Preparation method and application of marine fish-derived methacrylated gelatin gelma hydrogel

Abstract

The application discloses a preparation method of marine fish source methacrylated gelatin GelMA hydrogel and application thereof, and successfully prepares GelMA by using marine fish source gelatin, forms the hydrogel by ultraviolet curing through a photo initiator lithium phenyl-2, 4, 6-trimethyl benzoyl phosphonate (LAP), and successfully realizes 3D culture of cells. The marine source GelMA has the characteristics of sustainable source and high biological safety, the process of photo curing into gelatin is mild, fast, small in cell damage, simple in operation, and can be applied to the fields of cell culture, tissue engineering, drug screening and personalized treatment, and the like, and at present, marine source GelMA is less reported, and has a good market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing marine fish-derived methacrylamide gelatin (GelMA) hydrogel and its application. Background Technology

[0002] Methacryloyl gelatin (GelMA) is a photosensitive biomaterial obtained through chemical modification of natural gelatin, holding a central position in tissue engineering and biomanufacturing. Its basic raw material, gelatin, is derived from the partial hydrolysis of collagen. As a natural hydrogel matrix, gelatin possesses a structure and function similar to the extracellular matrix (ECM), such as excellent biocompatibility, controllable enzymatic degradation, and the presence of RGD active sequences that promote cell adhesion. However, gelatin itself has limitations such as weak mechanical strength and thermal reversibility, restricting its direct application as a stable scaffold. By grafting methacrylamide groups onto its molecular chain, GelMA undergoes a rapid and mild photocrosslinking reaction under UV light initiation, forming a covalently crosslinked hydrogel with a three-dimensional network structure. This modification process not only significantly enhances the material's mechanical properties, anti-swelling ability, and structural stability but also retains the inherent activity and biocompatibility of gelatin, achieving a combination of "bioactivity" and "controllable physical properties." With its precisely controllable mechanical properties, cell-growth-friendly three-dimensional microstructure, and mild and user-friendly handling process, GelMA hydrogel has shown broad application prospects in cutting-edge fields such as 3D cell culture, organoid construction, tissue engineering scaffolds, drug screening, and tumor model construction.

[0003] Currently, gelMA is mainly derived from mammalian gelatin (such as pig and cow), which faces challenges such as religious and cultural restrictions, potential zoonotic disease risks, and is complex and costly to process. In recent years, fish gelatin has emerged as a promising alternative raw material. GelMA hydrogels mimic the structure and function of the natural extracellular matrix (ECM), providing cells with a three-dimensional growth microenvironment, supporting tissue formation, and maintaining cell behaviors (such as proliferation, migration, and differentiation). In the field of tumor therapy, GelMA hydrogels mimic the tumor cell microenvironment. GelMA 3D hydrogels have been used to evaluate the response of MDA-MB-231 cells to the chemotherapy drug doxorubicin (DOX), revealing key drug resistance mechanisms that are difficult to reproduce using traditional models. The GelMA system constructs a vascularized tumor microenvironment through high-throughput, utilizing the characteristic of HUVEC cells to form a functional endothelial network, elucidating the mechanism of vascular-related tumor microenvironment (TME) resistance. The GelMA / SF system simultaneously drives osteogenic and chondrogenic differentiation of MSCs, achieving integrated regeneration of articular cartilage and subchondral bone defects, overcoming the challenge of tissue repair imbalance in the treatment of knee osteoarthritis. GelMA hydrogels can maintain the continuous liver-specific function of HepG2 cells, significantly improve the sensitivity of cells to drug-induced toxicity, and support the dynamic toxicity assessment of "liver microarrays" through a biomimetic three-dimensional microenvironment. It significantly enhances the viability of HepG2 cells and promotes the expression of hepatocyte-specific functions, reconstructing the 3D liver tumor microenvironment. This application presents marine-derived GelMA prepared from fish gelatin. The raw materials are stable and readily available, low in cost, and easy to process. It has higher biosafety, wider public acceptance, and environmental value for sustainable resource utilization, providing a high-performance and source-friendly next-generation material option for biomedical applications.

[0004] Currently available commercially available methacrylamide gelatin (GelMA) products require preheating to above 37°C to completely dissolve before preparing hydrogels. This is due to the thermally reversible gelation properties of the gelatin raw material: when the temperature is below its gel-sol transition temperature, the material spontaneously forms a physical gel. This phenomenon is closely related to the amino acid composition and sequence of the gelatin. Traditional mammalian gelatin (such as porcine and bovine gelatin) is rich in proline and hydroxyproline (usually ≥20%), and its molecular chains are relatively rigid, easily forming a stable triple helix structure. Therefore, its gelation temperature is relatively high, and it is in a gel state at room temperature. It must be heated to dissolve and be used, and subsequent operations still require heating to maintain the dissolved state. During mixing with cells, the high mixing temperature can easily damage cell activity, while insufficient heating can prevent uniform mixing with cells. This not only makes the operation cumbersome and time-consuming, but also increases the possibility of failure during the operation, increases the dependence on temperature control equipment, and limits its application. Summary of the Invention

[0005] The present invention aims to provide a method for preparing marine fish-derived methacrylamide gelatin (GelMA) hydrogel and its application in 3D cell culture. The method utilizes cold-water fish gelatin as a raw material to prepare GelMA. This material has a low imino acid content (typically ≤15%), high molecular chain flexibility, and exhibits significant "cold-adaptive" characteristics. Therefore, the GelMA hydrogel precursor solution remains in a sol state at room temperature (25°C) and can be directly mixed with the photoinitiator LAP, forming a hydrogel through photocuring without heating. This characteristic eliminates the need for heating and insulation in the entire preparation and subsequent operations, greatly simplifying the process and reducing reliance on temperature control equipment. The resulting hydrogel was used for 3D cell culture, and the results showed excellent cell proliferation and viability. By day seven, the cell proliferation rate reached 374%, and the cell viability reached 256%. This invention significantly improves the convenience and efficiency of 3D culture experiments while maintaining good gel performance and cell compatibility.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first objective of this invention is to provide a method for preparing marine fish-derived methacrylamide gelatin (GelMA) hydrogel. The methacrylamide gelatin (GelMA) is derived from cold-water marine fish, including but not limited to cod, salmon, and trout. The preparation method is as follows: gelatin obtained from the cold-water marine fish is dissolved in phosphate-buffered saline (PBS) to obtain a gelatin solution. Methacrylamide anhydride is added, and the reaction is terminated by adding PBS. Dialysis is then performed. After dialysis, one or more active proteins such as silk fibroin, sea cucumber peptide, and sericin are added for compounding. After pre-freezing, the mixture is freeze-dried to obtain methacrylamide gelatin (GelMA) sponge. The GelMA sponge is dissolved in a solution containing a photoinitiator (LAP) at room temperature (25°C) in the dark. After dissolution, the mixture is cross-linked and cured by ultraviolet light irradiation to form a hydrogel.

[0007] Furthermore, the dissolution of marine fish-derived gelatin in phosphate-buffered saline (PBS) was carried out at room temperature (25°C), and methacrylic anhydride was added to the gelatin solution, with the reaction proceeding at 20-30°C.

[0008] Furthermore, when terminating the reaction, add 4 times the volume of PBS solution, and then dialyze the reaction product. The reaction solution is dialyzed with purified water at 35-65°C for 2-5 days, with the purified water being changed every 2 hours during this period.

[0009] Furthermore, active protein was added to the dialysis solution at a ratio of gelatin to active protein of 10:1 to 5:1, followed by freeze-drying to obtain methacrylamide gelatin sponge.

[0010] Furthermore, the GelMA sponge was dissolved in a solution containing LAP photoinitiator to form a hydrogel precursor solution, which was dissolved at room temperature (25°C) under light-protected conditions. The hydrogel solution was then crosslinked under ultraviolet light for 30 seconds to form a hydrogel.

[0011] The second objective of this invention is to propose an application of a hydrogel in 3D cell culture.

[0012] The third objective of this invention is to provide a method for co-culturing hydrogels and cells, the method being as follows: the hydrogel precursor solution prepared according to any one of claims 1-5 is filtered through a 0.22 μm filter membrane in a clean bench, stored in the dark, and used for cell culture; then the cells are treated to a concentration of 1 × 10⁻⁶. 6 Cells were collected at a density of 100 cells / mL to obtain a cell pellet. The cell pellet was mixed thoroughly with the hydrogel precursor solution and transferred to wells. The hydrogel was irradiated with UV light to crosslink and solidify. Cell culture medium was added, and the cells were incubated at 37°C in a 5% CO2 incubator with daily medium changes. The cells were treated on the third and seventh days to lyse the hydrogel, obtain and count the cells, calculate the cell proliferation rate, and determine cell viability using CCK-8 reagent. The hydrogel cells were stained with propidium iodide (PI) staining solution and calcein AM staining solution, and cell live / dead markers were observed under a laser confocal microscope.

[0013] The above technical solution can achieve the following beneficial effects: Preparation of Marine Fish-Derived Methacrylated Gelatin (GelMA) Hydrogel: The gelatin is derived from cold-water marine fish. Its gelatin contains low levels of proline and hydroxyproline, which have stable protein rigid structures, and can dissolve at room temperature. Therefore, the preparation process of GelMA hydrogel and the subsequent mixing process with cells can both be carried out at room temperature (25°C). It is currently the only GelMA product that can dissolve at this temperature, greatly increasing the safety of use and the effect of cell culture.

[0014] This invention successfully prepared GelMA using marine fish-derived gelatin. The hydrogel was formed by UV curing using the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), enabling successful 3D cell culture. Marine fish-derived GelMA offers significant advantages in its preparation process. It can be directly dissolved and photocured at room temperature without heating, making the process gentle, rapid, and greatly simplifying the operation. Furthermore, this material exhibits excellent biocompatibility and proliferation-promoting ability in 3D cell culture, outperforming commercially available similar products. In addition, marine-derived GelMA is characterized by its sustainable source and high biosafety, making it widely applicable in cell culture, tissue engineering, drug screening, and personalized medicine. Currently, there are few reports on this material, indicating promising market prospects and application potential. Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation process of marine fish-derived GelMA; Figure 2 This is a flowchart of the process of co-culturing marine fish-derived GelMA hydrogel with cells; Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the marine fish source GelMA in Example 1; Figure 4 This refers to the biocompatibility of marine fish-derived GelMA at different concentrations in Example 1; Figure 5 This is the biocompatibility of marine fish-derived GelMA hydrogel under different concentrations of extract in Example 2; Figure 6 This is a 3D image of marine fish-derived GelMA hydrogel and NIH 3T3 cells co-cultured in Example 3; Figure 7 This refers to the cell proliferation rate of marine fish-derived GelMA hydrogel co-cultured with NIH 3T3 cells in Example 3. Figure 8 The cell viability of marine fish-derived GelMA hydrogel co-cultured with NIH 3T3 cells in Example 3; Figure 9 This is a live / dead staining of cells from a co-culture of marine fish-derived GelMA hydrogel and NIH 3T3 cells in Example 3. Detailed Implementation

[0016] The following is in conjunction with the appendix Figure 1-9 The present invention will be further illustrated by the following examples: A method for preparing marine fish-derived methacrylamide gelatin (GelMA) hydrogel is described below, covering three parts: GelMA preparation, GelMA hydrogel preparation method, and mixing of GelMA hydrogel with cells. First: The preparation method of GelMA is as follows: 1. Weigh out cod gelatin and dissolve it in PBS solution with a pH of 7.4. Stir magnetically until completely dissolved.

[0017] 2. Add methacrylic anhydride to the gelatin solution and stir magnetically to mix thoroughly.

[0018] 3. React at room temperature (20-30℃) for 4 hours, stirring continuously during the process.

[0019] 4. Add 4 times the volume of PBS solution (pH 7.4) to the reaction system.

[0020] 5. Continue stirring until the solution is evenly dispersed.

[0021] 6. Place the sample into a dialysis bag, change the purified water every 2 hours, and perform dialysis for 2 days.

[0022] 7. Add the active protein to the dialysis solution at a ratio of gelatin to silk fibroin (sea cucumber peptides, sericin, and other active proteins) of 10:1 to 5:1. 8. After dialysis, the sample was pre-frozen at -20°C for 24 hours and then freeze-dried using a vacuum freeze dryer to obtain GelMA sponge samples.

[0023] 9. Determine the degree of substitution of GelMA by TNBS method.

[0024] 10. The modification was verified by nuclear magnetic resonance hydrogen spectroscopy.

[0025] The specific method for step 9 above is as follows: Dissolve GelMA in 1 mL of distilled water to prepare a 0.5 mg / mL solution. Add 1 mL of 4% NaHCO3 and 1 mL of 0.01% TNBS solution to the GelMA solution, respectively, and react at 25°C for 2 h. After the reaction is complete, add 1 mL of 10% SDS and 0.5 mL of 1M HCl to the solution to terminate the reaction, and let it stand for 15 min. Measure the absorbance at 335 nm. The procedure for the gelatin solution is the same as above.

[0026] Standard curve preparation: Prepare glycine standard solutions with concentrations of 0, 0.005, 0.01, 0.015, 0.02, and 0.025 mg / mL. Measure the absorbance using the method described above to obtain the glycine standard curve. Calculate the degree of substitution of the sample using the standard curve and the formula. The DS calculation formula is as follows:

[0027] Among them, A GM GelMA concentration, A 明 This refers to the concentration of gelatin.

[0028] The specific method in step 10 above is as follows: The sample is loaded into an NMR tube, followed by the addition of 0.5 mL of heavy water. The synthesized polymer is analyzed using a 400 MHz NMR spectrometer (AVANCE3HD). The degree of substitution of GelMA is determined by comparing the structures of gelatin and GelMA. The degree of methacrylation substitution is defined as the percentage of ε-amino groups (lysine and hydroxylysine) modified in the gelatin molecule. When quantifying DS by 1H NMR, the spectra are normalized to the phenylalanine signal (6.9-7.5 ppm), which represents the concentration of gelatin. Subsequently, the methylene signal of lysine (2.8-2.95 ppm) in both the gelatin and GM spectra is integrated, and the degree of substitution is calculated using the formula: DS formula: .

[0029] Prepare the photoinitiator LAP solution. Weigh out a GelMA sponge and dissolve it in the LAP solution. In the dark, allow it to dissolve by shaking at room temperature. Irradiate the solution with a 365nm, 6W UV lamp to solidify it.

[0030] The specific method for preparing the photoinitiator LAP solution is as follows: Weigh 0.05 g of LAP powder, dissolve it in 20 ml of PBS solution, and heat at 40°C in the dark to dissolve. Store at 4°C.

[0031] 1. Prepare the hydrogel according to the above method and store it at room temperature away from light.

[0032] 2. Pass the filter through a 0.22μm filter membrane in a clean bench, and protect it from light with aluminum foil.

[0033] 3. Treat the cells to a concentration of 1×10⁻⁶. 6 Cells / mL, centrifuged to obtain cell pellet.

[0034] 4. Mix the hydrogel solution and cell pellet thoroughly, and pipette 100 μL into a 96-well plate.

[0035] 5. Irradiate with a 365nm ultraviolet lamp for 30 seconds.

[0036] 6. Add 100 μL of culture medium to each well and place in an incubator at 37°C.

[0037] 7. After 5 minutes, remove the culture medium, add 100 μL of fresh culture medium, and incubate in a 37°C, 5% CO2 incubator.

[0038] 8. Observe the cell and hydrogel status every 24 hours and change the culture medium.

[0039] 9. Lyse the hydrogel using GelMA lysis buffer in an incubator. After lysis, remove the cells, centrifuge, and count them using a cell counting chamber. Calculate the cell proliferation rate.

[0040]

[0041] Where Nt is the number of cells after time t; N0 is the initial number of cells.

[0042] 10. After lysis, add CCK-8 reagent to each well and measure the absorbance at 450 nm using a microplate reader to obtain cell viability.

[0043] 11. For cell live / dead staining, use a laser confocal microscope to observe the cell live / dead labeling.

[0044] For the specific method in step 9: Aspirate the culture medium from the 96-well plate and add 100 μL of lysis buffer to lyse the gel. Observe the hydrogel and cell status under an inverted microscope every 15 minutes until the hydrogel is completely lysed and no suspended cells are observed under the inverted microscope. Add 20 μL of CCK-8 reagent to each well and incubate in the dark for 2 hours. After the reaction, measure the absorbance at 450 nm using a microplate reader.

[0045] For the specific method in step 10: co-culture cells with a hydrogel and perform cell viability / death staining on days 0, 3, and 7. The procedure is as follows: aspirate the culture medium from the hydrogel and wash the hydrogel with PBS. Add PI staining solution and incubate at room temperature in the dark for 10 min. Aspirate the PI staining solution, gently wash with PBS, add AM staining solution, incubate at room temperature for 20-45 min, add PBS, and observe the cell viability / death labeling using a laser confocal microscope.

[0046] like Figure 1 , 2 The diagram illustrates the complete and detailed method for preparing marine fish-derived GelMA, as well as its application in co-culturing with cells. The specific steps are as follows: 1. Weigh out cod gelatin and dissolve it in PBS solution. Stir magnetically at room temperature (25°C) until completely dissolved.

[0047] 2. Add methacrylic anhydride to the solution and stir magnetically for 4 hours.

[0048] 3. After the reaction is complete, add 4 times the volume of PBS solution to the solution to terminate the reaction.

[0049] 4. Use a dialysis bag with a molecular weight of 14kDa for dialysis for 2 days, changing the purified water every 2 hours.

[0050] 5. After dialysis, add silk fibroin (SF) to the solution in a certain proportion and mix thoroughly.

[0051] 6. Pre-freeze at -20℃ for 24 hours, freeze-dry for 2 days to obtain GelMA sponge.

[0052] 7. Prepare LAP photoinitiator by weighing 0.05g of LAP powder and dissolving it in 20ml of PBS solution, then shaking at 40℃ in the dark to dissolve.

[0053] 8. Weigh out the GelMA sponge and dissolve it in the LAP photoinitiator solution at room temperature (25°C) in the dark.

[0054] 9. Filter the hydrogel precursor solution through a 0.22 μm filter membrane in a clean bench and store it in the dark for use in cell culture.

[0055] 10. Treat the cells to a concentration of 1×10⁻⁶. 6 Cells / mL were collected to obtain cell pellet.

[0056] 11. Mix the cell pellet with the hydrogel precursor solution until homogeneous, and then transfer the mixture into a 96-well plate.

[0057] 12. Irradiate the hydrogel with 365nm, 6W ultraviolet light to crosslink and solidify it.

[0058] 13. Add cell culture medium and incubate in a 37℃, 5% CO2 incubator, changing the medium daily.

[0059] 14. Treat the cells on the third and seventh days respectively to lyse the hydrogel, obtain cells, and perform cell counting. The cell proliferation rate was calculated, and cell viability was determined using the CCK-8 reagent.

[0060] 15. Use propidium iodide (PI) staining solution and calcein (AM) staining solution to stain hydrogel cells, and observe cell live and dead markings under a laser confocal microscope.

[0061] The reagents, instruments, and their sources used in the examples are as follows: Gelatin was provided by the Collagen Research Laboratory of Jiangsu Ocean University. Phosphate-buffered saline (PBS), trypsin, DMEM culture medium, fetal bovine serum, CCK-8 reagent, trypan blue staining solution, and glycine were purchased from Sangon Biotech (Shanghai) Co., Ltd. Methacrylamide (MAA) and TNBS solution were purchased from Sigma (Shanghai) Trading Co., Ltd. Lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) and GelMA (EFL) were purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. (China). GelMA (LG) was purchased from Shanghai Lingjiu Medical Technology Co., Ltd. Silk fibroin was purchased from Bloomage Biotechnology Co., Ltd., and mouse embryonic fibroblasts (NIH 3T3) cells were purchased from the Kunming Wildlife Cell Bank of the Chinese Academy of Sciences.

[0062] Biochemical incubator, clean bench, carbon dioxide incubator, centrifuge, electric thermostatic water bath, rotational rheometer, universal testing machine, Fourier transform infrared spectrometer, differential calorimeter, ultraviolet spectrophotometer, microplate reader, Comwell shaker, thermostatic incubator, magnetic stirrer, ultraviolet curing lamp (365nm), laser confocal microscope.

[0063] Example 1: Preparation of GelMA from Marine Fish 1. Weigh 1.5g of cod gelatin and add it to 10ml of PBS solution to prepare a 15% (w / w) gelatin solution. Stir on a magnetic stirrer until completely dissolved.

[0064] 2. Add 100 μL of methacrylic anhydride (MAA) dropwise to the gelatin solution at a rate of 0.5 mL / min, until the final concentration of MAA is 1%. Stir continuously at 25 °C for 4 h.

[0065] 3. Place the solution into a dialysis bag with a molecular weight of 14 kDa, set the dialysis temperature to 50°C, change the purified water every 2 hours, and perform dialysis for 2 days.

[0066] 4. After dialysis, add silk fibroin to the solution. The ratio of gelatin to silk fibroin is 1:10. Stir magnetically to mix thoroughly.

[0067] 5. Pre-freeze at -20℃ for 24 hours, freeze-dry for 2 days to obtain GelMA sponge (named GF).

[0068] 6. Biocompatibility: Using complete cell culture medium, GF was added to prepare sample solutions with concentrations of 2.5, 1.25, 0.625, 0.3125, and 0.15625 mg / ml. Zero-adjustment and blank groups were set up. The zero-adjustment group contained 100 μL of culture medium, and the blank group contained the same number of cells and culture medium as the sample groups. Each group was designed with three replicates. 100 μL of cell suspension was transferred to a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After removing the culture medium, 100 μL of sample solution was transferred to each well and incubated for 24 h. Then, 10 μL of CCK-8 solution was added to each well, and the reaction was allowed to proceed for 1 h. The absorbance was measured at 450 nm using a microplate reader. The cell proliferation rate (RGR) was calculated using the formula:

[0069] Where A is the average absorbance of the experimental group, A0 is the average absorbance of the zeroing group, and A1 is the average absorbance of the blank group.

[0070] Figure 3 The image shows the 1H NMR spectrum of marine fish-derived GelMA from Example 1. Compared to cod gelatin, the integral area of ​​the characteristic proton peak in the GelMA sample changed significantly, and the lysine methylene signal changed in the 2.8-2.95 ppm range. The calculated degree of substitution was 31.4%, indicating that MAA was successfully grafted onto the side chain of the FSG molecule. The TNBS method determined the degree of substitution to be 30%, and the 1H NMR results were basically consistent with the TNBS results.

[0071] Figure 4This study examines the biocompatibility of marine fish-derived GelMA at different concentrations in Example 1. Within the test concentration range of 2.5–0.15625 mg / mL, GF, similar to the control groups EFL and LG, exhibited good biocompatibility. Cell proliferation rates in all experimental groups were above 100%, with no cytotoxicity observed. However, the effects of different materials and concentrations on cell proliferation varied. GF showed the highest cell proliferation rate at all concentrations, especially at 0.625 mg / mL, where the proliferation rate reached 121%, demonstrating its excellent and stable cell proliferation-promoting potential. The LG sample also showed similar activity (119%) at 0.625 mg / mL, while the EFL sample showed a more similar proliferation rate, averaging around 110%.

[0073] 1. Preparation of photoinitiator LAP: Weigh 0.05g of LAP powder and dissolve it in 20ml of PBS solution. Shake at 40℃ in the dark to dissolve.

[0074] 2. Weigh 0.1g of marine fish-derived GelMA (GF) and dissolve it in 1ml of LAP solution. Shake at room temperature (25℃) to dissolve, and wrap in aluminum foil to protect from light.

[0075] 3. After fully dissolving, irradiate with a 365nm, 6W UV lamp for 30 seconds to solidify the hydrogel.

[0076] 4. Immerse the hydrogel sample in serum-containing culture medium at a ratio of 0.2 g / mL (add 1 mL of serum-containing culture medium to every 0.2 g of hydrogel).

[0077] 5. The solution after 24 hours of extraction was 100% concentration. Experimental groups were designed with concentrations of 75%, 50%, and 25%. The extract was centrifuged to remove impurities, and the supernatant was filtered through a 0.22 μm filter membrane.

[0078] 6. Simultaneously set up a blank group with no sample, only cells and culture medium, and a zeroing group with only culture medium and no cells. Each group has three replicates.

[0079] 7. Transfer 100 μL of cell suspension to a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 24 h. Aspirate the culture medium and transfer 100 μL of sample solution to the well plate.

[0080] 8. After culturing for 24 hours, add 10 μL of CCK-8 solution to each well and incubate for 1 hour. Measure the absorbance at 450 nm using a microplate reader. Calculate the cell proliferation rate according to the formula.

[0081] Figure 5This section describes the biocompatibility of marine fish-derived GelMA hydrogels at different concentrations in Example 2. Based on cell proliferation results, the GelMA hydrogel extracts exhibited good biocompatibility at various concentrations. As shown in the figure, within the extract concentration range of 25% to 100%, the cell proliferation rate of all sample groups remained above 96%, with most samples showing no toxicity and exceeding 100%. Most hydrogel extracts reached their maximum cell proliferation rate at a concentration of 75%. The cell proliferation rates of the hydrogel extracts were all close to or exceeded 100%.

[0082] Example 3: 3D culture of marine fish-derived GelMA hydrogel mixed with NIH 3T3 cells 1. Observe the NIH 3T3 cells in the culture flask. After the cells reach 90% confluence, carry out the experiment. Add 1 ml of trypsin to digest the cells and incubate at 37°C for 45-50 seconds. Remove the culture flask and observe the cell morphology under an inverted microscope. When the cells change from fibrous to round, add 2 ml of culture medium to stop the digestion.

[0083] 2. Count the cells and remove 1*10 cells from the culture flask. 6 Cells were collected at a density of 1000 cells / ml in a 15ml centrifuge tube. After centrifugation, the supernatant was aspirated. The cell pellet was resuspended in culture medium and centrifuged again at 1000 rpm for 3 min.

[0084] 3. Weigh 0.1g of GF sponge and dissolve it in 1ml of LAP solution to prepare a hydrogel precursor solution, and store it at room temperature. Filter the solution through a 0.22μm filter membrane in a clean bench and protect it from light with aluminum foil.

[0085] 4. Preparation of control samples: Weigh 0.05g of EFL and dissolve it in 1ml of LAP photoinitiator. Place it in a water bath and heat at 60-70℃ to dissolve. After complete dissolution, store at 37℃ in the dark. Weigh 0.07g of LG and dissolve it in 1ml of LAP photoinitiator. Place it in a water bath and heat at 37-45℃ to dissolve. After complete dissolution, store at 37℃ in the dark.

[0086]

[0087] 5. Mix the hydrogel solution and cell pellet thoroughly, and pipette 100 μL into a 96-well plate, with three replicates per sample. Irradiate at 365 nm for 30 s. Add 100 μL of culture medium to each well and incubate at 37°C. After 5 min, remove the culture medium, add 100 μL of fresh culture medium, and incubate at 37°C in a 5% CO2 incubator.

[0088] 6. Observe the state of cells and hydrogel every 24 hours and change the culture medium.

[0089] 7. On days 3 and 7, lyse the hydrogel using GelMA lysis buffer in an incubator for 2.5 hours, observing the hydrogel and cell status every half hour. No suspended cells should be observed under an inverted microscope. After lysis, centrifuge and resuspend the cells in 1 ml of complete culture medium. Count the cells using a cell counting chamber. Calculate the cell proliferation rate using the following formula:

[0090] Where Nt is the number of cells after time t; N0 is the initial number of cells.

[0091] 8. After the hydrogel is completely lysed, add 20 μL of CCK-8 reagent to each well and incubate in the dark for 2 hours. After the reaction, measure the absorbance at 450 nm using a microplate reader.

[0092] 9. Co-culture cells with hydrogel and perform cell viability / death staining on days 0, 3, and 7. First, add PI staining solution and incubate at room temperature in the dark for 10 min. Then, add AM staining solution and incubate at room temperature for 20-45 min. Aspirate the AM staining solution, then gently wash once with PBS and aspirate the supernatant. Add PBS and observe the cell viability / death labeling using a laser confocal microscope.

[0093] Figure 6 This is a 3D image of the co-culture of marine fish-derived GelMA hydrogel and NIH 3T3 cells in Example 3. The image shows the three-dimensional (3D) culture and distribution of NIH 3T3 cells within the marine fish-derived GelMA hydrogel. The GelMA hydrogel matrix constitutes a complete three-dimensional culture space, in which the uniformly distributed NIH 3T3 cells appear bright green under fluorescent labeling and are evenly dispersed throughout the three-dimensional network structure. This image visually demonstrates that the marine fish-derived GelMA hydrogel possesses excellent cell compatibility and three-dimensional support capabilities, providing a biomimetic and permeable microenvironment for cells, making it suitable for constructing in vitro 3D cell culture and tissue engineering models.

[0094] Figure 7This refers to the cell proliferation rate of NIH 3T3 cells co-cultured with marine fish-derived GelMA hydrogels in Example 3. Under three-dimensional culture conditions, all GelMA hydrogels (GF-marine fish-derived GelMA, EFL-GelMA produced by Suzhou Yongqinquan Intelligent Equipment Co., Ltd., and LG-GelMA produced by Shanghai Lingjiu Medical Technology Co., Ltd.) supported the adhesion and proliferation of NIH 3T3 cells. By day 3 of culture, the cell proliferation rate of all groups was greater than 100%, with GF and LG showing higher initial proliferation rates (173% and 158%, respectively). By day 7, the cell proliferation rate of all samples significantly increased, reaching 374% to 341%, showing a good long-term proliferation trend. These results indicate that all GelMA materials possess good cell compatibility and can support continuous cell growth in a three-dimensional environment, with the GF sample exhibiting the best cell proliferation-promoting ability.

[0095] Figure 8 The cell viability results for the co-culture of marine fish-derived GelMA hydrogel and NIH 3T3 cells in Example 3 are as follows: According to the cell viability results, the cell viability of all groups was significantly higher than 100% on Day 3 and Day 7. GF hydrogel exhibited the highest cell viability, reaching 159% on Day 3 and 256% on Day 7, significantly exceeding the other groups. This indicates that NIH 3T3 cells have good compatibility within GF hydrogel, consistent with the cell proliferation experiment results. LG hydrogel showed a large increase in viability from Day 3 to Day 7 (from 146% to 195%), indicating that LG hydrogel is more conducive to long-term cell growth and functional maintenance. EFL hydrogel showed a more gradual increase in viability from Day 3 to Day 7 compared to other groups.

[0096] Figure 9This is a cell viability staining study of marine fish-derived GelMA hydrogels and NIH 3T3 cells co-cultured in Example 3. The study used Calcein-AM / PI staining to observe the survival and distribution of NIH 3T3 cells within GelMA hydrogels (GF, EFL, LG) on days 0, 3, and 7. Throughout the culture period, all samples showed predominantly green fluorescently labeled live cells, with no obvious red fluorescence signal observed, indicating that the cells maintained good activity and membrane integrity within these three-dimensional scaffolds. In terms of cell proliferation and distribution density, different materials exhibited distinguishable trends. The GF and LG groups showed a larger number and denser distribution of live cells in the later stages of culture (day 7), demonstrating good cell support performance. In contrast, the EFL group had a relatively smaller number of cells and a sparser distribution. These morphological observations are highly consistent with the quantitatively measured cell viability data. Cell viability remained high in both GF and LG on days 3 and 7. Viability staining can directly reflect the survival status and growth trend of cells within the three-dimensional hydrogel.

[0097] The marine-derived GelMA of this invention completely avoids the heating step required by traditional GelMA, thereby greatly simplifying the operation process and improving preparation efficiency. In 3D cell culture applications, this material exhibits excellent biocompatibility and proliferation-promoting ability. Experimental data show that by the seventh day of culture, the cell proliferation rate and cell viability reached 374% and 256%, respectively, both superior to similar commercially available products.

[0098] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. A method for preparing a marine fish-derived methacrylated gelatin (GelMA) hydrogel, characterized by: Methacrylated gelatin (GelMA) hydrogel is derived from cold-water marine fish, including but not limited to cod, salmon, and trout. The preparation method is as follows: gelatin obtained from cold-water marine fish is dissolved in phosphate-buffered saline (PBS) to obtain a gelatin solution. Methacrylic anhydride is added, followed by the addition of PBS to terminate the reaction. Dialysis is then performed. After dialysis, one or more active proteins, such as silk fibroin, sea cucumber peptides, and sericin, are added for compounding. The mixture is then freeze-dried after pre-freezing to obtain marine fish-derived methacrylated gelatin (GelMA) sponge. The GelMA sponge is dissolved in a solution containing a photoinitiator (LAP). After dissolution in the dark, it is cross-linked and cured by ultraviolet light irradiation to form a hydrogel.

2. The preparation method of marine fish-derived methacrylated gelatin (GelMA) hydrogel according to claim 1, characterized in that: The gelatin derived from marine fish was dissolved in phosphate-buffered saline (PBS) at room temperature (25°C). Methacrylic anhydride was added to the gelatin solution, and the reaction was carried out at 20-30°C.

3. The method for preparing a marine fish-derived methacrylamide gelatin (GelMA) hydrogel according to claim 1, characterized in that: When the reaction is terminated, add 4 times the volume of PBS solution, and then dialyze the reaction product. The reaction solution is dialyzed with purified water at 35-65℃ for 2-5 days, and the purified water is changed every 2 hours during this period.

4. The method for preparing a marine fish-derived methacrylamide gelatin (GelMA) hydrogel according to claim 1, characterized in that: Active protein was added to the dialysis solution at a ratio of gelatin to active protein of 10:1 to 5:1, followed by freeze drying to obtain methacrylamide gelatin sponge.

5. The method for preparing a marine fish-derived methacrylamide gelatin (GelMA) hydrogel according to claim 1, characterized in that: The GelMA sponge was dissolved in a solution containing LAP photoinitiator to form a hydrogel precursor solution. The solution was dissolved at room temperature (25°C) under light-protected conditions. The hydrogel solution was then crosslinked under UV light for 30 seconds to form a hydrogel.

6. The application of a hydrogel prepared by the method of any one of claims 1-5 in 3D cell culture.

7. A method of co-culturing a hydrogel with cells, comprising: The method is as follows: The hydrogel precursor solution prepared according to any one of claims 1-5 is filtered through a 0.22 μm filter membrane in a clean bench, stored in the dark, and used for cell culture; then the cells are treated to a concentration of 1 × 10⁻⁶. 6 Cells were collected at a density of 100 cells / mL to obtain a cell pellet. The cell pellet was mixed thoroughly with the hydrogel precursor solution and transferred to wells. The hydrogel was irradiated with UV light to crosslink and solidify. Cell culture medium was added, and the cells were incubated at 37°C in a 5% CO2 incubator with daily medium changes. The cells were treated on the third and seventh days to lyse the hydrogel, obtain and count the cells, calculate the cell proliferation rate, and determine cell viability using CCK-8 reagent. The hydrogel cells were stained with propidium iodide (PI) staining solution and calcein AM staining solution, and observed under a laser confocal microscope.

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