Use of a collagen peptoid in the preparation of an organoid culture system

Abstract

This invention relates to the field of organoid culture technology, specifically to the application of a collagen peptide in the preparation of organoid culture systems. This invention discovers that a GER-containing, electrically neutral collagen peptide with a GPO monomer content as high as 40-80% can significantly promote organoid growth, exhibiting a marked promoting effect on organoid proliferation and differentiation. It can effectively combine with biomimetic gels to form organoid culture systems, promoting organoid formation. This invention has good industrialization prospects and broader application value in the field of medical regeneration.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to the application of a collagen peptide in the preparation of organoid culture systems. Background Technology

[0002] Traditional organoid culture uses matrix gels, but the composition of matrix gels is unclear, and batch instability limits their application. Collagen, with its well-defined chemical properties and clinical-grade availability, supports its application in organoid clinical translation. Hans Clever's team combined scTS2 / 16 with clinical-grade type I collagen, significantly increasing organoid yield in gastrointestinal epithelial organoid culture; however, its high cost impacts clinical application. Compared to collagen, a natural extracellular matrix, synthetic biomimetic gels can achieve organoid culture and maintain clinical consistency. Furthermore, biomimetic gels with well-defined components have greater potential for clinical translation, and chemically synthesized components offer advantages in component standardization, production control, and cost.

[0003] In biomimetic gel systems, collagen peptides are often incorporated into the hydrogel framework by introducing reactive groups such as cysteine, thereby mimicking the function of collagen in the extracellular matrix, providing biocompatibility and biomechanical support, and promoting the formation, maintenance, and functional maturation of organoids. The core sequence of classic collagen peptides is GFOGER, derived from type I collagen. This sequence directly mimics the integrin binding site of type I collagen, supporting cell receptor interactions and promoting cell adhesion and organoid proliferation. For example, Matthias P. Lutolf's team used GFOGER to culture intestinal organoids in a biomimetic gel system. However, challenges remain, including high synthesis difficulty, insufficient adaptability, and unsuitability for large-scale production, hindering their industrial application in the field of regenerative medicine. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide an application of collagen peptides in the preparation of organoid culture systems. This invention is achieved through the following technical solution: This invention provides the application of collagen peptides in the preparation of organoid culture systems. The collagen peptides are obtained by random polycondensation of amino and carboxyl groups from multiple identical or different mimicry monomers to yield collagen polypeptide polymers. The polymers are then hydrogenated and deprotected to prepare the collagen peptides.

[0005] Preferably, the mimicry monomer is selected from: glycine-proline-hydroxyproline tripeptide (GPO), glycine-proline-aspartic acid tripeptide (GPD), glycine-proline-glutamic acid tripeptide (GPE), glycine-proline-lysine tripeptide (GPK), glycine-proline-arginine tripeptide (GPR), glycine-glutamic acid-arginine tripeptide (GER), glycine-proline-histidine tripeptide (GPH), glycine-proline-glutamine tripeptide (GPQ), glycine-proline-asparagine tripeptide (GPN), and glycine-proline-tryptophan tripeptide (GPW).

[0006] Preferably, the collagen peptide contains GPO monomers and GER monomers. The molar ratio of GPO monomers in the collagen peptide is 40%-80%; the molar ratio of GER monomers is 20%. More preferably, the molar ratio of GPO monomers is 60%.

[0007] Preferably, the collagen peptide is a GER-neutral collagen peptide with the following amino acid sequence: (GPO)a-co-(GPD)b-co-(GPE)c-co-(GPR)d-co-(GPK)e-co-(GER)f, where a:b:c:d:e:f=4~8:1:1:1:1:2.

[0008] More preferably, the collagen peptide amino acid sequence is: (GPO)a-co-(GPD)b-co-(GPE)c-co-(GPR)d-co-(GPK)e-co-(GER)f, a:b:c:d:e:f=6:1:1:1:1:2.

[0009] The collagen peptide is modified with glycidyl methacrylate (GMA) to introduce carbon-carbon double bonds into the collagen peptide. The epoxy groups in GMA will undergo ring-opening reactions with the secondary hydroxyl, carboxyl, amino, or guanidine groups of the collagen peptide to complete the GMA modification.

[0010] Preferably, the GMA modification rate is 5%-40%; secondary hydroxyl, carboxyl, amino, and guanidine groups all have a chance of undergoing ring-opening reactions.

[0011] The content of collagen peptides in the organoid culture system is 0.005%-0.05%.

[0012] This invention provides a collagen peptide mimicry, which is prepared by: randomly polycondensing amino and carboxyl groups of mimicry monomers GPO, GPD, GPE, GPR, GPK, and GER to obtain a collagen polypeptide polymer; hydrogenating the polymer and removing the protecting group to prepare a neutral collagen peptide mimicry containing GER, which is then modified with glycidyl methacrylate; the molar ratio of GPO, GPD, GPE, GPR, GPK, and GER is 6:1:1:1:1:2. The collagen peptide mimicry can be combined with biomimetic gels and used in the preparation of organoid culture materials to achieve organoid culture.

[0013] The organoids are selected from at least one of lung cancer organoids, intestinal organoids, gastric organoids, pancreatic organoids, liver organoids, and kidney organoids.

[0014] Compared with the prior art, the technical advantages of the present invention are as follows: (1) The present invention found that GER-containing neutral collagen peptides with a GPO monomer ratio of up to 40-80% can significantly promote the growth of organoids, and show obvious promoting effects on the proliferation and differentiation of organoids. They can be effectively combined with biomimetic gels to form a culture system that helps organoid formation.

[0015] (2) The collagen peptide preparation provided by the present invention is carried out in a homogeneous solution, which is different from the conventional solid phase synthesis method of collagen peptide. The intermediate can be taken out for recrystallization, extraction or chromatographic purification at each step, and by-products, racemates and residual protecting groups can be removed one by one. The purity of the crude product is often higher than the cumulative impurity mode of "washing to the endpoint" on solid phase resin, and the pressure of subsequent ultrafiltration or preparative chromatography will also be lower. The final condensed collagen peptide is a simplified collagen protein, and the molecular structure can be designed according to different application scenarios.

[0016] (3) The monomer raw material provided by this invention has a structure that conforms to the major amino acid cycle sequence (GXY) of natural collagen, does not contain structures with excessively high activity, and the prepared collagen peptides do not contain easily sensitizing structures in natural collagen. It has good industrialization prospects and has broader application value in the field of medical regeneration. Attached Figure Description

[0017] Figure 1 This is a graph showing the cytotoxicity test results of collagen peptides CMP4, CMP7, and CMP8 on HUVECs. Figure 2 This is a graph showing the cytotoxicity test results of collagen peptides CMP4, CMP7, and CMP8 on CAF. Figure 3 These are morphological images of intestinal organoids from the CMP7 group, the negative control group, and the Collagen Type I group. Figure 4 These are morphological feature diagrams of intestinal organoids from groups CMP4, CMP7, and CMP8. Figure 5 These are growth status diagrams of intestinal organoids from groups CMP4, CMP7, and CMP8. Figure 6 These are morphological images of gastric organoids from the CMP7 group, the negative control group, and the GFOGER group. Figure 7 These are morphological feature diagrams of organoids in hydrogels from groups CMP4, CMP7, and CMP8. Figure 8 This is a comparison of the growth status of organoids in hydrogels from groups CMP4, CMP7, and CMP8. Figure 9 These are morphological images of lung cancer organoids from the CMP7 group, the negative control group, and the GFOGER group. Figure 10 These are morphological feature images of lung cancer organoids from CMP4, CMP7, and CMP8 groups. Figure 11 This is a comparison chart of the growth status of lung cancer organoids in the CMP4, CMP7, and CMP8 groups. Detailed Implementation

[0018] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solution of this invention and the actual situation. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known chemical reagents and chemicals in the prior art; they can all be purchased commercially. Unless otherwise specified, percentages in this invention refer to mass percentages. Unless otherwise specified, solutions in this invention refer to aqueous solutions with water as the solvent. Room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.

[0019] Reagents: Corning® Collagen I was from Corning Incorporated; intestinal organoid culture medium, gastric organoid culture medium, lung cancer organoid culture medium, Hep-NB, EGF, FGF2, R-Spondin1, Gel-NB, 4-PEG-SH, MMP, HGF, and GFOGER peptides were all from Suzhou Xianjue Biotechnology Co., Ltd.

[0020] Example 1: Preparation of collagen peptides The synthetic route for collagen peptides is as follows: Wherein, G represents glycine, P represents proline, O represents hydroxyproline, D represents aspartic acid, E represents glutamic acid, R represents arginine, and K represents lysine. Obzl represents benzyl ester protecting group, NO2 represents nitro protecting group, and Z represents benzyloxycarbonyl protecting group; af represents the proportion of each monomer, and af is selected from 0-8; co It represents random aggregation.

[0021] Preparation of S1 and GER electrically neutral collagen peptides S1-1: Aggregation (1) Weigh out collagen monomers GPO (0.30 g, 0.40 eq), GPD (0.09 g, 0.10 eq), GPE (0.09 g, 0.10 eq), GPK (0.09 g, 0.10 eq), GPR (0.10 g, 0.10 eq), GER (0.23 g, 0.20 eq), DIPEA (N,N-diisopropylethylamine, 1.0 mL, 3.00 eq, used to provide an alkaline environment and ensure the nucleophilicity of the amino group), and HOBt (0.28 g, 1.10 eq) into a reaction flask. Add 15 mL of ultrapure DMF (N,N-dimethylformamide) and stir to dissolve. Purge with nitrogen three times. Place the reaction flask in an ice-salt bath (a mixture of crushed ice and NaCl, at a temperature of approximately -10 to -15°C) and treat at low temperature for 30 min. EDC (0.54 g, 1.50 eq) was slowly added to the reaction flask, and the mixture was purged with nitrogen twice before being stirred for 48 h.

[0022] (2) Most of the DMF and DIPEA were removed by rotary evaporation, followed by vacuum distillation for 1 h. 2 mL of methanol was added to the bottle to dissolve the sample, and then precipitated in 30 mL of ethyl acetate. The precipitate was centrifuged at 5000 rpm for 10 min. The dissolution, precipitation, and centrifugation were repeated 3 times. The sample was dissolved in an appropriate amount of methanol, transferred to a sample bottle, and dried using an oil pump on a double-row tube. After drying, the drying process was continued for more than 3 h to ensure complete solvent removal, finally yielding 0.83 g of GER-containing neutral collagen peptides, with a yield of 119% (including solvent).

[0023] S1-2: Hydrogenation Catalysis (1) Dissolve 0.83 g of GER-containing neutral collagen polymer in 15 mL of methanol, purging with nitrogen three times. Add 25 mg of palladium on carbon to the system, purging with nitrogen twice. Turn on the hydrogen generator to prepare a hydrogen balloon with a volume of about 2 L, connecting a syringe and needle to the bottom of the balloon. Connect the hydrogen balloon to the reaction system, purging with hydrogen three times. Stir the reaction for 2-5 days.

[0024] (2) Stop stirring and purge with nitrogen once. Using a long-needle syringe, extract the system containing palladium on carbon under anhydrous and oxygen-free conditions, and filter out the palladium on carbon under anhydrous and oxygen-free conditions. Neutralize the filtrate with 2 mL of HCl methanol solution, and then remove the methanol from the system to obtain 0.59 g of GER electrically neutral collagen peptide (abbreviated as CMP1), a white bubbly solid with a yield of 86% and a deprotection rate of 95%.

[0025] Preparation of S2 and GMA-modified GER electronegative collagen peptides (1) Dissolve 0.05 g of GER neutral collagen peptide in 500 mL of anhydrous ethanol and stir until homogeneous. Add 10 mL of glycidyl methacrylate (GMA) to the system and stir overnight at room temperature.

[0026] (2) Take the reaction system solution, precipitate it in 30 mL of ethyl acetate, and centrifuge (8000 rpm, 10 min) to obtain a white flocculent solid. Remove the supernatant, dissolve the product in 2 mL of methanol, and dry it on an oil pump for 3 hours. Dissolve the product in 1 mL of deionized water, freeze-dry for 1 day, and obtain 29.3 mg of light yellow solid GMA-modified GER electrically neutral collagen peptide (CMP4), with a yield of 50%, a molecular weight (kDa) of 1.65, and a GMA grafting rate of 32%.

[0027] CMP7 was prepared by increasing the GPO monomer content by 20% and CMP8 was prepared by increasing the GPO content by 40% according to the preparation method of CMP4. Table 1 shows the performance data of the prepared collagen peptides CMP4, CMP7 and CMP8.

[0028] Table 1. Performance data of the prepared collagen peptides CMP4, CMP7, and CMP8 The cytotoxicity of the prepared collagen peptides CMP4, CMP7, and CMP8 was tested using the CCK8 assay. Figure 1 This is a graph showing the cytotoxicity test results of collagen peptides CMP4, CMP7, and CMP8 on HUVECs. Figure 2 This is a graph showing the cytotoxicity test results of collagen peptides CMP4, CMP7, and CMP8 against CAF; (The graph is from...) Figure 1 , Figure 2As can be seen, CMP4, CMP7, and CMP8 did not exhibit significant cytotoxicity against fibroblasts (CAF) at all tested concentrations (12.5, 25, 50, 100, 200, and 400 μg / mL), and cell viability was not statistically different from the blank control group after 24 h of treatment. CMP4, CMP7, and CMP8 did not exhibit significant cytotoxicity against human umbilical vein endothelial cells (HUVECs). CMP4 and CMP7 had no significant effect on HUVEC cell viability at low concentrations (≤200 μg / mL), while CMP8 showed no significant cytotoxicity against either HUVECs or CAFs at all tested concentrations, demonstrating good biocompatibility. These results indicate that the prepared collagen-mimicking peptides CMP4, CMP7, and CMP8 all possess good biocompatibility, and modification with GMA and / or increasing the GPO monomer content in the collagen-mimicking peptides can help improve their cellular safety.

[0029] Example 2: Intestinal organoid culture Norbornene-modified heparin (Hep-NB), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), R-Spondin 1, gelatin-norbornene (Gel-NB), thiol tetraarm polyethylene glycol (4-PEG-SH), matrix metalloproteinase mimic peptide (MMP), dextran, collagen-mimicking peptide CMP7, and photoinitiator LAP were dissolved in intestinal organoid culture medium, and the above component solutions were thoroughly mixed to prepare a precursor solution. The precursor solution contained 1% Hep-NB, 100 ng / mL EGF, 200 ng / mL FGF2, 1 μg / mL R-Spondin 1, 10% Gel-NB, 0.5% 4-PEG-SH, 0.05% MMP, 5% dextran, 100 μg / mL collagen-mimicking peptide CMP7, and 0.1% photoinitiator LAP. Mix the precursor solution with 2×10 5 The hydrogel precursor solution was prepared by mixing mouse intestinal cell suspension at a density of 10 mW / mL. The hydrogel precursor solution was then subjected to light at a wavelength of 365 nm and an intensity of 10 mW / cm². 2Cure it under ultraviolet light for 3 min. The obtained bionic hydrogel system was used as the experimental group (Group C). Prepare a negative control group and a positive control group. The difference from the experimental group is that in the negative control group (Group A), CMP or Collagen Type I is not added, and in the positive control group (Group B), natural collagen Collagen Type I with a concentration of 0.06% is added to replace the collagen-mimicking peptide CMP7. 200 μL of intestinal organoid medium was added to each group, and the medium was changed every 2 - 3 days according to the growth state of the organoids. Place the 96-well plate in a cell culture incubator for culture, and observe and record the original data on the growth state of the intestinal organoids in each group on the 7th day. On the 7th day of culture, there were significant differences in the growth state of the intestinal organoids among the groups. Record the morphological characteristics of the organoids in the hydrogels of each group through bright-field microscopy imaging. Figure 3 are the morphological diagrams of the intestinal organoids in the CMP7 group, the negative control group, and the Collagen Type I group; in the figure, A is the negative control group, B is the Collagen Type I group, and C is the CMP7 group; Figure 3 It can be seen that the intestinal organoids in the negative control group were almost completely dissociated into single cells, indicating that the organoids could not maintain their structural integrity without the support of collagen components. On the contrary, the intestinal organoids in the positive control group with the addition of natural collagen Collagen Type I grew well and had a complete morphology, confirming the key role of collagen in maintaining the structure and supporting the growth of the organoids. The CMP7 group showed a certain ability to support organoids and could maintain the structural integrity of the intestinal organoids. It can be seen that the electro-neutral collagen-mimicking peptide CMP7 containing GER provided by the present invention has the potential to partially replace natural collagen and can support the normal growth and morphological maintenance of intestinal organoids.

[0030] Replace CMP7 in the precursor solution of this example with CMP4 and CMP8 of the same concentration respectively, and investigate the organoid formation ability of CMP4, CMP7, and CMP8 combined with the bionic gel. On the seventh day, record the morphological characteristics of the organoids in the hydrogels of each group through bright-field microscopy imaging. Figure 4 are the morphological characteristic diagrams of the organoids in the CMP4, CMP7, and CMP8 hydrogels; Figure 4 It can be seen that the promoting effect of CMP4 and CMP8 on the growth of intestinal organoids is weaker than that of CMP7, indicating that an appropriate molecular weight is crucial for maintaining the function of the polypeptide.

[0031] Take the roundness and area of the organoids as quantitative evaluation indicators, and comprehensively evaluate the growth state of the organoids in each group; Figure 5 are the growth state diagrams of the organoids in the CMP4, CMP7, and CMP8 hydrogels; in the figure, A is the surface area diagram of the organoids, and B is the roundness diagram of the organoids; Figure 5As can be seen, there was no significant difference in the roundness of the intestinal organoids among the hydrogels of different groups, and the addition of CMP4, CMP7, and CMP8 could all maintain the structural integrity of the intestinal organoids. Compared with CMP4 and CMP8, CMP7 significantly increased the area of ​​the intestinal organoids, indicating that increasing the GPO content in collagen peptides is beneficial to the growth of intestinal organoids.

[0032] Example 3: Culture of gastric organoids Hep-NB, HGF, EGF, Gel-NB, 4-PEG-SH, MMP, dextran, collagen-mimicking peptide CMP7, and photoinitiator LAP were dissolved in gastric organoid culture medium, and the above components were thoroughly mixed to prepare a precursor solution. The precursor solution contained 1% Hep-NB, 500 ng / mL HGF, 200 ng / mL EGF, 10% Gel-NB, 3% 4-PEG-SH, 0.01% MMP, 5% dextran, 100 μg / mL collagen-mimicking peptide CMP7, and 0.1% photoinitiator LAP. The precursor solution was then mixed with 2 × 10⁻⁶... 5 The hydrogel precursor solution was prepared by mixing mouse gastric cell suspension at a concentration of 10 mW / mL. The hydrogel precursor solution was then subjected to light at a wavelength of 365 nm and an intensity of 10 mW / cm². 2 The cells were cured into a biomimetic gel by irradiation under ultraviolet light for 3 minutes. The resulting biomimetic hydrogel system served as the experimental group. A negative control group and a positive control group were also prepared. The difference between the positive and negative control groups was that the negative control group did not contain CMP or GFOGER peptides, while the positive control group contained 600 μg / mL GFOGER peptides instead of the collagen-mimicking peptide CMP7. Each group was added to 200 μL of gastric organoid culture medium, with the medium changed every 2-3 days depending on the growth status. The 96-well plates were incubated in a cell culture incubator. On day 7, the growth status of the gastric organoids in each group was observed and photographed for analysis.

[0033] The morphological characteristics of organoids in each group of hydrogels were recorded using bright-field microscopy. Figure 6 These are morphological images of gastric organoids in the CMP7 group, negative control group, and GFOGER group; in the image, A is the negative control group, B is the GFOGER group, and C is the CMP7 group; Figure 6 As can be seen, there were significant differences in the growth status of gastric organoids among the groups on day 7 of culture. Compared with the negative control group, both the CMP7 group and the positive control group with added GFOGER significantly promoted the growth of gastric organoids. The CMP7 group not only effectively supported the growth of gastric organoids, but its growth-promoting effect was even better than that of the positive control group with added GFOGER. Therefore, the GER-containing neutral collagen peptide CMP7 provided by this invention can significantly promote the growth of gastric organoids and shows a significant promoting effect on the proliferation and differentiation of gastric organoids.

[0034] CMP4 and CMP8 with the same concentration were used to replace CMP7 in the precursor solution of this example respectively, and the ability of CMP4, CMP7, and CMP8 to combine with the biomimetic gel to generate organoids was investigated. On the seventh day, the morphological characteristics of the organoids in each hydrogel were recorded by bright-field microscopy imaging. Figure 7 are the morphological characteristic diagrams of the organoids in the CMP4 group, CMP7 group, and CMP8 group hydrogels; Figure 7 It can be seen that CMP4, CMP7, and CMP8 can all promote the growth of gastric organoids. The promoting effect of CMP4 on the growth of gastric organoids is weaker than that of CMP7, suggesting that an appropriate molecular weight is crucial for maintaining the function of the polypeptide.

[0035] The roundness and area of the organoids were used as quantitative evaluation indicators to comprehensively evaluate the growth status of the organoids in each group; Figure 8 are the comparison diagrams of the growth status of the organoids in the CMP4 group, CMP7 group, and CMP8 group hydrogels; in the figure, A is the surface area diagram of the organoids, and B is the roundness diagram of the organoids; Figure 8 It can be seen that there is no significant difference in the roundness of the gastric organoids in each hydrogel group, and adding CMP4, CMP7, and CMP8 can all maintain the structural integrity of the gastric organoids. Compared with CMP4 and CMP8, CMP7 more significantly increases the area of the gastric organoids, indicating that appropriately increasing the GPO content is helpful for the growth of gastric organoids.

[0036] Example 4 Culture of lung cancer organoids Hep-NB, HGF, EGF, R-Spondin 1, Gel-NB, 4-PEG-SH, MMP, dextran, collagen-mimicking peptide CMP7, and photoinitiator LAP were dissolved in the lung cancer organoid culture medium, and the above component solutions were fully mixed to prepare a precursor solution. The concentration of Hep-NB in the precursor solution was 1%, the concentration of HGF was 250 ng / mL, the concentration of EGF was 100 ng / mL, the concentration of R-Spondin 1 was 1 μg / mL, the concentration of GFOGER was 650 μg / mL, the concentration of collagen-mimicking peptide CMP7 was 100 μg / mL, the concentration of Gel-NB was 10%, the concentration of 4-PEG-SH was 3%, the MMP content was 0.01%, the concentration of dextran was 5%, and the concentration of photoinitiator LAP was 0.1%. Each precursor solution was mixed with 2×10 5 cells / mL of human lung cancer cell suspension to obtain a hydrogel precursor solution. Each hydrogel precursor solution was irradiated at a wavelength of 365 nm and a light intensity of 10 mW / cm 2It was cured into a biomimetic gel by irradiating with ultraviolet light for 3 min. The obtained biomimetic hydrogel system was used as the experimental group. At the same time, a negative control group, a positive control group, a CMP4 group, and a CMP8 group were prepared. The difference from the experimental group was that CMP or GFOGER was not added in the negative control group, and GFOGER polypeptide with a concentration of 650 μg / mL was added in the positive control group to replace the collagen-mimicking peptide CMP7. In the CMP4 group and the CMP8 group, CMP4 and CMP8 with the same concentration were used to replace the collagen-mimicking peptide CMP7, respectively. 200 μL of lung cancer organoid medium was added to each group, 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 7th day, the growth status of lung cancer organoids in each group was observed and photographed for analysis.

[0037] The morphological characteristics of lung cancer organoids in the hydrogels of each group were recorded by bright-field imaging under a microscope. On the 7th day of culture, there were significant differences in the growth status of lung cancer organoids in each group. Figure 9 are the morphological characteristic diagrams of lung cancer organoids in the CMP7 group, the negative control group, and the GFOGER group; in the figure, A is the negative control group, B is the GFOGER group, and C is the CMP7 group; from Figure 9 it can be seen that compared with the negative control group, both the positive control group with added GFOGER and the CMP7 group can significantly promote the growth of lung cancer organoids. The CMP7 group not only effectively supports the growth of lung cancer organoids, but its growth-promoting effect is even better than that of the positive control group. Thus, the electroneutral collagen-mimicking peptide CMP7 containing GER provided by the present invention can significantly promote the growth of lung cancer organoids, and the effect is better than that of the positive control GFOGER polypeptide. Figure 10 are the morphological characteristic diagrams of lung cancer organoids in the CMP4 group, the CMP7 group, and the CMP8 group; from Figure 10 it can be seen that the promoting effect of CMP4 and CMP8 on the growth of lung cancer organoids is weaker than that of CMP7. Adding CMP4 can promote the growth of lung cancer organoids, but the promoting effect of CMP4 on the growth of lung cancer organoids is weaker than that of CMP7, suggesting that an appropriate molecular weight is crucial for maintaining the function of the polypeptide.

[0038] The roundness and area of the organoids were used as quantitative evaluation indexes to comprehensively evaluate the growth status of organoids in each group. Figure 11 are the comparison diagrams of the growth status of lung cancer organoids in the CMP4 group, the CMP7 group, and the CMP8 group; in the figure, A is the surface area diagram of the organoids, and B is the roundness diagram of the organoids; from Figure 11 it can be seen that there was no significant difference in the roundness of lung cancer organoids in the hydrogels of each group, indicating that the heparin hydrogel system containing the GER-containing electroneutral collagen-mimicking peptide of the present invention can maintain the structural integrity of lung cancer organoids. Compared with CMP4 and CMP8, the CMP7 group significantly increased the area of lung cancer organoids, indicating that appropriately increasing the GPO content is helpful for the growth of lung cancer organoids.

[0039] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of a collagen peptide in the preparation of organoid culture systems, characterized in that, The collagen peptides are obtained by random polycondensation of amino and carboxyl groups of multiple identical or different mimicry monomers to obtain collagen polypeptide polymer products; the obtained polymer products are then hydrogenated and deprotected to prepare collagen peptides.

2. The application according to claim 1, characterized in that, The collagen peptide contains GPO monomers and GER monomers; the molar ratio of GPO monomers is 40%-80%; the molar ratio of GER monomers is 20%.

3. The application according to claim 1, characterized in that, The collagen peptide is a GER-neutral collagen peptide with the following amino acid sequence: (GPO)a-co-(GPD)b-co-(GPE)c-co-(GPR)d-co-(GPK)e-co-(GER)f, where a:b:c:d:e:f=4~8:1:1:1:1:

2.

4. The application according to claim 1, characterized in that, The content of collagen peptides in the organoid culture system is 0.005-0.05%.

5. The application according to claim 1, characterized in that, The organoids are selected from at least one of lung cancer organoids, intestinal organoids, gastric organoids, pancreatic organoids, liver organoids, and kidney organoids.

6. The application according to claim 1, characterized in that, The mimicry monomers are selected from: GPO, GPD, GPE, GPK, GPR, GER, GPH, GPQ, GPN and GPW.

7. The application according to claim 1, characterized in that, The collagen peptides are modified with GMA, and carbon-carbon double bonds are introduced into the collagen peptides; the modification rate of GMA modification is 5%-40%.

8. A collagen peptide for use in organoid culture systems, characterized in that, The preparation method of the collagen peptide is as follows: random polycondensation of amino and carboxyl groups of the mimic monomers GPO, GPD, GPE, GPR, GPK, and GER to obtain collagen polypeptide polymer products; hydrogenation catalysis and removal of protecting groups of the obtained polymer products to prepare GER-containing neutral collagen peptides, and modification of the peptides with glycidyl methacrylate; the molar ratio of GPO, GPD, GPE, GPR, GPK, and GER is 6:1:1:1:1:

2.

9. The collagen peptide according to claim 8, characterized in that, The collagen peptides are combined with a biomimetic gel for organoid culture.

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