A composite hydrogel composition and its preparation method and application

Abstract

The present invention provides a composite hydrogel composition, its preparation method, and its application. The composition comprises an ionic compound, methacryloylated hyaluronic acid (HAMA), a photoinitiator, and a solvent. The ionic compound comprises one or both of carboxymethyl cellulose and sodium alginate. The ink dots produced by the composite hydrogel exhibit high adhesion to the substrate, stability, resistance to washout, and high elasticity, and can be used for drug screening, drug toxicity testing, and other aspects.

Description

Technical Field

[0001] The present invention belongs to the technical field of gel, and in particular relates to a composite hydrogel composition, a preparation method and an application thereof. Background Art

[0002] Cell 3D printing (also known as bioprinting) is a technology that creates three-dimensional cellular structures or tissues by printing cells together with biomaterials (such as hydrogels and extracellular matrices). Compared to traditional cell culture techniques, 3D bioprinting can precisely control the distribution and arrangement of cells in space, providing powerful technical support for tissue engineering, regenerative medicine, and personalized medicine.

[0003] Common technologies used in cell 3D printing include inkjet printing, extrusion printing, and stereolithography. The core of these technologies lies in the ability to deposit "bio-ink" containing living cells layer by layer to form complex three-dimensional cellular tissues. The printing material (usually a bio-hydrogel) must not only be printable and mechanically strong but also ensure biocompatibility with cells to maintain cell activity and function.

[0004] The widespread application of cell 3D printing technology is bringing innovations to multiple disciplines, mainly including the following aspects: Tissue engineering: 3D printing technology can generate complex cell scaffolds, create an ideal growth environment for in vitro tissue engineering, and promote the regeneration of tissues such as cartilage, skin, blood vessels, and bones. Drug screening and disease models: By printing patient-specific three-dimensional tissue structures, disease states can be simulated for drug screening, toxicity assessment, and disease research. This personalized model can provide more accurate results than traditional two-dimensional cell culture. Personalized transplant organs: The ultimate goal of cell 3D printing technology is to overcome the current shortage of donated organs by printing functional cell tissues or even entire organs for transplantation.

[0005] In the cell 3D printing process, ink dots (bio-ink) are usually hydrogels or other biomaterials containing cells. When they are deposited on the substrate, the following problems may occur:

[0006] (1) Poor adhesion between ink dots and substrate

[0007] Due to insufficient adhesion between the ink dots and the substrate surface, the ink dots may slip or become unfixed during printing, affecting printing accuracy. Even if the initial ink dot deposition is successful, insufficient interlayer adhesion can lead to unstable deposition of subsequent layers, resulting in delamination, collapse, or other structural problems.

[0008] (2) Ink dot instability during printing

[0009] The rheological properties of bio-inks (such as viscosity and elasticity) affect their ability to maintain a stable shape during printing. If the ink viscosity is too low, the ink dots may not maintain their shape and easily diffuse or disperse, which can lead to the inaccurate formation of the printed three-dimensional structure. For example, if the printed cell structure cannot immediately form a self-supporting structure upon deposition, it may deform or collapse, affecting the formation of the three-dimensional structure.

[0010] (3) Ink dots are easily dispersed

[0011] In various bioprinting technologies (such as inkjet and extrusion), ink dots may be affected by external forces during ejection or deposition, causing them to disperse, move away from their intended location, or be washed away. For example, during inkjet printing, if printing parameters (such as pressure and speed) are not optimized, the force of ink ejection will be too great, causing the ink dots to separate from the substrate or disperse. Alternatively, if the viscosity or elasticity of the bio-ink is insufficient, it can easily lose stability due to external interference (such as vibration and air flow) during printing, affecting the deposition quality.

[0012] (4) Poor ink dot elasticity

[0013] Ink dots with poor elasticity may not maintain their shape after deposition and may easily collapse or deform, affecting printing accuracy and functionality. This is primarily manifested by the inability of ink dots to maintain their shape: poor elasticity means they lack sufficient restoring force to maintain the desired shape when deposited onto the substrate or previous layer. Ink dots may flow or spread excessively, resulting in blurred boundaries of the printed structure and distorted overall shape. Especially when printing complex three-dimensional structures, inelastic ink dots are unable to withstand the effects of gravity or other external forces, easily causing the structure to collapse.

[0014] Poor ink adhesion, instability, easy dispersion, and poor elasticity are common challenges in cell 3D printing, impacting printing accuracy and structural stability. Therefore, a new technical approach is needed to enhance the adhesion of ink dots to the substrate, increase their elasticity and stability, ensure precise formation of printed structures, and maintain a favorable cell survival environment. Summary of the Invention

[0015] In response to the shortcomings of the prior art, the present invention provides a composite hydrogel composition, composite hydrogel, and preparation methods and applications thereof, which address the problems of poor ink adhesion, instability, easy dispersion, and poor elasticity in the prior art.

[0016] In one aspect, the present invention provides a composition for composite hydrogel, comprising an ionic compound, methacryloyl hyaluronic acid (HAMA), a photoinitiator and a solvent, wherein the ionic compound comprises one or two of carboxymethyl cellulose and sodium alginate.

[0017] In one embodiment of the present invention, the composition does not include a hydrogel adhesion enhancer, and the hydrogel adhesion enhancer includes but is not limited to at least one of polydopamine, polymethacrylamide, polyethylene glycol, and polyvinyl alcohol.

[0018] In one embodiment of the present invention, the composition comprises carboxymethyl cellulose, methacryloyl hyaluronic acid (HAMA) and a photoinitiator.

[0019] In one embodiment of the present invention, the composition comprises the following ingredients in the following percentages: 2% carboxymethyl cellulose (CMC), 0.5-3% methacryloyl hyaluronic acid (HAMA), and 0.05-0.25% photoinitiator.

[0020] Illustratively, the percentage of the methacryloylated hyaluronic acid is any value between 0.5% and 3%, specifically 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, and 3%. When the percentage of the methacryloylated hyaluronic acid is less than 0.5%, the adhesion of the ink dots formed by the hydrogel composition to the substrate is significantly reduced, and the ink droplet stability is reduced. When the percentage of the methacryloylated hyaluronic acid is greater than 3%, the cell survival rate is significantly reduced.

[0021] Exemplarily, the percentage content of the photoinitiator is any point value between 0.05% and 0.25%, such as 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, and 0.25%.

[0022] In one embodiment of the present invention, the molecular weight of the methacryloylated hyaluronic acid is 100KDa to 1,000KDa. Exemplarily, the molecular weight of the methacryloylated hyaluronic acid is 100K Da, 150K Da, 200K Da, 250K Da, 300K Da, 350K Da, 400K Da, 450K Da, 500K Da, 550K Da, 600K Da, 650K Da, 700K Da, 750K Da, 800K Da, 850K Da, 900K Da, 950K Da, or 1000K Da. Preferably, the molecular weight of the methacryloylated hyaluronic acid is 150K Da to 400K Da, more preferably 150K Da.

[0023] In one embodiment of the present invention, the percentage of the methacryloyl hyaluronic acid can be adjusted according to the molecular weight of the methacryloyl hyaluronic acid. For example, when the molecular weight of the methacryloyl hyaluronic acid increases, the percentage of the methacryloyl hyaluronic acid can be appropriately reduced.

[0024] In one embodiment of the present invention, the composition comprises sodium alginate, carboxymethyl cellulose, methacryloyl hyaluronic acid (HAMA) and a photoinitiator.

[0025] In one embodiment of the present invention, the composition comprises the following ingredients in the following percentages: 3-5% sodium alginate, 2% carboxymethyl cellulose, 0.5-3% methacryloyl hyaluronic acid (HAMA), and 0.05-0.25% photoinitiator.

[0026] Exemplarily, the percentage content of sodium alginate is any point value between 3-5%, for example, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, and 5%.

[0027] Exemplarily, the percentage content of the methacryloylated hyaluronic acid is any point value between 0.5-3%, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, and 3%.

[0028] Exemplarily, the percentage content of the photoinitiator is any point value between 0.05% and 0.25%, such as 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, and 0.25%.

[0029] In one embodiment of the present invention, the sodium alginate comprises a combination of low-viscosity sodium alginate and medium-viscosity sodium alginate.

[0030] In one embodiment of the present invention, the mass ratio of the low-viscosity sodium alginate to the medium-viscosity sodium alginate is 2:1.

[0031] In one embodiment of the present invention, the viscosity of the medium-viscosity sodium alginate is 500-1500 mPa·s, and the viscosity of the low-viscosity sodium alginate is 5-500 mPa·s.

[0032] In one embodiment of the present invention, the composition comprises 2% of low-viscosity sodium alginate, 1% of medium-viscosity sodium alginate, 2% of carboxymethyl cellulose, 0.5% of methacryloyl hyaluronic acid 150K (HAMA 150K) and 0.25% of photoinitiator.

[0033] In one embodiment of the present invention, the composition comprises 2% carboxymethyl cellulose, 1% methacryloyl hyaluronic acid 150K (HAMA 150K) and 0.25% photoinitiator.

[0034] In one embodiment of the present invention, the composition comprises 2% carboxymethyl cellulose, 0.5% methacryloyl hyaluronic acid 400K (HAMA 150K) and 0.25% photoinitiator.

[0035] In one embodiment of the present invention, the composition further comprises a protein / polypeptide, such as at least one of polylysine, RGD polypeptide, and laminin.

[0036] In one embodiment of the present invention, the content of the protein / polypeptide is 0.5-15 μg / mL. Preferably, the content of the protein / polypeptide is 5-15 μg / mL. More preferably, the content of the protein / polypeptide is 10 μg / mL.

[0037] In one embodiment of the present invention, the solvent comprises water and / or culture medium. Preferably, the culture medium is a complete culture medium. More specifically, the complete culture medium is a complete organoid culture medium.

[0038] In one embodiment of the present invention, nutrients are added to the complete culture medium; specifically, the nutrients include: one or more of amino acids, buffers, vitamins, growth factors, signal transduction inhibitors, lipids, and antibiotics.

[0039] In one embodiment of the present invention, the nutritional ingredients include: GlutaMAX TM 、HEPES、B-27 TM At least one of additives, cell growth factors, N-acetyl-L-cysteine, R-Spondin, gastrin I human, A83-01, SB202190, prostaglandin E2, nicotinamide, and antibiotics.

[0040] In one embodiment of the present invention, the culture medium includes 2mM GlutaMAX TM , 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27 TM Supplements, 100 ng / mL Wnt3a cell growth factor, 1.25 mM N-acetyl-L-cysteine, 500 ng / mL R-Spondin, 100 ng / mL mNoggin cell growth factor, 50 ng / mL EGF cell growth factor, 10 nM Gastrin I human, 0.5 μM A83-01, 3 μM SB202190, 10 nM prostaglandin E2, 10 mM nicotinamide, and 100 μg / mL Primocin TM Primary cells were cultured in antibiotic-free DMEM / F12 medium or Nearshore human intestinal organoid medium (OCMHC01-M500).

[0041] In a second aspect, the present invention provides a composite hydrogel, the raw material of which is the above-mentioned composition.

[0042] In the present invention, the inventors conducted multiple screenings and experiments on the ingredients in the composite hydrogel composition, including sodium alginate, carboxymethyl cellulose, polydopamine, polyethylene glycol-polydopamine, polyvinyl alcohol-polydopamine, xanthan gum, gellan gum, hyaluronic acid, methacrylated hyaluronic acid, sodium methacrylated alginate, and many other components. It was unexpectedly discovered that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacrylated hyaluronic acid in combination can significantly improve the adhesion of ink droplets to the substrate. The adhesion rate of ink droplets to the substrate reaches 100%, and the adhesion time can be up to 10 days or more.

[0043] Research has found that composite hydrogels formed by combining sodium alginate and / or carboxymethyl cellulose with methacryloylated hyaluronic acid are significantly superior to composite hydrogels formed by combining sodium alginate and / or carboxymethyl cellulose. For example, composite hydrogels formed by combining very low-viscosity sodium alginate and carboxymethyl cellulose exhibit an adhesion rate of less than 10% within one day. Composite hydrogels formed by combining low-viscosity sodium alginate, medium-viscosity sodium alginate, and carboxymethyl cellulose exhibit an adhesion rate of less than 50% within five days.

[0044] Studies have found that composite hydrogels formed by combining sodium alginate and / or carboxymethyl cellulose with methacryloylated hyaluronic acid are significantly better than composite hydrogels formed by combining sodium alginate and / or carboxymethyl cellulose or other gels. For example, composite hydrogels formed by combining very low-viscosity sodium alginate, carboxymethyl cellulose, and polydopamine have an adhesion rate of less than 10% within one day. Composite hydrogels formed by combining low-viscosity sodium alginate, medium-viscosity sodium alginate, carboxymethyl cellulose, and xanthan gum have an adhesion rate of less than 50% within five days. Composite hydrogels formed by combining low-viscosity sodium alginate, medium-viscosity sodium alginate, carboxymethyl cellulose, and gellan gum have an adhesion rate of less than 50% within one day. Composite hydrogels formed by combining low-viscosity sodium alginate, medium-viscosity sodium alginate, carboxymethyl cellulose, polyethylene glycol-polydopamine, polyvinyl alcohol-polydopamine, or hyaluronic acid have an adhesion rate of less than 10% within one day.

[0045] Research has found that composite hydrogels created by combining sodium alginate and / or carboxymethyl cellulose with methacrylated hyaluronic acid are significantly better than those created by combining sodium alginate and / or carboxymethyl cellulose with methacrylated sodium alginate. For example, the composite hydrogels created by combining sodium alginate and / or carboxymethyl cellulose with methacrylated sodium alginate achieved an ink droplet breakup rate of 90-100% within one day.

[0046] Research has found that composite hydrogels made from a combination of sodium alginate and / or carboxymethyl cellulose and methacrylated hyaluronic acid perform significantly better than composite hydrogels made from a combination of sodium alginate and / or carboxymethyl cellulose, methacrylated sodium alginate, and polydopamine. For example, the composite hydrogel made from a combination of sodium alginate and / or carboxymethyl cellulose, methacrylated sodium alginate, and polydopamine exhibited an adhesion rate of less than 50% within one day.

[0047] Studies have found that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacrylated hyaluronic acid in combination has a cell survival rate and cell / organoid proliferation rate that match those of commercial matrix glue, and can replace commercial matrix glue.

[0048] Studies have found that adding protein / peptide composite hydrogels can significantly increase the proliferation rate of cells / organoids.

[0049] In a third aspect, the present invention provides a method for preparing the composite hydrogel, comprising mixing an ionic compound, methacryloyl hyaluronic acid (HAMA), a photoinitiator and a solvent to obtain the composite hydrogel.

[0050] In one embodiment of the present invention, the preparation method comprises: dissolving an ionic compound, methacryloyl hyaluronic acid (HAMA), and a photoinitiator in a solvent, and stirring to obtain the composite hydrogel.

[0051] In a fourth aspect, the present invention provides a bio-ink comprising the composite hydrogel described above, and also comprising cells, tissues or organoids.

[0052] In a fifth aspect, the present invention provides a cell chip comprising the above-mentioned biological ink.

[0053] In one embodiment of the present invention, the above-mentioned bio-ink is printed on a substrate to obtain the cell chip.

[0054] In a sixth aspect, the present invention provides the use of the above-mentioned composite hydrogel composition or composite hydrogel in improving the adhesion between bio-ink and substrate, improving the stability of bio-ink, and improving the elasticity of bio-ink.

[0055] A seventh aspect of the present invention is to improve the application of the above-mentioned composite hydrogel composition, composite hydrogel, bio-ink, or cell chip in drug screening, drug toxicity and efficacy testing, organ model construction or tissue engineering.

[0056] In an eighth aspect of the present invention, a method for improving the adhesion of bio-ink to a substrate, improving the stability of bio-ink and / or improving the elasticity of bio-ink is provided, comprising using the above-mentioned composite hydrogel to improve the adhesion of bio-ink to a substrate, improving the stability of bio-ink and / or improving the elasticity of bio-ink.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] (1) The composite hydrogel of the present invention has high stability and high elasticity, a more stable shape, resistance to deformation due to external pressure, and high rebound ability; it has strong adhesion to the substrate and can be used for long-term cell culture (at least 10 days), drug screening, and drug toxicity and efficacy testing.

[0059] (2) The proportion of living cells in the bio-ink obtained from the composite hydrogel of the present invention is greater than 90%, and the organoid area (cell density) increases by about 0.83-0.87 times that in Matrigel, which is close to the effect of commercial Matrigel and can replace Matrigel. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1Graph showing cell viability during cell culture in 2% NaA (low viscosity)-1% NaA (medium viscosity)-2% CMC-0.5% HAMA (150K)-0.25% LAP hydrogel and commercial Matrigel;

[0061] Figure 2 The figure shows the proliferation of cells cultured in 2% NaA (low viscosity)-1% NaA (medium viscosity)-2% CMC-0.5% HAMA (150K)-0.25% LAP hydrogel and commercial Matrigel on day 4;

[0062] Figure 3 This is a graph showing the proliferation of cells cultured in 1% HAMA (150K)-2% CMC-0.25% LAP hydrogels containing different laminin concentrations on day 4;

[0063] Figure 4 The figure shows the comparison of the survival rate and proliferation rate of cells / organoids cultured in 1% HAMA (150K)-2% CMC-0.25% LAP hydrogel and 0.5% HAMA (400K)-2% CMC-0.25% LAP hydrogel;

[0064] FIG5 is a graph showing the activity and proliferation of CRC5 / CRC7 / CRC15 cells during cell culture in hydrogel and commercial Matrigel;

[0065] Figure 6 Statistical graph of cell survival rate in hydrogel after adding different concentrations of pentafluorouracil (5-Fu).

[0066] Figure 7 Mechanical property tests of hydrogels. (a) Compression test of hydrogels. None of the hydrogels tested contained CMC. (b) Compression modulus of hydrogels. The compression modulus was calculated based on the 0-20% compression curve. NaA-1: 5% NaA (veryLow)-2% CMC; HAMA-1: 1% HAMA-2% CMC-0.25% LAP; HAMA-2: 0.75% HAMA-2% CMC-0.25% LAP; NaA-1: 2% NaA (Low)-1% NaA (Medium)-2% CMC. None of the hydrogels tested contained CMC. (c) Rheological property test of hydrogel precursor solutions. All of the hydrogels tested contained CMC.

[0067] Figure 8Fluorescence images of a 1% HAMA (150K)-2% CMC-0.25% LAP hydrogel ink droplet and a 5% NaA (very low)-2% CMC hydrogel ink droplet after demolding (after CMC degradation). 10 μg / mL of fluorescent nanospheres (10 nm) were added to the hydrogel, and the ink droplet was forcibly separated from the substrate for in situ optical characterization of the substrate. DETAILED DESCRIPTION

[0068] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0069] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0070] Reagents used in the examples:

[0071] The separation solution is supplemented with 2mM GlutaMAX TM , 25 mM HEPES, 1% streptomycin and penicillin in DMEM / F12 medium.

[0072] Complete intestinal organoid culture medium supplemented with 2 mM GlutaMAX TM , 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27 TM Additives, 100 ng / mL Wnt3a cell growth factor, 1.25 mM N-acetyl-L-cysteine, 500 ng / mL R-Spondin, 100 ng / mL mNoggin cell growth factor, 50 ng / mL EGF cell growth factor, 10 nM gastrin Ihuman, 0.5 μM A83-01, 3 μM SB202190, 10 nM prostaglandin E2, 10 mM nicotinamide, 100 μg / mL Primocin TM Primary cells were cultured in antibiotic-free DMEM / F12 medium or Nearshore human intestinal organoid medium (OCMHC01-M500).

[0073] The digestion solution was supplemented with 10 μM Y-27632 and 100 μg / mL Primocin. TM Primary cell antibiotics and 2mg / mL Collagenase Type 2 enzyme separation solution.

[0074] Example 1 Organoid Culture and Passaging

[0075] 1. Organoid Culture

[0076] Organoids were seeded in Matrigel (50 μL per well) in 24-well plates and cultured and expanded in a 37°C, 5% CO2 incubator. Complete intestinal organoid medium (500 μL per well) was used, with medium replacement every two days and passaged every seven days (this number can be adjusted based on the specific growth rate of the individual sample).

[0077] 2. Enzymatic Passaging of Organoids

[0078] (1) Remove the culture medium and add 1 mL of separation solution to each well. Use a pipette (1 mL tip with a 10 μL tip) to break up the matrix gel by pipetting and aspirating. Repeat this process for more than 20 times and transfer all the liquid to a centrifuge tube. Rinse each well with 1 mL of separation solution and transfer all the liquid to the above centrifuge tube.

[0079] (2) The above liquid was centrifuged at 2000 RMP for 5 minutes, the supernatant was removed, and TrypLE was added TM Resuspend the cells with express enzyme (250 μL per tube, containing 10 μM Y-27632 dihydrochloride) and digest in a 37°C water bath for 5 min.

[0080] (3) After digestion, the liquid was pipetted for more than 20 times, centrifuged at 2000 rpm for 5 min, and the supernatant was removed.

[0081] (4) Wash the cells with 2 mL of separation solution, add an appropriate amount of Matrigel, mix by pipetting, and seed into a 24-well plate (50 μL per well). Solidify at 37°C for 30 min, add culture medium, and place in an incubator.

[0082] 3. Mechanical Passaging of Organoids

[0083] (1) Remove the culture medium and add 1 mL of separation solution to each well. Use a pipette (1 mL tip with a 10 μL tip) to break up the matrix gel by pipetting and aspirating. Repeat this process for more than 20 times and transfer all the liquid to a centrifuge tube. Rinse each well with 1 mL of separation solution and transfer all the liquid to the above centrifuge tube.

[0084] (2) The above liquid was centrifuged at 2000 RMP for 5 min, the supernatant was removed, and an appropriate amount of Matrigel was added. After mixing by pipetting, the mixture was seeded into a 24-well plate (50 μL per well), solidified at 37°C for 30 min, and culture medium was added and placed in an incubator.

[0085] Note: Matrigel solidifies at temperatures above 4°C and should be placed and mixed on ice.

[0086] Example 2 Preparation of hydrogel

[0087] 1. Preparation of carboxymethyl cellulose (CMC)-sodium alginate (NaA)-methacrylated hyaluronic acid (HAMA) hydrogel (experimental group 1)

[0088] (1) Use weighing paper (10×10 cm, avoid creases) to weigh 0.1 g (2%) of low-viscosity NaA powder, 0.05 g (1%) of medium-viscosity NaA powder, and 0.1 g (2%) of CMC powder. Spread the paper completely flat and sterilize under ultraviolet light for 3 h.

[0089] (2) Hydrogel Dissolution and Dispersion: Weigh 0.0125 g (0.25%) of photoinitiator (LAP) and 0.0025 g (0.5%) of HAMA (150K) into a 25 mL centrifuge tube. Add 5 mL of organoid complete culture medium, dissolve LAP and HAMA, and filter through a bacterial filter. Add NaA powder and stir well (avoid clumping). Then add CMC powder and stir well (avoid clumping). Ultrasonicate for 30-60 min.

[0090] (3) Centrifuge to remove gas, setting: 1500-2000 RMP, 5-10 min.

[0091] (4) Use a pipette to add 100 μL of laminin (final concentration 10 μg / mL) and stir well.

[0092] (5) Store at 2-4°C.

[0093] Note: The above operations were performed under yellow light. Use a 1 mL pipette tip for stirring.

[0094] 2. Preparation of carboxymethyl cellulose (CMC)-methacrylated hyaluronic acid (HAMA) hydrogel (experimental group 2)

[0095] The same as step 1, except that: in step (1), 0.1 g (2%) of low-viscosity NaA powder and 0.05 g (1%) of medium-viscosity NaA powder are not added, and the amount of HAMA (150K) is modified. The details are as follows:

[0096] (1) Weigh 0.1 g (2%) of CMC powder on a weighing paper (10 × 10 cm, avoid creases), spread it completely flat, and sterilize it under ultraviolet light for 3 h.

[0097] (2) Hydrogel Dissolution and Dispersion: Weigh 0.0125 g (0.25%) of photoinitiator (LAP) and 0.005 g (1%) of HAMA (150K) into a 25 mL centrifuge tube. Add 5 mL of complete organoid culture medium to dissolve the LAP and HAMA, and filter through a bacterial filter. Add CMC powder, stir well (avoid clumping), and sonicate for 30-60 min.

[0098] (3) Centrifuge to remove bubbles, setting: 1500-2000 RMP, 5-10 min.

[0099] (4) Use a pipette to add 100 μL of laminin (final concentration 10 μg / mL) and stir well.

[0100] (5) Store at 2-4°C.

[0101] 3. Preparation of carboxymethyl cellulose (CMC)-methacrylated hyaluronic acid (HAMA) hydrogel (experimental group 3)

[0102] The process is the same as step 2, except that the molecular weight and amount of HAMA are different. The molecular weight and amount of HAMA used in this step are 0.5% HAMA (400K).

[0103] The hydrogels of the control group were prepared according to the above method. The hydrogel compositions of experimental groups 1-3 and control groups 1-11 are shown in Table 1.

[0104] Table 1 Hydrogel composition

[0105] Group Hydrogel ingredients Experimental Group 1 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 0.5% HAMA (150K) - 0.25% LAP Control group 1 5% NaA (very low viscosity)-2% CMC Control group 2 5% NaA (very low viscosity)-2% CMC-PDA (2 mg / mL) Control group 3 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC Control group 4 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 0.1% xanthan gum Control group 5 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 0.1% gellan gum Control group 6 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 1% PEG-PDA Control group 7 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 1% PVA-PDA Control group 8 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 1% hyaluronic acid Control group 9 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 0.5% AlgMA - 0.25% LAP Control group 10 2% NaA (low viscosity) - 1% NaA (medium viscosity) - 2% CMC - 0.25% PDA - 0.5% AlgMA - 0.25% LAP Experimental Group 2 1%HAMA(150K)-2%CMC-0.25%LAP Experimental Group 3 0.5%HAMA(400K)-2%CMC-0.25%LAP Control group 11 0.75%HAMA(150K)-2%CMC-0.25%LAP

[0106] Example 3 Cell Ink Configuration and Printing Ink Filling (Conventional Needle)

[0107] 1. Seal the 3cc ink tube, 200μm needle, and pressure plug in a sterilization bag and place them in a high-pressure steam sterilizer for sterilization (110-120℃, 40-60min). Soak the tail cap and head plug in 75% alcohol for 30min.

[0108] 2. Precool the low-temperature centrifuge to 1°C.

[0109] 3. Lay the 3cc ink tube flat. Take a 1mL graduated disposable syringe and draw 200μL of CMC-NaA-HAMA hydrogel or CMC-HAMA hydrogel. Slowly inject it near the inlet of the ink tube. Use a pipette to inject 100μL of the organoid cell suspension prepared in Example 1 into the hydrogel, stirring while injecting. Stir for 2 minutes. (The hydrogel concentration is set to 1.5 times the target concentration. Then, mix the 1.5x concentration hydrogel and the cell suspension in a volume ratio of 2:1 to obtain a cell-containing hydrogel at the target concentration.)

[0110] 4. Add a pressure plug from the ink tube inlet and slowly push the pressure plug to push the ink to the ink tube outlet. Be careful not to squeeze out the ink and do not leave any gaps.

[0111] 5. Replace the tail cap and head cap, with the ink tube outlet facing upward, and centrifuge in a low-temperature centrifuge to remove air bubbles. Settings: 1500–2000 RPM, 5–10 minutes.

[0112] Note: When adding liquid to a 1mL disposable syringe, remove the metal needle or use a metal needle with a diameter greater than 1mm. To avoid residual hydrogel in the syringe, the syringe can be slightly overdrawn by 20μL above the target volume. Stirring until bubbles are uniform can confirm that the mixture is well mixed. Plastic materials should be sterilized at a temperature not exceeding 120°C.

[0113] Example 5 Printer programming and printing preparation

[0114] 1. Expose the printing environment and equipment to ultraviolet light for 30-60 minutes.

[0115] 2. Start the cooling system and circulation system of the cooling water of the needle tube and printing platform, set it to 1-4℃, and keep the temperature stable after about 30 minutes.

[0116] 3. Use the built-in program of the printing device to program the printing process. Taking a 96-well plate dot matrix as an example, set the coordinates of a dot drop (xyz - 20, 20, 61mm), dot matrix mode (x-axis spacing is 9mm, x-axis row number is 10, y-axis spacing is 9mm, y-axis row number is 6), needle movement speed is 30-50mm / s, dot interval needle lift height is 12-15mm, unit dot inkjet time is 0-1s, and inkjet pressure is set to 10-50psi.

[0117] 4. Assemble the air valve, ink tube, and needle, place them on the hanging plate, and place the 96-well plate at the corresponding coordinate position.

[0118] 5. Use a 50 mL centrifuge tube to weigh 0.7-0.8 g of CaCl2(s) and dissolve it in 50 mL of PBS (pH = 7.0) to make a 1.5% solution. Shake to dissolve and filter sterilize using a bacterial filter.

[0119] 6. Use a 50 mL centrifuge tube to weigh 0.045-0.055 g of cellulase powder and dissolve it in 50 mL of DMEM medium to make a 0.1% solution. Shake to dissolve and filter sterilize using a bacterial filter.

[0120] 7. Prepare complete culture medium solution and separation medium.

[0121] Note: The printing tip should be 20-100 μm above the substrate. Prepare 100 μL of CaCl₂ solution, cellulase solution, and complete culture medium per well. Prepare 200 μL of separation buffer per well.

[0122] Example 6 Printing and Cell / Organoid Culture Using CMC-NaA-HAMA Hydrogel

[0123] 1. Start the printing process and complete the printing, 6×10 about 2-5 minutes.

[0124] 2. After printing is completed, use a blue light lamp (405nm) to irradiate for 30 seconds, use a spray gun to add 100μL of 1.5% CaCl2 solution to each well, and then aspirate the solution after curing reaction for 5-10 minutes.

[0125] 3. Use a dispenser to add 100 μL of 0.1% cellulase solution to each well and aspirate the solution after reacting for 5-10 minutes.

[0126] 4. Use a dispenser to add 100 μL of separation solution to each well, soak for 10-20 seconds, and then aspirate the solution.

[0127] 5. Use a dispenser to add 100 μL of complete culture medium to each well and place in a cell culture incubator at 37°C.

[0128] 6. After printing, the cell / organoid chip is cultured in a 37°C, 5% CO2 incubator, and the culture medium is replaced every 3-4 days.

[0129] Note: Avoid ink spots during addition and aspiration.

[0130] Example 7 Printing using CMC-HAMA hydrogel

[0131] Similar to Example 6, except that the step of adding 100 μL of 1.5% CaCl 2 solution to each well using a dispenser and aspirating the solution after the solidification reaction for 5-10 minutes was omitted.

[0132] Test Example 1: Ink dot fall-off test

[0133] 1. Use a printer or pipette to place hydrogel ink dots (1-2 mm) on the surface of the well plate. After completing the curing and enzymatic hydrolysis steps, add separation solution and place it in a shaker (37°C). Take it out every 24 hours and perform normal liquid changes 10-20 times to check the adhesion of the ink droplets to the substrate.

[0134] 2. Use a printer or pipette to deposit cell-containing hydrogel ink dots (1-2 mm) onto the surface of the well plate. After the solidification and enzymatic hydrolysis steps, add complete culture medium and incubate in a 37°C, 5% CO2 incubator. Check the adhesion of the ink droplets to the substrate every two days. The results are shown in Tables 2 and 3.

[0135] Table 2 Ink dot falling off test results

[0136]

[0137]

[0138] Table 3 Ink dot adhesion rate test during cell culture (ink dot size: 1-2 mm, 10th day)

[0139]

[0140] Conclusion: As shown in the test results in Tables 1 and 2, the adhesion of the hydrogel ink dots prepared in experimental groups 1 and 3 was 100% after 10 days; the adhesion of the hydrogel ink dots prepared in experimental group 2 was 100% for more than 10 days, and there was no problem of falling off, which met the requirements of long-term cell experiments.

[0141] Experimental Example 2: Cell / Organoid Drug Testing

[0142] After 4 days of culture of printed cells / organoids, drugs were added (old culture medium was removed and 100 μL of complete organoid culture medium containing the drug or drug combination to be tested at a specific concentration was added to each well). Cell / organoid morphology was characterized and viability and mortality were tested 72 hours after drug addition.

[0143] 1. The experimental method is as follows:

[0144] (1) Preparation of dye solution: Use DMEM medium to dilute the stock solutions of calcein-AM (AM, 4 mM) and ethidium homodimer-1 (EthD-1, 2 mM) to prepare a dye solution containing 0.5 μM AM and 6 μM EthD-1.

[0145] (2) Remove the culture medium and add 100 μL of DMEM culture medium to each well. Soak for 10-20 seconds and then aspirate the solution.

[0146] (3) Add 100 μL of dye solution to each well and incubate at 37°C in the dark for 30 min.

[0147] (4) Remove the dye solution and add 100 μL of DMEM medium to each well. Soak for 10-20 seconds and then aspirate the solution.

[0148] (5) Add 100 μL of DMEM medium to each well. Place the plate under a fluorescence confocal microscope and perform multi-channel imaging with 488 nm / 561 nm excitation light. Record and photograph the images. Images are processed using Image J. The cell / organoid live / death ratio is calculated as the green fluorescence area / (red fluorescence area + green fluorescence area) in the same image.

[0149] (6) The data were statistically analyzed and plotted using GraphPad Prism.

[0150] 2. Experimental results

[0151] (1) The cell survival rate of hydrogel-cultured organoids prepared in experimental group 1 was as follows Figure 1 shown. Figure 1a is a fluorescence staining image of cells cultured in hydrogels on the fourth day (AM: living cells; EthD-1: dead cells). Figure 1 b is the fluorescence staining image of cells cultured in matrix gel on the fourth day. Figure 1 c is a statistical graph of cell survival rate.

[0152] Conclusion: After 4 days of culture, the proportion of living cells in hydrogel-mixed cells was greater than 90%, which is close to the effect of matrix gel. It can be seen that the cell activity is high during the culture process.

[0153] (2) Cell proliferation rate test of hydrogel cultured organoids prepared in experimental group 1 Figure 2 shown. Figure 2 a is the bright field image of cells cultured in CMC-NaA-HAMA hydrogel on the fourth day. Figure 2 b is the bright field image of cells cultured in matrix gel on the fourth day. Figure 2 c is the statistical graph of organoid area on the fourth day of culture.

[0154] Conclusion: After 4 days of culture using hydrogel-mixed cells, the organoid area (cell density) was approximately 0.4 times that in matrix gel (with the same initial cell density).

[0155] (3) Effect of laminin concentration in the hydrogel prepared in experimental group 2 on the proliferation rate of cultured organoids Figure 3 shown.

[0156] Conclusion: The optimal laminin concentration for promoting the proliferation of colorectal cancer cells is 10 μg / mL.

[0157] (4) Comparison of the survival rate and proliferation rate of cells / organoids cultured in the hydrogels prepared in experimental group 2 and those prepared in experimental group 3 Figure 4 shown.

[0158] Conclusion: In terms of cell viability, 1% HAMA (150K)-2% CMC-0.25% LAP and 0.5% HAMA (400K)-2% CMC-0.25% LAP had similar effects. Over time (from D0 to D4), cell viability was not significantly affected by the different components.

[0159] In terms of organoid growth, 1% HAMA (150K)-2% CMC-0.25% LAP was more conducive to the growth or expansion of organoids than 0.5% HAMA (400K)-2% CMC-0.25% LAP, promoting the formation of organoids over a larger area, while the growth of organoid area with 0.5% HAMA (400K)-2% CMC-0.25% LAP was more limited within the same period of time.

[0160] (5) Cell survival rate and proliferation rate of hydrogel-cultured organoids prepared in experimental group 2

[0161] Multiple repeated experiments were conducted on different patient samples, different hydrogel batches, and different organoid culture passages, and compared with commercial matrix gel. Figure 5-1 、 5-2 , 5-3, 5-4, and 5-5.

[0162] Figure 5-1 、 5-2 In 5-3, 5-4, and 5-5, a is the survival rate statistics of cells cultured in hydrogel and matrix gel on the fourth day, b is the statistical graph of organoid area on the fourth day after cells are cultured in hydrogel and matrix gel (normalized using the area on day 0), c is the fluorescence staining image of cells cultured in hydrogel on the fourth day (AM: live cells; EthD-1: dead cells), and d is the fluorescence staining image of cells cultured in matrix gel on the fourth day.

[0163] Conclusion: After four days of culture, the proportion of viable cells in hydrogel-mixed cells was greater than 90%, comparable to the effect of Matrigel, indicating high cell activity during culture. After four days of culture, the organoid area (cell density) increased approximately 0.83-0.87 times that of Matrigel-mixed cells.

[0164] Experimental Example 3: Drug Sensitivity Test of Hydrogel-Cultivated Organoids

[0165] The hydrogel prepared in experimental group 2 was used as an example for drug sensitivity testing. The statistical graph of cell survival rate in the hydrogel after adding different concentrations of 5-FU is shown in the figure below. Figure 6 As shown. a) CRC7 test 1 b) CRC7 test 2 c) CRC7 test 3 d) CRC3 test 4

[0166] Conclusion: The cell viability followed a decreasing trend with increasing drug concentration.

[0167] Test Example 4: Hydrogel Mechanical Properties Test

[0168] The hydrogel prepared in experimental group 2 was used as an example for testing. A hydrogel block (length: 3 cm, width: 3 cm, height: 3 mm) was prepared using a mold, and the compression performance test was performed after curing and degrading the CMC.

[0169] Depend on Figure 7 a It can be seen that 5% NaA (very low)-2% CMC hydrogel (control group 1) undergoes inelastic deformation when subjected to external force. After the hydrogel solidifies and CMC is enzymatically degraded, the main component is Ca 2+The cross-linked network of cross-linked NaA molecules, ionic bonds are dynamic bonds, which are easily broken by external forces. After adding PDA to the 5% NaA (very Low)-2% CMC hydrogel (control group 2), it cannot change the phenomenon that the material is non-elastically deformed under external force. 1% HAMA (150K)-2% CMC hydrogel (experimental group 2) undergoes elastic deformation when subjected to external force. 2% NaA (Low)-1% NaA (Medium)-2% CMC hydrogel (control group 3) undergoes elastic deformation when subjected to external force, but its elastic modulus is smaller than that of 1% HAMA (150K)-2% CMC hydrogel (experimental group 2), and the material strength is too low to meet the requirements of cell culture for up to 10 days. Figure 7 Figure b compares the compression moduli of four different hydrogels. Human tissue has a low compression modulus, so using hydrogels with lower compression moduli is beneficial for cell culture. The 0.75% HAMA (150K)-2% CMC hydrogel (Control Group 11) has a low compression modulus and poor stability in aqueous environments. Therefore, the compression modulus of 1% HAMA (150K)-2% CMC (Experimental Group 2) is the minimum value that ensures the stability of ink droplets in aqueous environments for long periods of time (greater than 10 days).

[0170] 10 μg / mL of fluorescent nanospheres (10 nm) were added to the hydrogel, and the ink droplet was forcibly separated from the substrate, and the substrate was optically photographed in situ ( Figure 8 The ink droplets of 5% NaA (very low)-2% CMC hydrogel (control group 1) deformed and broke during the separation process, leaving more residue on the substrate; while the ink droplets of 1% HAMA (150K)-2% CMC hydrogel (experimental group 2) could completely detach from the substrate, leaving less residue on the substrate.

[0171] like Figure 7 As shown in Figure c, due to the low viscosity of the 1% HAMA (150K) hydrogel precursor solution, cell sedimentation is prone to occur during the printing process, resulting in uneven distribution of cells in the hydrogel ink. This leads to uneven cell density distribution in the printed ink droplets, reducing cell activity. Therefore, it is necessary to add 2% CMC to the 1% HAMA (150K) to increase the viscosity of the printing ink (hydrogel precursor solution). At the same time, CMC has significant shear-thinning properties, which can prevent cells from being damaged by shear forces during the printing process. After printing and curing, the CMC is completely degraded by cellulase.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A composite hydrogel composition, characterized in that: The composition comprises an ionic compound, methacrylated hyaluronic acid, a photoinitiator and a solvent, wherein the ionic compound comprises one or two of carboxymethyl cellulose and sodium alginate; the composition does not comprise a hydrogel adhesion enhancer, and the hydrogel adhesion enhancer includes but is not limited to at least one of polydopamine, polymethacrylamide, polyethylene glycol and polyvinyl alcohol; The composition comprises the following ingredients in percentage: 2% carboxymethyl cellulose, 0.5-3% methacrylated hyaluronic acid and 0.05-0.25% photoinitiator; The molecular weight of the methacryloyl hyaluronic acid is 100KDa-1,000KDa.

2. The composite hydrogel composition according to claim 1, wherein: The molecular weight of the methacryloyl hyaluronic acid is 150K Da-400K Da.

3. The composite hydrogel composition according to claim 1, wherein: The composition also includes sodium alginate.

4. The composite hydrogel composition according to claim 3, wherein: The percentage content of sodium alginate in the composition is 3-5%.

5. The composite hydrogel composition according to claim 4, wherein: The sodium alginate includes a combination of low-viscosity sodium alginate and medium-viscosity sodium alginate.

6. The composite hydrogel composition according to claim 5, wherein: The mass ratio of the low-viscosity sodium alginate to the medium-viscosity sodium alginate is 2:

1.

7. A composite hydrogel composition according to claim 5 or 6, characterized in that: The viscosity of the medium-viscosity sodium alginate is 500-1500 mPa·s, and the viscosity of the low-viscosity sodium alginate is 5-500 mPa·s.

8. A composite hydrogel, characterized in that: The raw material is the composition according to any one of claims 1 to 7.

9. A method for preparing the composite hydrogel according to claim 8, characterized in that: The method comprises mixing an ionic compound, methacrylated hyaluronic acid, a photoinitiator and a solvent to obtain the composite hydrogel.

10. A bio-ink, characterized in that: The composite hydrogel according to claim 8 further comprises cells, tissues or organoids.

11. A cell chip, characterized in that: Including the biological ink according to claim 10.

12. The cell chip according to claim 11, wherein: The biological ink is printed on a substrate to obtain the cell chip.

13. Use of the composite hydrogel composition according to any one of claims 1 to 7, or the composite hydrogel according to claim 8, in improving the adhesion of a bio-ink to a substrate, improving the stability of the bio-ink, and / or improving the elasticity of the bio-ink.

14. Use of the composition for composite hydrogel according to any one of claims 1 to 7, the composite hydrogel according to claim 8, the bio-ink according to claim 10, or the cell chip according to claim 11 in drug screening, drug toxicity and efficacy testing, or organ model construction or tissue engineering.

15. A method for improving the adhesion of bio-ink to a substrate, improving the stability of bio-ink, and / or improving the elasticity of bio-ink, comprising using the composite hydrogel according to claim 8 to improve the adhesion of bio-ink to a substrate, improving the stability of bio-ink, and / or improving the elasticity of bio-ink.

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