Three-dimensional culture method for tumor organoids based on hydrogel and micro-scaffold

Abstract

The invention relates to the technical field of organoid culture, and discloses a three-dimensional culture method for tumor organoids based on hydrogel and a micro-scaffold, the culture method comprises the following steps: mixing the hydrogel with a tumor organoid basic culture medium to obtain a mixture of the hydrogel and the culture medium; 2, mixing a cell suspension containing tumor organoid cells with the micro-scaffold, centrifuging, and mixing an obtained centrifugal product with the mixture of the hydrogel and the culture medium in the step 1 to obtain a composite system based on the hydrogel and the micro-scaffold and a cell mixture; placing the composite system based on the hydrogel and the micro-scaffold and the cell mixture at low temperature to form soft gel; and covering the surface of the soft gel with a tumor organoid basal culture medium, culturing, and carrying out liquid change and observation according to experimental requirements in the culture process to obtain the tumor organoid. The hydrogel and the micro-scaffold are used in a composite mode, the three-dimensional culture method for the tumor organoid is provided, and new possibility is provided for in-vitro culture of the tumor organoid.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a three-dimensional culture method for tumor organoids based on hydrogels and microscaffolds. Background Technology

[0002] In recent years, research on organoid technology in oncology and cancer has increased rapidly, particularly in drug screening and precision medicine. Patient-driven organoids (PDOs) provide effective preclinical cancer models that better mimic the components of tumor tissue and can be effectively established from patient samples. The intratumoral diversity in PDOs captures tumor heterogeneity at the single-cell level and provides a valuable resource for cancer research. The culture of patient-driven tumor organoids enables routine primary culture of resected human tumor tissue. Currently, numerous PDO models have been established using patient tumor tissue, including models of colon, liver, stomach, lung, bladder, breast, pancreatic cancer, and head and neck squamous cell carcinoma. Tumor organoids, as excellent cancer models for drug development and personalized treatment, offer possibilities for patient-driven cancer therapy.

[0003] However, the in vitro culture conditions for tumor organoids are not yet mature, the culture environment requirements are very strict, and the construction of in vitro culture systems is quite complex. Therefore, developing novel 3D cell culture materials, constructing more complete culture systems, and exploring new culture methods are of great significance for the research of tumor organoids. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds, comprising the following steps: Step 1: Mix the hydrogel with the basic culture medium for tumor organoids to obtain a mixture of hydrogel and culture medium; Step 2: Mix the cell suspension containing tumor organoid cells with the microscaffold, centrifuge, and mix the centrifuged product with the mixture of hydrogel and culture medium from Step 1 to obtain a composite system based on hydrogel and microscaffold and a cell mixture. Step 3: Place the hydrogel- and microscaffold-based composite system and cell mixture at low temperature to form a soft gel; cover the surface of the soft gel with tumor organoid basal culture medium, and culture. During the culture process, change the medium and observe according to the experimental requirements to obtain tumor organoids.

[0005] Preferably, in step one, the preparation method of the mixture of hydrogel and culture medium is as follows: use a pipette to dispense 1 mL of hydrogel into a 2 mL EP tube and place it in a 37°C incubator for 30 min to rewarm; take two sterile syringes, one of which directly draws 500 μL of the rewarmed hydrogel according to the syringe scale, and the other uses a pipette to transfer 500 μL of tumor organoid basal culture medium from the syringe needle tip to the syringe. After purging the air from both syringes, connect them with a Luer tube connector and push back and forth 15-20 times to mix evenly.

[0006] Preferably, in step two, the preparation method of the hydrogel- and microscaffold-based composite system and cell mixture is as follows: Transfer 1 mL of cell suspension containing 100,000 to 1,000,000 tumor organoid cells to a 2 mL EP tube, add 10 μL of microscaffold, pipette and vortex 10 times, then centrifuge at 1500 rpm for 3 min. After centrifugation, discard most of the supernatant, leaving 100 μL of supernatant to obtain a centrifuged product containing resuspended tumor organoid cell precipitate and microscaffold. Transfer the centrifuged product to a syringe containing the mixture of hydrogel and culture medium described in step one, and gently push back and forth 15-20 times to mix thoroughly.

[0007] Preferably, in step three, the method for preparing the tumor organoids is as follows: Transfer 50 μL of the hydrogel- and microscaffold-based composite system and cell mixture into a 96-well low-absorption plate using a 200 μL wide-bore pipette tip. Gently tilt / rotate the plate to ensure that each well is evenly covered with a layer of the hydrogel- and microscaffold-based composite system and cell mixture. Seal the culture plate and transfer it to a refrigerator at 2-8°C. Wait 30 min for a stable soft gel to form. Add 100 μL of tumor organoid basal culture medium to cover the surface of the soft gel. Incubate the culture plate in an incubator and change the medium and observe according to experimental requirements.

[0008] Preferably, in step one, the basal culture medium for tumor organoids comprises the following components: basal culture medium Advanced DMEM / F12, and other additives as follows: 1×B27, 2mM GlutaMAX. TM 10mM HEPES, 10mM Nicotinamide, 1.5mM N-acetyl-L-cysteine, 1% Penicillin-Streptomycin, 1×N21-MAX Supplement, 100ng / mL Noggin, 500ng / mL R-Spondin 1, 10-100ng / mL EGF, 10μM Y-27632.

[0009] Preferably, in step one, the method for preparing the hydrogel includes the following steps: Step A1, Preparation of working solutions: Prepare 1X EDC / NHS activation solution, RGD peptide working solution, QK peptide working solution, VN peptide working solution and YGGFM peptide working solution respectively; the sequence of the RDG peptide is GGGGRGDASSP-NH2, the sequence of the QK peptide is KLTWQELYQLKYKGI-NH2, the sequence of the VN peptide is GDCPWKPWC-NH2, and the sequence of the YGGFM peptide is YGGFM-NH2; Step A2: Preparation of polypeptide-grafted sodium alginate solution: The RGD peptide working solution was added to the activated sodium alginate solution, so that the molar ratio of RGD peptide to sodium alginate in the mixture was in the range of 1:15 to 1:20. The mixture was stirred at 1000 rpm for 1 h at room temperature, then the stirring was stopped and the mixture was placed in a refrigerator at 4 °C for 20 h to obtain sodium alginate-RGD solution, denoted as AR solution. Sodium alginate-QK solution, sodium alginate-VN solution and sodium alginate-YGGFM solution were prepared according to the same method, and denoted as AQ solution, AV solution and AY solution, respectively. Step A3, Hydrogel preparation: Transfer AR solution, AQ solution, AV solution, and AY solution in a volume ratio of 10:10:50:30 to a 2000mL sterile reaction flask and stir at 250rpm for 4h at 25℃ to obtain hydrogel.

[0010] Furthermore, the method for preparing the activated sodium alginate solution is as follows: 30g of ultrapure sodium alginate was soaked in 300mL of tumor organoid basal culture medium for 10min, then stirred at 50rpm for 30min, and then 700mL of tumor organoid basal culture medium was added. The mixture was stirred at 250rpm for 6h to obtain sodium alginate solution. Add 3500 μL of 1X EDC / NHS activation solution to 1000 mL of sodium alginate solution, stir at 250 rpm for 1 h at room temperature, and then let stand at room temperature for 3 h to obtain activated sodium alginate solution.

[0011] Preferably, in step two, the method for preparing the microscaffold includes the following steps: Step B1, Preparation of working solutions: Prepare 1X EDC / NHS activation solution, RGD peptide working solution, QK peptide working solution, VN peptide working solution and YGGFM peptide working solution respectively; the sequence of the RDG peptide is GGGGRGDASSP-NH2, the sequence of the QK peptide is KLTWQELYQLKYKGI-NH2, the sequence of the VN peptide is GDCPWKPWC-NH2, and the sequence of the YGGFM peptide is YGGFM-NH2; Step B2, Preparation of polypeptide grafted with sodium alginate: The RGD peptide working solution was added to the activated 2% SA solution, so that the molar ratio of RGD peptide to sodium alginate in the mixture was in the range of 1:15 to 1:20. The mixture was stirred at 1000 rpm for 1 h at room temperature, then the stirring was stopped and the mixture was placed in a refrigerator at 4 °C for 20 h to obtain a 2% sodium alginate-RGD solution, denoted as 2% AR solution. Following the same method, 2% sodium alginate-QK solution, 2% sodium alginate-VN solution and 2% sodium alginate-YGGFM solution were prepared, denoted as 2% AQ solution, 2% AV solution and 2% AY solution, respectively. Step B3, Microscaffold fabrication: The crosslinking solution was added to a glass dish, which was then placed directly below the nozzle of the spray freeze-drying granulation device. The distance between the nozzle and the liquid surface was adjusted to 12 cm. The peristaltic pump was started and the pressure valve was opened to make the pressure 0.02 Pa. The microscaffold preparation solution was pumped in at a rate of 15 rpm / min. The microscaffold preparation solution formed a mist droplet through the nozzle and fell into the crosslinking solution below to form microspheres. After solidification for 1 hour, the supernatant was discarded, and the product was washed three times with deionized water. The solid product was first rapidly frozen with liquid nitrogen and then freeze-dried for 3 days to obtain the microscaffold.

[0012] Furthermore, the method for preparing the activated 2% SA solution is as follows: Add 2g of purified sodium alginate to 100mL of tumor organoid basal culture medium and stir at 1000rpm for 4h at room temperature to obtain a 2% SA solution. Add 350 μL of 1X EDC / NHS activation solution to 100 mL of 2% SA solution, stir at 1000 rpm for 1 h at room temperature, and then let stand at room temperature for 3 h to obtain the activated 2% SA solution.

[0013] Furthermore, the microscaffold preparation solution is obtained by mixing 2% AR solution, 2% AQ solution, 2% AV solution and 2% AY solution in a volume ratio of 10:10:50:30 and stirring evenly at 600 rpm; the crosslinking solution is obtained by adding 60g of anhydrous calcium chloride to 1000mL of 50% ethanol solution at room temperature and filtering it through a 0.22μm filter.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a three-dimensional culture method for tumor organoids based on hydrogels and microscaffolds. The method uses hydrogels and microscaffolds as carriers for three-dimensional culture. The hydrogels and microscaffolds are prepared from natural polysaccharide sodium alginate and various synthetic peptides. The hydrogels of this invention are characterized by good stability and low strength, making them easy to handle and conducive to cell proliferation and growth. The microscaffolds of this invention have a certain adsorption capacity, allowing for rapid adsorption of cells onto the microscaffold when mixed with cell slurry. They can also rapidly adsorb growth factors and other protein substances, providing support and nutrients for long-term three-dimensional cell culture. Therefore, this invention combines hydrogels and microscaffolds to construct a three-dimensional composite culture technology system with clearly defined components and no animal origin, replacing commonly used mouse-derived Matrigel and other related products in tumor organoid culture. This provides new possibilities for in vitro culture of tumor organoids and offers an opportunity to study the occurrence and treatment of tumors. Attached Figure Description

[0015] Figure 1 This is a flowchart of the three-dimensional culture method for tumor organoids based on hydrogels and microscaffolds according to the present invention; Figure 2 These are the viscosity test results of the hydrogel prepared in Example 1 of the present invention after being placed at 2-8°C for 1, 8, and 30 days; Figure 3 These are the strength test results of the hydrogel prepared in Example 1 of the present invention after being placed at 2-8°C for 1, 8, and 30 days; Figure 4 These are particle size distribution diagrams of the microscaffolds prepared in Example 2 of the present invention after curing (without freeze-drying) and after reconstitution; Figure 5 Here is a SEM image of the lyophilized powder of the microscaffold prepared in Example 2 of this invention; Figure 6 This is a particle size distribution diagram of the freeze-dried microscaffold powder prepared in Example 2 of the present invention; Figure 7 These are the adsorption test results of the microscaffold freeze-dried powder prepared in Example 2 of the present invention; Figure 8 These are the test results of the microscaffold prepared in Example 2 of the present invention adsorbing methylene blue; Figure 9 This is a fluorescence image of the microscaffold prepared in Example 2 of the present invention after adsorbing BSA-FITC; Figure 10 This describes the proliferation of human gastric cancer organoids in a hydrogel- and microscaffold-based composite system, as shown in Example 3 of the present invention. Figure 11 This is the cell liveness and death fluorescence staining of human gastric cancer organoids in Example 3 of the present invention after 1, 4 and 7 days of culture in a composite system based on hydrogel and microscaffold; Figure 12 This is Example 4 of the present invention, showing the proliferation of human lung adenocarcinoma organoids in a composite system based on hydrogel and microscaffold; Figure 13 This is the cell liveness and death fluorescence staining of human lung adenocarcinoma organoids cultured in a hydrogel and microscaffold-based composite system for 1, 4, and 7 days in Example 4 of the present invention. Figure 14 The ATP content of the human lung adenocarcinoma organoid in the hydrogel and microscaffold-based composite system of Example 4 of the present invention; Figure 15 The results of immunohistochemical (IHC) staining of human lung adenocarcinoma organoids in a hydrogel and microscaffold-based composite system are from Example 4 of the present invention. Figure 16 The results of H&E staining of human lung adenocarcinoma organoids in a hydrogel- and microscaffold-based composite system are from Example 4 of this invention. Figure 17 This describes the proliferation of human colorectal cancer organoids in a hydrogel- and microscaffold-based composite system, as described in Example 5 of the present invention. Figure 18 The results of cell liveness and death fluorescence staining of human colorectal cancer organoids in a hydrogel and microscaffold-based composite system are from Example 5 of the present invention. Figure 19 These are the cell viability and mortality staining results of human colorectal cancer organoids in the Masturbate system on day 7. Figure 20 These are the results of Ki67 and DAPI immunofluorescence staining of human colorectal cancer organoids in the Masturbin system; Figure 21 The results of Ki67 and DAPI immunofluorescence staining of human colorectal cancer organoids in a hydrogel and microscaffold-based composite system are from Example 5 of the present invention. Figure 22 These are the results of E-cadherin and DAPI immunofluorescence staining of human colorectal cancer organoids in the Masturbin system; Figure 23The results of E-cadherin and DAPI immunofluorescence staining of human colorectal cancer organoids in a hydrogel- and microscaffold-based composite system are from Example 5 of this invention. Figure 24 These are the results of Vimentin and DAPI immunofluorescence staining of human colorectal cancer organoids in the Masturbin system; Figure 25 The results of Vimentin and DAPI immunofluorescence staining of human colorectal cancer organoids in a hydrogel and microscaffold-based composite system are from Example 5 of the present invention. Figure 26 This is the proliferation of human breast cancer organoids in a hydrogel and microscaffold-based composite system according to Example 6 of the present invention; Figure 27 The results of fluorescence staining for cell viability and mortality of human breast cancer organoids cultured in a hydrogel and microscaffold-based composite system for 1, 4, and 7 days, according to Example 6 of the present invention; Figure 28 The results of CCK8 assay on cell proliferation of human breast cancer organoids in a hydrogel and microscaffold-based composite system, as described in Example 6 of this invention. Figure 29 The results of H&E staining of human breast cancer organoids in a hydrogel and microscaffold-based composite system are from Example 6 of the present invention. Figure 30 This is the immunohistochemical (IHC) staining result of human breast cancer organoids in a hydrogel and microscaffold-based composite system, as described in Example 6 of the present invention. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0017] Example 1 This embodiment discloses a method for preparing a hydrogel, including the following steps: Step A1, Preparation of working solution and culture medium: Dissolve 4.88g of MES in 225mL of deionized water, stir until completely dissolved, add 1N NaOH to adjust the pH to 6.8, and bring the volume to 250mL with deionized water to obtain a 0.1mol / L MES solution. 0.06 g N-hydroxysuccinimide (NHS) and 0.2 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were dissolved in 20 mL of 0.1 mol / L MES solution. The solution was filtered through a 0.22 μm filter in a clean bench and then packaged into sterile reagent bottles / centrifuge tubes to obtain 100X EDC / NHS working solution. Take 1 mL of 100X EDC / NHS working solution and mix it with 99 mL of 0.1 mol / L MES solution filtered through a 0.22 μm filter to obtain 1X EDC / NHS activation solution; RGD peptide, QK peptide, VN peptide, and YGGFM peptide were dissolved in DPBS to obtain working solutions of RGD peptide, QK peptide, VN peptide, and YGGFM peptide, each with a concentration of 20 mmol / L. The sequence of the RGD peptide is GGGGRGDASSP-NH2, the sequence of the QK peptide is KLTWQELYQLKYKGI-NH2, the sequence of the VN peptide is GDCPWKPWC-NH2, and the sequence of the YGGFM peptide is YGGFM-NH2. Preparation of serum-free basal culture medium for tumor organoids: Mix the raw materials in the specified proportions to obtain the basal culture medium for tumor organoids; the basal culture medium for tumor organoids includes the following components: basal culture medium Advanced DMEM / F12, and other additives as follows: 1×B27, 2mM GlutaMAX TM 10mM HEPES, 10mM Nicotinamide, 1.5mM N-acetyl-L-cysteine, 1% Penicillin-Streptomycin, 1×N21-MAX Supplement, 100ng / mL Noggin, 500ng / mL R-Spondin 1, 10-100ng / mL EGF, 10μM Y-27632; Step A2: Preparation of polypeptide-grafted sodium alginate solution: 30g of ultrapure sodium alginate was soaked in 300mL of tumor organoid basal culture medium for 10min, then stirred at 50rpm for 30min, and then 700mL of tumor organoid basal culture medium was added. The mixture was stirred at 250rpm for 6h to obtain sodium alginate solution. Add 3500 μL of 1X EDC / NHS activation solution to 1000 mL of sodium alginate solution, stir at 250 rpm for 1 h at room temperature, and then let stand at room temperature for 3 h to obtain activated sodium alginate solution. The RGD peptide working solution was added to the activated sodium alginate solution, so that the molar ratio of RGD peptide to sodium alginate in the mixture was 1:15. The mixture was stirred at 1000 rpm for 1 hour at room temperature, and then the stirring was stopped. The solution was transferred to a capped glass reagent bottle or PET square bottle and placed in a refrigerator at 4°C for 20 hours to obtain sodium alginate-RGD solution, denoted as AR solution. Sodium alginate-QK solution, sodium alginate-VN solution and sodium alginate-YGGFM solution were prepared according to the same method, and denoted as AQ solution, AV solution and AY solution, respectively. Step A3, Hydrogel preparation: Transfer AR solution, AQ solution, AV solution, and AY solution in a volume ratio of 10:10:50:30 to a 2000mL sterile reaction flask and stir at 250rpm for 4h at 25℃ to obtain hydrogel.

[0018] Example 2 This embodiment discloses a method for preparing a microscaffold, including the following steps: Step B1, Preparation of working solution and culture medium: Dissolve 4.88g of MES in 225mL of deionized water, stir until completely dissolved, add 1N NaOH to adjust the pH to 6.8, and bring the volume to 250mL with deionized water to obtain a 0.1mol / L MES solution. 0.06 g N-hydroxysuccinimide (NHS) and 0.2 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were dissolved in 20 mL of 0.1 mol / L MES solution. The solution was filtered through a 0.22 μm filter in a clean bench and then packaged into sterile reagent bottles / centrifuge tubes to obtain 100X EDC / NHS working solution. Take 1 mL of 100X EDC / NHS working solution and mix it with 99 mL of 0.1 mol / L MES solution filtered through a 0.22 μm filter to obtain 1X EDC / NHS activation solution; RGD peptide, QK peptide, VN peptide, and YGGFM peptide were dissolved in DPBS to obtain working solutions of RGD peptide, QK peptide, VN peptide, and YGGFM peptide, each with a concentration of 20 mmol / L. The four peptide working solutions were filtered through a 0.22 μm filter and then used for further processing. The sequences of the RGD peptide, QK peptide, VN peptide, and YGGFM peptide are: GGGGRGDASSP-NH2, KLTWQELYQLKYKGI-NH2, GDCPWKPWC-NH2, and YGGFM peptide. Preparation of serum-free basal culture medium for tumor organoids: Mix the raw materials in the specified proportions to obtain the basal culture medium for tumor organoids; the basal culture medium for tumor organoids includes the following components: basal culture medium Advanced DMEM / F12, and other additives as follows: 1×B27, 2mM GlutaMAX TM 10mM HEPES, 10mM Nicotinamide, 1.5mM N-acetyl-L-cysteine, 1% Penicillin-Streptomycin, 1×N21-MAX Supplement, 100ng / mL Noggin, 500ng / mL R-Spondin 1, 10-100ng / mL EGF, 10μM Y-27632; Step B2, Preparation of polypeptide grafted with sodium alginate: Add 2g of purified sodium alginate to 100mL of tumor organoid basal culture medium and stir at 1000rpm for 4h at room temperature to obtain a 2% SA solution. Add 350 μL of 1X EDC / NHS activation solution to 100 mL of 2% SA solution, stir at 1000 rpm for 1 h at room temperature, and then let stand at room temperature for 3 h to obtain the activated 2% SA solution. The RGD peptide working solution was added to the activated 2% SA solution, so that the molar ratio of RGD peptide to sodium alginate in the mixture was 1:20. The mixture was stirred at 1000 rpm for 1 hour at room temperature, and then the stirring was stopped. The solution was transferred to a capped glass reagent bottle or PET square bottle and placed in a refrigerator at 4°C for 20 hours to obtain a 2% sodium alginate-RGD solution, denoted as 2% AR solution. Following the same method, 2% sodium alginate-QK solution, 2% sodium alginate-VN solution and 2% sodium alginate-YGGFM solution were prepared, denoted as 2% AQ solution, 2% AV solution and 2% AY solution, respectively. Step B3, Microscaffold fabrication: 2% AR solution, 2% AQ solution, 2% AV solution and 2% AY solution were mixed in a volume ratio of 10:10:50:30 and stirred evenly at 600 rpm to obtain microscaffold preparation solution; At room temperature, 60g of anhydrous calcium chloride was added to 1000mL of 50% ethanol solution and filtered through a 0.22μm filter to obtain a crosslinked solution; The crosslinking solution was added to a glass dish (23cm×10cm), and the glass dish was placed directly below the nozzle of the spray freeze-drying granulation device. The distance between the nozzle of the granulation device and the liquid surface was adjusted to 12cm. The peristaltic pump was started and the pressure valve was opened to make the pressure 0.02Pa. The microscaffold preparation solution was pumped in at a rate of 15rpm / min. The microscaffold preparation solution formed a mist droplet through the nozzle and fell into the crosslinking solution below to form microspheres. After solidification for 1 hour, the supernatant was poured off, and deionized water was added to wash three times (to remove the crosslinking solution). The solid product was first quick-frozen with liquid nitrogen and then freeze-dried for 3 days to obtain the microscaffold.

[0019] Example 3 This embodiment discloses a method for culturing human gastric cancer organoids using a three-dimensional tumor organoid culture method based on hydrogels and microscaffolds, comprising the following steps: Step 1: Using a pipette, dispense 1 mL of the hydrogel prepared in Example 1 into a 2 mL EP tube and incubate at 37°C for 30 min. Take two sterile syringes. Use one syringe to directly draw 500 μL of the rewarmed hydrogel according to the syringe mark. Use the other syringe to transfer 500 μL of tumor organoid basal culture medium from the syringe needle tip to the syringe. After purging the air from both syringes, connect them with Luer tube connectors (mother-mother) and push back and forth 18 times to mix evenly and remove air bubbles from the syringes to obtain a mixture of hydrogel and culture medium. Step 2: Transfer 1 mL of cell suspension containing 1 million human gastric cancer organoid cells to a 2 mL EP tube, add 10 μL of the microscaffold powder prepared in Example 2, pipette and blow 10 times, then centrifuge at 1500 rpm for 3 min. After centrifugation, discard most of the supernatant, leaving 100 μL of supernatant to obtain a centrifuged product containing resuspended human gastric cancer organoid cell precipitate and microscaffold. Transfer the centrifuged product to a syringe containing the mixture of hydrogel and culture medium described in Step 1, and gently push back and forth, avoiding excessive force that could damage the cells or generate air bubbles. Push back and forth 18 times to mix evenly, to obtain a composite system based on hydrogel and microscaffold and a cell mixture. Step 3: Using a 200μL wide-bore pipette tip, transfer 50μL of the hydrogel- and microscaffold-based composite system and cell mixture into a 96-well low-absorption plate; gently tilt / rotate the plate to ensure that each well is evenly covered with a layer of the hydrogel- and microscaffold-based composite system and cell mixture; seal the culture plate and transfer it to a 5°C freezer, waiting 30 minutes for a stable soft gel to form; add 100μL of tumor organoid basal culture medium to cover the surface of the soft gel, handling carefully to avoid damaging the soft gel surface; place the culture plate in an incubator for incubation, changing the medium and observing according to experimental requirements to obtain human gastric cancer organoids; if medium changes are necessary, replace approximately 80% of the top layer of culture medium from the top, avoiding touching or aspirating the soft gel layer.

[0020] Example 4 Compared with Example 3, Example 4 replaces the human gastric cancer organoid cells in the culture process with human lung adenocarcinoma organoid cells, thereby culturing human lung adenocarcinoma organoids.

[0021] Example 5 Compared with Example 3, Example 5 replaces the human gastric cancer organoid cells in the culture process with human colorectal cancer organoid cells, thereby culturing human colorectal cancer organoids.

[0022] Example 6 Compared with Example 3, Example 6 replaces the human gastric cancer organoid cells in the culture process with human breast cancer organoid cells, thereby culturing human breast cancer organoids.

[0023] Experimental Example Test 1: Testing the hydrogel prepared in Example 1: 1. Physical and chemical properties (1) pH value measurement The pH values ​​of hydrogels and tumor organoid basal culture media were tested using a Mettler Seven Excellence multi-parameter analyzer. 5 mL of hydrogel and 5 mL of tumor organoid basal culture media were placed in 15 mL centrifuge tubes, and the pH values ​​were measured using a pre-calibrated pH meter. In addition, the pH value of hydrogels was tested using precision pH test paper. 20 μL of hydrogel was dropped onto the pH test paper, and the pH range was read within 30 seconds. (2) Osmotic pressure measurement The osmotic pressure of hydrogel and tumor organoid basal culture medium was tested using an osmometer. 100 μL of hydrogel and tumor organoid basal culture medium were placed in a specific EP tube for osmotic pressure testing, and the osmotic pressure of hydrogel and tumor organoid basal culture medium was tested using a calibrated osmometer. The test results are shown in Table 1: Table 1 2. Rheological properties The rheological properties of the hydrogel were tested using a rheometer (Anton Paar, MCR102e): (1) Viscosity test Test method: Hydrogel sample preparation (take out of the refrigerator at 4℃, let stand at room temperature for 30 min, and place the hydrogel stock solution in a metal bath at 37℃ for 30 min). Turn on the rheometer and initialize it; Select the corresponding template for viscosity testing and edit it (rotation mode - time scan). Select a suitable rotor based on the sample viscosity and set zero gap; Sample loading, ready for testing; Naming, testing; Export the results and repeat the test three times; Specific operation: Turn on the air compressor of the rheometer, wait for the pressure value to reach the set value, turn on the rheometer, turn on the computer; open the rheological testing software, initialize the rheometer, select the test template (rotation mode - time scan), install the rotor (PP25, PP50), set the zero gap, load the sample, and test (the specific test conditions need to be adjusted according to the hydrogel properties), the test temperature is 37℃; Test results are as follows Figure 2 As shown, Figure 2 The viscosity test results of the hydrogel prepared in Example 1 after being placed at 2-8°C for 1, 8, and 30 days are as follows: Figure 2 As can be seen, the viscosity changes over time when placed at 2-8℃. The viscosity of the hydrogel is between 3938-4652 mPa·s, with relatively small changes, indicating that the hydrogel has relatively good stability.

[0024] (2) Strength test Test method: Hydrogel sample preparation (take out of the refrigerator at 4℃, let stand at room temperature for 30 min, and place the hydrogel stock solution in a metal bath at 37℃ for 30 min). Turn on the rheometer and initialize it; Select the corresponding template for the strength test and edit it (oscillation mode - temperature scan). Select a suitable rotor based on the sample and set zero clearance; Sample loading, ready for testing; Naming, testing; Export the results and repeat the test three times; Specific operation: Turn on the air compressor of the rheometer, wait for the pressure value to reach the set value, turn on the rheometer, turn on the computer; open the rheological testing software, initialize the rheometer, select the test template (oscillation mode - temperature scan), install the rotor (PP25), set the zero gap, load the sample, and test (the specific test conditions need to be adjusted according to the hydrogel properties), the test temperature is 25-37℃; Figure 3 The strength test results of the hydrogel prepared in Example 1 were compared after being placed at 2-8℃ for 1, 8 and 30 days. The highest storage modulus was between 4.4-6.1 Pa. The strength changed with the passage of time. After 30 days, the hydrogel tended to be stable. The hydrogel has low strength, is easy to handle, and is conducive to cell proliferation and growth.

[0025] Test 2: Performance testing of the microscaffold prepared in Example 2 1. Morphological characteristics (1) Observation of the state of microscaffold after curing: The solution of microscaffold immediately after curing (i.e., before freeze-drying the cured product) was observed and photographed using a microscope. The particle size distribution of the microscaffold was then statistically analyzed using Nano Measure and OriginPro 2024 software. (2) Observation of the state of microscaffolds after reconstitution: Weigh 3 μg of freeze-dried microscaffold powder and place it in a culture dish. Add 5 mL of tumor organoid basal culture medium. After 5 min, observe the microscaffolds in the culture dish under a microscope and take pictures (for subsequent analysis of the particle size of the microscaffolds after reconstitution). (3) State of microscaffolds after freeze-drying: Take an appropriate amount of collected freeze-dried microscaffold powder and place it in a petri dish for observation and photography; (4) Scanning electron microscope (SEM): Take an appropriate amount of collected microscaffold freeze-dried powder and place it on a conductive adhesive strip, then fix it on the sample stage, spray a gold coating on the sample for 30 seconds, and finally use SEM to observe and photograph the morphology of the microscaffold freeze-dried powder. Use Nano Measure and OriginPro 2024 software to calculate and analyze the particle size distribution of the microscaffold dry powder. Results analysis: like Figure 4 In the image, (a) shows a microscopic photograph and particle size distribution of the microscaffold immediately after solidification. Data analysis shows that the size range of the solidified microscaffold is 10-130 μm, mainly concentrated in the 10-0 μm range. (b) shows a microscopic photograph and particle size distribution of the microscaffold after resolidification. Data analysis shows that the size range of the resolidified microscaffold is 20-220 μm, mainly concentrated in the 20-100 μm range. In comparison, the particle size of the resolidified microscaffold is basically doubled compared to that of the immediately solidified microscaffold, making it suitable for three-dimensional cell culture. like Figure 5 The surface structure of the freeze-dried microscaffolds was investigated by SEM. Figures (a) and (b) show that the freeze-dried powder of the microscaffolds attached to the conductive adhesive was well dispersed and did not stick together. The surface of two microscaffolds was magnified and observed. It can be seen that the surface of the freeze-dried microscaffolds was uneven and a large number of wrinkles appeared on some surfaces. like Figure 5 Based on the scanning electron microscope (SEM) observations, the particle size of the lyophilized microscaffold powder was analyzed and calculated using Nano Measure software, and the length of the lyophilized powder particles was statistically analyzed. Figure 6 (a) and (b) show that the size range of the freeze-dried microscaffold powder is 5-90μm, mainly concentrated in the range of 5-50μm, and the size is basically consistent with the particle length of the microscaffold after curing.

[0026] 2. BET Analysis like Figure 7BET testing was used to obtain data and curves related to the pore structure, i.e. Figure 8 (a), Figure 8 The adsorption amount in (a) is negative, which is presumably due to the small specific surface area of ​​the sample, resulting in a large area of ​​negative adsorption amount of nitrogen. Negative adsorption amount generally indicates that the sample is non-porous, that is, the number of 0-2nm micropores and 2-50nm mesopores is very small, or the sample is a material mainly composed of macropores above 100nm. The mass, density, pore volume, and pore size of the microscaffold are integrated into a structure like... Figure 8 (b): The mass of the microscaffold freeze-dried powder is 0.0774 g, and the density is 1.000 g / cm³. 3 The average adsorption pore volume is 0.002476 cm³. 3 / g, with an average pore size of 8.1564nm and an average mesopore size of 104.6246nm; Porosity can be calculated using the following formula: Porosity = Pore volume / (Sample volume + Pore volume) = Pore volume / (Sample mass / Density + Pore volume) * 100% The calculated porosity of the microscaffold is 3.1%.

[0027] 3. Adsorption performance test To investigate the adsorption capacity of the microscaffolds, methylene blue (MB) and bovine serum albumin (BSA-FITC) were selected as model drugs to study the adsorption behavior of the microscaffolds. Preparation of MB standard solution: Weigh 30 mg of MB powder into a 15 ml centrifuge tube, add 10 mL of tumor organoid basal culture medium, and wait for dissolution to complete to obtain a 3 mg / mL MB solution. Then dilute the 3 mg / mL MB solution to 15 mg / L for later use. First, take 100 μL, 95 μL, 90 μL, 85 μL, 80 μL, 75 μL, 70 μL, 65 μL, 60 μL, 55 μL, and 50 μL of the 3 mg / mL MB solution into a 96-well plate, and then add 0 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, and 50 μL of tumor organoid basal culture medium in sequence and mix well to obtain a series of MB solutions with different concentrations. Determination of the standard curve: A 96-well plate containing MB solutions of varying concentrations was placed in a microplate reader. The absorbance (A) was measured within the wavelength range of 500-780 nm. A standard working curve was plotted based on the relationship between concentration and absorbance. To ensure the accuracy of the results, a linear regression method was used, employing OriginPro 2024. The relationship between absorbance and MB concentration was obtained based on the correction mechanism. MB adsorption process: Weigh 3 microscaffolds and place them in 5ml centrifuge tubes. Add 15mg / L MB solution to prepare microscaffold reconstitution solutions of 1mg / mL, 2mg / mL and 4mg / mL. Then, pipette the solutions evenly (pipette 2-3 times) and place them at room temperature. Remove the corresponding centrifuge tubes at 5min, 1h and 3h respectively, aspirate the supernatant, and test the UV absorbance in the range of 500-780nm. The concentrations of the control group and the experimental group can be calculated from the regression curve, and the adsorption content can be calculated using the adsorption capacity formula. Adsorption capacity (%) = (Concentration of control group - Concentration of experimental group) / Concentration of control; Fluorescent bovine serum albumin (BSA-FITC, 5 mg / mL) adsorption process: First, weigh 1.5 mg of microscaffold and place it in a 5 mL centrifuge tube, add 1.35 mL of culture medium, and then add 150 μL of BSA-FITC solution in the dark. Use a pipette to mix the microscaffold and solution evenly (pipette 2-3 times), and then let it stand at room temperature in the dark for 2.5-3 h. After the reaction is complete, aspirate the supernatant to elute the microscaffold, and observe the adsorption of the microscaffold using a fluorescence microscope. Results analysis: such as Figure 8 In the figure, (ab) are the standard curve and adsorption curve of MB; (c) are the adsorption photos of the blank group and the experimental group. It can be clearly observed that the bottom microscaffold is darker in color after adsorbing MB, and the supernatant of the experimental group is lighter in color than that of the blank group; (d) is the adsorption curve. The adsorption amount reaches the maximum of 38.19% at 5 min, 28.23% at 1 h, and 23.87% at 3 h. The microscaffold reaches the maximum adsorption amount at 5 min. This is because the osmotic pressure of the culture medium is relatively high. During the swelling process, the microscaffold has the greatest adsorption force on MB. Basically, the microscaffold has reached the swelling equilibrium at 5 min. This result was confirmed during the reconstitution observation. After the swelling equilibrium, the MB adsorbed by the microscaffold by physical adsorption begins to gradually diffuse into the solution. At this time, the adsorption amount begins to decrease. This characteristic is beneficial to the rapid adsorption of cells onto the microscaffold when the microscaffold and cell fluid are mixed. like Figure 9 This is a fluorescence image of the microscaffold after bovine serum albumin (BSA)-FITC has been adsorbed onto it. Figure 9 It can be observed that most of the microscaffold particles have high fluorescence intensity and obvious adsorption of bovine serum albumin (BSA-FITC). The fluorescence results show that the microscaffolds have the ability to adsorb macromolecules. Qualitative and quantitative analyses of the above adsorption experiments lead to the conclusion that the microscaffold has a certain adsorption capacity and a relatively fast adsorption rate. It can adsorb protein substances such as growth factors, providing support and nutrients for long-term three-dimensional cell culture.

[0028] Test 3: Detection of human gastric cancer organoids prepared in Example 3 1. Morphological observation of human gastric cancer organoids The proliferation of human gastric cancer organoids after 1, 4, and 7 days of three-dimensional culture in a hydrogel- and microscaffold-based composite system is shown in the figure. Figure 10 As shown, images were acquired using three objective lenses: 4x, 10x, and 20x. Figure 10 (A, D, G) are images acquired with a 4x objective lens. Figure 10 (A, B, C) are images collected after one day of cultivation. Figure 10 (B, E, H) are images acquired with a 10x objective lens. Figure 10 (D, E, F) are images collected after 4 days of cultivation. Figure 10 (C, F, I) are images acquired with a 20x objective lens. Figure 10 (G, H, I) are images collected after 7 days of cultivation; The results showed that the proliferation rate of human gastric cancer organoids cultured in a three-dimensional system based on hydrogel and microscaffolds increased with the extension of culture time, and the size and volume of the organoids showed the same growth trend.

[0029] 2. Detection of cell viability in human gastric cancer organoids To further assess the cell viability of human gastric cancer organoids in a hydrogel- and microscaffold-based composite system, we used a cell viability and cytotoxicity assay kit to perform fluorescence staining analysis on the cell viability and death of human gastric cancer organoids in the hydrogel- and microscaffold-based composite system. The results of fluorescence staining analysis on the cell viability and death of human gastric cancer organoids after 1, 4, and 7 days of three-dimensional culture in the hydrogel- and microscaffold-based composite system are as follows: Figure 11 As shown, Figure 11 (A, D, G) Green fluorescence is due to staining with calcein acetoxymethyl ester. Figure 11 (B, E, H) The red fluorescence is due to propidium iodide staining. Figure 11 (A, B, C) are the staining results after 1 day of incubation. Figure 11 (D, E, F) are the staining results after 4 days of culture. Figure 11 (G, H, I) represent the staining results after 7 days of culture; The results from confocal microscopy showed that the number of live cells in human gastric cancer organoids far exceeded the number of dead cells, indicating that the cells in the human gastric cancer organoids exhibited good cell viability after 1, 4, and 7 days of three-dimensional culture in a hydrogel- and microscaffold-based composite system, and that the human gastric cancer organoids showed a significant proliferation rate; combined with Figure 9 The bright-field observation results of the inverted optical microscope can prove that the three-dimensional culture of human gastric cancer organoids is supported in the composite system based on hydrogel and microscaffold. The three-dimensional culture method of tumor organoids based on hydrogel and microscaffold according to the present invention can realize the culture of human colorectal cancer organoids.

[0030] Test 4: Detection of human lung adenocarcinoma organoids prepared in Example 4 1. Morphological observation of human lung adenocarcinoma organoids The proliferation of human lung adenocarcinoma organoids after 1, 4, and 7 days of three-dimensional culture in a hydrogel- and microscaffold-based composite system is shown in the figure. Figure 12 As shown, images were acquired using three objective lenses: 4x, 10x, and 20x. Figure 12 (A, D, G) are images acquired with a 4x objective lens. Figure 12 (A, B, C) are images collected after one day of cultivation. Figure 12 (B, E, H) are images acquired with a 10x objective lens. Figure 12 (D, E, F) are images collected after 4 days of cultivation. Figure 12 (C, F, I) are images acquired with a 20x objective lens. Figure 12 (G, H, I) are images collected after 7 days of cultivation; The results showed that the proliferation rate of human lung adenocarcinoma organoids increased with prolonged culture time, and the size of the organoids exhibited the same growth trend. Figure 12 As can be seen from -I, the maximum size of organoids can reach about 200μm, and the volume of most organoids is about 100μm.

[0031] 2. Detection of cell viability in human lung adenocarcinoma organoids To further detect cell viability in human lung adenocarcinoma organoids within a hydrogel- and microscaffold-based composite system, human lung adenocarcinoma organoids were cultured in the composite system for 1, 4, and 7 days. We then performed live / dead fluorescence staining analysis on the organoids using a cell viability and cytotoxicity assay kit. Calcein AM, a cell staining reagent capable of fluorescently staining live cells, is an additive to Calcein (calcein) with the addition of an acetoxymethyl ester (AM) group, enhancing its hydrophobicity and allowing it to easily penetrate cell membranes. Calcein AM itself is non-fluorescent; after entering the cell, it is hydrolyzed by endogenous esterases in the living cell to generate a strongly negatively charged, non-membrane-permeable polar molecule, Calcein, which is then retained within the cell. Calcein emits strong green fluorescence. Because dead cells lack esterases or have very low esterase activity, Calcein... Once AM enters the cell, live cells containing esterases can produce Calcein, while dead cells cannot or produce very little Calcein. Therefore, only live cells are stained with strong green fluorescence, while dead cells are not stained or are stained very weakly. Propidium iodide (PI), a red fluorescent dye for nucleic acids, cannot penetrate the cell membrane of live cells and can only stain dead cells whose cell membrane integrity has been disrupted. Therefore, the combined use of Calcein AM and PI provides dual fluorescent staining of both live and dead cells for the detection of cell viability and cytotoxicity. Test results as follows Figure 13 As shown; Figure 13 (A, D, G) The green fluorescence is due to staining with Calcein AM (calcein acetoxymethyl ester). Figure 13 (B, E, H) The red fluorescence is due to propidium iodide (PI) staining. Figure 13 (A, B, C) show the cell viability and mortality staining results after 1 day of culture. Figure 13 (D, E, F) show the cell viability staining results after 4 days of culture. Figure 13 (G, H, I) represent the cell viability and mortality staining results after 7 days of culture; like Figure 13 As shown in the confocal microscopy results, there are far more live cells than dead cells in the lung adenocarcinoma organoids. The number and volume of surviving organoids both show an increasing trend with the extension of culture time. This indicates that the composite system based on hydrogel and microscaffold is suitable for the three-dimensional culture of lung adenocarcinoma organoids. During the culture process, live cells account for the majority of human lung adenocarcinoma organoids, the size of the tumor organoids shows an increasing trend, and the lung adenocarcinoma organoids exhibit a good proliferation rate.

[0032] 3. Detection of relative ATP content in human lung adenocarcinoma organoid cells during proliferation. Cell viability was assessed by measuring intracellular ATP levels using a chemiluminescence immunoassay kit. ATP, as the most important energy molecule, plays a crucial role in various physiological and pathological processes of cells. It is an important indicator of cellular metabolism and a key marker of metabolically active cells, exhibiting a strong linear relationship with the number of viable cells. Therefore, ATP levels effectively reflect the number of viable cells, allowing for cell counting or viability assessment using this kit. The relative ATP levels of human lung adenocarcinoma organoids cultured in a hydrogel- and microscaffold-based composite system for 1, 4, and 7 days were measured using a chemiluminescence ATP assay kit to study the proliferation of these organoids. The results of the assay after 1, 4, and 7 days of culture in the hydrogel- and microscaffold-based composite system are shown below. Figure 14 As shown, the absorbance values ​​measured by the ELISA reader indicate that the relative ATP content of lung adenocarcinoma organoids increases in a time-dependent manner. Furthermore, a significance analysis of the measured values ​​showed that the relative ATP content on days 4 and 7 of culture was significantly higher than that on day 1, exhibiting an increasing trend. Figure 12 , Figure 13 The results are consistent with those in the previous section.

[0033] 4. Immunohistochemical (IHC) and H&E staining analysis of human lung adenocarcinoma organoids To further evaluate the histological characteristics of human lung adenocarcinoma organoids in a hydrogel- and microscaffold-based composite system, the same human lung adenocarcinoma organoid cells were simultaneously cultured in three dimensions for 10 days in both the hydrogel- and microscaffold-based composite system and the conventionally used Matrix system. After fixation with paraformaldehyde in both systems, the cultured human lung adenocarcinoma organoids were subjected to immunohistochemical (IHC) staining. The results are as follows: Figure 15 As shown, Figure 15 (A, B, C) are the staining results of human lung adenocarcinoma organoids in the Matrix Gel system. Figure 15 (D, E, F) show the staining results of human lung adenocarcinoma organoids in a hydrogel- and microscaffold-based composite system. Figure 15 (A, D) are the results of antibody CK7 staining. Figure 15 (B, E) are the results of Naspin A staining. Figure 15 (C, F) show the TTF1 staining results. According to the IHC staining results, the expression of three antibodies CK7, Naspin A and TTF1 in human lung adenocarcinoma organoids in the matrix gel system and the hydrogel and microscaffold-based composite system are positive, positive and weakly positive, respectively. This indicates that the biological characteristics of human lung adenocarcinoma organoids cultured in the hydrogel and microscaffold-based composite system are consistent with those in the traditional matrix gel system. Finally, hematoxylin-eosin (H&E) staining analysis was performed. Hematoxylin is a basic dye that primarily stains acidic substances in the cell nucleus (such as DNA and RNA) blue-violet, while eosin is an acidic dye that primarily stains basic proteins in the cytoplasm and extracellular matrix pink. Figure 16 A shows the H&E staining of human lung adenocarcinoma organoids after culture in the Mastearin system. Figure 16 B shows the H&E staining results of human lung adenocarcinoma organoids cultured in a hydrogel- and microscaffold-based composite system. The above data further indicate that the human lung adenocarcinoma organoids cultured in the hydrogel- and microscaffold-based composite system of the present invention are not significantly different in histological morphology from those cultured in matrix gel, and have the histological characteristics of tumor organoids. In summary, the results show that the hydrogel and microscaffold-based composite system of the present invention can effectively support the survival and proliferation of human lung adenocarcinoma organoids, and its effect on culturing human lung adenocarcinoma organoids is consistent with that of commercially available mouse-derived matrix gel products. The immunohistochemical and H&E staining results of human lung adenocarcinoma organoids cultured in the two systems are also consistent.

[0034] Test 5: Detection of human colorectal cancer organoids prepared in Example 5 1. Morphology of human colorectal cancer organoids The proliferation of human colorectal cancer organoids after 1, 4, and 7 days of three-dimensional culture in a hydrogel- and microscaffold-based composite system is compared as follows: Figure 17 As shown, images were acquired using three objective lenses: 4x, 10x, and 20x, from an inverted optical microscope. Figure 17 (AI) Proliferation of human colorectal cancer organoids after 1, 4, and 7 days of culture in a microscaffold / hydrogel 3D system. Figure 17 (A, B, C) are images acquired with a 4x objective lens. Figure 17 (A, D, G) are images collected after 1 day of cultivation. Figure 17 (D, E, F) are images acquired with a 10x objective lens. Figure 14 (B, E, H) are images collected after 4 days of cultivation. Figure 17 (G, H, I) are images acquired with a 20x objective lens. Figure 17 (C, F, I) are images collected after 7 days of cultivation. Figure 17 (JR) Proliferation of human colorectal cancer organoids after 1, 4, and 7 days of culture in a Matrigel 3D system. Figure 17 (J, K, L) are images acquired with a 4x objective lens. Figure 17 (J, M, P) are images collected after 1 day of cultivation. Figure 17 (M, N, O) are images acquired with a 10x objective lens. Figure 17(K, N, Q) are images collected after 4 days of cultivation. Figure 17 (P, Q, R) are images acquired with a 20x objective lens. Figure 17 (L, O, R) are images collected after 7 days of culture; The results showed that the proliferation rate of human colorectal cancer organoids in three-dimensional culture in a hydrogel- and microscaffold-based composite system increased with the extension of culture time, and the size of the human colorectal cancer organoids showed the same growth trend, which is consistent with the proliferation in commercially available mouse-derived matrix gel systems.

[0035] 2. Detection of cell viability in human colorectal cancer organoids To further assess the cell viability of colorectal cancer organoids, we used a cell viability and cytotoxicity assay kit to perform fluorescence staining analysis on human colorectal cancer organoids cultured in a hydrogel- and microscaffold-based composite system for 1, 4, and 7 days to determine cell viability and death. The results are as follows: Figure 18 As shown: Figure 18 (A, D, G) The green fluorescence is due to staining with Calcein AM (calcein acetoxymethyl ester). Figure 18 (B, E, H) The red fluorescence is due to propidium iodide (PI) staining. Figure 18 (A, B, C) show the cell viability and mortality staining results after 1 day of culture. Figure 18 (D, E, F) show the cell viability staining results after 4 days of culture. Figure 18 (G, H, I) represent the cell viability staining results after 7 days of culture. The results of human colorectal cancer organoid culture in the Matter gel system after 7 days are shown below. Figure 19 As shown; The results from confocal microscopy show that there are far more live cells than dead cells in the colorectal cancer organoids, indicating that live cells make up the majority of the human colorectal cancer organoids in the hydrogel- and microscaffold-based composite system. The size of the colorectal cancer organoids shows an increasing trend, exhibiting a good proliferation rate, which proves that the hydrogel- and microscaffold-based composite system of the present invention supports three-dimensional culture of human colorectal cancer organoids.

[0036] 3. Immunofluorescence staining identification and result analysis of human colorectal cancer organoids To further evaluate the expression of relevant biomarkers Ki67, E-cadherin, and Vimentin in human colorectal cancer organoids in a hydrogel- and microscaffold-based composite system, immunofluorescence staining analysis was performed. The same human colorectal cancer organoid cells were simultaneously cultured in three dimensions for 10 days in both the hydrogel- and microscaffold-based composite system and the conventionally used Matrice system. After the human colorectal cancer organoids cultured in both systems were fixed with paraformaldehyde, immunofluorescence staining analysis of relevant biomarkers Ki67, E-cadherin, and Vimentin was performed on the colorectal cancer organoids. Figure 20 The staining results of colorectal cancer organoids after culture in Matrigel. Figure 21 The staining results of colorectal cancer organoids after culture in Matrigel are shown. Figure 20 A and 21A blue fluorescence represent the nuclei of live cells stained with DAPI. Figure 20 B and 21B green fluorescence represent cell nuclei stained with Ki67 during proliferation. According to the immunofluorescence staining results, human colorectal cancer organoids exhibit good proliferative activity in both the matrix gel system and the hydrogel- and microscaffold-based composite system, proving that the hydrogel- and microscaffold-based composite system can effectively support the survival and proliferation of human colorectal cancer organoids. Figure 22 The results of E-cadherin immunofluorescence staining of colorectal cancer organoids after culture in Mastiff. Figure 23 The images show the E-cadherin immunofluorescence staining results of colorectal cancer organoids cultured in a hydrogel- and microscaffold-based composite system. Figure 22 A and 23A blue fluorescence indicate cell nuclei stained with DAPI. Figure 22 B and 23B green fluorescence are E-cadherin staining. According to the immunofluorescence staining results, human colorectal cancer organoids can express E-cadherin in both hydrogel-based and microscaffold-based composite systems, and there is no significant difference between the two systems. This indicates that the biological characteristics of human colorectal cancer organoids cultured in the hydrogel-based and microscaffold-based composite system are consistent with those in the traditional Matrices system. Figure 24 The images show the immunofluorescence staining results of vimentin antibody on colorectal cancer organoids after culture in Mastiff. Figure 25 The results of immunofluorescence staining with the antibody Vimentin are shown for colorectal cancer organoids cultured in a hydrogel- and microscaffold-based composite system. Figure 24 A. 25A blue fluorescence indicates cell nuclei stained with DAPI. Figure 24B. 25B green fluorescence indicates Vimentin staining. According to the immunofluorescence staining results, human colorectal cancer organoids can express Vimentin in both the hydrogel- and microscaffold-based composite system and the Matricel system, and there is no significant difference between the two systems. This shows that the biological characteristics of colorectal cancer organoids cultured in the hydrogel- and microscaffold-based composite system are consistent with those in the traditional Matricel system.

[0037] Test 6: Detection of human breast cancer organoids prepared in Example 6 1. Morphology of human breast cancer organoids The proliferation of human breast cancer organoids after 1, 4, and 7 days of three-dimensional culture in a hydrogel- and microscaffold-based composite system is compared as follows: Figure 26 As shown, images were acquired using 4x, 10x, and 20x objective lenses from an inverted optical microscope. Figure 26 (A, D, G) are images acquired with a 4x objective lens. Figure 26 (A, B, C) are images collected after one day of cultivation. Figure 26 (B, E, H) are images acquired with a 10x objective lens. Figure 26 (D, E, F) are images collected after 4 days of cultivation. Figure 26 (C, F, I) are images acquired with a 20x objective lens. Figure 26 (G, H, I) are images collected after 7 days of cultivation; The results showed that the proliferation rate of human breast cancer organoids cultured in three dimensions in a hydrogel- and microscaffold-based composite system increased with the extension of culture time, and the size of the organoids showed the same growth trend.

[0038] 2. Human breast cancer organoid cell viability detection To further assess the cell viability of human breast cancer organoids cultured in a hydrogel- and microscaffold-based composite system, we performed live-and-death fluorescence staining analysis on human breast cancer organoids cultured in the three-dimensional hydrogel- and microscaffold-based composite system using a cell viability and cytotoxicity assay kit. The results of human breast cancer organoids cultured in the hydrogel- and microscaffold-based composite system for 1, 4, and 7 days are shown below. Figure 27 As shown, Figure 27 (A, D, G) The green fluorescence is due to staining with Calcein AM (calcein acetoxymethyl ester). Figure 27 (B, E, H) The red fluorescence is due to propidium iodide (PI) staining. Figure 27 (A, B, C) are the cell viability and mortality staining results of human breast cancer organoids after 1 day of three-dimensional culture in a hydrogel- and microscaffold-based composite system. Figure 27 (D, E, F) are the cell viability staining results of human breast cancer organoids after 4 days of three-dimensional culture in a hydrogel- and microscaffold-based composite system. Figure 27(G, H, I) are the cell viability and death staining results of human breast cancer organoids after 7 days of three-dimensional culture in a hydrogel and microscaffold-based composite system; The results from confocal microscopy show that the number of live cells in human breast cancer organoids far exceeds the number of dead cells, indicating that human breast cancer organoids can survive well in the hydrogel- and microscaffold-based composite system and exhibit a good proliferation rate.

[0039] 3. CCK8 assay for the proliferation of human breast cancer organoid cells The relative CCK8 content of human breast cancer organoids in a hydrogel- and microscaffold-based composite system was determined using a cell CCK8 assay kit. The results of the assay after 1, 4, and 7 days of culture in the hydrogel- and microscaffold-based composite system are shown below. Figure 28 As shown; based on the absorbance values ​​measured by the ELISA reader, it can be seen that the relative CCK8 content of breast cancer organoids increased in a time-dependent manner after culturing in the hydrogel-microscaffold-based composite system for 1, 4, and 7 days. Furthermore, a significance analysis of the measured values ​​showed that the relative content of CCK8 on days 4 and 7 was significantly increased compared to day 1, exhibiting an increasing trend. Figure 26 , Figure 27 The results are consistent with those in the previous studies; this demonstrates that the composite system based on hydrogels and microscaffolds supports three-dimensional culture of human breast cancer organoids.

[0040] 4. H&E and IHC staining analysis of human breast cancer organoids To further evaluate the histological characteristics of human breast cancer organoids in a hydrogel- and microscaffold-based composite system, we performed hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis. The H&E staining results are shown below. Figure 29 As shown, the staining results indicate that human breast cancer organoids possess the histological characteristics of tumor organoids in a hydrogel- and microscaffold-based composite system. Figure 30 IHC staining results of human breast cancer organoids in a hydrogel- and microscaffold-based composite system. Figure 30 A shows the immunohistochemical staining results for antibody-mediated cytokeratin 5 / 6 (CK5 / 6). Ki-67 is a nuclear protein associated with cell proliferation and is present in the nuclei of all actively proliferating cells. Figure 30 B represents the results of Ki67 immunohistochemical staining. A positive progesterone receptor (PR) result indicates that tumor growth may be influenced by progesterone. Figure 30 C represents the immunohistochemical staining results of antibody PR. All three antibodies showed positive results, indicating that the composite system based on hydrogel and microscaffold can effectively support the growth of human breast cancer organoids and maintain their histological characteristics.

[0041] In summary, the data above indicate that a composite matrix and its composite system based on hydrogels and microscaffolds, using natural polysaccharide sodium alginate and various synthetic peptides as raw materials, can be prepared to create a new carrier for three-dimensional culture of tumor organoids. This system can construct a three-dimensional composite culture technology system with well-defined components and no animal origin, which can be used to replace the mouse-derived Matrigel and other related products commonly used in tumor organoid culture.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds, characterized in that, Includes the following steps: Step 1: Mix the hydrogel with the basic culture medium for tumor organoids to obtain a mixture of hydrogel and culture medium; Step 2: Mix the cell suspension containing tumor organoid cells with the microscaffold, centrifuge, and mix the centrifuged product with the mixture of hydrogel and culture medium from Step 1 to obtain a composite system based on hydrogel and microscaffold and a cell mixture. Step 3: Place the hydrogel- and microscaffold-based composite system and cell mixture at low temperature to form a soft gel; cover the surface of the soft gel with tumor organoid basal culture medium, and culture. During the culture process, change the medium and observe according to the experimental requirements to obtain tumor organoids.

2. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 1, characterized in that, In step one, the preparation method of the mixture of hydrogel and culture medium is as follows: use a pipette to dispense 1 mL of hydrogel into a 2 mL EP tube and place it in a 37°C incubator for 30 min to rewarm; take two sterile syringes, use one syringe to directly draw 500 μL of the rewarmed hydrogel according to the syringe scale, and use the other syringe to transfer 500 μL of tumor organoid basal culture medium from the syringe needle tip to the syringe. After expelling air from both syringes, connect them with Luer tube connectors and push back and forth 15-20 times to mix evenly.

3. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 1, characterized in that, In step two, the preparation method of the hydrogel- and microscaffold-based composite system and cell mixture is as follows: Transfer 1 mL of cell suspension containing 100,000 to 1,000,000 tumor organoid cells to a 2 mL EP tube, add 10 μL of microscaffold, pipette and vortex 10 times, then centrifuge at 1500 rpm for 3 min. After centrifugation, discard most of the supernatant, leaving 100 μL of supernatant to obtain a centrifuged product containing resuspended tumor organoid cell precipitate and microscaffold. Transfer the centrifuged product to a syringe containing the mixture of hydrogel and culture medium described in step one, and gently push back and forth 15-20 times to mix thoroughly.

4. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 1, characterized in that, In step three, the method for preparing the tumor organoids is as follows: Transfer 50 μL of the hydrogel- and microscaffold-based composite system and cell mixture into a 96-well low-adsorption plate using a 200 μL wide-bore pipette tip; gently tilt / rotate the plate to ensure that each well is uniformly covered with a layer of the hydrogel- and microscaffold-based composite system and cell mixture; seal the culture plate and transfer it to a refrigerator at 2-8°C for 30 min to allow a stable soft gel to form. Add 100 μL of tumor organoid basal culture medium to cover the surface of the soft gel; place the culture plate in an incubator for culture, and change the medium and observe according to the experimental requirements.

5. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 1, characterized in that, In step one, the basal culture medium for tumor organoids comprises the following components: basal culture medium AdvancedDMEM / F12, and other additives as follows: 1×B27, 2mM GlutaMAX. TM 10mM HEPES, 10mM Nicotinamide, 1.5mM N-acetyl-L-cysteine, 1% Penicillin-Streptomycin, 1×N21-MAX Supplement, 100ng / mL Noggin, 500ng / mL R-Spondin 1, 10-100ng / mL EGF, 10μM Y-27632.

6. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 1, characterized in that, In step one, the method for preparing the hydrogel includes the following steps: Step A1, Preparation of working solutions: Prepare 1X EDC / NHS activation solution, RGD peptide working solution, QK peptide working solution, VN peptide working solution and YGGFM peptide working solution respectively; the sequence of the RDG peptide is GGGGRGDASSP-NH2, the sequence of the QK peptide is KLTWQELYQLKYKGI-NH2, the sequence of the VN peptide is GDCPWKPWC-NH2, and the sequence of the YGGFM peptide is YGGFM-NH2; Step A2: Preparation of polypeptide-grafted sodium alginate solution: The RGD peptide working solution was added to the activated sodium alginate solution, so that the molar ratio of RGD peptide to sodium alginate in the mixture was in the range of 1:15 to 1:

20. The mixture was stirred at 1000 rpm for 1 hour at room temperature, then the stirring was stopped and the mixture was placed in a refrigerator at 4°C for 20 hours to obtain sodium alginate-RGD solution, denoted as AR solution. Following the same method, sodium alginate-QK solution, sodium alginate-VN solution and sodium alginate-YGGFM solution were prepared, denoted as AQ solution, AV solution and AY solution, respectively. Step A3, Hydrogel preparation: Transfer AR solution, AQ solution, AV solution and AY solution into a 2000mL sterile reaction bottle at a volume ratio of 10:10:50:30, and stir at 250rpm for 4h at 25℃ to obtain hydrogel.

7. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 6, characterized in that, The method for preparing the activated sodium alginate solution: 30g of ultrapure sodium alginate was soaked in 300mL of tumor organoid basal culture medium for 10min, then stirred at 50rpm for 30min, and then 700mL of tumor organoid basal culture medium was added. The mixture was stirred at 250rpm for 6h to obtain sodium alginate solution. Add 3500 μL of 1X EDC / NHS activation solution to 1000 mL of sodium alginate solution, stir at 250 rpm for 1 h at room temperature, and then let stand at room temperature for 3 h to obtain activated sodium alginate solution.

8. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 1, characterized in that, In step two, the method for preparing the microscaffold includes the following steps: Step B1, Preparation of working solutions: Prepare 1X EDC / NHS activation solution, RGD peptide working solution, QK peptide working solution, VN peptide working solution and YGGFM peptide working solution respectively; the sequence of the RDG peptide is GGGGRGDASSP-NH2, the sequence of the QK peptide is KLTWQELYQLKYKGI-NH2, the sequence of the VN peptide is GDCPWKPWC-NH2, and the sequence of the YGGFM peptide is YGGFM-NH2; Step B2, Preparation of polypeptide grafted with sodium alginate: The RGD peptide working solution was added to the activated 2% SA solution, so that the molar ratio of RGD peptide to sodium alginate in the mixture was in the range of 1:15 to 1:

20. The mixture was stirred at 1000 rpm for 1 h at room temperature, then the stirring was stopped and the mixture was placed in a refrigerator at 4 °C for 20 h to obtain a 2% sodium alginate-RGD solution, denoted as 2% AR solution. Following the same method, 2% sodium alginate-QK solution, 2% sodium alginate-VN solution and 2% sodium alginate-YGGFM solution were prepared, denoted as 2% AQ solution, 2% AV solution and 2% AY solution, respectively. Step B3, Microscaffold fabrication: The crosslinking solution was added to a glass dish, which was then placed directly below the nozzle of the spray freeze-drying granulation device. The distance between the nozzle and the liquid surface was adjusted to 12 cm. The peristaltic pump was started and the pressure valve was opened to make the pressure 0.02 Pa. The microscaffold preparation solution was pumped in at a rate of 15 rpm / min. The microscaffold preparation solution formed a mist droplet through the nozzle and fell into the crosslinking solution below to form microspheres. After solidification for 1 hour, the supernatant was discarded, and the product was washed three times with deionized water. The solid product was first rapidly frozen with liquid nitrogen and then freeze-dried for 3 days to obtain the microscaffold.

9. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 8, characterized in that, The method for preparing the activated 2% SA solution: Add 2g of purified sodium alginate to 100mL of tumor organoid basal culture medium and stir at 1000rpm for 4h at room temperature to obtain a 2% SA solution. Add 350 μL of 1X EDC / NHS activation solution to 100 mL of 2% SA solution, stir at 1000 rpm for 1 h at room temperature, and then let stand at room temperature for 3 h to obtain the activated 2% SA solution.

10. The method for three-dimensional culture of tumor organoids based on hydrogels and microscaffolds according to claim 8, characterized in that, The microscaffold preparation solution is obtained by mixing 2% AR solution, 2% AQ solution, 2% AV solution and 2% AY solution in a volume ratio of 10:10:50:30 and stirring evenly at a speed of 600 rpm. The crosslinking solution was obtained by adding 60g of anhydrous calcium chloride to 1000mL of 50% ethanol solution at room temperature and filtering it through a 0.22μm filter.

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