Photocurable hydrogel capable of binding cytokines for tumor organoid culture and its application

Abstract

The present invention discloses a photocurable hydrogel that can bind cytokines and is used for culturing tumor organoids, and its application, and belongs to the technical field of organoid culture. The present invention develops a hydrogel that can fix cells, bind cytokines, and achieve organoid recovery by using norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP, and photoinitiator. Organoids can be directly prepared using this hydrogel, and a variety of structural heparins can be used, which reduces costs. Modified heparin can bind factors or matrix proteins and controllably release factors. Modified gelatin provides amino acid energy for collagen hydrolysate. The hydrogel is formed by photocuring in a step-by-step polymerization manner, and has a controllable structure and adjustable biomimetic modulus and adjustable biomimetic stress relaxation. The hydrogel of the present invention can be used for the cultivation of a variety of organoids, overcoming the problems of low repeatability and instability of current technologies, and is suitable for fields such as tumor drug evaluation.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and in particular relates to a photocurable hydrogel capable of binding to cytokines for use in tumor organoid culture and its application. Background Art

[0002] Organoids are three-dimensional cultures formed by culturing precursor cells or stem cells derived from human tissue. These 3D cultures possess cellular composition, structure, morphology, and function similar to real human organs and can be cultured long-term. Over the past 15 years, organoid technology has rapidly advanced, with the creation of normal and tumor organoids from the intestine, stomach, heart, brain, liver, lung, kidney, and breast. The extracellular matrix (ECM) required for organoid culture is a hot topic in research and development within this field.

[0003] Traditional organoid culture relies on Matrigel, but its composition is complex and batch-to-batch unstable. Furthermore, because it contains many aqueous-insoluble components, the product is difficult to use in a freeze-dried form, hindering transportation and storage. Negatively charged glycosaminoglycans and positively charged proteoglycans, the main components of the ECM, form hydrogels through electrostatic interactions. The charges on their side chains maintain the osmotic pressure required for cell growth and facilitate the delivery of growth factors. Heparin, a naturally occurring glycosaminoglycan with a high negative charge, can bind and stabilize a wide range of growth factors, such as fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF). Gelatin, a natural polymer derived from collagen, contains numerous reactive functional groups, including amino, carboxyl, and hydroxyl groups. Type A gelatin, derived from acid treatment, has an isoelectric point between 6 and 9. During cell culture, the pH of the culture medium is around 6-7, and the amino groups in gelatin are protonated, exhibiting positive charge behavior. However, current gel systems still have limitations. For example, WO2017198258A1 discloses a heparin-polyethylene glycol system, in which all the heparin used is standard heparin, which is relatively expensive. Furthermore, the hydrogel formation process is too rapid, making it difficult for cells to disperse during aliquoting. Factors or peptides must be added to the hydrogel to achieve organoid culture. MR Arkenberg et al. (Heparinized Gelatin-Based Hydrogels for Differentiation of Induced Pluripotent Stem Cells) developed a heparin-gelatin system. Heparin and gelatin are modified using different systems. The resulting hydrogel has a modulus far higher than the 100 Pa modulus of Matrigel (a non-bionic structure). The hydrogel degrades in 1.5 hours, which is slow and not conducive to cell recovery, affecting cell activity and preventing its use in tumor organoid culture. Summary of the Invention

[0004] To address these issues, the present invention provides a novel and efficient method for constructing tumor organoids. This method features a simple construction process, repeatability, and product stability. The resulting organoids possess stable components and morphology, mimicking the development and migration of tumors under physiological conditions, and possess significant potential for tumor drug screening and testing. More importantly, the bare hydrogels prepared by the present invention can be used to culture organoids, while hydrogels bound to factors or peptides can be used to cultivate difficult-to-cultivate organoids and enable organoid recovery.

[0005] The first object of the present invention is to provide a method for preparing tumor organoids, comprising the following steps:

[0006] S1. Mixing a tumor cell suspension with a gel precursor composition to obtain a hydrogel precursor solution, and irradiating the solution with light to obtain a hydrogel;

[0007] S2. mixing the hydrogel described in S1 with a tumor organoid culture medium, and incubating to obtain the tumor organoid;

[0008] The gel precursor composition contains norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP and a photoinitiator.

[0009] Furthermore, the gel precursor composition also contains cytokines and / or matrix proteins.

[0010] Furthermore, the cytokines include one or more of R-spondin 1, R-spondin 3, organoid cytokine Noggin protein (a secretory glycoprotein), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and nerve growth factor (NGF).

[0011] Furthermore, the matrix protein includes one or more of laminin, collagen, fibronectin and matrix protein polypeptide.

[0012] Furthermore, the matrix protein polypeptides include RGD polypeptide (SEQ ID NO.1 or SEQ ID NO.2), YIGSR polypeptide (SEQ ID NO.4), IKVAV polypeptide (SEQ ID NO.5) and GFOGER polypeptide (SEQ ID NO.6).

[0013] Furthermore, the hydrogel precursor solution contains norbornene-modified heparin (Hep-NB) at a concentration of 0.2-2% (w / v), norbornene-modified gelatin (Gel-NB) at a concentration of 0.5-5% (w / v), thiol-containing four-arm polyethylene glycol (4-PEG-SH) at a concentration of 0.3-3% (w / v), dextran at a concentration of 0.1%-5% (w / v), and a photoinitiator at a concentration of 0.01%-0.1% (w / v). There is no specific limit on the concentration of the matrix metalloprotein peptidomimetic (MMP); for organoid recovery purposes, an appropriate amount, such as 0.5-2 mg / mL, can be added.

[0014] Furthermore, the matrix protein mimicking peptide MMP includes the sequence shown in SEQ ID NO.3.

[0015] Furthermore, in the hydrogel precursor solution, the concentration of cytokines is 0.01-10 μg / mL, and the concentration of matrix protein polypeptide is 0.01%-1% (w / v).

[0016] Furthermore, it contains at least one of the following characteristics:

[0017] (1) The number average molecular weight of heparin is 8000-15000;

[0018] (2) Heparin is standard heparin, acetylated heparin or deacetylated heparin; preferably fully N-acetylated or partially N-acetylated;

[0019] (3) The number average molecular weight of gelatin is 50,000-100,000;

[0020] (4) The number average molecular weight of dextran is 40,000-70,000;

[0021] (5) The number average molecular weight of thiol four-arm polyethylene glycol is 8000-20000.

[0022] Furthermore, the tumor organoids can be any solid tumor organoids, and the tumor cells include but are not limited to lung cancer, intestinal cancer, liver cancer, gastric cancer, pancreatic cancer, bile duct cancer, breast cancer and ovarian cancer.

[0023] A second object of the present invention is to provide a gel precursor composition for preparing a hydrogel, wherein the gel precursor composition contains a first raw material; the first raw material includes:

[0024] Norbornene-modified heparin, wherein each 1L of the gel precursor composition contains 2-20g of norbornene-modified heparin, i.e., its concentration is 0.2-2% (w / v);

[0025] Norbornene-modified gelatin, wherein each 1L of the gel precursor composition contains 5-50g of norbornene-modified gelatin, i.e., its concentration is 0.5-5% (w / v);

[0026] Thiol four-arm polyethylene glycol, each 1L of the gel precursor composition contains 3-30g of thiol four-arm polyethylene glycol, that is, its concentration is 0.3-3% (w / v);

[0027] Dextran, wherein each 1 L of the gel precursor composition contains 1-50 g of dextran, i.e., its concentration is 0.1%-5% (w / v);

[0028] Matrix metalloprotein peptidomimetics MMP, there is no special requirement for the concentration, just add it according to actual requirements;

[0029] The photoinitiator contains 0.1-1 g of photoinitiator per 1 L of the gel precursor composition, that is, the concentration is 0.01%-0.1% (w / v).

[0030] Furthermore, in the gel precursor composition, the molar ratio of the thiol four-arm polyethylene glycol to the norbornene-modified heparin is 0.6-1.0.

[0031] Furthermore, the gel precursor composition contains a second raw material; the second raw material includes cytokines and / or matrix proteins.

[0032] Furthermore, the gel precursor composition contains 0.01-10 mg of cytokines per 1 L of the gel precursor composition, that is, a concentration of 0.01-10 μg / mL; and contains 0.1-10 g of matrix protein polypeptide per 1 L of the gel precursor composition, that is, a concentration of 0.01%-1% (w / v).

[0033] Furthermore, the gel precursor composition contains a third raw material; the third raw material includes a cell suspension (preferably a tumor cell suspension).

[0034] Furthermore, the photoinitiator includes phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt LAP.

[0035] The third object of the present invention is to provide a photocurable hydrogel, which contains norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP and a photoinitiator. Preferably, the photocurable hydrogel is prepared by photocuring the gel precursor composition, and contains a cross-linked network structure formed by norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP and a photoinitiator.

[0036] Furthermore, the photocurable hydrogel contains at least one of cells (such as tumor cells), growth factors and matrix proteins fixed in a cross-linked network structure.

[0037] A fourth object of the present invention is to provide a method for preparing the photocurable hydrogel, comprising the following steps:

[0038] A gel precursor composition containing norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP and a photoinitiator is mixed with a buffer solution or an organoid culture medium to obtain a hydrogel precursor solution, and the hydrogel precursor solution is cured under light conditions to obtain the photocurable hydrogel.

[0039] Furthermore, the gel precursor composition further includes at least one of cells, cytokines and matrix proteins.

[0040] Furthermore, the preparation method of norbornene-modified heparin is: using a norbornene material containing an amino group (such as 5-norbornene-2-methylamine) to carry out an amide condensation reaction with the carboxyl group of heparin itself to obtain norbornene-modified heparin.

[0041] Furthermore, the preparation method of norbornene-modified gelatin is as follows: using a norbornene material containing a carboxyl group (such as 5-norbornene-2-carboxylic acid) to carry out an amide condensation reaction with the amino group of gelatin itself to obtain norbornene-modified gelatin.

[0042] Furthermore, the illumination conditions are: wavelength 365-405 nm (ultraviolet light or visible light), light intensity 3-10 mW / cm 2 , illumination time 3-10 min.

[0043] A fifth object of the present invention is to provide use of the gel precursor composition or the photocurable hydrogel in organoid culture.

[0044] Furthermore, the organoid is preferably a tumor organoid.

[0045] Furthermore, the application includes: recovering the organoids (when containing MMP) using a recovery reagent after the organoid culture is completed; the recovery reagent includes type I collagenase.

[0046] Beneficial effects of the present invention:

[0047] (1) Unlike heparin and gelatin with electrostatic effects, the norbornene-modified heparin and norbornene-modified gelatin used in the present invention can be cross-linked with thiol-modified multi-arm polyethylene glycol through a thiol-norbornene click reaction and then photocured to form a hydrogel. The hydrogel of the present invention is formed by photocuring in a step-by-step polymerization manner, has a controllable structure, and has an adjustable biomimetic modulus and adjustable biomimetic stress relaxation. Moreover, this heparin-gelatin hydrogel with covalent bonds can bind to growth factors and continuously and stably output the growth factors required for cell growth, thereby maintaining the proliferation of organoids and reducing the dosage of growth factors required for the culture medium during the organoid fluid replacement process.

[0048] (2) The present invention can use heparin with various structures (such as deacetylated and acetylated heparin), which reduces the cost compared to using only standard heparin.

[0049] (3) The hydrogel containing pores of several to tens of microns prepared by the specific composition of the present invention is more conducive to the 3D culture of cells. The introduction of a certain concentration of sticky glucan can regulate the structure of the hydrogel and improve the stress relaxation of the hydrogel, which helps the rearrangement of cells and is beneficial to the culture of organoids.

[0050] (4) The present invention introduces matrix proteins, especially matrix metalloprotein peptidomimetics (MMPs), which, on the one hand, improve the stress relaxation ability of the hydrogel. On the other hand, its enzymatic hydrolysis gives the hydrogel the function of organoid recovery, which is beneficial to the passage and preservation of organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the hydrogel preparation process of the present invention.

[0052] Figure 2 The storage modulus (G') and loss modulus (G'') test results of heparin gelatin hydrogels containing different components.

[0053] Figure 3 The stress relaxation performance test results of the hydrogel.

[0054] Figure 4 The nuclear magnetic resonance spectra of standard heparin, acetylated heparin and deacetylated heparin used in the present invention.

[0055] Figure 5 Results of culturing lung cancer organoids on heparin gelatin hydrogels with or without the addition of dextran.

[0056] Figure 6 Results of culturing lung cancer organoids on bare heparin gelatin hydrogels without added factors or peptides.

[0057] Figure 7 Results of culturing refractory lung cancer organoids on heparin gelatin hydrogels supplemented with R-Spondin 1.

[0058] Figure 8 For ELISA quantification, a hydrogel-binding factor for R-Spondin 1 was added.

[0059] Figure 9 Results of culturing refractory lung cancer organoids on heparin gelatin hydrogels supplemented with EGF.

[0060] Figure 10 The hydrogel binding factor with EGF was added for ELISA quantification.

[0061] Figure 11 Results of culturing lung cancer organoids on heparin hydrogels combined with laminin and RGD peptides.

[0062] Figure 12 Results of culturing lung cancer organoids on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs).

[0063] Figure 13 Results of culturing refractory lung cancer organoids on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) for R-spondin 1 binding.

[0064] Figure 14 Results of lung cancer organoids cultured on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) for YIGSR binding.

[0065] Figure 15 The results show that heparin gelatin hydrogel with added matrix metalloprotein peptidomimetic MMP can achieve efficient recovery of lung cancer organoids.

[0066] Figure 16 Results of HGF-binding to gastric cancer organoids cultured on heparin-gelatin hydrogels and Matrigel-based addition of matrix metalloprotein peptidomimetics (MMPs).

[0067] Figure 17 Roundness of gastric cancer organoids cultured in hydrogel and Matrigel.

[0068] Figure 18 Results of colorectal cancer organoids cultured on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) for EGF binding.

[0069] Figure 19 Results of HGF-binding hepatocellular carcinoma organoids cultured on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs).

[0070] Figure 20 Results of pancreatic cancer organoids cultured on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) for binding to IKVAV peptide.

[0071] Figure 21 Results of culturing cholangiocarcinoma organoids on heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) in combination with EGF. DETAILED DESCRIPTION

[0072] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0073] The materials involved in the present invention are:

[0074] (1) Name definition

[0075] Hep-NB: norbornene-modified heparin

[0076] Gel-NB: norbornene-modified gelatin

[0077] 4-PEG-SH: thiol four-arm polyethylene glycol

[0078] MMP: Matrix metalloproteinase mimetic peptide

[0079] (2) Raw materials and sources

[0080]

[0081] (3) Peptides and sequences

[0082]

[0083] Example 1: Preparation of modified materials and hydrogels

[0084] Preparation of Hep-NB: Dissolve 500 mg of heparin sodium in 25 mL of deionized water with a pH of 5-6, add the dissolved 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride dropwise to the reaction system, react at room temperature for 15 min, then add 70 μL of 5-norbornene-2-methylamine dropwise, react overnight in the dark, dialyze, and freeze-dry to obtain a sample for storage.

[0085] Preparation of Gel-NB: 35 mg of 5-norbornene-2-carboxylic acid was dissolved in 20 mL of deionized water with a pH of 5-6, and a mixed solution containing 98 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 31 mg of N-hydroxysuccinimide was added dropwise. The mixture was reacted at 50°C for 15 min, 1 g of gelatin was added, the mixture was reacted at 50°C overnight, dialyzed, and freeze-dried to obtain a sample for storage.

[0086] Preparation of light-curable hydrogel: Hep-NB (number average molecular weight 14,000, the same below), Gel-NB (number average molecular weight 50,000-100,000, the same below), dextran (number average molecular weight 40 kDa, the same below), 4-PEG-SH (number average molecular weight 10,000, the same below), MMP (number average molecular weight 1700, the same below), and photoinitiator LAP (phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt) were dissolved in HyClone phosphate buffered saline. The above component solutions were thoroughly mixed. The concentration of Hep-NB was 0.7% (w / v, referring to the addition of 0.7% per 100 mL of the solution). g, the same below), the concentration of Gel-NB was 2.1%, the concentration of dextran was 0.5%, the concentration of 4-PEG-SH was 1.2% and the concentration of photoinitiator LAP was 0.045% to obtain a hydrogel precursor solution (wherein: when dextran and MMP were both present, MMP was added to the system, the concentration of MMP was 0.1%, and the concentration of 4-PEG-SH was adjusted to 0.6%). The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to obtain a photocurable degradable hydrogel.

[0087] The modulus of the hydrogel was tested using a rheometer. 600 μL of the hydrogel precursor solution was dropped into a 2 cm polytetrafluoroethylene mold and the rheometer was used to measure the modulus of the hydrogel. 2 The hydrogel disc was prepared by irradiating the sample under ultraviolet light for 2 min. The sample was placed on the sample stage of the rheometer for rheological testing. Figure 2 As shown in the figure, the storage modulus (G') of heparin gelatin hydrogels containing different components is around 100 Pa; at different frequencies, the G' of heparin gelatin hydrogels containing different components is higher than the loss modulus (G''), indicating that the hydrogels form a stable three-dimensional network structure under ultraviolet light, and the storage modulus is comparable to that of matrix gel.

[0088] The stress relaxation properties of the hydrogels were tested using a rheometer, e.g. Figure 3 As shown in the results, the addition of dextran helps to shorten the stress relaxation time. The simultaneous addition of dextran and degradable matrix metalloprotein peptidomimetics (MMPs) can shorten the half stress relaxation time to about 3 min, which helps the migration and rearrangement of cells in the hydrogel.

[0089] Example 2: Non-standard heparin hydrogels can be used to culture organoids

[0090] The different acetylation (acetylation>80%) and deacetylation (acetylation<5%) Figure 4) Hep-NB, Gel-NB, 4-PEG-SH, and photoinitiator LAP were dissolved in PBS and the above component solutions were thoroughly mixed to obtain a precursor solution with a concentration of 0.7% Hep-NB, 3.3% Gel-NB, 1.0% 4-PEG-SH, and 0.05% photoinitiator LAP. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by using a single lung cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 5 mW / cm 2 The heparin gelatin hydrogel was prepared by irradiating the sample under ultraviolet light for 5 min.

[0091] 150 μL of lung cancer organoid culture medium was added, and the medium was changed every 2-3 days depending on the growth status. The 96-well plate was placed in a cell culture incubator and incubated. On day 9, the organoids in each group were similar in size and number, with no significant difference, indicating that the non-standard heparin hydrogel is suitable for organoid culture.

[0092] Example 3: Hydrogels with added dextran can be used to culture organoids

[0093] Acetylated Hep-NB, Gel-NB, 4-PEG-SH, photoinitiator LAP, and dextran were dissolved in PBS and the above component solutions were thoroughly mixed to obtain a precursor solution with a concentration of 0.7% Hep-NB, 3.3% Gel-NB, 1.2% 4-PEG-SH, 0.05% (w / v) photoinitiator LAP, and 1% dextran. The precursor solution was mixed with 1×10 5 The hydrogel precursor solution was obtained by irradiating the hydrogel precursor solution with a wavelength of 365 nm and a light intensity of 5 mW / cm 2 The heparin gelatin hydrogel was prepared by irradiating the sample under ultraviolet light for 5 minutes. No dextran was added to the control group, and all other conditions were the same.

[0094] Add 300 μL of lung cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 24-well plate in a cell culture incubator and incubate. Figure 5 As shown in the figure, on the third day, the size of the organoids in the hydrogel with added dextran component was close to 100 μm, and the culture effect was significantly better than that of the control group without added dextran, indicating that the hydrogel can promote the culture of organoids.

[0095] Example 4: Cultivation of lung cancer organoids using bare hydrogels without added factors or peptides

[0096] Acetylated Hep-NB, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in lung cancer culture medium, and the above component solutions were thoroughly mixed to obtain a precursor solution with a concentration of 0.6% for Hep-NB, 4% for Gel-NB, 1.2% for 4-PEG-SH, 0.5% for dextran, and 0.1% for photoinitiator LAP. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by using a single lung cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to prepare heparin gelatin hydrogel.

[0097] Add 150 μL of lung cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On day 14, the organoid size reaches approximately 200 μm, indicating that bare heparin gelatin hydrogels without added factors or peptides can culture lung cancer organoids ( Figure 6 ).

[0098] Example 5: Heparin gelatin hydrogels combined with R-Spondin 1 can culture refractory lung cancer organoids

[0099] Acetylated Hep-NB, R-Spondin 1 (R-spondin 1), Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in lung cancer culture medium, and the above component solutions were thoroughly mixed to obtain a precursor solution with a concentration of 0.7% Hep-NB, 1 μg / mL R-Spondin 1, 3.5% Gel-NB, 1.6% 4-PEG-SH, 1% dextran, and 0.05% photoinitiator LAP. The precursor solution was mixed with 3×10 5 The hydrogel precursor solution was obtained by using a single lung cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 2 min to prepare heparin gelatin hydrogel.

[0100] Add 150 μL of lung cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On the 6th day, the size of lung cancer organoids cultured in Matrigel was about 50 μm, while the size of lung cancer organoids cultured in heparin gelatin hydrogels supplemented with R-Spondin 1 was close to 100 μm, indicating that heparin gelatin hydrogels supplemented with R-Spondin 1 can culture difficult-to-cultivate lung cancer organoids ( Figure 7 ).

[0101] By quantitatively studying the hydrogel binding factors through Elisa, it was found that after 1 hour, 99% of R-Spondin1 was bound to the hydrogel and could be stably released for one week ( Figure 8 ).

[0102] Example 6: Heparin gelatin hydrogel combined with EGF can culture difficult-to-cultivate lung cancer organoids

[0103] Deacetylated Hep-NB, EGF (epidermal growth factor), Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in lung cancer culture medium, and the above component solutions were thoroughly mixed to obtain a precursor solution with a concentration of 0.7% Hep-NB, 50 ng / mL EGF, 3.5% Gel-NB, 1.6% 4-PEG-SH, 1% dextran, and 0.05% photoinitiator LAP. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by using a single lung cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 2 min to prepare heparin gelatin hydrogel.

[0104] Add 150 μL of lung cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On the 6th day, the size of the lung cancer organoids cultured in Matrigel was about 50 μm, while the size of the lung cancer organoids cultured in heparin gelatin hydrogel supplemented with EGF was close to 150 μm, indicating that heparin gelatin hydrogel supplemented with EGF can culture difficult-to-cultivate lung cancer organoids ( Figure 9 ).

[0105] By quantitatively studying the hydrogel binding factors through Elisa, it was found that after 1 hour, 99% of EGF was bound to the hydrogel and could be stably released for one week ( Figure 10 ).

[0106] Example 7: Heparin hydrogels combined with laminin and RGD peptides can be used to culture lung cancer organoids

[0107] Deacetylated Hep-NB, RGD peptide (sequence shown in SEQ ID NO.1), laminin, 4-PEG-SH, dextran, and photoinitiator LAP were dissolved in lung cancer culture medium, and the above component solutions were thoroughly mixed to obtain a precursor solution with a concentration of 2.8% Hep-NB, 2 mg / mL RGD, 0.5 mg / mL laminin, 0.8-2% 4-PEG-SH, 0.5% dextran, and 0.05% photoinitiator LAP. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by using a single lung cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 2 min to prepare heparin gelatin hydrogel.

[0108] 150 μL of lung cancer organoid culture medium was added and the medium was changed every 2-3 days depending on the growth status. The 96-well plate was placed in a cell culture incubator and incubated. On the 6th day, when the molar ratio of the crosslinker was adjusted to 0.6-1.0, the hydrogel storage modulus was 80-500 Pa and the roundness of the lung cancer organoids was greater than or equal to 0.9, indicating that the modulus of the heparin hydrogel combined with laminin and RGD peptide is adjustable and can be used to culture lung cancer organoids ( Figure 11 ).

[0109] The RGD polypeptide was replaced with the sequence shown in SEQ ID NO. 2, and the above steps were repeated. The culture results were not significantly different from those of the polypeptide shown in SEQ ID NO. 1.

[0110] Example 8: Heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) can be used to culture lung cancer organoids

[0111] Acetylated Hep-NB, MMP (see SEQ ID NO. 3), Gel-NB, 4-PEG-SH, dextran, and LAP were dissolved in lung cancer culture medium and thoroughly mixed. The concentration of Hep-NB was 0.8%, the content of MMP was 1.5 mg / mL, the concentration of Gel-NB was 3.8%, the concentration of 4-PEG-SH was 0.8%, the concentration of dextran was 1.2%, and the concentration of LAP was 0.06%, thereby obtaining a precursor solution. The precursor solution was mixed with 4×10 5 The hydrogel precursor solution was then irradiated with ultraviolet light at a wavelength of 365 nm and an intensity of 10 mW / cm² for 3 minutes to successfully produce a heparin-gelatin hydrogel incorporating a matrix metalloproteinase (MMP) mimetic peptide.

[0112] 150 μL of lung cancer organoid culture medium was added to the above hydrogel system, and the medium was changed every 2-3 days depending on the cell growth status. The 96-well plate was placed in a cell culture incubator for incubation. On the 4th day, it was observed that the size of the lung cancer organoids cultured in matrix gel was about 100 μm, and the size of the lung cancer organoids cultured in heparin gelatin hydrogel with matrix metalloprotein mimetic peptide MMP was also about 100 μm. This shows that heparin gelatin hydrogel with matrix metalloprotein mimetic peptide MMP can effectively culture lung cancer organoids ( Figure 12 ).

[0113] Example 9: Heparin gelatin hydrogels containing matrix metalloprotein peptidomimetics (MMPs) combined with R-Spondin 1 can culture refractory lung cancer organoids

[0114] Hep-NB (standard heparin), R-Spondin 1, MMP, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were fully dissolved in lung cancer culture medium and mixed evenly. The concentration of Hep-NB was 0.6%, the concentration of the matrix metalloprotein peptidomimetic MMP that binds to R-Spondin 1 was 1.2 mg / mL, the concentration of Gel-NB was 3.2%, the concentration of 4-PEG-SH was 1.4%, the concentration of dextran was 0.8%, and the concentration of photoinitiator LAP was 0.04%. This resulted in a precursor solution. Subsequently, this solution was mixed with 3.5×10 5 The hydrogel precursor solution was prepared by mixing a single lung cancer cell suspension of 100 cells / mL with the solution. This solution was then irradiated with ultraviolet light at a wavelength of 365 nm and an intensity of 10 mW / cm² for 3 minutes, successfully creating a heparin-gelatin hydrogel containing a matrix metalloproteinase (MMP)-containing peptide that binds to R-Spondin 1.

[0115] 150 μL of lung cancer organoid culture medium was added to the above hydrogel system, and the medium was changed every 2-3 days according to the cell growth status. The 96-well plate was placed in a cell culture incubator for incubation. On the 6th day, it was observed that the size of the lung cancer organoids cultured in matrigel was about 80 μm, while the size of the lung cancer organoids cultured using heparin gelatin hydrogels with matrix metalloprotein peptide MMP combined with R-Spondin 1 was close to 100 μm, which was slightly larger than the result of matrigel culture. This shows that heparin gelatin hydrogels with matrix metalloprotein peptide MMP combined with R-Spondin1 can effectively culture difficult-to-cultivate lung cancer organoids ( Figure 13 ).

[0116] Example 10: Heparin gelatin hydrogels combined with laminin peptidomimetics and matrix metalloprotein peptidomimetics (MMPs) can be used to culture lung cancer organoids

[0117] Acetylated Hep-NB, laminin mimetic peptide YIGSR, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in lung cancer culture medium, and the above component solutions were thoroughly mixed, wherein the concentration of Hep-NB was 0.7%, the sequence of YIGSR was shown in SEQ ID NO.4, the content of YIGSR was 0.5 mg / mL, the concentration of Gel-NB was 3.5%, the concentration of 4-PEG-SH was 1.6%, the concentration of MMP was 1.2 mg / mL, the concentration of dextran was 1%, and the concentration of photoinitiator LAP was 0.05% to obtain a precursor solution. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by using a single lung cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to prepare heparin gelatin hydrogel.

[0118] 200 μL of organoid lung cancer culture medium was added and the medium was changed every 2-3 days depending on the growth status. The 96-well plate was placed in a cell culture incubator and incubated. On the 6th day, the size of the lung cancer organoids cultured in the two heparin gelatin hydrogels (with or without the addition of laminin mimetic YIGSR) and matrigel was greater than 100 μm. Morphologically, there were more vacuolar structures in the two heparin gelatin hydrogels, and the heparin gelatin hydrogel combined with YIGSR showed some solid cystic structures. The lung cancer organoids cultured in matrigel had more solid structures, indicating that the heparin gelatin hydrogel combined with YIGSR and the addition of matrix metalloprotein mimetic MMP is conducive to the cultivation of lung cancer organoids with solid cystic structures, while the heparin gelatin hydrogel without YIGSR and the addition of matrix metalloprotein mimetic MMP is conducive to the cultivation of lung cancer organoids with vacuolar structures ( Figure 14 ).

[0119] Example 11: Heparin gelatin hydrogels with matrix metalloprotein peptidomimetics (MMPs) can be used to recover lung cancer organoids

[0120] Dilute type I collagenase with MEM medium (essential basal medium) to a 5 mg / mL solution. Mix the diluted collagenase solution thoroughly and preheat for 15 minutes. Gently transfer the hydrogel cells from Example 9 to a weighed centrifuge tube. Reweigh the centrifuge tube and hydrogel cells again, then add 500 μL of collagenase. Gently pipette 30 times to break up the cell hydrogel. Place the centrifuge tube in a 37°C incubator to dissolve for 15 minutes, gently pipetting five times every 5 minutes to aid digestion.

[0121] After digestion, the cell suspension was removed, the centrifuge tube was weighed, and 500 μL of DMEM / F12 medium was added to the cell suspension to terminate the reaction. gCentrifuge for 5 minutes. After centrifugation, carefully discard the supernatant and observe the cell recovery rate. It was found that the cell recovery efficiency of the hydrogel can reach about 90%, and the hydrogel solubility can reach 99%, indicating that heparin gelatin hydrogel with matrix metalloprotein peptide MMP can achieve efficient recovery of lung cancer organoids ( Figure 15 ).

[0122] Example 12: Heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) combined with HGF can be used to culture gastric cancer organoids

[0123] Deacetylated Hep-NB, HGF (hepatocyte growth factor), MMP, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP were fully dissolved in gastric cancer organoid culture medium and mixed evenly. The concentration of Hep-NB was 0.8%, the concentration of HGF was 200 ng / mL, the concentration of MMP was 1.2 mg / mL, the concentration of Gel-NB was 3.0%, the concentration of 4-PEG-SH was 1.4%, the concentration of dextran was 0.8%, and the concentration of photoinitiator LAP was 0.04%. This resulted in a precursor solution. Subsequently, this solution was mixed with 3×10 5 The researchers then mixed a single gastric cancer cell suspension (1000 cells / mL) with the hydrogel precursor solution. This solution was then irradiated with ultraviolet light at a wavelength of 365 nm and an intensity of 10 mW / cm² for 3 minutes, successfully creating a heparin-gelatin hydrogel containing a matrix metalloprotein peptide (MMP) that binds to HGF.

[0124] 150 μL of gastric cancer organoid culture medium was added to the hydrogel system, and the medium was changed every 2-3 days depending on the cell growth status. The 96-well plate was placed in a cell culture incubator and incubated on the 8th day. It was found that the size of gastric cancer organoids cultured in heparin gelatin hydrogels supplemented with matrix metalloprotein peptides (MMPs) combined with HGF and matrigel was about 100 μm ( Figure 16 ), and the circularity of gastric cancer organoids cultured in hydrogel was 0.89, which was significantly higher than that of gastric cancer organoids cultured in matrigel (0.76). Figure 17 ), indicating that heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) combined with HGF can effectively culture spherical gastric cancer organoids.

[0125] Example 13: Heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) combined with EGF can be used to culture colorectal cancer organoids

[0126] Deacetylated Hep-NB, EGF, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in the intestinal cancer organoid culture medium, and the above component solutions were thoroughly mixed. The concentration of Hep-NB was 0.7%, the concentration of EGF was 300 ng / mL, the concentration of Gel-NB was 3.5%, the concentration of 4-PEG-SH was 1.6%, the content of MMP was 1.0 mg / mL, the concentration of dextran was 1%, and the concentration of photoinitiator LAP was 0.05% to obtain a precursor solution. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by mixing the colon cancer cell suspension of 1000 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to prepare heparin gelatin hydrogel.

[0127] Add 200 μL of colorectal cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On the 6th day, the size of colorectal cancer organoids cultured in heparin gelatin hydrogel and matrigel was about 100 μm, and both showed real cystic structures in morphology ( Figure 18 ).

[0128] Example 14: Heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) combined with HGF can be used to culture liver cancer organoids

[0129] Acetylated Hep-NB, HGF, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in liver cancer organoid culture medium, and the above component solutions were thoroughly mixed. The concentration of Hep-NB was 0.7%, the concentration of HGF was 100 ng / mL, the concentration of Gel-NB was 3.5%, the concentration of 4-PEG-SH was 1.6%, the content of MMP was 1.0 mg / mL, the concentration of dextran was 1%, and the concentration of photoinitiator LAP was 0.05% to obtain a precursor solution. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by mixing the liver cancer cell suspension of 100 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to prepare heparin gelatin hydrogel.

[0130] Add 200 μL of liver cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On day 6, the size of liver cancer organoids cultured in heparin gelatin hydrogel and matrigel is about 100 μm ( Figure 19 ).

[0131] Example 15: Heparin gelatin hydrogels containing matrix metalloprotein peptidomimetics (MMPs) combined with IKVAV polypeptides can be used to culture pancreatic cancer organoids

[0132] Acetylated Hep-NB, IKVAV polypeptide, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in pancreatic cancer organoid culture medium, and the above component solutions were thoroughly mixed, wherein the concentration of Hep-NB was 0.7%, the sequence of IKVAV polypeptide was shown in SEQ ID NO.5, the concentration of IKVAV was 0.28 mg / mL, the concentration of Gel-NB was 3.5%, the concentration of 4-PEG-SH was 1.6%, the content of MMP was 1.2 mg / mL, the concentration of dextran was 1%, and the concentration of photoinitiator LAP was 0.05% to obtain a precursor solution. The precursor solution was mixed with 2×10 5 The hydrogel precursor solution was obtained by mixing pancreatic cancer cell suspension with 100 / mL of PBS. The hydrogel precursor solution was irradiated under a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to prepare heparin gelatin hydrogel.

[0133] Add 200 μL of pancreatic cancer organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On day 6, the size of pancreatic cancer organoids cultured in heparin gelatin hydrogel and matrigel is about 80 μm ( Figure 20 ).

[0134] Example 16: Heparin gelatin hydrogels supplemented with matrix metalloprotein peptidomimetics (MMPs) combined with EGF can be used to culture cholangiocarcinoma organoids

[0135] Deacetylated Hep-NB, EGF, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in cholangiocarcinoma culture medium, and the above component solutions were thoroughly mixed. The concentration of Hep-NB was 0.7%, the concentration of EGF was 100 ng / mL, the concentration of Gel-NB was 3.5%, the concentration of 4-PEG-SH was 1.6%, the content of MMP was 1.0 mg / mL, the concentration of dextran was 1%, and the concentration of photoinitiator LAP was 0.05% to obtain a precursor solution. The precursor solution was mixed with 1×10 5 The hydrogel precursor solution was obtained by mixing the bile duct cancer cell suspension of 100 cells / mL. The hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 The samples were irradiated under ultraviolet light for 3 min to prepare heparin gelatin hydrogel.

[0136] Add 200 μL of cholangiocarcinoma organoid culture medium and change the medium every 2-3 days depending on the growth status. Place the 96-well plate in a cell culture incubator and incubate. On day 8, the size of cholangiocarcinoma organoids cultured in heparin gelatin hydrogel and matrigel is about 80 μm ( Figure 21 ).

[0137] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing tumor organoids, characterized in that: The following steps are involved: S1. Mixing a tumor cell suspension with a gel precursor composition to obtain a hydrogel precursor solution, and irradiating the solution with light to obtain a hydrogel; S2. mixing the hydrogel described in S1 with a tumor organoid culture medium, and incubating to obtain the tumor organoid; The gel precursor composition contains norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP with a sequence as shown in SEQ ID NO.3, and a photoinitiator; The preparation method of the norbornene-modified heparin comprises: dissolving heparin sodium and reacting it with dissolved 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, adding 5-norbornene-2-methylamine and performing the reaction in the dark to obtain the norbornene-modified heparin; The preparation method of the norbornene-modified gelatin comprises: dissolving 5-norbornene-2-carboxylic acid, reacting the solution with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adding gelatin and reacting the solution again to obtain the norbornene-modified gelatin; The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate; The tumor is one of lung cancer, intestinal cancer, liver cancer, stomach cancer, pancreatic cancer and bile duct cancer; When the tumor is intestinal cancer, the gel precursor composition further contains epidermal growth factor; When the tumor is liver cancer, the gel precursor composition further contains hepatocyte growth factor; When the tumor is gastric cancer, the gel precursor composition further contains hepatocyte growth factor; When the tumor is pancreatic cancer, the gel precursor composition further contains an IKVAV polypeptide having a sequence as shown in SEQ ID NO.5; When the tumor is bile duct cancer, the gel precursor composition further contains epidermal growth factor.

2. The preparation method according to claim 1, characterized in that When the tumor is lung cancer, the gel precursor composition further contains cytokines and / or matrix proteins. The cytokine is R-spondin 1 or epidermal growth factor, The matrix protein is a combination of laminin and the RGD polypeptide shown in SEQ ID NO.1, or is the laminin mimetic peptide YIGSR shown in SEQ ID NO.

4.

3. The preparation method according to claim 2, characterized in that Contains at least one of the following characteristics: (1) The concentration of the cytokine in the hydrogel precursor solution is 0.01-10 μg / mL; (2) In the hydrogel precursor solution, the concentration of the matrix protein polypeptide is 0.01%-1% (w / v).

4. The preparation method according to claim 1, characterized in that In the hydrogel precursor solution, the concentration of norbornene-modified heparin is 0.2-2% (w / v), the concentration of norbornene-modified gelatin is 0.5-5% (w / v), the concentration of thiol four-arm polyethylene glycol is 0.3-3% (w / v), the concentration of dextran is 0.1%-5% (w / v), and the concentration of the photoinitiator is 0.01%-0.1% (w / v).

5. The preparation method according to claim 1, characterized in that Contains at least one of the following characteristics: (1) The number average molecular weight of heparin is 8000-15000; (2) Heparin is one or more of standard heparin, acetylated heparin and deacetylated heparin; (3) The number average molecular weight of gelatin is 50,000-100,000; (4) The number average molecular weight of dextran is 40,000-70,000; (5) The number average molecular weight of thiol four-arm polyethylene glycol is 8000-20000.

6. A gel precursor composition, characterized in that The gel precursor composition contains a first raw material; the first raw material includes: Norbornene-modified heparin, wherein each 1L of the gel precursor composition contains 2-20g of norbornene-modified heparin; the norbornene-modified heparin is prepared by: dissolving heparin sodium and reacting it with dissolved 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, adding 5-norbornene-2-methylamine, and then reacting in the dark to obtain the norbornene-modified heparin; Norbornene-modified gelatin, wherein each liter of gel precursor composition contains 5-50 g of norbornene-modified gelatin; the norbornene-modified gelatin is prepared by: dissolving 5-norbornene-2-carboxylic acid, reacting the solution with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adding gelatin, and reacting the solution again to obtain the norbornene-modified gelatin; Thiol four-arm polyethylene glycol, each 1L of the gel precursor composition contains 3-30g of thiol four-arm polyethylene glycol; Dextran, containing 1-50 g of dextran per 1 L of the gel precursor composition; A photoinitiator, wherein each 1L of the gel precursor composition contains 0.1-1g of the photoinitiator; the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt; The sequence of the matrix metalloprotein peptidomimetic MMP is shown in SEQ ID NO.

3.

7. The gel precursor composition according to claim 6, characterized in that The gel precursor composition contains a second raw material; the second raw material includes cytokines and / or matrix proteins.

8. A photocurable hydrogel, characterized in that: The photocurable hydrogel contains norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metalloprotein peptidomimetic MMP with a sequence as shown in SEQ ID NO.3, and a photoinitiator; The preparation method of the norbornene-modified heparin comprises: dissolving heparin sodium and reacting it with dissolved 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, adding 5-norbornene-2-methylamine and performing the reaction in the dark to obtain the norbornene-modified heparin; The preparation method of the norbornene-modified gelatin comprises: dissolving 5-norbornene-2-carboxylic acid, reacting the solution with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adding gelatin and reacting the solution again to obtain the norbornene-modified gelatin; The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

9. The light-curable hydrogel according to claim 8, characterized in that The light-curable hydrogel contains growth factors and / or matrix proteins.

10. A method for preparing a photocurable hydrogel, characterized in that: The following steps are involved: The gel precursor composition according to claim 6 or 7 is mixed with a buffer solution or an organoid culture medium to obtain a hydrogel precursor solution, and the hydrogel precursor solution is cured under light conditions to obtain the light-cured hydrogel.

11. The preparation method according to claim 10, characterized in that: The illumination conditions are: wavelength 365-405 nm, light intensity 3-10 mW / cm 2 , illumination time 3-10 min.

12. Use of the gel precursor composition according to claim 6 or 7, the photocurable hydrogel according to claim 8 or 9, or the photocurable hydrogel prepared by the preparation method according to claim 10 or 11 in organoid culture, characterized in that: The organoid is a tumor organoid, and the tumor is one of lung cancer, intestinal cancer, liver cancer, gastric cancer, pancreatic cancer and bile duct cancer.

13. The use according to claim 12, characterized in that The application includes: recovering the organoids using a recovery reagent after the organoid culture is completed; the recovery reagent includes type I collagenase.

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