A ternary dynamic glycopeptide hydrogel bio-ink, its preparation method and application in colorectal organoid culture

Abstract

This invention discloses a method for preparing a ternary dynamic glycopeptide hydrogel bio-ink, comprising the following steps: dissolving methacrylamide gelatin, aldehyde-modified hyaluronic acid, hydrazide-modified gelatin, and a photoinitiator together in a solvent and mixing them evenly to obtain the bio-ink. This bio-ink constructs a dynamic covalent cross-linking network through reversible acylhydrazone bonds formed between aldehyde-modified hyaluronic acid and hydrazide-modified gelatin, providing a remodelable three-dimensional microenvironment for cells. Simultaneously, the photocrosslinking properties of methacrylic anhydride-modified gelatin are introduced to rapidly solidify and maintain overall shape stability during the printing stage, providing a remodelable cellular microenvironment during subsequent in vitro culture. This establishes an organoid culture platform with both good printability and excellent cell compatibility, providing a reliable material basis for subsequent drug screening and functional studies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a ternary dynamic glycopeptide hydrogel bio-ink, its preparation method, and its application in colorectal organoid culture. Background Technology

[0002] With the widespread application of colorectal organoid technology in disease model construction and drug screening, higher demands are being placed on bio-inks in terms of printability, cellular microenvironment regulation, and functional maintenance. The success of colorectal organoid culture is highly dependent on the accurate simulation of the intestinal stem cell nest microenvironment, including the synergistic effects of basement membrane composition, matrix stiffness, and tissue-specific biochemical signals. Currently used matrix gels suffer from problems such as unclear composition and large batch-to-batch variability. While traditional synthetic hydrogels such as methacrylamide gelatin (GelMA) have good biocompatibility, they have technical drawbacks such as a narrow printable temperature window (typically 23-28°C) and a long stress relaxation half-life (typically >1000 s). Although simply introducing dynamic crosslinking can improve stress relaxation, it sacrifices the structural stability after photocrosslinking. At the same time, excessively high dynamic crosslinking density can reduce printability due to mechanical enhancement (e.g., high extrusion stress leads to low cell viability).

[0003] To address the aforementioned issues, an ideal organoid culture hydrogel should possess a wide printable temperature window to ensure structural fidelity in high-throughput printing, while its mechanical network should exhibit appropriate dynamics—allowing cells to undergo local remodeling through traction forces to support stem cell proliferation and organoid morphogenesis. Furthermore, the system should simplify operational steps as much as possible to adapt to standardized production processes. However, existing material systems struggle to simultaneously meet these requirements. Therefore, developing a novel bio-ink system that overcomes the limitations of traditional GelMA while possessing excellent printability and a suitable microenvironment for organoid development is crucial for promoting high-throughput, automated culture of intestinal organoids and other stem cell-derived organoids. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a ternary dynamic glycopeptide hydrogel bio-ink that addresses the shortcomings of the prior art.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the ternary dynamic glycopeptide hydrogel bio-ink.

[0006] The final technical problem to be solved by this invention is to provide the application of the ternary dynamic glycopeptide hydrogel bioink in colorectal organoid culture.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0008] The first aspect of this invention provides a ternary dynamic glycopeptide hydrogel bio-ink, which is made by mixing the following raw materials: methacrylamide gelatin, aldehyde-modified hyaluronic acid, hydrazide-modified gelatin, photoinitiator and solvent.

[0009] Wherein, the aldehyde-modified hyaluronic acid is obtained by reacting hyaluronic acid or its sodium salt with 3-amino-1,2-propanediol and then oxidizing it with periodate; and / or, the degree of modification of the aldehyde-modified hyaluronic acid is 10-30%, preferably 13-16%, and most preferably 13.6%.

[0010] The method for preparing the aldehyde-modified hyaluronic acid is as follows:

[0011] Step 1: Under ice bath and stirring conditions, sulfo-NHS and EDC·HCl are added to a solution of hyaluronic acid or its sodium salt to carry out the first reaction and obtain the first reaction solution.

[0012] Step 2: Add 3-amino-1,2-propanediol solution to the first reaction solution to carry out the second reaction, obtain the second reaction solution, perform the first dialysis, freeze dry, and obtain the first product;

[0013] Step 3: Dissolve the first product in water and add periodate aqueous solution to carry out the third reaction to obtain the third reaction solution. Add ethylene glycol to the solution to carry out the fourth reaction to obtain the fourth reaction solution. Perform a second dialysis and freeze dry to obtain the final product.

[0014] In step 1, the solvent for the solution of hyaluronic acid or its sodium salt is MES buffer or sodium acetate buffer; the concentration of the MES buffer is 0.05 ~ 0.2 M, and the pH is 5 ~ 5.5; preferably, the concentration of the MES buffer is 0.1 M, and the pH is 5.5; the concentration of the sodium acetate buffer is 0.05 ~ 0.2 M, and the pH is 4.5 ~ 5.5.

[0015] In step 1, the amount of sulfo-NHS added is 1 times the amount of carboxyl groups contained in the hyaluronic acid or its sodium salt; the amount of EDC·HCl added is 2 times the amount of carboxyl groups contained in the hyaluronic acid or its sodium salt; the first reaction is carried out for 30 min under ice bath and stirring conditions.

[0016] In step 2, the solvent for the 3-amino-1,2-propanediol solution is a phosphate buffer solution with a concentration of 0.05-0.2 M and a pH of 7.2-7.4; preferably, the concentration of the phosphate buffer solution is 0.1 M; the amount of 3-amino-1,2-propanediol solution added is controlled such that the amount of 3-amino-1,2-propanediol added is at least twice the amount of carboxyl groups contained in the hyaluronic acid or its sodium salt, preferably twice; preferably, the concentration of the 3-amino-1,2-propanediol solution is 0.5 M; the 3-amino-1,2-propanediol solution is added dropwise to the first reaction solution at a flow rate of 3-6 mL / min.

[0017] In step 2, the second reaction is carried out at room temperature in the dark with stirring for 3 to 6 hours; when performing the first dialysis, the dialysis bag used has a molecular weight cutoff of 8000 to 10000 Da, the dialysate is water, the temperature is 0 to 4°C, the dialysis duration is 3 to 4 days, and the dialysate is changed every 6 to 8 hours.

[0018] In step 3, the concentration of the first solution obtained after dissolving the first product in water is 5-15 mg / mL, preferably 8 mg / mL; the concentration of the sodium periodate aqueous solution is 107-214 mg / mL, preferably 214 mg / mL; the volume ratio of the first solution to the sodium periodate aqueous solution is 20-100:1, preferably 50:1; the periodate aqueous solution is added dropwise to the first solution at a flow rate of 0.25-0.6 mL / min under light-protected conditions.

[0019] In step 3, the third reaction is carried out for 5 minutes under light-protected and stirred conditions; the amount of ethylene glycol added is 10 to 20 times the amount of sodium periodate, preferably 20 times; the fourth reaction is carried out for 2 hours under light-protected and stirred conditions; when performing the second dialysis, the dialysis bag used has a molecular weight cutoff of 8000 to 10000 Da, the dialysate is water, the temperature is room temperature, the dialysis duration is 1 to 2 days, and the dialysate is changed every 6 to 8 hours.

[0020] Wherein, the degree of modification of the hydrazide-modified gelatin is 10-30%, preferably 17-20%, and most preferably 18.8%; and / or, the hydrazide-modified gelatin is prepared according to the following method:

[0021] Step I: Under stirring conditions, sulfo-NHS and EDC·HCl are added to the gelatin aqueous solution to carry out the fifth reaction and obtain the fifth reaction solution;

[0022] Step II: Add an aqueous solution of dihydrazide carbonate to the fifth reaction solution to carry out the sixth reaction, obtain the sixth reaction solution, perform the third dialysis, and dry to obtain the final product.

[0023] In step I, the amount of sulfo-NHS added is 0.1 times the mass of the gelatin; the amount of EDC·HCl added is 0.17 times the mass of the gelatin; and the fifth reaction is carried out by stirring at a temperature of 37°C for 30 minutes.

[0024] In step II, the amount of dihydrazide carbonate aqueous solution added is controlled so that the amount of dihydrazide carbonate added is 0.09 times the mass of the gelatin. Preferably, the concentration of the dihydrazide carbonate aqueous solution is 0.162 M. The sixth reaction is carried out by stirring at a temperature of 37°C for 4 hours. When performing the third dialysis, the dialysis bag used has a molecular weight cutoff of 3500 Da, the dialysis solution is water, the temperature is room temperature, the dialysis duration is 3 to 4 days, and the dialysis solution is changed every 6 to 8 hours.

[0025] The degree of modification of the methacrylamide gelatin is 20-40%, preferably 28-32%, and most preferably 30%. The methacrylamide gelatin can be purchased commercially or prepared by a method including the following steps: preparing a 100 g / L gelatin aqueous solution, adding methacrylic anhydride dropwise at a rate of 0.2-0.3 mL / min under stirring, continuously stirring and reacting at 50°C in the dark for 6 hours, and then dialyzing and freeze-drying the resulting solution. The amount of gelatin aqueous solution and methacrylic anhydride used is controlled such that the mass ratio of gelatin to methacrylic anhydride is 1 g:1 mL; during dialysis, the dialysis bag used has a molecular weight cutoff of 8000-14000 Da, the dialysis solution is water, the temperature is room temperature, the dialysis duration is 3-4 days, and the dialysis solution is changed every 6-8 hours.

[0026] The mass ratio of the methacrylamide gelatin, aldehyde-modified hyaluronic acid, and hydrazide-modified gelatin is (0.6~6):1:1, preferably 3:0.5:0.5, 2:1:1, or 1:1.5:1.5, more preferably 2:1:1 or 1:1.5:1.5, and most preferably 1:1.5:1.5; the ratio of the sum of the masses of the methacrylamide gelatin, aldehyde-modified hyaluronic acid, and hydrazide-modified gelatin to the volume of the solvent is (40~50) g:1 L, preferably 40 g:1 L; the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate; the concentration of the photoinitiator in the bio-ink is 0.5~5 g / L, preferably 1 g / L. g / L; the solvent is a phosphate buffer, preferably a phosphate buffer with a pH of 7.4 and a concentration of 0.01 M.

[0027] To address the limitations of existing GelMA hydrogels, which suffer from a narrow printable temperature window and a long stress relaxation half-life that cannot be overcome through simple modifications, this invention employs a unique ternary dynamic glycopeptide hydrogel synergistic mechanism. In this mechanism, GelMA provides the photocrosslinking backbone, AHA provides the aldehyde crosslinking sites, and GelCDH provides the acylhydrazine crosslinking sites. This achieves the technical effect of simultaneously widening the printable temperature window to 23-37°C and shortening the stress relaxation half-life to approximately 100 seconds, an effect that conventional bio-ink systems cannot achieve.

[0028] The second aspect of the present invention provides a method for preparing the ternary dynamic glycopeptide hydrogel bio-ink, comprising the following steps: dissolving methacrylamide gelatin, aldehyde-modified hyaluronic acid, hydrazide-modified gelatin and a photoinitiator together in a solvent, mixing them evenly to obtain the bio-ink.

[0029] The third aspect of this invention provides the application of the aforementioned ternary dynamic glycopeptide hydrogel bioink in colorectal organoid culture.

[0030] The method for culturing colorectal organoids using the aforementioned ternary dynamic glycopeptide hydrogel bio-ink includes: adding colorectal crypts to the ternary dynamic glycopeptide hydrogel bio-ink, mixing them evenly to obtain crypt-containing bio-ink, loading it into the barrel of an extrusion-type bio-3D printer for 3D printing to obtain crypt-loaded hydrogel microspheres, and culturing them in vitro.

[0031] Preferably, the density of colorectal crypts in the crypt-containing bio-ink is 1×10⁻⁶. 3 ~ 1×10 4 per mL.

[0032] More preferably, the density of colorectal crypts in the crypt-containing bio-ink is 5000 crypts / mL.

[0033] Preferably, during 3D printing, the temperature of the crypt-containing bio-ink is controlled at 23~37℃, and a pneumatic pressure of 0.5~2 bar is applied to the crypt-containing bio-ink to extrude it from the printing needle of the extrusion-type bio 3D printer to form droplets, which then settle on the culture dish or culture well plate on the receiving platform of the extrusion-type bio 3D printer, are exposed and cured to obtain crypt-loaded hydrogel microspheres.

[0034] More preferably, during 3D printing, the temperature of the crypt-containing bio-ink is controlled at 30~37℃, which meets the optimal temperature requirements for cell culture.

[0035] More preferably, during 3D printing, the pneumatic pressure applied to the crypt-containing bio-ink is 1 bar.

[0036] Preferably, the temperature of the culture dish or culture plate is controlled at 4~20℃; the wavelength of the exposed light is 340~405 nm, and the light power density is 0.5~3 W / cm². 2 The exposure time is 10 to 120 seconds.

[0037] More preferably, the temperature of the culture dish or culture plate is controlled at 10°C; the wavelength of the exposed light is 405 nm, and the light power density is 1.6 W / cm². 2 The exposure time is 30 seconds.

[0038] After the droplets are deposited on a culture dish or culture plate on a receiving platform with a low temperature, they achieve self-support and solidification by relying on the viscoelasticity and temperature sensitivity of GelMA. Subsequently, they are cured by exposure to complete secondary photocrosslinking and form stable crypt-carrying gel microspheres.

[0039] Preferably, the inner diameter of the printing needle is 210 μm.

[0040] Preferably, the extrusion time of the droplet is 0.5 to 2.5 s, and the diameter of the droplet is controlled to be 1000 to 3000 μm (the longer the extrusion time, the larger the droplet diameter); the roundness of the droplet is controlled by controlling the temperature of the bio-ink during 3D printing to be >0.9.

[0041] Preferably, the in vitro culture is carried out in a cell culture incubator at 37°C and a CO2 concentration of 5% for 3 to 7 days.

[0042] Droplet printing typically uses inkjet bioprinters, but this method can only use ultra-low viscosity inks (<20 mPa·s), and the available bioinks have low cell densities (<10). 6 (cells / mL), otherwise it is easy to cause printer clogging. The droplet-type bioprinting crypt gel microspheres provided by this invention use an extrusion-type bioprinter (controlled by a pneumatic piston). The bio-ink prepared by this invention is always in a "weak gel" state in the temperature range of 23~37℃. Unlike the lines usually printed by conventional extrusion-type bioprinters, this "weak gel" state allows the cell-carrying bio-ink to be extruded in droplet form and deposited on the receiving platform. This also avoids the problems of ink selection and cell density in traditional droplet printing such as inkjet bioprinting.

[0043] This invention aims to develop a ternary dynamic glycopeptide bioink (MAC) suitable for high-throughput organoid construction. This bioink constructs a dynamic covalent cross-linking network through reversible acylhydrazone bonds formed between aldehyde-modified hyaluronic acid (AHA) and carbapenem-modified gelatin (GelCDH), providing a remodelable three-dimensional microenvironment for cells. Simultaneously, the photocross-linking properties of methacrylic anhydride-modified gelatin (GelMA) are introduced to rapidly solidify and maintain overall shape stability during the printing stage, providing a remodelable cellular microenvironment during subsequent in vitro culture. This establishes an organoid culture platform with both good printability and excellent cell compatibility, providing a reliable material basis for subsequent drug screening and functional studies.

[0044] This invention focuses on the construction and application of a ternary dynamic glycopeptide bioink (MAC), systematically evaluating its physicochemical properties, rheological and mechanical properties, printability, and its impact on the growth, activity, and differentiation characteristics of intestinal organoids. By adjusting the ratio of the three components, the influence of the dynamic cross-linking network on the rheological behavior of the bioink and the three-dimensional growth of organoids is explored. Furthermore, combined with a droplet-based bioprinting strategy, the feasibility and stability of this system in high-throughput organoid construction are verified. The research objective is to establish a dynamic bioink platform suitable for organoid printing and culture, providing a reliable material and process foundation for subsequent organoid drug screening and functional studies.

[0045] Beneficial effects:

[0046] (1) This invention overcomes the problems of narrow printable temperature window and long stress relaxation half-life of traditional GelMA bioinks, which are not conducive to organoid growth and development. The MAC system, through a unique ternary synergistic mechanism, uses GelMA as the photocrosslinking backbone and introduces dynamic acylhydrazone crosslinking between AHA and GelCDH to construct a dual crosslinking mechanism. This achieves the technical effect of widening the printable temperature window to 23~37℃ and shortening the stress relaxation half-life to about 100 seconds, overcoming the limitations of pure GelMA bioinks. Moreover, the acylhydrazone bonds in the MAC system are dynamic covalent bonds, which have both the high strength of covalent bonds and the reversibility of physical crosslinking. Shear thinning and self-healing can be achieved without additional steps, resulting in high structural stability after printing.

[0047] (2) The bio-ink constructed using the MAC system of this invention can be used for high-throughput printing of stem cells and organoids, solving the problems of long operation time and large batch-to-batch variation in the traditional manual dispensing method for organoid culture. The printed hydrogel microenvironment supports the spreading and proliferation of stem cells and can support the growth and development of organoids. It provides a universal platform for stem cell culture and organoid culture. This chemical heterogeneity simulates the complex signaling environment of the intestinal epithelial-matrix interface, which can facilitate the differentiation of multiple lineages of absorptive cells (Aldob+), secretory cells (Mucin 2+), and endocrine cells (Chromogranin A+).

[0048] (3) This invention uses extrusion-type droplet bioprinting technology, which can achieve high-throughput and high-precision organoid array deposition on 96-well plates. The droplets are uniform in size and accurate in position, which significantly improves the reproducibility and throughput of organoid construction and overcomes the limitations of traditional methods such as large batch differences and cumbersome operation.

[0049] (4) This system integrates three major elements: printability, culture stability, and functional maintenance, forming a complete technology chain from bio-ink preparation, droplet printing, three-dimensional culture to drug evaluation. It is particularly suitable for standardized, high-throughput organoid chip construction, overcoming the bottleneck of traditional organoid technology in large-scale application. Attached Figure Description

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0051] Figure 1 The image shows the 1H NMR spectrum of the intermediate product hyaluronic acid-aminodiol derivative obtained in Example 1.

[0052] Figure 2 The image shows the 1H NMR spectrum of the aldehyde-modified hyaluronic acid prepared in Example 1.

[0053] Figure 3 The 1H NMR spectrum of the product obtained when quantifying the degree of substitution of aldehyde-modified hyaluronic acid in Example 1.

[0054] Figure 4 The concentration-absorbance standard curve of hydrazide gelatin and the UV-Vis absorption spectra of gelatin and hydrazide gelatin are used to quantify the degree of substitution of hydrazide gelatin in Example 1; wherein, Figure a is the concentration-absorbance standard curve of hydrazide gelatin, and Figure b is the UV-Vis absorption spectrum of gelatin and hydrazide gelatin.

[0055] Figure 5 The image shows the 1H NMR spectrum of the methacrylamide gelatin prepared in Example 1.

[0056] Figure 6 The following are statistical graphs showing the rheological properties of the MAC hydrogels prepared in Example 2: Figure a shows the frequency scan results of different MAC hydrogels; Figure b shows the viscosity-temperature curves of different MAC hydrogels; Figure c shows the shear thinning test results of different MAC hydrogels; Figure d shows the shear thinning test results of M2A1C1 hydrogel at 30-37℃; Figure e shows the shear thinning test results of M1A... 1.5 C 1.5 Figure f shows the statistical results of shear thinning tests of hydrogels at 30 ~ 37℃; Figure g shows the statistical results of stress relaxation tests of different MAC hydrogels; Figure g shows the statistical results of half-life of different MAC hydrogels.

[0057] Figure 7 Figure 3 shows a schematic diagram of the high-throughput droplet bio-3D printing process and the forming stability of the resulting gel microspheres. Figure 4a shows a schematic diagram of the high-throughput droplet bio-3D printing process; Figure 5b shows the high-throughput deposition of droplet bio-3D printing on a 96-well plate; and Figure 6c shows the morphology of gel microspheres obtained by droplet bio-3D printing with different MAC hydrogel solutions after in vitro culture for 48 h.

[0058] Figure 8 The results of evaluating the activity, proliferation, and migration abilities of MSCs loaded with different MAC hydrogel microspheres after in vitro culture for 12–48 h are shown in Example 4. Figure a shows the live / dead cell fluorescence staining images of MSCs loaded with different MAC hydrogel microspheres after in vitro culture for 12 h, 24 h, and 48 h; Figure b shows the OD values ​​of MSCs loaded with different MAC hydrogel microspheres as detected by CCK-8 after in vitro culture for 12 h, 24 h, and 48 h. 450Statistical graphs; Figure c shows the Western blotting results of pFAK (Tyr397) and Cyclin D1 in MSCs after 48 h of in vitro culture on different MAC hydrogel microspheres loaded with MSCs; Figure d shows the statistical graph of CTGF mRNA expression levels in MSCs after 48 h of in vitro culture on different MAC hydrogel microspheres loaded with MSCs; Figure e shows the statistical graph of CYR61 mRNA expression levels in MSCs after 48 h of in vitro culture on different MAC hydrogel microspheres loaded with MSCs.

[0059] Figure 9 The image shows DAPI and Phalloidine immunofluorescence staining of MSCs cultured in different MAC hydrogels in Example 5.

[0060] Figure 10 This is a multiplex immunofluorescence staining image of DAPI, Phalloidine, and Vinculin on MSCs cultured in different MAC hydrogels in Example 5.

[0061] Figure 11 This is a multiplex immunofluorescence staining image of DAPI, Phalloidine, and YAP on MSCs cultured in different MAC hydrogels in Example 5.

[0062] Figure 12 The effects of different MAC hydrogels on the formation and growth of colorectal organoids in Example 6 are shown in Figure a. Bright-field micrographs of colorectal organoids cultured in different MAC hydrogels at different stages. Figure b is a normalized area graph of colorectal organoids cultured in different MAC hydrogels at different stages. Figure c is an area graph of colorectal organoids cultured in different MAC hydrogels for 7 days.

[0063] Figure 13 Figure 7 shows the results of live / dead cell staining and cell viability statistics for colorectal organoids cultured in different MAC hydrogels in Example 7; Figure a shows the results of live / dead cell staining; Figure b shows the cell viability statistics.

[0064] Figure 14 M1A in Example 8 1.5 C 1.5 Multiplex immunofluorescence staining images of DAPI, Phalloidine, Chromogranin A, Mucin 2, and Aldob in colorectal organoids cultured on hydrogels for 48 h. Detailed Implementation

[0065] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0066] The extrusion-type bio-3D printer used in the following examples was purchased from EnvisionTec, product number 3D-Bioplotter production model.

[0067] Example 1

[0068] Preparation and characterization of AHA, GelCDH and GelMA materials.

[0069] Preparation of aldehyde-modified hyaluronic acid (AHA): 1.0 g of HA (weight-average molecular weight 200 kDa, containing 2.50 mmol of carboxyl groups) was dissolved in 100 mL of pre-chilled 0.1 M MES buffer (pH=5.5). The reaction system was placed in an ice-water bath and stirred at a low temperature. While stirring continuously, 0.54 g of sulfo-NHS (2.49 mmol) and 0.96 g of EDC·HCl (5.01 mmol) were added to the HA solution, and the reaction was continued to be stirred in the ice bath for 30 min. 3-Amino-1,2-propanediol (0.46 g, 5.00 mmol) was dissolved in 10 mL of 0.1 M phosphate buffer (pH=7.4), and then the solution was added dropwise to the above reaction system at a flow rate of 6 mL / min using a constant pressure dropping funnel. After the addition was complete, the ice bath was removed, and the reaction system was allowed to reach room temperature and stirred in the dark for 4 h. The reaction solution was transferred to a dialysis bag (8000 ~ 10000 Da) and dialyzed in pure water at 4°C for 3 days, changing the water 3 ~ 4 times a day. Finally, the dialysis product was freeze-dried to obtain a white flocculent product, which is the intermediate product hyaluronic acid-aminodiol derivative (with a vicinal diol side chain introduced onto sodium hyaluronate) obtained by amidation reaction of sodium hyaluronate and 3-amino-1,2-propanediol. 0.2 g of the above white flocculent product was weighed and dissolved in 25 mL of deionized water. Then, under light-protected conditions, 0.5 mL of sodium periodate (0.107 g, 0.5 mmol) aqueous solution was added dropwise at a flow rate of 0.6 mL / min. After stirring in the dark for 5 min, 0.6 mL of ethylene glycol (10 mmol) was added to the reaction system to quench the excess sodium periodate. The mixed solution was stirred and reacted in the dark for 2 h. The reaction solution of the reaction system was placed in a dialysis bag (8000 ~ 10000 Da), dialyzed in deionized water at room temperature for 1 day, and the dialysate was changed every 8 hours. The dialysate was then freeze-dried to obtain aldehyde-modified hyaluronic acid (AHA). Figure 1 This is the 1H NMR spectrum of a hyaluronic acid-aminodiol derivative. Figure 2 The hydrogen NMR spectrum of AHA shows a new characteristic peak in the 2-3 ppm region, confirming the successful modification of HA.

[0070] Quantitative method for determining the degree of aldehyde substitution in aldehyde-modified hyaluronic acid (AHA): 100 mg AHA was dissolved in 10 mL of deionized water (10 mg / mL), and 0.66 g (5.0 mmol) of tert-butylcarbazine was added. The mixture was stirred for 1 hour. Subsequently, 0.31 g (5.0 mmol) of sodium cyanoborohydride (NaBH3CN) was dissolved in 10 mL of deionized water and added to the reaction system. The mixture was stirred for another 24 hours. The product was purified by dialysis with deionized water for 3 days (MWCO 1000 Da) and then freeze-dried. The degree of aldehyde substitution was calculated by the integral ratio of the tert-butyl proton (δ 1.44 ppm, 9H) to the N-acetylmethyl proton (δ 1.97 ppm, 3H) in the 1H NMR spectrum. Figure 3 Calculations show that the degree of aldehyde modification in AHA is 13.6%.

[0071] Preparation of hydrazide-modified gelatin (GelCDH): 1.50 g of gelatin (Gel, weight average molecular weight 50-100 kDa) was dissolved in 150 mL of deionized water at 37 °C. Under continuous stirring, 0.67 mmol sulfo-NHS (0.146 g) and 1.35 mmol EDC·HCl (0.259 g) were added to the gel solution, and the reaction was continued at 37 °C for 30 min. After the reaction was complete, 1.57 mmol dihydrate dicarbonate (CDH) (0.141 g) dissolved in 5 mL of pure water was added dropwise to the mixed solution, and the reaction was continued at 37 °C for 4 h. The product was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in pure water for 3 days, and then freeze-dried to obtain hydrazide-modified gelatin (GelCDH).

[0072] Quantitative analysis of the degree of substitution of carbazide in gelCDH was performed using the ninhydrin method: the free amino group content was determined to ascertain the degree of substitution of carbazide. First, 0.4 g of ninhydrin and 0.06 g of reduced ninhydrin were dissolved in 15 mL of DMSO; then, lithium acetate solution (1.32 g dissolved in 5 mL of deionized water) was added and mixed thoroughly to prepare the colorimetric reagent. Gelatin or gelCDH samples were separately prepared into 0.5 mg / mL solutions (total volume 1 mL) with deionized water and placed in glass bottles. 1 mL of the colorimetric reagent was added to each bottle. The glass bottles were heated in a boiling water bath for 15 min, then transferred to a 25°C water bath and kept at that temperature for 30 min. Then, 5 mL of 50% v / v ethanol solution was added and the mixture was stirred vigorously for 20 s. Finally, the absorbance was measured using a UV-Vis spectrophotometer (V-770, JASCO) in the wavelength range of 400–700 nm. A standard curve was plotted using 2-chloroethylamine as a standard, and the degree of substitution of carbazide was calculated based on the amino content. The amino contents of gelatin and GelCDH were measured to be 0.217 mmol / g and 0.367 mmol / g, respectively, corresponding to a degree of substitution of 18.8% for carbazide in GelCDH. Figure 4 ).

[0073] Preparation of methacrylamide gelatin (GelMA): 1 g of gelatin was dissolved in pure water and magnetically stirred in a water bath at 50-60°C until completely dissolved, preparing a gelatin solution with a concentration of 100 g / L. 1.0 mL of methacrylic anhydride was added dropwise to the gelatin solution at a rate of 0.2-0.3 mL / min under stirring. The reaction was carried out continuously at 50°C in the dark for 6 hours. After the reaction, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 8000-14000 Da) and dialyzed in deionized water for 3 days, changing the dialysate every 8 hours to remove unreacted methacrylic anhydride and byproducts. Methacrylamide gelatin (GelMA) was obtained by freeze-drying. Figure 5 The 1H NMR spectrum of GelMA revealed new characteristic peaks in the 5–6 ppm region, confirming the successful modification of the gelatin. The methacryloylated gelatin prepared in this example had a methacryloyl group substitution rate of 30%.

[0074] Example 2

[0075] Rheological properties testing of MAC bio-ink.

[0076] Methacrylamide gelatin (GelMA), aldehyde-modified hyaluronic acid (AHA), and carbapenem-modified gelatin (GelCDH) were mixed in mass ratios of 4:0:0 (i.e., only GelMA was dissolved in PBS), 3:0.5:0.5, 2:1:1, and 1:1.5:1.5, and then dissolved in phosphate buffered saline (PBS, pH 7.4, concentration 0.01 M) to prepare a mixed solution with a total concentration of 40 g / L. Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) photoinitiator was added to achieve a concentration of 1 g / L in the mixed solution. After thorough mixing, bioinks M4 and M3A were prepared. 0.5 C 0.5 M2A1C1, M1A 1.5 C 1.5 The above four groups of bio-inks were poured into a 200 μL mold and tested with light at a wavelength of 405 nm and an intensity of 1.6 W / cm². 2 The gel was formed by irradiation with light for 30 seconds, yielding M4 and M3A0.5C respectively. 0.5 M2A1C1, M1A 1.5 C 1.5 Hydrogel.

[0077] The rheological properties of the above four groups of hydrogels were tested (frequency scan test, viscosity-temperature curve test, shear thinning test, stress relaxation test) to explore suitable conditions for subsequent 3D printing.

[0078] Frequency scan: frequency 0.1 ~ 10 Hz, 10 points in each interval, fixed strain 1%, and the modulus at 1 Hz was selected for statistical analysis. Results are as follows: Figure 6 As shown in Figure a, the stiffness of the four groups of MAC hydrogels with different ratios gradually increases, with M1A... 1.5 C 1.5 Hydrogels have the strongest stiffness.

[0079] The viscosity-temperature curve was tested in a temperature range of 10 ~ 40℃, with an equilibrium time of 180 s. The test results are as follows: Figure 6 As shown in b, a significant viscosity decrease was observed in the M4 hydrogel around 25°C, corresponding to the gel-sol transition temperature. After adding AHA and GelCDH components, the gel-sol transition of the MAC hydrogel was significantly weakened. The supporting effect provided by the acylhydrazone crosslinking network kept the MAC hydrogel in a "weak gel" state throughout the temperature range of 23–37°C. Compared to traditional GelMA bio-inks, MAC bio-inks in this state exhibit greater morphological stability and are less prone to cell deposition during cell-loaded printing.

[0080] The shear thinning test was conducted at 25℃ with an equilibration time of 180 s. The test results are as follows: Figure 6As shown in c. Additionally, M2A1C1 and M1A were subjected to temperature variations at 30-37°C. 1.5 C 1.5 The hydrogel underwent shear thinning tests, and the results are as follows: Figure 6 As shown in d and 6e. The results indicate that all four groups of MAC hydrogels are shear-thinning fluids, and M2A1C1 and M1A 1.5 C 1.5 Hydrogels all exhibit shear-thinning behavior and can be used for bio-3D printing.

[0081] The rheological performance tests described above demonstrate that the ternary dynamic glycopeptide hydrogel bio-ink prepared in this invention exhibits excellent temperature adaptability when used for 3D printing, with an effective printing temperature window of 23~37℃ (spanning 14℃), significantly better than the typical allowable range of only a few degrees (23~28℃) for traditional GelMA bio-inks (M4). Within this temperature window, the bio-ink maintains ideal rheological equilibrium and meets the optimal temperature range for cell survival (30~37℃).

[0082] Because matrices with suitable stress relaxation properties can better simulate the mechanical environment of in vivo tissues, they can support the formation of more complex structures and functions in intestinal organoids. Stress relaxation tests were performed on the above four groups of MAC hydrogels, with a total volume of 200 μL and a gap of 600 μm. The test temperature was 37℃, the equilibration time was 180 s, the strain was 10%, and the stress decay process was monitored at sampling intervals of 0.01 s and 10 Hz. The time required for the stress to decay to half of the initial normalized stress was recorded as the stress relaxation half-life (τ) of the hydrogel. d1 / 2 The results showed that M1A 1.5 C 1.5 The stress relaxation rate is the fastest. Figure 6 f), and has the shortest half-life (approximately 100 s) ( Figure 6 g), M4 has the longest half-life (approximately 3000 seconds), indicating that the acylhydrazone bond formed by GelCDH and AHA endows the bio-ink with good stress relaxation ability, among which M1A 1.5 C 1.5 Hydrogels exhibit the best stress relaxation ability, followed by M2A1C1 hydrogels, making these hydrogels better able to meet the growth requirements of organoids.

[0083] Example 3

[0084] The appearance of droplets printed with bio-ink at different temperatures during droplet-based bio-3D printing.

[0085] Preparation of bio-ink: AHA, GelCDH, GelMA, and the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were dissolved together in phosphate buffer (0.01 M, pH=7.4) to prepare a homogeneous solution with a total concentration of GelMA, AHA, and GelCDH of 0.04 g / mL and a LAP concentration of 1 g / L, thus obtaining the cell-free bio-ink. Five different ratios of MAC bio-ink were prepared, with the mass ratios of GelMA, AHA, and GelCDH being 4:0:0, 3:0.5:0.5, 2:1:1, 1:1.5:1.5, and 0:2:2, respectively.

[0086] Droplet-based bio-3D printing: The prepared bio-ink is loaded into the barrel of an extrusion-type bio-3D printer. A temperature control system connected to the barrel precisely controls the temperature of the M4 bio-ink at 25℃ (this temperature falls within the sol-gel transition temperature window of GelMA bio-ink; within this window, the ratio tanδ of the storage modulus G' to the loss modulus G'' of the bio-ink is adjusted to 0.3 ~ 0.5, ensuring that the M4 bio-ink is in a weak gel state with shear-thinning properties, while preventing cell sedimentation in the barrel and resulting in uneven cell density, allowing the control group M4 bio-ink to print successfully). M3A 0.5 C 0.5 M2A1C1, M1A 1.5 C 1.5 The temperature of the bio-ink was precisely controlled at 35℃ (a temperature range suitable for cell culture). Under temperature control, a pneumatic pressure of 1 bar was applied to the bio-ink, causing it to be extruded as droplets from a 27G 0.4 specification printing needle (outer diameter 0.4 mm, inner diameter 0.21 mm) under shear force, settling on the receiving platform. The droplet diameter was controlled to approximately 1000 μm by adjusting the extrusion time to 0.5 s, and the droplet sphericity was controlled to >0.9 by temperature control. Under extrusion-driven conditions, the MAC bio-ink stably formed uniformly sized droplets and achieved a regularly arranged droplet array structure. No obvious tailing or droplet fusion was observed during printing. After printing, the droplets deposited on the receiving platform (platform temperature 10℃) and achieved self-supporting curing through the viscoelasticity and thermosensitivity of GelMA and the Schiff base reaction of GelCDH and AHA. Subsequently, light was emitted at a wavelength of 405 nm and an intensity of 1.6 W / cm². 2 Irradiation with light for 30 seconds completes secondary photocrosslinking, and the crosslinked bio-ink forms a stable hydrogel scaffold. This indicates that the system has good droplet formation stability. Figure 7 a). Further high-throughput printing experiments were conducted on a 96-well plate ( Figure 7(b) The results showed that the droplet deposition positions were accurate and uniformly distributed, with good repeatability and positional controllability. MAC bio-inks with different component ratios could all form droplet structures with clear boundaries and complete morphology during the printing process, and could maintain their good morphology for up to 48 hours. Figure 7 c) indicates that the bio-ink system has good process adaptability and stability to changes in composition.

[0087] Example 4

[0088] Bio-ink cell compatibility and gene expression test.

[0089] GelMA, AHA, GelCDH, and the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were dissolved in phosphate buffer (0.01 M, pH=7.4) to prepare a homogeneous solution with a total concentration of 0.04 g / mL for GelMA, AHA, and GelCDH and a LAP concentration of 1 g / L, thus obtaining the cell-free bioink. Four groups of MAC bioinks with different ratios were prepared, with the mass ratios of GelMA, AHA, and GelCDH being 4:0:0 (M4 bioink), 3:0.5:0.5 (M3A), and 3:0.5:0.5, respectively. 0.5 C 0.5 Bio-ink), 2:1:1 (M2A1C1 bio-ink) and 1:1.5:1.5 (M1A 1.5 C 1.5 (Bio-ink). The prepared unloaded cell-rich bio-ink was filtered through a 0.22 μm filter for sterilization. Passaged mesenchymal stem cells (MSCs) were digested, centrifuged, and counted before being added to the sterilized unloaded cell-rich bio-ink. The final bio-ink contained an MSC cell density of 1 × 10⁻⁶ cells. 6 ~ 1×10 8 cells / mL.

[0090] Following the method described in Example 3, MSC-loaded bio-ink was used for droplet-based bio-3D printing. The resulting four groups of cell-loaded gel microspheres were cultured in a cell culture incubator at 37°C and 5% CO2 for 12 h, 24 h, and 48 h, respectively. After culture, the viability of MSCs in the gel microspheres was detected using a Cell Counting Kit-8 (CCK-8). Live / dead cell staining of the MSCs in the gel microspheres was performed using Calcein-AM / PI. Furthermore, Western blotting was used to detect the protein expression levels of pFAK (Tyr397) (a cell proliferation and spreading indicator whose activation level reflects the cell's adhesion, spreading, and migration ability under specific matrix conditions) and Cyclin D1 (a cell cycle regulatory marker whose level directly reflects cell proliferation activity) in the four groups of cell-loaded gel microspheres cultured in vitro for 48 h. RT-qPCR was used to analyze the expression of CTGF (connective tissue growth factor, which regulates cell adhesion, migration, proliferation and extracellular matrix synthesis, and whose expression can reflect changes in the mechanical properties of hydrogel) and CYR61 (which regulates cell adhesion, migration and angiogenesis) mRNA in the four groups of cell-carrying gel microspheres cultured in vitro for 48 h. Upregulation of CTGF or CYR61 expression usually indicates that the cells are in a more active state of proliferation and migration.

[0091] Cell viability test results as follows Figure 8 As shown in b, M1A 1.5 C 1.5 The MSC cell-loaded bio-ink obtained by printing exhibited the best cell viability, with OD200 measured by CCK-8 assay. 450 The cell viability was 1.3-1.4, significantly better than other groups of bio-ink formulations. Cell viability staining results showed that the MSC cell proliferation in the hydrogel obtained by MAC printing was better than that in the control group M4, and the cell viability remained above 80%. Figure 8 a) This bio-ink exhibits good cell compatibility. Western blot and RT-qPCR results showed that, with the same internal control GAPDH level, compared to pure GelMA bio-ink (M4 bio-ink), the protein expression levels of pFAK (Tyr397) (a cell proliferation and spreading indicator) and Cyclin D1 (a cell cycle regulatory marker) in cells printed with MSC gel microspheres using MAC bio-ink were upregulated. Figure 8 c), and the expression levels of CTGF mRNA and CYR61 mRNA were also significantly upregulated. The higher the content of AHA and GelCDH in MAC bio-ink, the higher the expression levels of pFAK (Tyr397), Cyclin D1, CTGF mRNA and CYR61 mRNA, and M1A.1.5 C 1.5 The mRNA expression levels of CTGF and CYR61 in the cells printed with MSC cell bio-ink were higher than those in the other groups, reaching 1.8 and 1.6, respectively. Figure 8 d and 8e).

[0092] The above results show that, compared with pure GelMA hydrogel, MSCs cultured in the MAC hydrogel of the present invention have stronger cell proliferation performance, as well as better adhesion and migration ability. Due to the stress relaxation effect of MAC hydrogel, cells can proliferate and differentiate better.

[0093] Calcein-AM / PI live / dead cell staining method: Under 37℃ and in the dark, 2.5 μL of calcein-AM and 3 μL of PI were added to each 500 μL of serum-free DMEM / F-12 medium, mixed thoroughly, and stored for later use. After MSC-loaded gel microsphere culture, the old medium in the wells was aspirated, and 500 μL of the pre-prepared AM / PI staining reagent was added to each well. The wells were incubated for 0.5 h, then washed twice with DPBS buffer. The growth of the organoids was observed under a microscope and photographed to record the results, thus observing the liveness and death status of MSC cells.

[0094] Example 5

[0095] Effects of MAC hydrogel on gene expression in MSC cells.

[0096] After fixing and permeabilizing MSCs cultured for 48 h in Example 4, the cells were stained sequentially with antibodies against DAPI (nucleus), Phalloidine (cytoskeleton), Vinculin (foci of adhesion), and YAP (YAP protein, a key effector in the Hippo signaling pathway that regulates cell proliferation, organ size, and stemness. When the pathway is inhibited, YAP remains in the cytoplasm (inactive); when the pathway is activated, YAP enters the nucleus, binds to transcription factors, and initiates the expression of pro-proliferation and anti-apoptotic genes). Immunofluorescence staining was used to verify the effects of different compositions of MAC bio-ink on gene expression in MSCs. The staining results are shown below. Figure 9 , 10 As shown in Figure 11, the results indicate that with the increase of GelCDH and AHA component content, the spreading effect of MSC cells gradually improved, the nucleocytoplasmic ratio decreased, and M1A... 1.5 C 1.5 The group with the best cell spreading effect ( Figure 9 Vinculin staining of MSC cells showed that almost no adhesion plaques appeared in group M4, while adhesion plaques appeared and gradually increased in subsequent groups. Figure 10YAP nuclear localization staining showed that with the introduction of GelCDH and AHA, YAP protein gradually entered the nucleus, the HiPPo pathway was inhibited, and cell proliferation and survival were promoted. Figure 11 These two sets of fluorescence staining experiments fully demonstrate that the biocompatibility of the bioink after the introduction of GelCDH and AHA is significantly improved, supporting the transformation of cells from simple adhesion to functional spreading and signal transduction.

[0097] Example 6

[0098] Hydrogels are used for intestinal organoid culture.

[0099] Prepare 6-week-old C57BL / 6 mice, euthanize them by cervical dislocation, and immerse them in alcohol. Place the mice in a sterile laminar flow hood and open the abdominal cavity. Cut approximately 10 cm of colorectal tissue 1-2 cm below the cecum. Place the colorectal tissue into a 10 cm bacterial culture dish containing 15 mL of cold DPBS buffer. Rinse the inside of the intestine with PBS buffer and remove external fatty tissue with forceps. Longitudinally cut the colorectal region with a small scalpel, then gently scrape away the villi on the inner side of the intestine with a coverslip, and rinse the intestine. Transfer the intestine to a new culture dish containing 15 mL of cold DPBS buffer, scrape away intestinal villi and feces with a coverslip, and repeat the intestinal rinsing at least 3 times. Place 15 mL of cold DPBS buffer into a 50 mL centrifuge tube, hold the colorectal tissue with forceps, and then cut it into small pieces with a small scalpel, placing it into the centrifuge tube. After wetting the pipette tip with DPBS buffer, repeatedly pipette the colon and rectal fragments about 10 times. After the colon and rectal fragments precipitate, discard the supernatant. Add 15 mL of cold, fresh DPBS buffer to the centrifuge tube and repeat this step 15 to 20 times until the supernatant is clear after washing. Discard the supernatant and add 2 mL of mild cell dissociation reagent to the centrifuge tube. Gently pipette the tube and let it stand at room temperature for 15 minutes, shaking it continuously during this time. After colorectal fragments precipitated, the supernatant was carefully aspirated. The colorectal fragments were then resuspended in 3 mL of DPBS buffer containing 0.1% FBS. The mixture was gently pipetted three times until the fragments precipitated. The supernatant was filtered through a 70 μm filter into a 50 mL centrifuge tube and centrifuged at 4°C and 300 g for 5 min. The supernatant was discarded, and the precipitate was resuspended in 10 mL of DPBS containing 0.1% FBS. The mixture was centrifuged at 4°C and 200 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 10 mL of cold DMEM / F12. 10 μL of the suspension was taken to count the number of crypts and calculate the crypt density. Four crypt suspensions were prepared, each containing 300 crypts. Each suspension was centrifuged at 4°C and 200 g for 5 min, and the supernatant was discarded to obtain four crypt pellets.

[0100] Take the bio-inks M4 and M3A prepared in Example 2 respectively. 0.5 C 0.5 M2A1C1, M1A 1.5 C 1.5 60 μL of each sample was filtered through a 0.22 μm filter for sterilization, and then added to the four portions of crypt precipitate mentioned above. After mixing, four groups of crypt-loaded bio-inks were prepared. These were printed into 48-well plates using the droplet-based bio-3D printing method described in Example 3. 300 μL of complete intestinal organoid culture medium was added to each well, and the plates were incubated at 37°C and 5% CO2 for 7 days. The complete intestinal organoid culture medium was replaced every 2-3 days. Cells embedded in the gel were observed and photographed daily under an inverted microscope. Furthermore, the growth rate and area of ​​the intestinal organoids in each hydrogel were evaluated.

[0101] The growth status of organoids from day 1 to day 7 is as follows: Figure 12 As shown in Figure a, firstly, the colorectal crypts extracted from all experimental groups acquired a spherical morphology within hours, exhibiting cystic luminal features. On the third day, the spherical bodies displayed irregular protrusions from the central cavity to the outer surface, a phenomenon known as "budding." Organoid budding is considered an important characteristic of subsequent maturation and differentiation. Subsequently, the intestinal organoids highly differentiated into larger spherical structures; this morphology is termed mature colorectal organoids. Figure 12 b represents the change in normalized area of ​​organoids in each group over time. The results show that, compared to the M4 system, MAC bio-inks containing AHA and GelCDH components (especially M2A1C1 and M1A) have a higher normalized area. 1.5 C 1.5 The organoids in the group (1) exhibited more complete structures, clearer boundaries, and a more significant growth rate, with a significantly larger area on day 7 compared to the low-component system (2). Figure 12 b); Simultaneously, in the high AHA / GelCDH content system, the organoid size distribution shifted towards the higher value range ( Figure 12 c). This indicates that by regulating the composition of MAC bio-ink, organoid expansion can be effectively promoted, resulting in a population of organoids with uniform size and stable structure.

[0102] Example 7

[0103] Cryptogenic bioinks M4 and M3A were prepared according to the method in Example 6. 0.5 C 0.5 M2A1C1 and M1A 1.5 C 1.5(Crypt density was 5000 cells / mL). After printing according to the method in Example 3, the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 48 h. After culture, the viability of colorectal organoids was detected using the Cell Counting Kit-8 (CCK-8), and the cell viability was observed using Calcein-AM / PI staining.

[0104] Cell viability test results as follows Figure 13 As shown in b, M2A1C1 and M1A 1.5 C 1.5 The cell viability of the printed colorectal organoid gel microspheres was the best, doubling compared to the control group M4, and significantly superior to the cell viability of other bio-ink formulations, with a relative activity of around 220%. Cell viability staining results showed that within this crypt density range (5000 crypts / mL), the cell survival rate of the printed organoids remained above 60%, and all printed organoids could proliferate and differentiate normally. Figure 13 a) Due to the obstruction of material exchange, some cells die in the central part of the organoid, while the organoids at the edge maintain good biological activity.

[0105] Example 8

[0106] Gene expression assay for colorectal organoids.

[0107] For M1A in Example 7 1.5 C 1.5 After 48 h of culture, colorectal organoids were fixed and permeabilized. They were then stained sequentially with antibodies against Chromogranin A (endocrine cells), Mucin 2 (goblet cells), and Aldob (absorbent epithelial cells). Immunofluorescence staining was used to verify the effects of different compositions of MAC bio-inks on gene expression in colorectal organoids. The staining results are shown below. Figure 14 As shown in the figure, the results indicate that intestinal organoids can form and maintain multi-lineage differentiation within the three-dimensional microenvironment constructed by MAC bio-ink. Positive expression of Chromogranin A (endocrine cells), Mucin 2 (goblet cells), and Aldob (absorbent epithelial cells) signifies the establishment of endocrine, mucus secretion, and nutrient absorption functions. Multichannel fluorescence superposition reveals an ordered spatial distribution of different markers, which, together with nuclear staining, constitute a structurally complete three-dimensional tissue morphology. MAC bio-ink provides an intestinal organoid with a three-dimensional microenvironment supporting multi-type cell differentiation, maintenance of functional phenotypes, and the formation of complex tissue structures, laying the foundation for its application in disease model construction and drug evaluation.

[0108] This invention provides a ternary dynamic glycopeptide hydrogel bio-ink, its preparation method, and its application in colorectal organoid culture. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A ternary dynamic glycopeptide hydrogel bio-ink, characterized in that, It is made from a mixture of the following raw materials: methacrylamide gelatin, aldehyde-modified hyaluronic acid, hydrazide-modified gelatin, photoinitiator and solvent.

2. The ternary dynamic glycopeptide hydrogel bio-ink according to claim 1, characterized in that, The aldehyde-modified hyaluronic acid is obtained by amidation of hyaluronic acid or its sodium salt with 3-amino-1,2-propanediol followed by oxidation with periodate; and / or, the degree of modification of the aldehyde-modified hyaluronic acid is 10 to 30%.

3. The ternary dynamic glycopeptide hydrogel bio-ink according to claim 2, characterized in that, The aldehyde-modified hyaluronic acid was prepared according to the following method: Step 1: Under ice bath and stirring conditions, sulfo-NHS and EDC·HCl are added to a solution of hyaluronic acid or its sodium salt to carry out the first reaction and obtain the first reaction solution. Step 2: Add 3-amino-1,2-propanediol solution to the first reaction solution to carry out the second reaction, obtain the second reaction solution, perform the first dialysis, freeze dry, and obtain the first product; Step 3: Dissolve the first product in water and add periodate aqueous solution to carry out the third reaction to obtain the third reaction solution. Add ethylene glycol to the solution to carry out the fourth reaction to obtain the fourth reaction solution. Perform a second dialysis and freeze dry to obtain the final product.

4. The ternary dynamic glycopeptide hydrogel bio-ink according to claim 1, characterized in that, The degree of modification of the hydrazide-modified gelatin is 10-30%; and / or, the hydrazide-modified gelatin is prepared according to the following method: Step I: Under stirring conditions, sulfo-NHS and EDC·HCl are added to the gelatin aqueous solution to carry out the fifth reaction and obtain the fifth reaction solution; Step II: Add an aqueous solution of dihydrazide carbonate to the fifth reaction solution to carry out the sixth reaction, obtain the sixth reaction solution, perform the third dialysis, and dry to obtain the final product.

5. The ternary dynamic glycopeptide hydrogel bio-ink according to claim 1, characterized in that, The degree of modification of the methacrylamide gelatin is 20-40%; and / or the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate; the solvent is a phosphate buffer, preferably a phosphate buffer with a pH of 7.4 and a concentration of 0.01 M.

6. The ternary dynamic glycopeptide hydrogel bio-ink according to claim 1, characterized in that, The mass ratio of the methacrylated gelatin, aldehyde-modified hyaluronic acid, and hydrazide-modified gelatin is (0.6 ~ 6): 1: 1; the ratio of the sum of the masses of the methacrylated gelatin, aldehyde-modified hyaluronic acid, and hydrazide-modified gelatin to the volume of the solvent is (40 ~ 50) g: 1 L; and the concentration of the photoinitiator in the bio-ink is 0.5 ~ 5 g / L.

7. A method for preparing the ternary dynamic glycopeptide hydrogel bio-ink according to any one of claims 1 to 6, characterized in that, Methacrylamide gelatin, aldehyde-modified hyaluronic acid, hydrazide-modified gelatin, and photoinitiator are dissolved in a solvent and mixed evenly to obtain the bio-ink.

8. The application of the ternary dynamic glycopeptide hydrogel bio-ink according to any one of claims 1 to 6 in colorectal organoid culture.

9. The application according to claim 8, characterized in that, The method for culturing the colorectal organoids using the aforementioned ternary dynamic glycopeptide hydrogel bio-ink includes: adding colorectal crypts to the ternary dynamic glycopeptide hydrogel bio-ink, mixing them evenly to obtain crypt-containing bio-ink, loading it into the barrel of an extrusion-type bio-3D printer for 3D printing to obtain crypt-loaded hydrogel microspheres, and culturing them in vitro to obtain the organoids.

10. The application according to claim 9, characterized in that, In the crypt-containing bio-ink, the density of colorectal crypts is 1×10⁻⁶. 3 ~ 1×10 4 1 / mL; and / or, the 3D printing conditions are as follows: the temperature of the crypt-containing bio-ink is 23 ~ 37℃, a pneumatic pressure of 0.5 ~ 2 bar is applied to the crypt-containing bio-ink, it is extruded to form droplets, which settle on a culture dish or culture plate, and then exposed to solidify to obtain crypt-loaded hydrogel microspheres.

11. The application according to claim 10, characterized in that, The temperature of the culture dish or culture plate is controlled at 4~20℃; and / or the wavelength of the exposed light is 340~405 nm, and the light power density is 0.5~3 W / cm². 2 The exposure time is 10 to 120 seconds.

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