ion concentrations.

[0025] Figure 10 This image shows the secretion levels of MMP-9 cells in HeLa cells cultured in composite hydrogels with different concentrations of calcium ions.

[0026] Figure 11 This image shows the HPV16 E6 secretion levels of HeLa cells cultured in composite hydrogels with different concentrations of calcium ions.

[0027] In the above figure, the calcium ion concentration of A is 5 mM, the calcium ion concentration of B is 10 mM, the calcium ion concentration of C is 20 mM, the calcium ion concentration of D is 40 mM, the calcium ion concentration of E is 60 mM, and the calcium ion concentration of F is 80 mM. 2D is two-dimensional. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0029] In the following examples, 2D refers to HeLa (human cervical cancer cells) (STR identification correct), product code CL-0101 Pronosai, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.

[0030] Example 1: Construction of HeLa cell culture model This embodiment provides a method for constructing a three-dimensional culture model of HeLa cells, including the following steps: 1) Preparation of hydrogel precursor solution: The collagen-sodium alginate composite hydrogel precursor solution was preheated to 37°C to form a cell culture substrate state and a three-dimensional cell culture scaffold was constructed. Preparation of collagen-sodium alginate composite hydrogel precursor solution: Col-NH2 (preparation method below) was dissolved in 0.25 mol / L acetic acid to prepare a 2 wt% Col-NH2 solution; OSA (preparation method below) was dissolved in 0.1 mol / L, pH 7.2 PBS to prepare a 16 wt% OSA solution. The above Col-NH2 solution and OSA solution were thoroughly mixed at a volume ratio of 1:1 to obtain the Col-NH2 / OSA precursor solution (i.e., the collagen-sodium alginate composite hydrogel precursor solution).

[0031] Preparation of Col-NH2 (aminated collagen): 3 g of collagen was dissolved in 740 mL of 0.02 mol / L acetic acid solution and stirred with a magnetic stirrer until fully dissolved. Then, 60 mL of 2.5 mol / L EDA aqueous solution at pH 6.0 was added to the solution and stirred until homogeneous. Then, 36 mL of 0.2 mol / L EDC solution was added to the above system and the reaction was stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was purified by dialysis using a 3500 Da dialysis bag and then freeze-dried under vacuum to obtain aminated collagen (Col-NH2).

[0032] The preparation of OSA (oxidized sodium alginate) was as follows: 2 g of sodium alginate was dissolved in 100 mL of distilled water at 60 °C. The solution was stirred with a magnetic stirrer until completely dissolved and then cooled to room temperature. Subsequently, 1.5 g of NaIO4 was weighed and dissolved in 20 mL of distilled water. The NaIO4 was added dropwise to the sodium alginate solution and reacted at room temperature in the dark for 48 h. After the reaction was completed, 1 mL of ethylene glycol was added and stirring was continued for 0.5 h to terminate the reaction and reduce the residual NaIO4. Dialysis was performed using a dialysis bag with a molecular weight cutoff of 3500 Da to remove unreacted reagents and small molecules. After vacuum freeze-drying, oxidized sodium alginate (OSA) was obtained.

[0033] 2) Preparation of cell suspension: When HeLa cells reach 80%-90% confluence, digest the cells with 0.25% trypsin, resuspend the cells in complete culture medium, and gently pipette to adjust the cell concentration to 1×10⁻⁶. 5 Cells / mL, concentrate the cell suspension according to experimental requirements, and pipette evenly; 3) Mixing and gel formation: Mix the collagen-sodium alginate composite hydrogel precursor solution and cell suspension at a volume ratio of 8:1 to obtain a cell-precursor mixture, and transfer it into a pre-cooled 1 mL Luerlock syringe.

[0034] CaCl2 was dissolved in distilled water to prepare sterile solutions of different concentrations, so that when mixed with the cell-precursor mixture, Ca²⁺... +The final concentrations were 5, 10, 20, 40, 60, and 80 mM, respectively. The specific procedure was as follows: For each gel sample, the corresponding concentration of CaCl2 solution was added to another 1 mL Luer-lock syringe. After shaking and homogenization, the syringe was connected to a syringe containing a cell-precursor mixture via a double-mother Luer-lock connector. The two solutions were rapidly mixed at a volume ratio of 3:1 (cell-precursor mixture: CaCl2 solution) and immediately transferred to a 96-well plate. The culture plate was then transferred to a 37°C, 5% CO2 incubator for 30 min to form a double-network hydrogel. Fresh complete culture medium containing 10% fetal bovine serum was added to each hydrogel to cover it, and the plate was returned to the incubator for further culture. The culture medium was changed every 2 days, and the culture was continued for 1-14 days. A three-dimensional culture model of HeLa cells was obtained, and the morphology, number, and state of the cell spheroids were observed.

[0035] During culture, cell morphology, viability, and proliferation curves are observed: Day 1: Represents the initial state. Cells are just encapsulated in the hydrogel and have not yet aggregated. At this stage, live / dead staining can assess the acute toxicity of the gelation process to the cells (e.g., Ca²⁺). + (Whether concentration and mixed shear forces damage cells). Day 3: Represents the early aggregation stage. Cells begin to migrate, come into contact with each other, and form small clusters. At this time, observe whether cells can proliferate and aggregate normally to determine whether the gel environment supports three-dimensional growth.

[0036] Day 7: Represents the mature spheroid stage. HeLa cells typically form well-defined, dense spheroids with high viability. This is the optimal period for functional experiments. Day 14: Represents the late stage of long-term culture. It allows observation of whether spheroids have excessively enlarged, central necrosis (hypoxia / nutrient deficiency), or gel degradation. This determines the maximum effective culture time for this culture system. HPVE6 and MMP-9 protein levels are only measured on day 7 because spheroids have formed, cell-cell contact and cell-matrix interactions are fully established, mimicking the in vivo microenvironment. At this point, the expression levels of HPVE6 and MMP-9 best reflect the malignant phenotype induced by three-dimensional culture.

[0037] The addition of doxorubicin (DOX) after 24 hours of culture was to evaluate the acute effect of DOX on rapidly proliferating cells without barrier interference.

[0038] In this example, in step 2) preparing the cell suspension, after the digested cells are centrifuged at 1000 rpm, all supernatant is removed. After washing with PBS, the quantity is adjusted with fresh culture medium, and then resuspended with hydrogel precursor solution. The volume ratio of hydrogel to cell suspension is 8:1. The mixed cell suspension needs to be quickly transferred to sterile well plates to avoid premature gelation.

[0039] In this example, during step 3) three-dimensional cell culture, the resuspended cell suspension can be placed in a cell culture incubator for pre-adaptation, allowed to stand until gelation occurs, and then complete culture medium is added to cover each well.

[0040] The more specific steps are as follows: Complete culture medium preparation: Preparation of culture medium for HeLa cell three-dimensional culture model: In a biosafety cabinet, use a pipette to draw 44.5 mL of DMEM high glucose cell culture medium into a sterile centrifuge tube, add 5 mL of fetal bovine serum and 0.5 mL of penicillin-streptomycin solution to prepare a complete culture medium containing 10% FBS. Gently invert and mix well, then place in a 4°C refrigerator for later use.

[0041] Construction of a three-dimensional culture model of HeLa cells: Frozen HeLa cells were removed from liquid nitrogen and rapidly thawed in a sterile water bath at 37°C, gently agitated until completely thawed. In a biosafety cabinet, the cell cryopreservation solution was transferred to a sterile centrifuge tube, 1 mL of fresh complete culture medium was added, and the mixture was gently pipetted and centrifuged at 1000 rpm for 3 min. After centrifugation, the supernatant was removed, freshly prepared culture medium was added, and the cells were transferred to a T25 culture flask and incubated in a 37°C, 5% CO2 incubator. The culture medium was observed and changed every 24 h. When the cells reached 80-90% confluence, they were digested with 0.25% trypsin, resuspended in complete culture medium, and gently pipetted to adjust the cell concentration to 1×10⁻⁶. 5 Cells / mL, concentrate and prepare cell suspension according to experimental requirements, vortex until homogeneous and ready for use.

[0042] The preheated hydrogel precursor solution to 37℃ was mixed with the cell suspension at a volume ratio of 8:1 and quickly transferred into sterile well plates. The plates were incubated at 37℃ and 5% CO2 for 30 min. After the hydrogel had completely solidified, 100 μL of fresh complete culture medium containing 10% fetal bovine serum was added to each well to cover it. The plates were then cultured for 1-14 days, and the culture medium was changed every 2 days. After 1-14 days of culture, a three-dimensional culture model of HeLa cells was obtained.

[0043] See results Figures 1-6 . Figure 1Bright-field images of HeLa cell spheroids cultured for 7 days under different calcium ion concentrations are shown. The calcium ion concentrations are: A (5 mM), B (10 mM), C (20 mM), D (40 mM), E (60 mM), and F (80 mM). After 7 days of culture, the cell spheroid diameters for the 5, 10, 20, 40, 60, and 80 mM calcium ion concentration groups were 52.36 μm, 50.37 μm, 46.08 μm, 43.86 μm, 35.70 μm, and 20.05 μm, respectively. It can be seen that the diameter of HeLa cell spheroids gradually decreases with increasing calcium ion concentration, indicating that the hydrogel stiffness can effectively regulate the cell spheroid size. Figure 2 The images show the green fluorescence staining of HeLa cell spheroids under different ion concentrations: It can be seen that after 14 days of culture, cell viability gradually increased and the green fluorescence became stronger and more uniformly distributed when the calcium ion concentration was 5, 10, 20, and 40 mM. However, when the concentration increased to 60 mM and 80 mM, cell viability significantly decreased and the fluorescence intensity weakened. This indicates that a suitable calcium ion concentration (around 40 mM) can effectively maintain cell viability, while excessively high concentrations inhibit cell survival; that is, the stiffness of the hydrogel can regulate cell viability. Figure 3 The image shows the red fluorescence staining of HeLa cell spheroids under different ion concentrations: After 14 days of culture, at calcium ion concentrations of 5, 10, 20, and 40 mM, the red fluorescence signal was weak, with only occasional sporadic dead cells; however, when the concentration increased to 60 mM and 80 mM, the red fluorescence was significantly enhanced, the number of dead cells increased significantly, and the distribution range expanded. Figure 2 The green fluorescence results further indicate that an appropriate calcium ion concentration (around 40 mM) can maintain high cell viability and inhibit cell death, while excessively high calcium ion concentrations induce massive cell death. Figure 4 Immunofluorescence staining images of HeLa cell spheroids under different ion concentrations: It can be seen that the colocalization of the cytoskeleton and nucleus is clear and the boundaries are distinct in each calcium ion concentration group, that is, the change of calcium ion concentration does not affect the clarity of colocalization. Figure 5 The images show DAPI immunofluorescence staining of HeLa cell spheroids under different ion concentrations. It can be seen that the cell nuclei in each calcium ion concentration group are round or oval and are evenly and densely distributed, which means that changes in calcium ion concentration did not cause abnormalities in cell nucleus morphology. Figure 6 Fluorescent staining images of phalloidin in HeLa cells under different ion concentrations: It can be seen that actin filaments are distributed in a continuous network in each calcium ion concentration group, and the cytoskeleton structure is intact, that is, the change in calcium ion concentration did not destroy the integrity of the cytoskeleton.

[0044] Conclusion: By adjusting the calcium ion concentration, precise control can be achieved over cell spheroid size, cell viability, and drug response behavior in a three-dimensional HeLa cell culture model.

[0045] Example 2: Determination of viability of HeLa cell spheroids in three-dimensional culture HeLa cells were cultured in three dimensions according to the above experimental method. Cell viability was detected on day 14. Cells were washed twice with PBS, and then Calcein / PI detection working solution was added. The cells were incubated at 37°C in the dark for 30 min. After incubation, residual Calcein / PI solution was washed away with PBS. The staining results were observed under a laser confocal microscope to assess cell viability during the cell culture process.

[0046] See results Figure 7 , Figure 7 The image shows the viability of HeLa cells cultured in hydrogels with different concentrations of calcium ions.

[0047] It can be seen that with the extension of the cell culture cycle, the diameter of HeLa cell spheroids in the matrix gel becomes too large. Cells in the central region may have difficulty obtaining sufficient nutrients due to metabolic restriction, resulting in a lower cell survival rate than HeLa cells in the hydrogel. Adding 5mM, 10mM, 20mM, 40mM, 60mM, and 80mM Ca... 2+ The cell viability rates in the collagen-sodium alginate hydrogels were 56.76±6.71%, 61.66±6.44%, 68.12±2.98%, 78.03±3.28%, 53.13±5.35%, and 45.79±8.30%, respectively. When the calcium ion concentration was too high, the hydrogel network became denser, the pore size decreased, and the internal nutrient exchange was restricted, leading to necrosis in the central region of the cell spheroids and a decrease in cell viability.

[0048] Based on experimental data, cell viability was quantitatively represented by the survival rate percentage after Calein / PI staining: Cell spheroids cultured for 14 days were fluorescently stained; live cells appeared green, and dead cells appeared red. The survival rate at different calcium ion concentrations was obtained by counting the proportion of live cells to the total number of cells (e.g., 5 mM group 56.76±6.71%, 10 mM group 61.66±6.44%, 20 mM group 68.12±2.98%, 40 mM group 78.03±3.28%, 60 mM group 53.13±5.35%, 80 mM group 45.79±8.30%). These values ​​directly reflect the differences in HeLa cell viability in the hydrogel, from IC50... 50 Numerical values ​​can directly reveal differences in drug response behavior: IC50 of HeLa cells in the two-dimensional culture group 50The concentration was 2.35 μg / mL (HeLa cells could not be cultured continuously for 14 days without passaging under two-dimensional culture conditions. Without passaging, cells would die in large numbers due to excessive fusion, contact inhibition, and rapid nutrient depletion; with passaging, the culture process would lose continuity, making it impossible to compare with three-dimensional culture at the same timeframe. Therefore, Figure 7 Cell viability data after 14 days of two-dimensional culture were not presented. To compare drug response, this example measured the IC50 of HeLa cells to doxorubicin under standard two-dimensional culture conditions (48-72 hours of culture, no passage required). 50 IC50 of HeLa cells in a three-dimensional culture model constructed under calcium ion concentrations of 5 mM, 10, 20, 40, 60, and 80 mM. 50 The concentrations were 18.84, 19.14, 20.52, 21.08, 22.14, and 24.76 μg / mL, respectively. IC50 50 A higher IC50 value indicates lower sensitivity and stronger resistance in cells to doxorubicin. Therefore, the drug response of HeLa cells to doxorubicin in the three-dimensional culture model differs significantly from that in two-dimensional culture, exhibiting a markedly increased resistance. This difference is precisely demonstrated by comparing the IC50 values ​​under different culture systems. 50 The value is used to illustrate that the three-dimensional culture model constructed in this invention can more realistically simulate the in vivo drug response behavior.

[0049] Example 3: Statistical analysis of the diameter of HeLa cell spheroids in three-dimensional culture HeLa cells were cultured in three dimensions using the above experimental method. The state of HeLa cell clusters on days 0, 1, 3, and 7 was observed and recorded using phase contrast microscopy, and the cell diameter was analyzed using ImageJ.

[0050] See results Figure 8 , Figure 8 The figure shows the average diameter of HeLa cells cultured in composite hydrogels with different concentrations of calcium ions for different culture times.

[0051] As can be seen, ImageJ was used to quantitatively analyze the diameter of cells cultured in three dimensions in matrix gels and hydrogels. Adding 5mM, 10mM, 20mM, 40mM, 60mM, and 80mM Ca... 2+ In the collagen-sodium alginate hydrogel series, the cell spheroid diameter gradually decreases with increasing calcium ion concentration. Example 4: Application of antitumor drug screening based on HeLa cell three-dimensional culture model An application of constructing an in vitro antitumor drug screening model based on the above-mentioned three-dimensional HeLa cell culture model includes the following steps: 1) constructing a three-dimensional HeLa cell culture model using the three-dimensional cell culture method described in Example 1; 2) after culturing for 24 hours, treating HeLa cells with different concentrations of doxorubicin solution to form a drug response; 3) after treatment for 24 hours, detecting cell viability using a CCK-8 kit, thereby achieving the evaluation and screening of drug efficacy.

[0052] In this example, in step 2) when treating the HeLa cell culture model with doxorubicin, sterilization is required when preparing the doxorubicin solution. Doxorubicin is prepared with distilled water, transferred to a biosafety cabinet, and filtered through a 0.22 μm sterile filter membrane for sterilization. It is then diluted to a certain concentration using complete culture medium and filtered again for sterilization to complete the preparation of doxorubicin. Doxorubicin is then added to the cell model to induce cell damage.

[0053] Validation example: Drug sensitivity validation of a three-dimensional culture model of HeLa cells. To determine the effect of doxorubicin on HeLa cells in the three-dimensional culture model constructed in this experiment, the following experimental setup was adopted: After passage, HeLa cells were cultured until they reached 80%-90% confluence. The cells were then digested with 0.25% trypsin and seeded into the three-dimensional culture model constructed in Example 1, and cultured in a constant temperature incubator for one day. Doxorubicin was first diluted to a certain concentration with distilled water, then filtered through a 0.22 μm sterile filter on a clean bench for sterilization. It was then diluted with two-dimensional and three-dimensional HeLa cell culture media (two-dimensional, i.e., 2D in the figure, refers to HeLa (human cervical cancer cells) (STR identification correct), product code CL-0101 Pronosai, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; three-dimensional, see Example 1), filtered again, and prepared fresh for use. The concentrations of doxorubicin used in the experiment were 5, 10, 20, 40, and 80 μg / mL, with an incubation time of 24 h. Three parallel wells were set up per well, and the experiment was repeated three times. The viability of HeLa cells was determined by measuring the absorbance at 450 nm using a CCK-8 cell viability assay kit and an ELISA reader. The IC50 of doxorubicin on HeLa cells was then calculated. 50 value.

[0054] The results are as follows Figure 9 As shown, Figure 9 The figure shows the effect of doxorubicin on the viability of HeLa cells under different ion concentrations. When different concentrations of doxorubicin were used to treat the three-dimensional culture model of HeLa cells, the cell viability gradually decreased with increasing concentration. The IC50 of HeLa cells in the 2D culture group was calculated. 50 The value was 2.35 μg / mL, while the IC50 of HeLa cells in the three-dimensional culture model constructed in this invention was...50 The IC50 value of HeLa cells was significantly improved in three-dimensional culture models constructed under calcium ion concentrations of 5 mM, 10, 20, 40, 60, and 80 mM. 50 The concentrations were 18.84, 19.14, 20.52, 21.08, 22.14, and 24.76 μg / mL, respectively. In the three-dimensional culture model, HeLa cells showed significantly higher resistance to doxorubicin than those in the two-dimensional culture model. The three-dimensional culture model of HeLa cells constructed in this invention can more accurately reflect the in vivo efficacy of drugs.

[0055] Example 5: An application of constructing an in vitro tumor microenvironment simulation model based on the above-mentioned HeLa cell three-dimensional culture model. The steps include: 1) constructing a three-dimensional cell culture model of HeLa cells using the three-dimensional cell culture method described in Example 1; 2) collecting the cell culture supernatant after one week of culture and detecting the secretion level of matrix metalloproteinase-9 (MMP-9) using an ELISA kit; 3) detecting the expression of HPV16 E6 oncogenic protein using an ELISA method to assess the ability of the model to maintain a malignant phenotype.

[0056] 1. Determination of MMP-9 secretion levels in three-dimensional cultured HeLa cells HeLa cells were cultured in three dimensions according to the above experimental method. The culture medium was collected and centrifuged at 1000 rpm for 20 min, and the supernatant was collected. Following the ELISA kit instructions, 50 μL of the corresponding test solution was added to each well. Then, 100 μL of HRP-labeled detection antibody was added to each well except for the blank well. The plates were sealed with a sealing membrane and incubated at 37℃ for 60 min. After the incubation period, the liquid in the wells was removed, and 350 μL of washing buffer was added to each well. After standing for 1 min, the buffer was removed, and any remaining liquid in the wells was removed with absorbent paper. This washing process was repeated 5 times. After the washing step, 50 μL each of chromogenic solution A and chromogenic solution B were added to each well sequentially. The mixture was thoroughly mixed and incubated at 37℃ in the dark for 15 min. To terminate the reaction, 50 μL of stop solution was added to each well to interrupt the reaction, and the absorbance of each well was measured at 450 nm using a microplate reader within 15 min.

[0057] The results are as follows Figure 10 As shown, Figure 10This figure shows the MMP-9 secretion levels in HeLa cells cultured in hydrogels with different calcium ion concentrations. After 7 days of culture in the hydrogel, the MMP-9 secretion level of HeLa cells increased with increasing hydrogel stiffness within the calcium ion concentration range of 5–40 mM, with the 40 mM calcium ion group showing a significantly higher level than the 5 mM group. However, when the calcium ion concentration increased to 60 mM and 80 mM, the MMP-9 secretion level decreased, but was still higher than that of the 2D culture group. Within the 5–40 mM range, the MMP-9 secretion level in the hydrogel increased with increasing calcium ion concentration, indicating that moderately increasing the calcium ion concentration can improve the hydrogel stiffness and promote the expression of HeLa cell invasion and metastasis-related proteins. However, when the calcium ion concentration increased to 60 mM and 80 mM, excessively high gel stiffness or an overly dense cross-linked network may restrict cell proliferation, diffusion, or even induce cell stress, leading to a decrease in MMP-9 secretion levels. This invasion is highly dependent on matrix remodeling, reflecting the regulatory role of the cellular microenvironment on the biological functions of tumor cells. Upregulation of MMP-9 expression suggests that tumor models constructed in hydrogels can enhance their invasive and metastatic capabilities.

[0058] 2. Determination of HPV16 E6 expression in three-dimensionally cultured HeLa cells HeLa cells were cultured in three dimensions according to the above experimental steps. The culture medium was collected, centrifuged at 1000 rpm for 20 min, and the supernatant of the test sample was collected for later use. Negative control wells, positive control wells, and sample wells were set up. 50 μL of the corresponding control standard was added to each of the negative control and positive control wells (positive control: solution containing known HPV16 E6 protein; negative control: solution without HPV16 E6 protein or antibody, both from Shanghai Keabob Biotechnology kit, product code: CB13752-Hu). To maintain consistency with the background of the test sample (cell culture supernatant), blank cell culture supernatant without E6 protein or buffer containing protein protectant is often used; blank control: usually only substrate and stop solution are added, without sample and detection antibody). 50 μL of the test sample was added to the sample wells, and no treatment was performed on the blank wells. Except for the blank wells, 100 μL of horseradish peroxidase-labeled detection antibody was added to each control well and sample well. The reaction wells were sealed with sealing film and incubated in a 37℃ incubator for 60 min. After incubation, discard the liquid in the wells and gently blot dry with absorbent paper. Add 350 μL of washing buffer to each well, let stand for 1 min, discard, and blot dry again with absorbent paper. Repeat washing 5 times. After washing, add 50 μL each of substrate A and substrate B to each well sequentially, and incubate at 37℃ in the dark for 15 min. Then add 50 μL of stop solution to each well, and measure the optical density (OD) of each well at 450 nm within 15 min. The results are interpreted as follows: the OD value of the negative control well should be below 0.2, and the OD value of the positive control well should be above 0.8. Add 0.25 to the negative control OD value as the cutoff value; a sample OD value above this cutoff value is considered positive, and a value below or equal to this cutoff value is considered negative.

[0059] The results are as follows Figure 11 As shown, Figure 11 This image shows the HPV16E6 secretion levels in HeLa cells cultured in hydrogels with different concentrations of calcium ions. It can be seen that HPV16E6 was positively expressed in all groups of HeLa cells. This indicates that culturing HeLa cells in calcium-rich environments does not interfere with the normal function of the HPV16E6 gene, and the cells remain typical malignant tumor cells carrying HPV oncogenes.

[0060] Conclusion: The HeLa cell three-dimensional culture model constructed by the above three-dimensional culture method can effectively simulate the tumor microenvironment in vivo. The cells maintain a malignant phenotype, secrete MMP-9 protein related to tumor invasion, and maintain good cell viability in long-term culture. It provides a reliable in vitro model platform for the study of cervical cancer mechanisms.

[0061] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for constructing a three-dimensional culture model of HeLa cells, characterized in that, Includes the following steps: HeLa cells were mixed with a collagen-sodium alginate composite hydrogel precursor solution to obtain a cell-precursor mixture. The cell-precursor mixture was then mixed with CaCl2 solution to obtain a mixture. The mixture was then placed in an incubator and incubated for 10-60 min to allow the hydrogel to crosslink and solidify, forming a three-dimensional culture system. Complete culture medium was added to the three-dimensional culture system, and the cells were cultured continuously to construct a three-dimensional culture model of HeLa cells. The preparation method of the collagen-sodium alginate composite hydrogel precursor solution is as follows: S1. Preparation of aminoated collagen: Collagen was fully dissolved in acetic acid solution, then EDA aqueous solution was added to the solution and mixed well, then EDC solution was added, and the reaction was stirred at room temperature. After the reaction was completed, the reaction solution was purified by dialysis using a dialysis bag, and then freeze-dried under vacuum to obtain aminoated collagen; it was then dissolved in dilute acetic acid to prepare an aminoated collagen solution with a mass fraction of 2±1wt%. S2. Preparation of oxidized sodium alginate: Sodium alginate was completely dissolved in hot distilled water and cooled to room temperature. NaIO4 aqueous solution was added dropwise to the sodium alginate solution and reacted in the dark at room temperature. Ethylene glycol was then added and stirring was continued to terminate the reaction and reduce the residual NaIO4. Dialysis was performed using a dialysis bag to remove unreacted reagents and small molecules. After vacuum freeze-drying, oxidized sodium alginate was obtained. The oxidized sodium alginate was then dissolved in PBS buffer solution with a pH of near neutral to prepare a 10-20 wt% oxidized sodium alginate solution. The order of steps S1 and S2 is not important; they can be performed simultaneously. The amino-collagen solution is mixed with the sodium oxidized alginate solution to obtain a collagen-sodium alginate composite hydrogel precursor solution. Preferably, the amino-collagen solution and the sodium oxidized alginate solution are mixed at a volume ratio of 1:(1±0.2).

2. The method for constructing the HeLa cell three-dimensional culture model as described in claim 1, characterized in that, The density of the HeLa cells was 0.5 × 10⁻⁶. 5 ~5×10 5 per mL.

3. The method for constructing the HeLa cell three-dimensional culture model as described in claim 1, characterized in that, The volume ratio of HeLa cells to the collagen-sodium alginate composite hydrogel precursor solution is 1:

8.

4. The method for constructing the HeLa cell three-dimensional culture model as described in claim 1, characterized in that, Ca in the mixture 2 + The concentration is 5-80 mM.

5. The method for constructing the HeLa cell three-dimensional culture model as described in claim 1, characterized in that, The continuous culture refers to replacing the complete culture medium every 48 hours and culturing for 1-14 days.

6. The method for constructing the HeLa cell three-dimensional culture model as described in claim 1, characterized in that, In step S1, the acetic acid solution is a 0.02 mol / L acetic acid solution, the EDA aqueous solution is a pH 6.0, 2.5 mol / L EDA aqueous solution, and the EDC solution is a 0.2 mol / L EDC solution. More preferably, the ratio of collagen to 0.02 mol / L acetic acid solution to pH 6.0, 2.5 mol / L EDA aqueous solution to 0.2 mol / L EDC solution is 3 g: 740 mL: 60 mL: 36 mL; Optionally, in step S1, the reaction time at room temperature with stirring is 8-20 hours, and the dialysis bag is a 3500 Da dialysis bag.

7. The method for constructing the HeLa cell three-dimensional culture model as described in claim 1, characterized in that, In step S2, the ratio of sodium alginate, distilled water, NaIO4 and ethylene glycol is 2 g: 100 mL: 1.5 g / 20 mL: 1 mL. Optionally, in step S2, the reaction time at room temperature in the dark is 24-60 hours, and the dialysis bag is a 3500 Da dialysis bag.

8. A three-dimensional culture model of HeLa cells constructed by the construction method according to any one of claims 1-7.

9. The application of the HeLa cell three-dimensional culture model according to claim 8 in the screening of antitumor drugs.

10. The application as described in claim 9, characterized in that, The antitumor drug mentioned is doxorubicin.

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