Construction method of bacteria and cancer cell co-culture model
By encapsulating bacteria in sodium alginate microcapsules and co-encapsulating them with cancer cells in sodium alginate gel beads, a bacteria-cancer cell co-culture model was constructed, which solved the problems of bacterial growth rate control and continuous action of metabolites in the existing system, and achieved ideal simulation of cell growth and investigation of bacterial influence.
Patent Information
- Application Number
- CN202411747809.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-03-28
AI Technical Summary
The existing co-culture system of Lactobacillus and cervical cancer cells is difficult to effectively control the growth rate of bacteria and the continuous effect of metabolites, resulting in the problem of competition inhibition of cell growth and reduction of metabolites.
By encapsulating bacteria in sodium alginate microcapsules, and then mixing bacteria-encapsulated microcapsules with cancer cells and jointly encapsulate them in sodium alginate gel beads, a bacteria-cancer cell co-culture model based on sodium alginate microcapsule-bead pendant encapsulation system was constructed.
The spatial isolation of bacterial growth and the effective release of metabolites is achieved, and the competitive inhibition of cell growth is avoided, and a more ideal three-dimensional model is provided to facilitate the exploration of the impact of bacteria on cancer cells.
Smart Images
Figure CN119709486A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medical three-dimensional models, and in particular relates to a method for constructing a bacteria and cancer cell co-culture model. Background Art
[0002] In recent years, more and more studies have shown that bacteria play an important role in the occurrence and development of cancer. For example, vaginal lactobacilli can inhibit the proliferation and migration of cervical cancer. To this end, researchers have established a co-culture system of lactobacilli and cervical cancer cells in order to further explore its mechanism of action. The existing co-culture systems of lactobacilli and cervical cancer cells mainly include two types: one is to directly co-culture lactobacilli with flat-cultured cervical cancer cells, but in the same living space, it is difficult to control the growth rate of lactobacilli, and cell growth is significantly inhibited by competition during long-term culture; the other is to use a culture medium with added lactobacilli supernatant to act on the cultured cells, but as the culture time increases, the bacterial metabolites in the supernatant gradually decrease, and it is difficult to observe its continued effect on cervical cancer cells. Therefore, it is urgent to establish an ideal lactobacillus-cervical cancer cell co-culture model.
[0003] Microencapsulation technology is a high-throughput preparation technology that uses different biomaterials to form a semipermeable membrane to encapsulate functional cells. The semipermeable membrane allows nutrients to enter and metabolites to be discharged. At the same time, it can effectively isolate immune molecules and cells in the body by adjusting its molecular weight cutoff. It has excellent immune isolation and good biocompatibility. Among them, sodium alginate microcapsules are favored because of their large specific surface area, good compression and friction resistance, controllable microsphere size, and non-invasive transplantation. However, to date, there has been no report on encapsulating bacteria in sodium alginate microcapsules, and then mixing the microcapsules encapsulated with bacteria with cancer cells and encapsulating them together in sodium alginate beads to construct a bacteria-cancer cell co-culture model based on the sodium alginate microcapsule-bead nesting encapsulation system. Summary of the invention
[0004] The present invention aims to solve the above-mentioned technical problems existing in the prior art and provides a method for constructing a bacteria and cancer cell co-culture model.
[0005] The technical solution of the present invention is: a method for constructing a bacteria and cancer cell co-culture model, which is carried out in the following steps: Step 1. Take a mixed solution of bacteria and sodium alginate to prepare calcium alginate beads; Step 2. further calcifying the calcium alginate beads; Step 3. forming a polycationic film on the surface of the calcified calcium alginate beads; Step 4. liquefying the calcium alginate beads with a polycationic film formed on the surface to obtain sodium alginate microcapsules encapsulating bacteria; Step 5. Take the sodium alginate microcapsules encapsulating bacteria, the cells and the sodium alginate mixed solution, prepare the calcium alginate beads encapsulating the sodium alginate microcapsules and cells, and calcify them.
[0006] Preferably, the step 1 is to mix the bacteria with a sodium alginate solution having a mass / volume concentration of 1.5% to prepare 1×10 7 CFU / mL of bacteria and sodium alginate mixed solution were used to prepare calcium alginate beads using an electrostatic droplet generator and a 24-gauge needle; Preferably, the step 2 is to place the calcium alginate beads in a 100 mM CaCl2 solution for calcification for 15 min; Preferably, the step 3 is to discard the excess CaCl2 solution after calcification, add a 0.05% PLL solution with a mass / volume concentration several times the volume of the calcium alginate beads, and react for 8-10 minutes under continuous shaking at room temperature.
[0007] Preferably, the step 4 is to fully wash the calcium alginate beads with a polycationic membrane formed on the surface with physiological saline, add a 55 mM sodium citrate solution several times the volume of the calcium alginate beads, and continuously shake at room temperature for 2-5 minutes to obtain sodium alginate microcapsules encapsulating bacteria; Preferably, the step 5 is to wash the sodium alginate microcapsules encapsulating bacteria with physiological saline, resuspend them with a sodium alginate solution with a mass / volume concentration of 1.5%, and then add cells to prepare a mixed solution of sodium alginate microcapsules, cells and sodium alginate, wherein the volume ratio of the sodium alginate solution with a mass / volume concentration of 1.5% to the sodium alginate solution in step 1 is 1-3:1, and the dosage ratio of the cells to the sodium alginate solution is 1×10 6 Cells / mL were prepared using an electrostatic droplet generator and a 20-gauge needle to prepare sodium alginate microcapsules encapsulating bacteria and calcium alginate beads encapsulating cells, which were then calcified in a 100 mM CaCl2 solution for 15 min.
[0008] Preferably, the bacteria are vaginal lactobacilli, and the cancer cells are human cervical squamous cell carcinoma cells.
[0009] The preferred operating conditions of the electrostatic droplet generator are: 5-5.5 Kv voltage, 130-150 Hz frequency, and a syringe pump speed of 30 mL / h.
[0010] The present invention is to encapsulate bacteria in sodium alginate microcapsules, and then mix the sodium alginate microcapsules encapsulated with bacteria with cancer cells and encapsulate them in sodium alginate beads, thereby constructing a bacteria-cancer cell co-culture model based on the sodium alginate microcapsule-bead nesting encapsulation system. It has the following advantages: first, by encapsulating bacteria in microcapsules, its growth space and growth rate are limited, and a relatively anaerobic environment is provided for bacteria at the same time, but the discharge of bacterial metabolites does not affect the effect on cancer cells; secondly, cancer cells grow in balls in the beads to form a bionic tumor-like structure; finally, the relatively independent space is convenient for recovering each component for detection and analysis. Therefore, the co-culture model of the present invention has the characteristics of high preparation flux, convenient separation and detection, isolation of bacteria-cell growth space, oxygen and nutrient gradient, etc., which provides a more ideal three-dimensional model for exploring the influence of bacteria on cancer cells, and can provide new strategies and theoretical basis for the prevention and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 It is a schematic diagram of the process of constructing a Lactobacillus jensenii-Siha cell co-culture model in Examples 1, 2, and 3 of the present invention.
[0012] Figure 2 Schematic diagrams of the co-culture model of Lactobacillus jensenii-Siha cells prepared in Examples 1, 2 and 3 of the present invention and statistical diagrams of the relative proportions of beads and microcapsules are respectively shown.
[0013] Figure 3 These are microscopic photographs of the growth changes of cells and bacteria in the Lactobacillus jensenii-Siha cell co-culture model constructed in Example 2 of the present invention and the models of Comparative Examples 1 and 2.
[0014] Figure 4 It is a diagram showing the proliferation and activity detection results of cells and bacteria in the Lactobacillus jensenii-Siha cell co-culture model constructed in Example 2 of the present invention and the models of Comparative Examples 1 and 2. DETAILED DESCRIPTION Example 1
[0015] A method for constructing a bacteria and cancer cell co-culture model of the present invention is as follows Figure 1 As shown, follow the steps below: Step 1. Mix Lactobacillus jensenii with 1.5% (w / v) sodium alginate solution to make 1×10 7 CFU / mL of bacteria and sodium alginate mixed solution were used to prepare calcium alginate beads using an electrostatic droplet generator and a 24-gauge needle; The preservation number of the Lactobacillus jensenii is CGMCC NO.22802.
[0016] The operating conditions of the electrostatic droplet generator are: 5-5.5 Kv voltage, 130-150 Hz frequency, and a syringe pump speed of 30 mL / h.
[0017] Step 2. Place the calcium alginate beads prepared in step 1 in a 100 mM CaCl2 solution for calcification for 15 min; Step 3. Discard the excess CaCl2 solution after calcification, add 0.05% PLL (poly-lysine) solution with a mass / volume concentration of 5 times the volume of calcium alginate beads, and react for 8-10 minutes at room temperature with continuous shaking, thereby forming a polycationic film on the surface of the calcified calcium alginate beads; Step 4. Wash the calcium alginate beads with polycationic membrane on the surface with saline for 3 times and add A 55 mM sodium citrate solution with a volume 5 times that of the calcium alginate beads was continuously shaken at room temperature for 2-5 min to liquefy the calcium alginate beads with a polycationic film formed on the surface, thereby obtaining sodium alginate microcapsules with a diameter of 250-300 µm encapsulating Lactobacillus jensenii; Step 5. The sodium alginate microcapsules encapsulating Lactobacillus jensenii were fully washed with physiological saline, resuspended with 1.5% (w / v) sodium alginate solution, and then Siha cells were added to prepare a mixed solution of sodium alginate microcapsules, Siha cells and sodium alginate, wherein the mass / volume concentration was 1.5% sodium alginate solution and the volume ratio of the sodium alginate solution used in step 1 was 1:1, and the dosage ratio of the cells to the sodium alginate solution was 1×10 6 cells / mL, an electrostatic droplet generator and a 20-gauge needle were used to prepare sodium alginate microcapsules encapsulating Lactobacillus jensenii and calcium alginate beads encapsulating Siha cells, which were then calcified in 100 mM CaCl2 solution for 15 min.
[0018] The sodium alginate microcapsules encapsulating Lactobacillus jensenii were washed three times with physiological saline, mixed with 1.5% (w / v) sodium alginate solution at a volume ratio of 1:1, and then Siha cells were added to prepare a mixed solution of sodium alginate microcapsules, Siha cells and sodium alginate. The dosage ratio of Siha cells to sodium alginate solution was 1×10 6 cells / mL, using an electrostatic droplet generator and a 20-gauge needle to make calcium alginate beads with a diameter of 450-550 µm to encapsulate microcapsules and cells, and then calcified in 100 mM CaCl2 solution for 15 min; The operating conditions of the electrostatic droplet generator are: 5-5.5 Kv voltage, 130-150 Hz frequency, and a syringe pump speed of 30 mL / h.
[0019] Embodiment 2: The operation method of Example 2 is basically the same as that of Example 1, except that the volume ratio of the sodium alginate solution with a mass / volume concentration of 1.5% in step 5 to the sodium alginate solution used in step 1 is 2:1.
[0020] Embodiment 3: The operation method of Example 3 is basically the same as that of Example 1, except that the volume ratio of the sodium alginate solution with a mass / volume concentration of 1.5% in Step 5 to the sodium alginate solution used in Step 1 is 3:1.
[0021] The schematic diagram of the co-culture model of Lactobacillus jensenii-Siha cells prepared in Examples 1, 2 and 3 of the present invention and the statistical diagram of the relative proportion of beads and microcapsules are shown in FIG. Figure 2 As shown, Figure 2 A, B, and C are schematic diagrams of the co-culture model of Lactobacillus jensenii-Siha cells prepared in Examples 1, 2, and 3, respectively, and D is a statistical diagram of the relative proportions of beads and microcapsules in each example. Figure 2 It can be seen that the overlapping encapsulation will result in one bead encapsulating multiple microcapsules, one microcapsule, and no microcapsule. When one bead encapsulates multiple microcapsules, the outer bead and the inner microcapsule will be deformed and damaged, and the proportion of a bead encapsulating a single microcapsule in Example 2 of the present invention is the highest, reaching 59%.
[0022] Comparative Example 1: A model was manufactured basically according to the method of Inventive Example 1, except that Lactobacillus jensenii was not added in step 1, and a gel bead nesting model encapsulating non-bacterial microcapsules and Siha cells was finally formed.
[0023] Comparative Example 2: The method of Example 1 of the invention was basically followed, except that Siha cells were not added in step 5, and a microcapsule-bead nesting encapsulation model encapsulating Lactobacillus jensenii was finally formed.
[0024] Experiments on the growth changes of cells and bacteria in the Lactobacillus jensenii-Siha cell co-culture model constructed in Example 2 of the present invention and the models of Comparative Examples 1 and 2: Cell viability assay: According to the reagent manual, use the Live / Dead staining kit to detect sample activity. The specific steps are as follows: After the sample is washed twice with PBS, it is placed in a 1.5 ml Eppendorf tube, and an appropriate amount of staining reagent is added (0.5 μL of calcein-AM reagent and 2 μL of EthD-1 reagent are added to 1 mL of basal culture medium). Stain for 2 hours in a constant temperature incubator at 37°C and 5% CO2 concentration, and be careful to avoid light; thoroughly wash with PBS to remove excess stain, and use a laser confocal microscope to take pictures.
[0025] Bacterial activity test: Sodium citrate solution was used to liquefy the macrocapsules to release Siha cells and Lactobacillus jensenii microcapsules. After rinsing with saline, Siha cells and Lactobacillus jensenii microcapsules were collected respectively. Lactobacillus jensenii microcapsules were placed in bacterial activity detection working solution (100 μL saline + 0.15 μL PI + 0.15 μL SYTO) and incubated in the dark for 15 min. After rinsing with saline, laser confocal microscopy was used to take pictures.
[0026] Cell proliferation assay: The calcium alginate beads encapsulating the microcapsules and cells were collected every 2 days, rinsed several times with saline and resuspended in culture medium, and plated on a 96-well plate with a 1ml pipette tip (to avoid damage to the microcapsules, the 1ml pipette tip can be cut off), minimizing the difference between wells, with the bottom of the well plate covered and no gaps as the standard. Incubate with CCK-8 reagent for 12 hours, and detect its absorbance at a wavelength of 450nm. Set at least 3 replicate wells for each group and perform 3 independent repeated experiments.
[0027] Magenta stain: Samples were collected at corresponding time points and fixed with 10% formalin. After being washed three times with ultrapure water, they were incubated in magenta dye at room temperature in the dark for 24 h. The samples were sequentially placed in 30%, 70%, 90%, and 100% ethanol solutions (20 min each) for gradient dehydration. After the xylene solution became transparent (20 min), the samples were moved to a glass slide for sealing and photographed under an optical microscope.
[0028] Microscopic photos of the growth changes of cells and bacteria in the Lactobacillus jensenii-Siha cell co-culture model constructed in Example 2 of the present invention and the models of Comparative Examples 1 and 2 are shown in FIG. Figure 3 As shown. The results show that the encapsulated Siha cells and Lactobacillus jensenii in Example 2 of the present invention are dispersed in the gel beads and capsules. As the culture time increases, the Siha cells gradually grow into clusters. However, compared with the single cell culture group in Comparative Example 1, after co-culture with Lactobacillus jensenii, the number and volume of Siha cell clusters are small, indicating that lactobacillus inhibits the growth of cervical cancer cells. At the same time, compared with the single bacterial culture group in Comparative Example 2, after co-culture with cells, the lactobacillus proliferates significantly.
[0029] Active staining and CCK8 were further used to clarify the effect of co-culture on the activity and proliferation of Siha cells and Janssen cells. The proliferation and activity detection results of cells and bacteria in the model of Example 2 of the present invention and Comparative Examples 1 and 2 are as follows: Figure 4 shown. Figure 4The CCK-8 results showed that compared with the single cell culture group of comparative example 1, the Siha cells in the co-culture group of Example 2 of the present invention proliferated slowly. The activity test results showed that after co-culture in Example 2 of the present invention, the volume and number of Siha cell clusters in the beads were smaller, the number of dead cells increased, and the Lactobacillus jensenii in the microcapsules was active and proliferated into larger clusters.
[0030] The experimental results suggest that after Siha cells were co-cultured with Lactobacillus jensenii, the proliferation and activity of Siha cells were inhibited.
Claims
1. A method for constructing a bacterial and cancer cell co-culture model, characterized in that Follow the steps below: Step 1. Take a mixed solution of bacteria and sodium alginate to prepare calcium alginate beads; Step 2. further calcifying the calcium alginate beads; Step 3. forming a polycationic film on the surface of the calcified calcium alginate beads; Step 4. liquefying the calcium alginate beads with a polycationic film formed on the surface to obtain sodium alginate microcapsules encapsulating bacteria; Step 5. Take the sodium alginate microcapsules encapsulating bacteria, the cells and the sodium alginate mixed solution, prepare the calcium alginate beads encapsulating the sodium alginate microcapsules and cells, and calcify them.
2. The method for constructing a bacteria and cancer cell co-culture model according to claim 1, characterized in that: The step 1 is to mix the bacteria with a sodium alginate solution having a mass / volume concentration of 1.5% to prepare 1×10 7 CFU / mL of bacteria and sodium alginate mixed solution were used to prepare calcium alginate beads using an electrostatic droplet generator and a 24-gauge needle.
3. The method for constructing a bacteria and cancer cell co-culture model according to claim 2, characterized in that: The step 2 is to place the calcium alginate beads in a 100 mM CaCl2 solution for calcification for 15 minutes.
4. The method for constructing a bacteria and cell co-culture model according to claim 3, characterized in that: The step 3 is to discard the excess CaCl2 solution after calcification, add a 0.05% PLL solution with a mass / volume concentration several times the volume of the calcium alginate beads, and react for 8-10 minutes under continuous shaking at room temperature.
5. The method for constructing a bacteria and cancer cell co-culture model according to claim 4, characterized in that: The step 4 is to fully wash the calcium alginate beads with a polycationic membrane formed on the surface with physiological saline, add a 55 mM sodium citrate solution several times the volume of the calcium alginate beads, and continuously shake at room temperature for 2-5 minutes to obtain sodium alginate microcapsules encapsulating bacteria.
6. The method for constructing a bacteria and cancer cell co-culture model according to claim 4, characterized in that: The step 5 is to fully wash the sodium alginate microcapsules encapsulating bacteria with physiological saline, resuspend them with a sodium alginate solution with a mass / volume concentration of 1.5%, and then add cells to prepare a mixed solution of sodium alginate microcapsules, cells and sodium alginate, wherein the volume ratio of the sodium alginate solution with a mass / volume concentration of 1.5% to the sodium alginate solution in step 1 is 1-3:1, and the dosage ratio of the cells to the sodium alginate solution is 1×10 6 Cells / mL were prepared using an electrostatic droplet generator and a 20-gauge needle to prepare sodium alginate microcapsules encapsulating bacteria and calcium alginate beads encapsulating cells, which were then calcified in a 100 mM CaCl2 solution for 15 min.
7. The method for constructing a bacteria and cancer cell co-culture model according to claim 1, 2, 3, 4, 5 or 6, characterized in that: The bacteria are vaginal lactobacilli, and the cancer cells are human cervical squamous cell carcinoma cells.
8. The method for constructing a bacteria and cancer cell co-culture model according to claim 2 or 6, characterized in that: The operating conditions of the electrostatic droplet generator are: 5-5.5 Kv voltage, 130-150 Hz frequency, and a syringe pump speed of 30 mL / h.