Photo-curing hydrogel capable of being combined with cytokines and used for culturing tumor organoids and application of photo-curing hydrogel

Abstract

The invention discloses photocuring hydrogel capable of being combined with cytokines and used for tumor organoid culture and application, and belongs to the technical field of organoid culture. According to the invention, the hydrogel which can fix cells, can be combined with cell factors and can realize organoid recovery is developed through norbornene modified heparin, norbornene modified gelatin, sulfydryl four-arm polyethylene glycol, glucan, matrix metal protein peptidomimetic MMP and a photoinitiator. The hydrogel can be used for directly preparing organoids, heparin of various structures can be used, the cost is reduced, the modified heparin can bind factors or matrix protein and can controllably release the factors, the modified gelatin provides amino acid energy for collagen hydrolysate, and the hydrogel is formed by photocuring in a stepwise polymerization mode, is controllable in structure and can be used for preparing collagen collagen. The adjustable bionic modulus and the adjustable bionic stress relaxation are realized. The hydrogel disclosed by the invention can be used for culturing various organs, overcomes the problems of low repeatability, instability and the like in the prior art, and is suitable for the fields of tumor drug evaluation and the like.

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 cytokines for 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 tissues. These three-dimensional cultures have cell compositions, structures, morphologies, and functions similar to those of real human organs and can be subcultured for a long time. In the past 15 years, organoid technology has developed rapidly, and normal organoids and tumor organoids such as those of the intestine, stomach, heart, brain, liver, lung, kidney, and breast have been established. Among them, the extracellular matrix (ECM) required for organoid culture is a research hotspot in the culture technology.

[0003] Traditional organoid culture relies on Matrigel. However, the components of Matrigel are complex and the batches are unstable. Secondly, due to its containing a variety of components that are difficult to dissolve in the aqueous phase, the product is difficult to use in the form of freeze-drying, which is not conducive to transportation and storage. Negatively charged glycosaminoglycans and positively charged proteoglycans, as the main components of ECM, can form hydrogels by electrostatic interaction and maintain the osmotic pressure required for cell growth and obtain the growth factors required by cells through the charges on the side chains. Heparin is a natural glycosaminoglycan with a high negative charge, which can bind and stabilize a large number of growth factors, such as fibroblast growth factor (FGF), transforming growth factor (TGF-β), and vascular endothelial growth factor (VEGF), etc. Gelatin is a natural polymer derived from collagen, containing a large number of active functional groups such as amino, carboxyl, and hydroxyl groups. Type A gelatin is derived from acid treatment, and its isoelectric point is between 6 and 9. During cell culture, the pH of the culture medium is about 6 to 7, and the amino groups in gelatin are protonated to show positive charge behavior. However, the current gel system still has defects. For example, a heparin-polyethylene glycol system disclosed in WO2017198258A1 uses all standard heparin, which has a high cost. Moreover, in the method of forming a hydrogel, the gelation speed is too fast, and it is not easy to disperse cells during cell sub-packaging. The hydrogel needs to add factors or polypeptides to achieve organoid culture. MR Arkenberg et al. (Heparinized Gelatin-Based Hydrogels for Differentiation of Induced Pluripotent Stem Cells) developed a heparin-gelatin system. The modification systems of heparin and gelatin are different, and the modulus of the synthesized hydrogel is much higher than the modulus of Matrigel of 100 Pa (non-bionic structure). The degradation time of the hydrogel is 1.5 h, which is too slow and not conducive to cell recovery, will affect cell activity, and cannot be used for culturing tumor organoids. Summary of the Invention

[0004] To solve the above problems, the present invention provides a new method for efficiently constructing tumor organoids. The construction process is simple, with advantages such as repeatability and product stability. The constructed organoids have stable components and morphology, can simulate the occurrence and migration of tumors under physiological conditions, and have great potential in tumor drug screening and drug detection. More importantly, the naked gel prepared by the present invention can be used to culture organoids, and the hydrogel combined with factors or polypeptides can be used to culture difficult-to-culture organoids, and organoid recovery can be achieved.

[0005] The first object of the present invention is to provide a method for preparing tumor organoids, comprising the following steps: S1. Mix a tumor cell suspension with a gel precursor composition to obtain a hydrogel precursor solution, and irradiate with light to obtain a hydrogel; S2. Mix the hydrogel obtained in S1 with a tumor organoid culture medium and incubate to obtain the tumor organoids; Wherein, the gel precursor composition contains norbornene-modified heparin, norbornene-modified gelatin, thiol-functionalized tetra-arm polyethylene glycol, dextran, matrix metalloproteinase mimetic MMP, and a photoinitiator.

[0006] Furthermore, the gel precursor composition further contains cytokines and / or matrix proteins.

[0007] Furthermore, the cytokines include one or more of R-spondin 1 (R-Spondin 1), R-spondin 3 (R-Spondin 3), the organoid cytokine Noggin protein (a secreted 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).

[0008] Furthermore, the matrix proteins include one or more of laminin, collagen, fibronectin, and matrix protein polypeptides.

[0009] 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).

[0010] Further, in the hydrogel precursor solution, the concentration of norbornene-modified heparin (Hep-NB) is 0.2-2% (w / v), the concentration of norbornene-modified gelatin (Gel-NB) is 0.5-5% (w / v), the concentration of thiol-functionalized tetra-arm polyethylene glycol (4-PEG-SH) is 0.3-3% (w / v), the concentration of dextran is 0.1%-5% (w / v), and the concentration of photoinitiator is 0.01%-0.1% (w / v). Among them, the concentration of the matrix metalloproteinase mimetic peptide MMP has no specific limitation. For the purpose of organoid recovery, appropriate addition is sufficient, such as 0.5-2 mg / mL.

[0011] Further, the matrix protein mimetic peptide MMP includes the sequence shown in SEQ ID NO.3.

[0012] Further, in the hydrogel precursor solution, the concentration of cytokine is 0.01-10 μg / mL, and the concentration of matrix protein polypeptide is 0.01%-1% (w / v).

[0013] Further, it has at least one of the following characteristics: (1) The number average molecular weight of heparin is 8000-15000; (2) Heparin is standard heparin, acetylated heparin or deacetylated heparin; preferably all N-acetylated or partially N-acetylated; (3) The number average molecular weight of gelatin is 50000-100000; (4) The number average molecular weight of dextran is 40000-70000; (5) The number average molecular weight of thiol-functionalized tetra-arm polyethylene glycol is 8000-20000.

[0014] Further, the tumor organoids can be any solid tumor organoids, and the tumor cells include but are not limited to lung cancer, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, etc.

[0015] The second object of the present invention is to provide a gel precursor composition for preparing a hydrogel. The gel precursor composition contains a first raw material; the first raw material includes: Norbornene-modified heparin, 2-20 g of norbornene-modified heparin is contained in every 1 L of the gel precursor composition, that is, its concentration is 0.2-2% (w / v); Norbornene-modified gelatin, 5-50 g of norbornene-modified gelatin is contained in every 1 L of the gel precursor composition, that is, its concentration is 0.5-5% (w / v); Thiol-functionalized tetra-arm polyethylene glycol, 3-30 g of thiol-functionalized tetra-arm polyethylene glycol is contained in every 1 L of the gel precursor composition, that is, its concentration is 0.3-3% (w / v); Dextran, containing 1 - 50 g of dextran per 1 L of the gel precursor composition, i.e., its concentration is 0.1% - 5% (w / v); Matrix metalloproteinase mimetic MMP, there is no special requirement for its concentration, and it can be added according to actual requirements; Photoinitiator, containing 0.1 - 1 g of photoinitiator per 1 L of the gel precursor composition, i.e., its concentration is 0.01% - 0.1% (w / v).

[0016] Furthermore, in the gel precursor composition, the molar ratio of thiol - terminated tetra - arm polyethylene glycol to norbornene - modified heparin is 0.6 - 1.0.

[0017] Furthermore, the gel precursor composition contains a second raw material; the second raw material includes cytokines and / or matrix proteins.

[0018] Furthermore, in the gel precursor composition, it contains 0.01 - 10 mg of cytokines per 1 L of the gel precursor composition, i.e., the concentration is 0.01 - 10 μg / mL; it contains 0.1 - 10 g of matrix protein polypeptides per 1 L of the gel precursor composition, i.e., the concentration is 0.01% - 1% (w / v).

[0019] Furthermore, the gel precursor composition contains a third raw material; the third raw material includes a cell suspension (preferably a tumor cell suspension).

[0020] Furthermore, the photoinitiator includes lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate LAP.

[0021] The third object of the present invention is to provide a photocurable hydrogel, which contains norbornene - modified heparin, norbornene - modified gelatin, thiol - terminated tetra - arm polyethylene glycol, dextran, matrix metalloproteinase mimetic MMP and a photoinitiator. Preferably, it is prepared by photocuring the gel precursor composition, and it contains a cross - linked network structure formed by norbornene - modified heparin, norbornene - modified gelatin, thiol - terminated tetra - arm polyethylene glycol, dextran, matrix metalloproteinase mimetic MMP and a photoinitiator.

[0022] Furthermore, the photocurable hydrogel contains at least one of cells (such as tumor cells), growth factors and matrix proteins fixed in the cross - linked network structure.

[0023] The fourth object of the present invention is to provide a preparation method of the photocurable hydrogel, including the following steps: Mix the gel precursor composition containing norbornene-modified heparin, norbornene-modified gelatin, mercapto tetra-arm polyethylene glycol, dextran, matrix metalloprotein mimetic MMP and photoinitiator with buffer or organoid culture medium to obtain a hydrogel precursor solution, and cure the hydrogel precursor solution under light conditions to obtain the photocured hydrogel.

[0024] Furthermore, the gel precursor composition further includes at least one of cells, cytokines and matrix proteins.

[0025] Furthermore, the preparation method of norbornene-modified heparin is: use 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.

[0026] Furthermore, the preparation method of norbornene-modified gelatin is: use 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.

[0027] Furthermore, the conditions of the light are: wavelength 365 - 405 nm (both ultraviolet light and visible light are acceptable), light intensity 3 - 10 mW / cm 2 , and the light irradiation time is 3 - 10 min.

[0028] The fifth object of the present invention is to provide the application of the gel precursor composition or the photocured hydrogel in organoid culture.

[0029] Furthermore, the organoid is preferably a tumor organoid.

[0030] Furthermore, the application includes: recovering the organoid (when containing MMP) with a recovery reagent after the organoid culture; the recovery reagent includes type I collagenase.

[0031] The beneficial effects of the present invention: (1) Different from heparin and gelatin with electrostatic action, the norbornene-modified heparin and norbornene-modified gelatin used in the present invention can be gradually crosslinked and photocured to form a hydrogel through a thiol-norbornene click reaction with thiol-modified multi-arm polyethylene glycol. The hydrogel of the present invention is photocured by a stepwise polymerization method, with a controllable structure, adjustable biomimetic modulus and adjustable biomimetic stress relaxation. Moreover, this heparin-gelatin hydrogel introducing covalent bonds can bind growth factors and continuously and stably output the growth factors required for cell growth, maintaining the proliferation of organoids to reduce the dose of growth factors required in the culture medium during the organoid medium change process.

[0032] (2) The present invention can use heparins with various structures (such as deacetylated and acetylated heparins), and the cost is reduced compared with only using standard heparin.

[0033] (3) The hydrogel containing pores in the range of several to dozens of micrometers prepared by the specific composition of the present invention is more conducive to 3D cell culture. Introducing a certain concentration of viscous dextran can regulate the structure of the hydrogel, improve the stress relaxation of the hydrogel at the same time, contribute to cell rearrangement, and is beneficial to the culture of organoids.

[0034] (4) The present invention introduces matrix proteins, especially matrix metalloproteinase mimetics (MMP). On the one hand, it improves the stress relaxation ability of the hydrogel. On the other hand, its own enzymatizability endows the hydrogel with the function of organoid recovery, which is beneficial to the passage and preservation of organoids. Description of the Drawings

[0035] Figure 1 It is a schematic diagram of the preparation process of the hydrogel of the present invention.

[0036] Figure 2 It is the test result of the storage modulus (G') and loss modulus (G'') of heparin-gelatin hydrogels containing different components.

[0037] Figure 3 It is the test result of the stress relaxation performance of the hydrogel.

[0038] Figure 4 It is the nuclear magnetic resonance spectra of standard heparin, acetylated heparin and deacetylated heparin used in the present invention.

[0039] Figure 5 It is the result of culturing lung cancer organoids with heparin-gelatin hydrogels with or without dextran added.

[0040] Figure 6 It is the result of culturing lung cancer organoids with bare heparin-gelatin hydrogels without adding factors and polypeptides.

[0041] Figure 7 It is the result of culturing refractory lung cancer organoids with heparin-gelatin hydrogels added with R-Spondin 1.

[0042] Figure 8 It is the situation of the hydrogel-binding factor added with R-Spondin 1 quantified by Elisa.

[0043] Figure 9 It is the result of culturing refractory lung cancer organoids with heparin-gelatin hydrogels added with EGF.

[0044] Figure 10 It is the situation of the hydrogel-binding factor added with EGF quantified by Elisa.

[0045] Figure 11 It is the result of culturing lung cancer organoids with heparin hydrogels combined with laminin and RGD polypeptides.

[0046] Figure 12 Results of culturing lung cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP

[0047] Figure 13 Results of culturing refractory lung cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with R - Spondin 1

[0048] Figure 14 Results of culturing lung cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with YIGSR

[0049] Figure 15 Results of heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP enabling efficient recovery of lung cancer organoids

[0050] Figure 16 Results of culturing gastric cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with HGF and Matrigel

[0051] Figure 17 Roundness of gastric cancer organoids cultured with hydrogel and Matrigel

[0052] Figure 18 Results of culturing colorectal cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with EGF

[0053] Figure 19 Results of culturing liver cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with HGF

[0054] Figure 20 Results of culturing pancreatic cancer organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with IKVAV polypeptide

[0055] Figure 21 Results of culturing cholangiocarcinoma organoids with heparin - gelatin hydrogel added with matrix metalloproteinase mimetic peptide MMP combined with EGF Detailed implementation manners

[0056] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the cited embodiments do not limit the present invention.

[0057] Materials related to the present invention: (1) Definition of names Hep - NB: Norbornene - modified heparin Gel - NB: Norbornene - modified gelatin 4-PEG-SH: Thiolated four-armed polyethylene glycol MMP: Matrix metalloproteinase mimetic peptide (2) Raw materials and sources

[0058] (3) Polypeptides and sequences

[0059] Example 1: Preparation of modified materials and hydrogels Preparation of Hep-NB: Dissolve 500 mg of sodium heparin in 25 mL of deionized water with a pH of 5 - 6. Dropwise add the dissolved 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride into the reaction system, react at room temperature for 15 min, then gradually add 70 μL of 5-norbornene-2-methylamine dropwise, react overnight in the dark, dialyze, and lyophilize to obtain a sample for storage.

[0060] Preparation of Gel-NB: Dissolve 35 mg of 5-norbornene-2-carboxylic acid in 20 mL of deionized water with a pH of 5 - 6. Dropwise add it into a mixed solution containing 98 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 31 mg of N-hydroxysuccinimide, react at 50 °C for 15 min, add 1 g of gelatin, react overnight at 50 °C, dialyze, and lyophilize to obtain a sample for storage.

[0061] Preparation of photocurable hydrogel: Dissolve Hep-NB (number average molecular weight 14000, the same below), Gel-NB (number average molecular weight 50000 - 100000, the same below), dextran (number average molecular weight 40KDa, the same below), 4-PEG-SH (number average molecular weight 10000, the same below), MMP (number average molecular weight 1700, the same below), and photoinitiator LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate) in HyClone phosphate buffered saline. Mix the above component solutions thoroughly. The concentration of Hep-NB is 0.7% (w / v, which means adding 0.7 g per 100 mL of the solution, the same below), the concentration of Gel-NB is 2.1%, the concentration of dextran is 0.5%, the concentration of 4-PEG-SH is 1.2%, and the concentration of photoinitiator LAP is 0.045% to obtain a hydrogel precursor solution (where: when both dextran and MMP are present, add MMP to the system, the concentration of MMP is 0.1%, and adjust the concentration of 4-PEG-SH to 0.6%). Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to obtain a photocurable and degradable hydrogel.

[0062] The modulus of the hydrogel was tested using a rheometer. 600 μL of the hydrogel precursor solution was dropped onto a 2 cm polytetrafluoroethylene mold and irradiated with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 2 min to prepare a hydrogel disc. The sample was placed on the rheometer sample stage for rheological testing. As Figure 2 shown, the storage modulus (G') of the heparin-gelatin hydrogels containing different components was around 100 Pa; at different frequencies, the G' of the heparin-gelatin hydrogels containing different components was higher than the loss modulus (G''), indicating that a stable three-dimensional network structure was formed in the hydrogel under ultraviolet light irradiation, and the storage modulus was comparable to that of the matrix gel.

[0063] The stress relaxation performance of the hydrogel was tested using a rheometer. As Figure 3 shown, the addition of dextran helped to shorten the stress relaxation time. The simultaneous addition of dextran and the degradable matrix metalloprotein mimetic MMP could shorten the half stress relaxation time to about 3 min, which was helpful for cell migration and rearrangement in the hydrogel.

[0064] Example 2: Non-standard heparin hydrogel can achieve the culture of organoids Hep-NB, Gel-NB, 4-PEG-SH, and the photoinitiator LAP with different degrees of acetylation (acetylation > 80%) and deacetylation (acetylation < 5%) ( Figure 4 ) were dissolved in PBS. The above component solutions were fully mixed, with the concentration of Hep-NB being 0.7%, the concentration of Gel-NB being 3.3%, the concentration of 4-PEG-SH being 1.0%, and the concentration of the photoinitiator LAP being 0.05% to obtain a precursor solution. The precursor solution was mixed with 2×10 5 single lung cancer cell suspensions / mL to obtain a hydrogel precursor solution. The hydrogel precursor solution was irradiated with ultraviolet light at a wavelength of 365 nm and a light intensity of 5 mW / cm 2 for 5 min to prepare a heparin-gelatin hydrogel.

[0065] 150 μL of lung cancer organoid culture medium was added, and the medium was changed every 2 - 3 days according to the growth status. The 96-well plate was incubated in a cell culture incubator. On the 9th day, the sizes of the organoids in each group were close and the number showed no significant difference, indicating that the non-standard heparin hydrogel was suitable for organoid culture.

[0066] Example 3: Hydrogel added with dextran can achieve the culture of organoids Dissolve acetylated Hep-NB, Gel-NB, 4-PEG-SH, photoinitiator LAP, and dextran in PBS. Mix the above component solutions well. The concentration of Hep-NB is 0.7%, the concentration of Gel-NB is 3.3%, the concentration of 4-PEG-SH is 1.2%, the concentration of photoinitiator LAP is 0.05% (w / v), and the concentration of dextran is 1% to obtain the precursor solution. Mix the precursor solution with 1×10 5 cells / mL lung cancer cell cluster suspension to obtain the hydrogel precursor solution. Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 5 mW / cm 2 for 5 min to prepare the heparinized gelatin hydrogel. Dextran was not added in the control group, and the rest was the same.

[0067] Add 300 μL of lung cancer organoid medium and change the medium every 2 - 3 days according to the growth status. Place the 24-well plate in the cell culture incubator. As Figure 5 shown, on the 3rd day, the size of the organoids in the hydrogel with the dextran component added was close to 100 μm, and the culture effect was significantly better than that of the control group without dextran addition, indicating that this hydrogel can promote the culture of organoids.

[0068] Example 4: Culturing lung cancer organoids with a naked hydrogel without factors and polypeptides added Dissolve acetylated Hep-NB, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP in lung cancer medium. Mix the above component solutions well. The concentration of Hep-NB is 0.6%, the concentration of Gel-NB is 4%, the concentration of 4-PEG-SH is 1.2%, the concentration of dextran is 0.5%, and the concentration of photoinitiator LAP is 0.1% to obtain the precursor solution. Mix the precursor solution with 2×10 5 cells / mL lung cancer single cell suspension to obtain the hydrogel precursor solution. Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to prepare the heparinized gelatin hydrogel.

[0069] Add 150 μL of lung cancer organoid medium and change the medium every 2 - 3 days according to the growth status. Place the 96-well plate in the cell culture incubator. On the 14th day, the size of the organoids reached about 200 μm, indicating that the naked heparinized gelatin hydrogel without factors and polypeptides added can culture lung cancer organoids ( Figure 6 ).

[0070] Example 5: Heparinized gelatin hydrogel combined with R-Spondin 1 factor can culture refractory lung cancer organoids Dissolve acetylated Hep-NB, R-Spondin 1 (R-spondin 1), Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP in lung cancer medium, and fully mix the above component solutions. The concentration of Hep-NB is 0.7%, the content of R-Spondin 1 is 1 μg / mL, the concentration of Gel-NB is 3.5%, the concentration of 4-PEG-SH is 1.6%, the concentration of dextran is 1%, and the concentration of photoinitiator LAP is 0.05% to obtain a precursor solution. Mix the precursor solution with 3×10 5 cells / mL of lung cancer single-cell suspension to obtain a hydrogel precursor solution. Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 2 min to prepare a heparinized gelatin hydrogel.

[0071] Add 150 μL of lung cancer organoid medium and change the medium every 2 - 3 days according to the growth status. Incubate the 96-well plate in a cell culture incubator. On the 6th day, the size of lung cancer organoids cultured in Matrigel is about 50 μm, while the size of lung cancer organoids cultured in heparinized gelatin hydrogel supplemented with R-Spondin 1 can be close to about 100 μm, indicating that heparinized gelatin hydrogel supplemented with R-Spondin 1 can culture difficult-to-culture lung cancer organoids ( Figure 7 ).

[0072] Quantitatively study the hydrogel-binding factors by Elisa. It can be found that after 1 h, 99% of R-Spondin1 is bound by the hydrogel and can be stably released for one week ( Figure 8 ).

[0073] Example 6: Heparinized gelatin hydrogel combined with EGF factor can culture difficult-to-culture lung cancer organoids Dissolve deacetylated Hep-NB, EGF (epidermal growth factor), Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP in lung cancer medium, and fully mix the above component solutions. The concentration of Hep-NB is 0.7%, the content of EGF is 50 ng / mL, the concentration of Gel-NB is 3.5%, the concentration of 4-PEG-SH is 1.6%, the concentration of dextran is 1%, and the concentration of photoinitiator LAP is 0.05% to obtain a precursor solution. Mix the precursor solution with 2×10 5 cells / mL of lung cancer single-cell suspension to obtain a hydrogel precursor solution. Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 2 min to prepare a heparinized gelatin hydrogel.

[0074] 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 matrix gel was about 50 μm, while the size of lung cancer organoids cultured in heparin gelatin hydrogel with EGF was close to 150 μm, indicating that heparin gelatin hydrogel with EGF can culture difficult-to-culture lung cancer organoids ( Figure 9 ).

[0075] Through ELISA quantitative study of the hydrogel binding factor, it was found that after 1 h, 99% of EGF was bound by the hydrogel and could be stably released for one week ( Figure 10 ).

[0076] Example 7: Heparin hydrogels combined with laminin and RGD peptides can culture lung cancer organoids Deacetylated Hep-NB, RGD polypeptide (sequence see 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 fully mixed, wherein the concentration of Hep-NB was 2.8%, the content of RGD was 2 mg / mL, the concentration of laminin was 0.5 mg / mL, the concentration of 4-PEG-SH was 0.8-2%, the concentration of dextran was 0.5%, 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 a single lung cancer cell suspension of 100 μg / mL. The hydrogel precursor solution was heated 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 obtain heparin gelatin hydrogel.

[0077] 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 organoid was greater than or equal to 0.9, indicating that the modulus of the heparin hydrogel combined with laminin and RGD peptide was adjustable and could culture lung cancer organoids ( Figure 11 ).

[0078] 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.

[0079] Example 8: Heparin gelatin hydrogels with matrix metalloprotein peptidomimetics MMP can be used to culture lung cancer organoids Dissolve acetylated Hep-NB, matrix metalloprotein mimetic peptide MMP (sequence shown in SEQ ID NO.3), Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP in lung cancer medium, and fully mix the component solutions. Among them, the concentration of Hep-NB is 0.8%, the content of matrix metalloprotein mimetic peptide MMP is 1.5 mg / mL, the concentration of Gel-NB is 3.8%, the concentration of 4-PEG-SH is 0.8%, the concentration of dextran is 1.2%, and the concentration of photoinitiator LAP is 0.06% to obtain a precursor solution. Mix the precursor solution with 4×10 5 cells / mL of lung cancer single-cell suspension to obtain a hydrogel precursor solution again. Then, irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm² for 3 min to successfully prepare a heparin hydrogel incorporated with matrix metalloprotein mimetic peptide MMP.

[0080] Add 150 μL of lung cancer organoid medium to the above hydrogel system, and change the medium once every 2 - 3 days according to the cell growth state. Place the 96-well plate in the cell culture incubator. On the 4th day, it was observed that the size of lung cancer organoids cultured in Matrigel was about 100 μm, and the size of lung cancer organoids cultured in the heparin hydrogel incorporated with matrix metalloprotein mimetic peptide MMP was also about 100 μm. This indicates that the heparin hydrogel incorporated with matrix metalloprotein mimetic peptide MMP can effectively culture lung cancer organoids ( Figure 12 ).

[0081] Example 9: Heparin hydrogel incorporated with matrix metalloprotein mimetic peptide MMP combined with R-Spondin 1 can culture refractory lung cancer organoids Fully dissolve Hep-NB (standard heparin), R-Spondin 1, MMP, Gel-NB, 4-PEG-SH, dextran, and photoinitiator LAP in lung cancer medium and mix evenly. Among them, the concentration of Hep-NB is 0.6%, the concentration of matrix metalloprotein mimetic peptide MMP combined with R-Spondin 1 is 1.2 mg / mL, the concentration of Gel-NB is 3.2%, the concentration of 4-PEG-SH is 1.4%, the concentration of dextran is 0.8%, and the concentration of photoinitiator LAP is 0.04% to obtain a precursor solution. Subsequently, mix this solution with 3.5×10 5 cells / mL of lung cancer single-cell suspension to obtain a hydrogel precursor solution again. Then, irradiate the solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm² for 3 min to successfully prepare a heparin hydrogel incorporated with matrix metalloprotein mimetic peptide MMP combined with R-Spondin 1.

[0082] Add 150 μL of lung cancer organoid culture medium to the above hydrogel system, and change the medium every 2 - 3 days according to the cell growth status. Place the 96-well plate in the cell culture incubator. 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 in heparinized gelatin hydrogel with added matrix metalloprotein mimetic MMP conjugated with R-Spondin 1 was close to 100 μm, slightly larger than the result of Matrigel culture. This indicates that the heparinized gelatin hydrogel with added matrix metalloprotein mimetic MMP conjugated with R-Spondin 1 can effectively culture refractory lung cancer organoids ( Figure 13 )

[0083] Example 10: Heparinized gelatin hydrogel with added matrix metalloprotein mimetic MMP conjugated with laminin mimetic can culture lung cancer organoids Dissolve acetylated Hep-NB, laminin mimetic YIGSR, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP in lung cancer culture medium, and fully mix the above component solutions. The concentration of Hep-NB is 0.7%, the sequence of YIGSR is shown in SEQ ID NO.4, the content of YIGSR is 0.5 mg / mL, the concentration of Gel-NB is 3.5%, the concentration of 4-PEG-SH is 1.6%, the concentration of MMP is 1.2 mg / mL, the concentration of dextran is 1% and the concentration of photoinitiator LAP is 0.05% to obtain the precursor solution. Mix the precursor solution with 2×10 5 cells / mL of lung cancer single cell suspension to obtain the hydrogel precursor solution. Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to prepare the heparinized gelatin hydrogel

[0084] Add 200 μL of organoid lung cancer culture medium, and change the medium every 2 - 3 days according to the growth status. Place the 96-well plate in the cell culture incubator. On the 6th day, the sizes of the lung cancer organoids cultured in 2 kinds of heparinized gelatin hydrogels (with or without added laminin mimetic YIGSR) and Matrigel were all larger than 100 μm. Morphologically, there were more vacuole-like structures in the two heparinized gelatin hydrogels. The heparinized gelatin hydrogel conjugated with YIGSR showed some solid cystic structures, and the lung cancer organoids cultured in Matrigel had more solid structures, indicating that the heparinized gelatin hydrogel with added matrix metalloprotein mimetic MMP conjugated with YIGSR is helpful for culturing lung cancer organoids with solid cystic structures, while the heparinized gelatin hydrogel with added matrix metalloprotein mimetic MMP without conjugated YIGSR is helpful for culturing lung cancer organoids with vacuole-like structures ( Figure 14 )

[0085] Example 11: Heparin - gelatin hydrogel added with matrix metalloprotein mimetic MMP can achieve the recovery of lung cancer organoids Dilute type I collagenase with MEM medium (essential basal medium) to a solution with a concentration of 5 mg / mL. Mix the diluted collagenase solution thoroughly and preheat it for 15 min. Gently pick the hydrogel cells from Example 9 above into a pre - weighed centrifuge tube. After weighing the centrifuge tube and the hydrogel cells again, add 500 μL of collagenase, and gently pipette 30 times to break the cell hydrogel. Place the centrifuge tube in an incubator at 37°C for 15 min, and gently pipette 5 times every 5 min to assist digestion.

[0086] After digestion, take out the cell suspension, weigh the centrifuge tube, add 500 μL of DMEM / F12 medium to the cell suspension to terminate the reaction, and centrifuge at 300 g rpm for 5 min. After centrifugation, carefully aspirate and discard the supernatant, and observe the cell recovery rate. It is found that the cell recovery efficiency of this hydrogel can reach about 90%, and the hydrogel solubility can reach 99%, indicating that the heparin - gelatin hydrogel added with matrix metalloprotein mimetic MMP can achieve the efficient recovery of lung cancer organoids ( Figure 15 )

[0087] Example 12: Heparin - gelatin hydrogel added with matrix metalloprotein mimetic MMP combined with HGF can culture gastric cancer organoids Fully dissolve deacetylated Hep - NB, HGF (hepatocyte growth factor), MMP, Gel - NB, 4 - PEG - SH, dextran, and photoinitiator LAP in gastric cancer organoid medium and mix evenly. Among them, the concentration of Hep - NB is 0.8%, the concentration of HGF is 200 ng / mL, the concentration of MMP is 1.2 mg / mL, the concentration of Gel - NB is 3.0%, the concentration of 4 - PEG - SH is 1.4%, the concentration of dextran is 0.8%, and the concentration of photoinitiator LAP is 0.04% to obtain a precursor solution. Subsequently, mix this solution with a single - cell suspension of gastric cancer cells at a density of 3×10 5 cells / mL to obtain a hydrogel precursor solution. Then, irradiate this solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm² for 3 min to successfully prepare a heparin - gelatin hydrogel added with matrix metalloprotein mimetic MMP combined with HGF.

[0088] Add 150 μL of gastric cancer organoid medium to the above hydrogel system, and change the medium every 2 - 3 days according to the cell growth condition. Place the 96 - well plate in a cell incubator. On the 8th day, it is observed that the sizes of gastric cancer organoids cultured with the heparin - gelatin hydrogel added with matrix metalloprotein mimetic MMP combined with HGF and Matrigel are both about 100 μm ( Figure 16), and the roundness 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 the heparinized gelatin hydrogel conjugated with HGF and added with the matrix metalloprotein mimetic MMP can effectively culture spherical gastric cancer organoids.

[0089] Example 13: Heparinized gelatin hydrogel conjugated with EGF and added with the matrix metalloprotein mimetic MMP can culture intestinal cancer organoids Deacetylated Hep-NB, EGF, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in the intestinal cancer organoid medium. The above component solutions were fully mixed, where 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 cells / mL of intestinal cancer cell suspension to obtain a hydrogel precursor solution. The hydrogel precursor solution was irradiated with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to prepare a heparinized gelatin hydrogel.

[0090] 200 μL of intestinal cancer organoid medium was added, and the medium was changed every 2 - 3 days according to the growth status. The 96-well plate was incubated in a cell culture incubator. At day 6, the sizes of intestinal cancer organoids cultured in heparinized gelatin hydrogel and Matrigel were both about 100 μm, and solid cystic structures appeared in both morphologies ( Figure 18 ).

[0091] Example 14: Heparinized gelatin hydrogel conjugated with HGF and added with the matrix metalloprotein mimetic MMP can culture liver cancer organoids Acetylated Hep-NB, HGF, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP were dissolved in the liver cancer organoid medium. The above component solutions were fully mixed, where 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 cells / mL of liver cancer cell suspension to obtain a hydrogel precursor solution. The hydrogel precursor solution was irradiated with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to prepare a heparinized gelatin hydrogel.

[0092] Add 200 μL of liver cancer organoid culture medium and change the medium every 2 - 3 days according to the growth status. Place the 96-well plate in the cell culture incubator. On the 6th day, the sizes of the liver cancer organoids cultured in heparinized gelatin hydrogel and Matrigel are both about 100 μm ( Figure 19 ).

[0093] Example 15: Heparinized gelatin hydrogel combined with IKVAV polypeptide and added matrix metalloproteinase mimetic MMP can culture pancreatic cancer organoids Dissolve acetylated Hep-NB, IKVAV polypeptide, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP in pancreatic cancer organoid culture medium. Mix the above component solutions well, where the concentration of Hep-NB is 0.7%, the sequence of IKVAV polypeptide is shown in SEQ ID NO.5, the concentration of IKVAV is 0.28 mg / mL, the concentration of Gel-NB is 3.5%, the concentration of 4-PEG-SH is 1.6%, the content of MMP is 1.2 mg / mL, the concentration of dextran is 1%, and the concentration of photoinitiator LAP is 0.05% to obtain a precursor solution. Mix the precursor solution with 2×10 5 cells / mL of pancreatic cancer cell suspension to obtain a hydrogel precursor solution. Irradiate the hydrogel precursor solution with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to prepare heparinized gelatin hydrogel.

[0094] Add 200 μL of pancreatic cancer organoid culture medium and change the medium every 2 - 3 days according to the growth status. Place the 96-well plate in the cell culture incubator. On the 6th day, the sizes of the pancreatic cancer organoids cultured in heparinized gelatin hydrogel and Matrigel are both about 80 μm ( Figure 20 ).

[0095] Example 16: Heparinized gelatin hydrogel combined with EGF and added matrix metalloproteinase mimetic MMP can culture cholangiocarcinoma organoids Dissolve deacetylated Hep-NB, EGF, Gel-NB, 4-PEG-SH, MMP, dextran, and photoinitiator LAP in cholangiocarcinoma culture medium. Mix the above component solutions well, where the concentration of Hep-NB is 0.7%, the concentration of EGF is 100 ng / mL, the concentration of Gel-NB is 3.5%, the concentration of 4-PEG-SH is 1.6%, the content of MMP is 1.0 mg / mL, the concentration of dextran is 1%, and the concentration of photoinitiator LAP is 0.05% to obtain a precursor solution. Mix the precursor solution with 1×10 5cells / mL cholangiocarcinoma cell suspension to obtain a hydrogel precursor solution. The hydrogel precursor solution was irradiated with ultraviolet light at a wavelength of 365 nm and a light intensity of 10 mW / cm 2 for 3 min to prepare a heparin-gelatin hydrogel.

[0096] Add 200 μL of cholangiocarcinoma organoid culture medium and change the medium every 2 - 3 days according to the growth status. Place the 96-well plate in a cell culture incubator. On the 8th day, the sizes of cholangiocarcinoma organoids cultured in the heparin-gelatin hydrogel and Matrigel were both about 80 μm ( Figure 21 ).

[0097] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope 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 metal protein peptidomimetic MMP and a photoinitiator.

2. The preparation method according to claim 1, characterized in that: The gel precursor composition also contains cytokines and / or matrix proteins.

3. The preparation method according to claim 2, characterized in that: Contains at least one of the following characteristics: (1) The cytokines include one or more of R-spondin 1, R-spondin 3, organoid cytokine Noggin protein, fibroblast growth factor 2, epidermal growth factor, hepatocyte growth factor, insulin-like growth factor, platelet-derived growth factor and nerve growth factor; (2) The matrix protein includes one or more of laminin, collagen, fibronectin and matrix protein polypeptide.

4. The preparation method according to claim 3, characterized in that: The matrix protein polypeptide includes one or more of RGD polypeptide, YIGSR polypeptide, IKVAV polypeptide and GFOGER polypeptide; wherein, The RGD polypeptide includes the sequence shown in SEQ ID NO.1 or SEQ ID NO.2; The YIGSR polypeptide includes the sequence shown in SEQ ID NO.4; The IKVAV polypeptide includes the sequence shown in SEQ ID NO.5; The GFOGER polypeptide includes the sequence shown in SEQ ID NO.

6.

5. The preparation method according to any one of claims 2 to 4, characterized in that: Contains at least one of the following characteristics: (1) In the hydrogel precursor solution, the concentration of cytokines 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).

6. 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).

7. 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 the thiol four-arm polyethylene glycol is 8000-20000; (6) The matrix protein mimetic peptide MMP includes the sequence shown in SEQ ID NO.

3.

8. 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; Norbornene-modified gelatin, wherein each 1L of the gel precursor composition contains 5-50g of 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, each 1L of the gel precursor composition contains 1-50g of dextran; A photoinitiator, wherein each 1L of the gel precursor composition contains 0.1-1g of the photoinitiator; Matrix metalloprotein peptidomimetics MMP.

9. The gel precursor composition according to claim 8, characterized in that Contains at least one of the following characteristics: (1) The gel precursor composition contains a second raw material; the second raw material includes cytokines and / or matrix proteins; (2) The gel precursor composition contains a third raw material; the third raw material includes cells; (3) The photoinitiator includes phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

10. A photocurable hydrogel, characterized in that: The photocurable hydrogel contains norbornene-modified heparin, norbornene-modified gelatin, thiol four-arm polyethylene glycol, dextran, matrix metal protein peptidomimetic MMP and a photoinitiator.

11. The photocurable hydrogel according to claim 10, characterized in that: The photocurable hydrogel contains at least one of cells, growth factors and matrix proteins fixed in a cross-linked network structure.

12. A method for preparing a photocurable hydrogel, characterized in that: The following steps are involved: The gel precursor composition according to claim 8 or 9 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.

13. The preparation method according to claim 12, characterized in that: Contains at least one of the following characteristics: (1) The preparation method of norbornene-modified heparin comprises: using a norbornene material containing an amino group to carry out an amide condensation reaction with a carboxyl group of heparin to obtain the norbornene-modified heparin; (2) The preparation method of norbornene-modified gelatin comprises: using a norbornene material containing a carboxyl group to carry out an amide condensation reaction with the amino group of gelatin to obtain the norbornene-modified gelatin; (3) The illumination conditions are: wavelength 365-405 nm, light intensity 3-10 mW / cm 2 , illumination time 3-10 min.

14. Use of the gel precursor composition according to claim 8 or 9, the photocurable hydrogel according to claim 10 or 11, or the photocurable hydrogel prepared by the preparation method according to claims 12-13 in organoid culture.

15. The use according to claim 14, characterized in that Contains at least one of the following characteristics: (1) The organoids include tumor organoids; (2) 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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