Use of a collagen peptoid in the preparation of a vascularized culture system

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to application of a collagen peptoid in preparation of a vascularized culture system. It is found for the first time that the electrically neutral collagen peptoid containing GER with high GPO monomer proportion has a stable 3D vascularized structure, can be effectively combined with a biomimetic gel component, and is helpful to formation of organoid vascularization. The collagen peptoid has a good industrialization prospect and has a wider application value in the field of medical regeneration.

Description

Technical Field

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

[0002] The "gold standard" material for inducing endothelial cells (ECs) to self-assemble into vascularized structures in three-dimensional space relies on the natural extracellular matrix, especially... Type II collagen and matrix collagen. However, the inherent defects of natural extracellular matrix—batch-to-batch variability, unclear composition, immunogenicity, and poor adjustability of mechanical properties—hinder its clinical translation.

[0003] In recent years, synthetic biomimetic gels incorporating collagen-like peptides are gradually replacing the natural extracellular matrix. By introducing reactive groups such as cysteine, collagen-like peptides can be precisely anchored in mechanically tunable hydrogel networks, mimicking the function of collagen in the extracellular matrix and thus finely regulating the adhesion, migration, proliferation, and lumen formation of endothelial cells. GFOGER is the classic core sequence of vascularized collagen-like peptides, and this collagen-specific polypeptide shows significant advantages in promoting vascular maturation. However, it has shortcomings in stability, preparation efficiency, and application suitability, limiting its development in organoid vascularization technology and its industrial application in the field of medical regeneration. 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 vascularized culture systems. This invention is achieved through the following technical solution: This invention provides an application of collagen peptide mimicry in the preparation of a vascularized culture system. The collagen peptide mimicry is obtained by random polycondensation of amino and carboxyl groups of multiple identical or different mimicry monomers to yield a collagen polypeptide polymer; the obtained polymer is then hydrogenated and catalyzed to prepare the collagen peptide mimicry; the mimicry monomers contain GPO monomers and GER monomers.

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

[0006] Preferably, 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 80%.

[0007] Preferably, the purpose of the hydrogenation catalysis is to remove protecting groups such as nitro, benzyl ester, and benzyloxycarbonyl groups from the polymerization product.

[0008] 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 of the product.

[0009] Preferably, the GMA modification rate is 5%-40%; secondary hydroxyl, carboxyl, amino, and guanidine groups all have a chance of undergoing ring-opening reactions.

[0010] 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, a:b:c:d:e:f=4~8:1:1:1:1:2.

[0011] More preferably, the amino acid sequence of the collagen peptide is: (GPO)a-co-(GPD)b-co-(GPE)c-co-(GPR)d-co-(GPK)e-co-(GER)f, a:b:c:d:e:f=8:1:1:1:1:2.

[0012] The content of collagen peptides in the vascularization culture system is 0.005%-0.05%.

[0013] The present invention provides a collagen peptide for vascular culture, which is prepared by: random polycondensation of amino and carboxyl groups of the mimic monomers GPO, GPD, GPE, GPR, GPK, and GER to obtain a collagen polypeptide polymer; hydrogenation catalysis and removal of protecting groups of the obtained polymer to prepare an electrically neutral collagen peptide containing GER, and modifying it with glycidyl methacrylate (GMA); the molar ratio of GPO, GPD, GPE, GPR, GPK, and GER is 8:1:1:1:1:2.

[0014] The collagen peptides provided by this invention can be combined with biomimetic gels to achieve vascularized culture.

[0015] Compared with the prior art, the technical advantages of the present invention are as follows: (1) This invention is the first to discover that GER-containing neutral collagen peptides with a high proportion of GPO monomers have a stable 3D vascularization structure, which can effectively combine with biomimetic gel components and help to form organoid vascularization.

[0016] (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.

[0017] (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

[0018] Figure 1 This is a graph showing the cytotoxicity test results of collagen peptide CMP1-CMP8 on HUVECs. Figure 2 This is a graph showing the cytotoxicity test results of collagen peptide CMP1-CMP8 on CAF. Figure 3 This is a diagram of the primary vascularized culture system of CMP4 and GFOGER. Figure 4 This is a vascularized culture image of the iPSC system after one day of culture; Figure 5 This is a vascularized culture image of the iPSC system after 5 days of culture; Figure 6 This is an immunofluorescence staining image of capillary lumen cultured with CMP8-bound biomimetic gel; Figure 7 This is a comparison image of vascularization culture in the CMP7 / CMP8 iPSC system; Figure 8 This is a co-culture image of vascularized lung cancer organoids from CMP4; Figure 9 This is a co-culture image of vascularized lung cancer organoids from CMP8; Figure 10 These are images showing the co-culture of hepatocellular carcinoma organoids at different time points. Detailed Implementation

[0019] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known in the prior art and can 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, and is generally defined as 25°C.

[0020] Reagents: Vascularization culture medium, lung cancer organoid culture medium, liver cancer organoid culture medium, GFOGER peptide, VEGF, FGF2, PDGF-BB, and SDF-1α were all from Suzhou Xianjue Biotechnology Co., Ltd.

[0021] 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.

[0022] Preparation of S1 and GER electrically neutral collagen peptides S1-1, GER Electron-neutral Collagen Peptide Polymerization (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. Open the reaction flask and slowly add EDC (0.54 g, 1.50 eq) into the reaction flask. Purge with nitrogen twice and stir the reaction for 2 days.

[0023] (2) Post-treatment: 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 process of dissolving, precipitating, and centrifuging was repeated 3 times.

[0024] (3) Product drying: Dissolve the sample in an appropriate amount of methanol, transfer it to a sample vial, and dry it using an oil pump on a double-row tube. Continue drying for at least 3 hours after drying to ensure complete solvent removal. The final yield was 0.83 g of GER-containing neutral collagen peptide, with a yield of 119% (including solvent).

[0025] Hydrogenation catalysis of S1-2 and GER electronegative collagen peptides (1) Reactant preparation: 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 approximately 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.

[0026] (2) Post-treatment: Stop stirring and purge with nitrogen once. Use a long-needle syringe to extract the palladium-containing system under anhydrous and oxygen-free conditions, and filter out the palladium-containing system under anhydrous and oxygen-free conditions.

[0027] (3) Product drying: The filtrate was neutralized with 2 mL of HCl methanol solution, and then the methanol in the system was dried to obtain 0.59 g of white bubbly solid GER electrically neutral collagen peptide (abbreviated as CMP1), with a yield of 86% and a deprotection rate of 95%.

[0028] Preparation of S2 and GMA-modified GER electronegative collagen peptides (1) Reactant addition: 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.

[0029] (2) Post-treatment: Take the reaction system solution, precipitate it in 30 mL of ethyl acetate, centrifuge (8000 rpm, 10 min) to obtain a white flocculent solid. Remove the supernatant, dissolve the product in 2 mL of methanol, and place it on an oil pump to dry for 3 hours.

[0030] (3) Product freeze-drying: Dissolve the product in 1 mL of deionized water and freeze-dry for 1 day to 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%.

[0031] Prepare collagen-mimetic peptides with different GPO contents according to the preparation method of CMP4. Increase the GPO monomer content by 20% based on the original CMP4 ratio to prepare CMP7, and increase the GPO content by 40% based on the original CMP4 ratio to prepare CMP8. Table 1 is the performance data table of the prepared collagen-mimetic peptides CMP4, CMP7, and CMP8.

[0032] Table 1. Performance data table of the prepared collagen-mimetic peptides CMP4, CMP7, and CMP8 Perform cytotoxicity tests on the prepared collagen-mimetic peptides CMP4, CMP7, and CMP8 using the CCK8 method. Figure 1 It is the cytotoxicity test result graph of collagen-mimetic peptides CMP4, CMP7, and CMP8 on HUVEC; Figure 2 It is the cytotoxicity test result graph of collagen-mimetic peptides CMP4, CMP7, and CMP8 on CAF; As can be seen from Figure 1 、 Figure 2 It can be seen that CMP4, CMP7, and CMP8 did not show obvious cytotoxicity to fibroblasts (CAF) at all test concentrations (12.5, 25, 50, 100, 200, 400 μg / mL). After 24 h of treatment, the cell viability was not statistically different from that of the blank control group. CMP4, CMP7, and CMP8 did not show significant cytotoxicity to human umbilical vein endothelial cells (HUVEC). CMP4 and CMP7 had no significant effect on the cell viability of human umbilical vein endothelial cells (HUVEC) at low concentrations (≤200 μg / mL). CMP8 did not show obvious cytotoxicity to HUVEC and CAF at all test concentrations and had good biocompatibility. The results show that modifying with GMA and / or increasing the GPO monomer content in the collagen-mimetic peptide helps to improve the cell safety of the collagen-mimetic peptide.

[0033] Example 2: Primary system vascularization culture of CMP4 Norbornene-modified heparin (Hep-NB), CMP4, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), platelet-derived growth factor-BB (PDGF-BB), gelatin-norbornene (Gel-NB), thiol tetra-arm polyethylene glycol (4-PEG-SH), matrix metalloproteinase mimic peptide (MMP), fibrinogen, and photoinitiator LAP were thoroughly mixed in proportion and dissolved in PBS solution (9%) to form a precursor solution. The concentrations of Hep-NB, CMP4, VEGF, FGF2, PDGF-BB, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were 0.05%. Mix the precursor solution with 1×10 6 / mL of RFP-HUVEC cell suspension, 1×10 5 A hydrogel precursor was formed by mixing CAF cell suspension at a concentration of / mL. The hydrogel precursor was then subjected to light at a wavelength of 365 nm and an intensity of 5 mW / cm². 2 Expose the sample to ultraviolet light at a specific intensity for 5 min, solidify it into a biomimetic gel, and then incubate it upside down in a 37 ℃ incubator for 10 min. Add vascularization medium and culture, changing the medium every 48 h. Use 600 μg / mL GFOGER peptide instead of CMP4 as a control group. Figure 3 This is a diagram of the primary vascularized culture system of CMP4 and GFOGER; by Figure 3 It was observed that, after the second day of culture, both the collagen peptide CMP4 group and the control group containing GFOGER peptide exhibited the formation of a reticular structure, with endothelial cells proliferating and elongating at their tips to form the reticular structure. These results indicate that GMA-modified, GER-containing, electrically neutral collagen peptide CMP4 can combine with biomimetic gels to form a vascularized reticular structure, facilitating the formation of 3D angiogenesis.

[0034] Example 3: Vascularization culture of CMP8 iPSC system Hep-NB, CMP8, VEGF, FGF2, PDGF-BB, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were thoroughly mixed in a specific ratio and dissolved in PBS to form a precursor solution. The concentrations of Hep-NB, CMP8, VEGF, FGF2, PDGF-BB, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and LAP were 0.05%, 0.35%, 0.5%, 0.08%, 0.1%, and 0.05%, respectively. The precursor solution was then mixed with 1.4 × 10⁻⁶ PBS solution. 6 / mL of RFP-iPSC-EC cell suspension, 1.4×10 5 A hydrogel precursor was prepared by mixing iPSC-MSC cell suspension at a concentration of / mL. The hydrogel precursor was then incubated under light at a wavelength of 365nm and an intensity of 5 mW / cm². 2 Expose the sample to ultraviolet light for 5 min to solidify it into a biomimetic gel. Then, invert the gel in a 37 ℃ incubator for 10 min and add vascularization medium for further culture. This group serves as the experimental group. Simultaneously, replace CMP8 in the precursor solution with 600 μg / mL GFOGER peptide, and use this as the control group. Figure 4 This is a vascularized culture image of the iPSC system after one day of culture; Figure 5 This is a vascularized culture image of the iPSC system after 5 days of culture; by Figure 4 , Figure 5 As observed, after day 1 of culture, iPSC-induced endothelial cell proliferation rapidly formed a network structure in both CMP8 and GFOGER biomimetic gels, with lumen diameters of approximately 20 μm. In the CMP8 group, after 5 days of culture, the vascularized network structure remained stable, with some lumen diameters approaching 70 μm. This indicates that the GER-containing, neutrally charged collagen peptide CMP8-bound biomimetic gel facilitates the rapid generation of 3D angiogenesis, forming a stable 3D vascularized structure.

[0035] Figure 6 This is an immunofluorescence staining image of capillary lumen cultured from a biomimetic gel bound to CMP8; by Figure 6 As can be seen, after 7 days of culture, the iPSC-derived endothelial capillary network with RFP labeling showed abundant CD31 expression, indicating that the vascular network was mature and stable. CD31 is a surface protein with extremely high endothelial cell specificity, and its expression is stable and abundant in mature vascular endothelial cells. Therefore, the biomimetic gel bound to collagen peptide CMP8 can form a stable 3D vascularized network.

[0036] Using the same ratio as CMP8, 100 ug / mL of CMP7 was used to replace CMP8 in the precursor solution of this embodiment, and the angiogenic ability of CMP7 and CMP8 combined with the biomimetic gel was investigated. Figure 7 These are comparative images of vascularized culture in the CMP7 and CMP8 iPSC systems; by Figure 7 It was observed that after one day of culture, the tips of endothelial cells in the CMP8 group elongated and interconnected, forming a prevascular network structure. Compared to CMP7, the collagen-mimicking peptide CMP8, with a molecular weight of approximately 4000, has a higher proportion of GPO monomers, which is more conducive to the rapid formation of angiogenesis.

[0037] Example 4: Vascularization culture of lung cancer organoids (primary helper cells) The precursor solution was prepared according to the proportions in Example 2, except that the concentration of Hep-NB was adjusted to 0.35% and the concentration of CMP4 was adjusted to 100 μg / mL. The precursor solution was then mixed with 1×10... 6 / mL of RFP-HUVEC cell suspension, 1×10 5 / mL of CAF cell suspension and 4×10 3 A biomimetic gel precursor solution was obtained by mixing the hydrogel precursor with lung cancer organoid culture medium at a concentration of / mL. The hydrogel precursor was then subjected to light at a wavelength of 365 nm and an intensity of 5 mW / cm². 2 Expose the sample to ultraviolet light for 5 min to solidify it into a biomimetic gel, then invert it in a 37°C incubator for 10 min. Add lung cancer organoid culture medium and vascularization culture medium at a concentration of 1:1, and adjust the final concentration of VEGF and FGF2 to 50 ng / mL. Change the medium every 48 h. Figure 8 This is a co-culture image of vascularized lung cancer organoids from CMP4. (By...) Figure 8 As observed, after the second day of culture, the round lung cancer organoids grew normally, with some endothelial cells sprouting and connecting at their tips to form a network structure, and some endothelial cells growing around the outer edge of the tumor organoids. This indicates that the collagen peptide CMP4 can effectively bind to the biomimetic gel components, contributing to the formation of angiogenesis in lung cancer organoids.

[0038] Example 5: Vascularization culture of lung cancer organoids (iPSC helper cells) Hep-NB, CMP8, VEGF, FGF2, SDF-1α, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were dissolved in PBS solution and thoroughly mixed. The concentrations of Hep-NB, CMP8, VEGF, FGF2, SDF-1α, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were 0.05%, 0.5%, 0.08%, 0.1%, and 0.05%, respectively. The precursor solution was then mixed with 1.4 × 10⁶ / mL RFP-iPSC-EC cell suspension, 1.4 × 10⁵ / mL iPSC-MSC cell suspension, and 4 × 10⁶ / mL PBS solution. 3 The biomimetic gel precursor solution was prepared by mixing lung cancer organoid culture medium at a concentration of / mL. The hydrogel precursor was exposed to ultraviolet light at a wavelength of 365 nm and an intensity of 5 mW / cm² for 5 min to solidify into a biomimetic gel. The gel was then incubated upside down at 37℃ for 10 min. Lung cancer organoid culture medium and vascularization culture medium at a concentration of 1:1 were added, and the final concentrations of VEGF and FGF2 were adjusted to 50 ng / mL. The medium was changed every 48 h. Figure 9 This is a co-culture image of vascularized lung cancer organoids from CMP8; by Figure 9 It was observed that, after 5 days of culture, some capillaries invaded the lung cancer organoids. The results indicate that the biomimetic gel containing the collagen-mimicking peptide CMP8 can replicate the effect of vascular invasion of tumors.

[0039] Example 6: Vascularization culture of liver cancer organoids (iPSC helper cells) Hep-NB, CMP8, VEGF, FGF2, PDGF-BB, Gel-NB, 4-PEG-SH, MMP, fibrinogen, and photoinitiator LAP were dissolved in PBS solution, and the above component solutions were thoroughly mixed. The concentrations of Hep-NB (0.35%), CMP8 (100 μg / mL), VEGF (2 μg / mL), FGF2 (2 μg / mL), SDF-1α (2 μg / mL), Gel-NB (2.5%), 4-PEG-SH (0.5%), MMP (0.08%), fibrinogen (0.1%), and photoinitiator LAP (0.05%) were mixed to a final volume of 2.5 × 10⁻⁶. 6 / mL of RFP-iPSC-EC cell suspension, 2.5×10 5 / mL of iPSC-MSC cell suspension and 5×10 3A biomimetic gel precursor solution was obtained from liver cancer organoids at a concentration of 5 mW / cm³. 2 Expose the sample to ultraviolet light for 5 min to solidify it into a biomimetic gel, then invert it in a 37°C incubator for 10 min. Add lung cancer organoid culture medium and vascularization culture medium at a concentration of 1:1, and adjust the final concentration of VEGF and FGF2 to 50 ng / mL. Change the medium every 48 h. Figure 10 These are images showing the vascularization of hepatocellular carcinoma organoids co-cultured at different time points; such as... Figure 10 As shown, the inoculated cystic hepatocellular carcinoma organoids maintained this morphology on day 2 of co-culture. On day 4 of co-culture, all cystic hepatocellular carcinoma organoids contracted inward and underwent substantial growth, with some showing epithelial-mesenchymal transition. This demonstrates that the biomimetic gel containing the collagen-mimicking peptide CMP8 can exhibit the promoting effect of angiogenesis on invasive tumor growth.

[0040] Although the present invention has been described in detail above with general descriptions and specific embodiments, 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 a vascularized culture system, characterized in that, The collagen peptide is 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 prepared by hydrogenation catalysis to obtain collagen peptides; the mimicry monomers contain GPO monomers and GER monomers.

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

3. The application according to claim 1, characterized in that, The collagen peptide contains 40%-80% GPO monomer and 20% GER monomer.

4. The application according to claim 1, characterized in that, The collagen peptides are modified with GMA to introduce carbon-carbon double bonds into the collagen peptides.

5. The application according to claim 4, characterized in that, The GMA modification rate is 5%-40%.

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

2.

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

2.

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

9. A collagen peptide analogue for use in vascularized culture, 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; the obtained polymer products are hydrogenated and catalyzed to prepare GER-containing neutral collagen peptides, which are then modified with glycidyl methacrylate to obtain the final product; the molar ratio of GPO, GPD, GPE, GPR, GPK, and GER is 8:1:1:1:1:

2.

10. The collagen peptide according to claim 9, characterized in that, The collagen peptides can be combined with biomimetic gels for vascularized culture.

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