Composition for composite hydrogel as well as preparation method and application of composition

Abstract

The invention provides a composition for composite hydrogel as well as a preparation method and application of the composition. Comprising an ionic compound, methacrylated hyaluronic acid (HAMA), a photoinitiator and a solvent, and the ionic compound comprises one or two of carboxymethyl cellulose and sodium alginate. The ink dots prepared from the composite hydrogel have high adhesion to a substrate, are stable, are not easy to disperse, have high elasticity, and can be used in the aspects of drug screening, drug toxicity and test and the like.

Description

Technical Field

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

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

[0003] The technologies commonly used in cell 3D printing include inkjet printing, extrusion printing and photocuring printing. The core of these technologies is the ability to deposit "bio-ink" containing living cells layer by layer to form three-dimensional cell tissues with complex structures. The printing material (usually bio-hydrogel) must not only have printability and mechanical strength, but also must ensure biocompatibility with cells to maintain cell activity and function.

[0004] The widespread application of cell 3D printing technology is bringing innovation to multidisciplinary fields, 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 process of cell 3D printing, ink dots (bio-ink) are usually hydrogels or other biological materials containing cells. When they are deposited on the substrate, there are the following problems:

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

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

[0008] (2) Ink dot instability during printing

[0009] The rheological properties of bio-ink (such as viscosity, elasticity, etc.) will affect whether it can maintain a stable shape during the printing process. If the viscosity of the ink is too low, the ink dots may not be able to maintain their shape and are easily diffused or dispersed, which will cause the printed three-dimensional structure to be unable to be accurately formed. For example, if the printed cell structure cannot form a self-supporting structure immediately when deposited, 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, extrusion, etc.), ink dots may be affected by external forces when they are ejected or deposited, causing them to disperse, move away from the expected position, or be washed away. For example, during inkjet printing, if the printing parameters (such as pressure and speed) are not optimized, the power of the ink dots when they are ejected will be too large, causing the ink dots to separate from the substrate or disperse. Or if the viscosity or elasticity of the bio-ink is insufficient, it is easy to lose stability due to external interference (such as vibration, air flow) during printing, affecting the deposition quality.

[0012] (4) Poor ink dot elasticity

[0013] Ink dots with poor elasticity may not be able to maintain their shape after deposition and may collapse or deform easily, thus affecting printing accuracy and functionality. This is mainly manifested in the inability of ink dots to maintain their shape: poor elasticity of ink dots means that they lack sufficient restoring force and cannot maintain the expected shape when deposited on the substrate or the 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, ink dots that lack elasticity have difficulty withstanding the effects of gravity or other external forces, which can easily lead to structural collapse.

[0014] Poor adhesion, instability, easy dispersion and poor elasticity of ink dots are common problems in cell 3D printing, which affect the printing accuracy and stability of the structure. Therefore, a new technical measure is needed to enhance the adhesion between the ink dots and the substrate, increase the elasticity and stability of the ink dots, ensure the precise molding of the printed structure, and maintain a good cell living environment. Summary of the invention

[0015] In view of the deficiencies in the prior art, the present invention provides a composite hydrogel composition, a composite hydrogel, and a preparation method and application thereof, which solve the problems of poor adhesion, instability, easy dispersion, and poor elasticity of ink dots in the prior art.

[0016] In one aspect, the present invention provides a composite hydrogel composition, which comprises 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 percentage: 2% carboxymethyl cellulose (CMC), 0.5-3% methacryloyl hyaluronic acid (HAMA) and 0.05-0.25% photoinitiator.

[0020] Exemplarily, the percentage content of the methacryloyl hyaluronic acid is any point value in the range of 0.5-3%, such as 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%, 3%. When the percentage content of the methacryloyl hyaluronic acid is less than 0.5%, the adhesion of the ink dots formed by the hydrogel composition to the matrix is ​​significantly reduced, and the stability of the ink drops is reduced. When the percentage content of the methacryloyl hyaluronic acid is greater than 3%, the survival rate of the cells 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 methacryloyl hyaluronic acid is 100KDa-1,000KDa. Exemplarily, the molecular weight of the methacryloyl hyaluronic acid is 100K Da, 150K Da, 200K Da, 250KDa, 300KDa, 350K Da, 400K Da, 450K Da, 500K Da, 550K Da, 600K Da, 650K Da, 700K Da, 750KDa, 800K Da, 850K Da, 900K Da, 950K Da, 1000K Da. Preferably, the molecular weight of the methacryloyl hyaluronic acid is 150K Da-400K Da. More preferably, it is 150K Da.

[0023] In one embodiment of the present invention, the percentage content 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 content 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 percentage: 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%, such as 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 methacryloyl 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 a specific 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 a specific embodiment of the present invention, the composition comprises 2% carboxymethyl cellulose, 1% methacryloyl hyaluronic acid 150K (HAMA 150K) and 0.25% photoinitiator.

[0034] In a specific 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 / peptide, such as at least one of poly-lysine, 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 an organoid complete 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 , 25mM HEPES, 1% streptomycin and penicillin, 2% B-27 TM Supplement, 100ng / mL Wnt3a cell growth factor, 1.25mM N-acetyl-L-cysteine, 500ng / mL R-Spondin, 100ng / mL mNoggin cell growth factor, 50ng / mL EGF cell growth factor, 10nM Gastrin I human, 0.5μM A83-01, 3μM SB202190, 10nM prostaglandin E2, 10mM nicotinamide, and 100μg / mL Primocin TM Primary cells were cultured in antibiotic-free DMEM / F12 medium or Nearctic 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 have screened and experimented for many times 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, methacryloyl hyaluronic acid, methacryloyl sodium alginate and many other components. It was unexpectedly found that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacryloyl hyaluronic acid in combination can significantly improve the adhesion between ink droplets and the matrix, the adhesion rate between the ink droplets and the matrix reaches 100%, and the adhesion time can be up to 10 days or more.

[0043] The study found that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacryloyl hyaluronic acid together is significantly better than the composite hydrogel formed by sodium alginate and / or carboxymethyl cellulose. For example, the composite hydrogel obtained by using very low viscosity sodium alginate and carboxymethyl cellulose together has an adhesion rate of less than 10% within 1 day. The composite hydrogel obtained by using low viscosity sodium alginate, medium viscosity sodium alginate and carboxymethyl cellulose together has an adhesion rate of less than 50% within 5 days.

[0044] The study found that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacryloyl hyaluronic acid together is significantly better than the composite hydrogel formed by sodium alginate and / or carboxymethyl cellulose and other gels. For example, the composite hydrogel obtained by using very low viscosity sodium alginate, carboxymethyl cellulose and polydopamine together has an adhesion rate of less than 10% within 1 day. The composite hydrogel obtained by using low viscosity sodium alginate, medium viscosity sodium alginate, carboxymethyl cellulose and xanthan gum together has an adhesion rate of less than 50% within 5 days. The composite hydrogel obtained by using low viscosity sodium alginate, medium viscosity sodium alginate, carboxymethyl cellulose and gellan gum together has an adhesion rate of less than 50% within 1 day. The composite hydrogel obtained by using low viscosity sodium alginate, medium viscosity sodium alginate, carboxymethyl cellulose, polyethylene glycol-polydopamine or polyvinyl alcohol-polydopamine or hyaluronic acid together has an adhesion rate of less than 10% within 1 day.

[0045] The study found that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacrylated hyaluronic acid together is significantly better than the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacrylated sodium alginate together. For example, the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacrylated sodium alginate together has an ink droplet breakage rate of 90-100% within 1 day.

[0046] The study found that the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose and methacrylated hyaluronic acid together is significantly better than the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose, methacrylated sodium alginate and polydopamine together. For example, the composite hydrogel obtained by using sodium alginate and / or carboxymethyl cellulose, methacrylated sodium alginate and polydopamine together has an adhesion rate of less than 50% within 1 day.

[0047] Studies have found that the composite hydrogel obtained by using the sodium alginate and / or carboxymethyl cellulose and methacryloyl 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 the ionic compound, methacryloyl hyaluronic acid (HAMA), and a photoinitiator in a solvent, and stirring to obtain the composite hydrogel.

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

[0052] A fifth aspect of 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 biological ink and a substrate, improving the stability of biological ink, and improving the elasticity of biological 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 biological ink to a substrate, improving the stability of biological ink and / or improving the elasticity of biological ink is provided, comprising using the above-mentioned composite hydrogel to improve the adhesion of biological ink to a substrate, improving the stability of biological ink and / or improving the elasticity of biological 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 resilience; 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 live cells in the bio-ink obtained by 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 the matrix gel, which is close to the effect of commercial matrix gel and can replace matrix gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1The results of cell activity during cell culture in 2% NaA (low viscosity)-1% NaA (medium viscosity)-2% CMC-0.5% HAMA (150K)-0.25% LAP hydrogel and commercial matrix gel;

[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 matrix gel on the 4th day;

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

[0063] Figure 4 The figure is a comparison result 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 matrix gel;

[0065] Figure 6 This is a statistical chart of cell survival rate in hydrogel after adding different concentrations of pentafluorouracil (5-Fu).

[0066] Figure 7 Mechanical property tests of hydrogels. (a) Compression property test of hydrogels. None of the hydrogels in the test 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 in the test contained CMC. (c) Rheological property test of hydrogel precursor solution. All of the hydrogels in the test contained CMC.

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

[0068] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions 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 included in the scope 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 , DMEM / F12 medium containing 25 mM HEPES, 1% streptomycin and penicillin.

[0072] Complete medium for intestinal organoids is supplemented with 2 mM GlutaMAX TM , 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27 TM Additives, 100ng / mL Wnt3a cell growth factor, 1.25mM N-acetyl-L-cysteine, 500ng / mL R-Spondin, 100ng / mL mNoggin cell growth factor, 50ng / mL EGF cell growth factor, 10nM gastrin Ihuman, 0.5μM A83-01, 3μM SB202190, 10nM prostaglandin E2, 10mM nicotinamide, 100μg / mL Primocin TM Primary cells were cultured in antibiotic-free DMEM / F12 medium or Nearctic 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, 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 a 24-well plate and cultured and expanded in a 37°C, 5% CO2 incubator. The culture medium used was complete intestinal organoid culture medium (500 μL per well), the culture medium was changed every two days, and the culture was passaged every seven days (which can be adjusted according to the specific growth rate of different samples).

[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 pipette tip with 10 μL pipette tip) to break up the matrix gel by blowing and aspirating. Repeat this process for more than 20 times and transfer all the liquid to a centrifuge tube. Take another 1 mL of separation solution to rinse each well and transfer all the liquid to the above centrifuge tube.

[0079] (2) The above liquid was centrifuged at 2000 rpm for 5 min, the supernatant was removed, and TrypLE 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, pipette the liquid for more than 20 times, centrifuge at 2000 rpm for 5 min, and remove the supernatant.

[0081] (4) Wash the cells with 2 mL of separation solution, add an appropriate amount of Matrigel, mix by pipetting, and then 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 pipette tip with 10 μL pipette tip) to break up the matrix gel by blowing and aspirating. Repeat this process for more than 20 times and transfer all the liquid to a centrifuge tube. Take another 1 mL of separation solution to rinse each well and transfer all the liquid to the above centrifuge tube.

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

[0085] Note: The matrix gel solidifies at above 4°C and needs to be placed on ice during the mixing process.

[0086] Example 2 Preparation of hydrogel

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

[0088] (1) 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 using weighing paper (10 × 10 cm, avoid creases), spread them completely flat, and sterilize them 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). Take a 25 mL centrifuge tube, add 5 mL of organoid complete culture medium solution, dissolve LAP and HAMA, and filter with a bacterial filter. Add NaA powder, stir evenly (avoid clumping), then add CMC powder, stir evenly (avoid clumping), and ultrasonicate for 30-60 minutes.

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

[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)-methacrylylated hyaluronic acid (HAMA) hydrogel (experimental group 2)

[0095] 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 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). Take a 25 mL centrifuge tube, add 5 mL of organoid complete culture medium solution, dissolve LAP and HAMA, and filter with a bacterial filter. Add CMC powder, stir evenly (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)-methacrylylated hyaluronic acid (HAMA) hydrogel (experimental group 3)

[0102] 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] According to the above method, the hydrogel of the control group was prepared. The hydrogel components of the experimental groups 1-3 and the 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 with 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. Place the 3cc ink tube flat, take a 1mL disposable syringe with a scale, draw 200μL of CMC-NaA-HAMA hydrogel or CMC-HAMA hydrogel, and slowly inject it near the entrance 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 for 2 minutes. (The substance concentration of the hydrogel is configured at 1.5 times the set concentration, and then the 1.5 times concentration hydrogel and the cell suspension are mixed at a volume ratio of 2:1 to finally obtain a cell-containing hydrogel of the set 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 leave no gaps.

[0111] 5. Put the tail cap and head cap on, with the ink tube outlet facing upward, and place in a low-temperature centrifuge to remove bubbles. Settings: 1500-2000RMP, 5-10min.

[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 errors in the hydrogel in the syringe, the amount drawn by the syringe can be slightly more than the target amount by 20μL. Stirring until the bubbles are uniform can be judged as uniform mixing. The sterilization temperature of plastic materials is not higher than 120℃.

[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. Take the 96-well plate dot matrix as an example, set a dot coordinate (xyz-20, 20, 61mm), dot matrix mode (x-axis interval is 9mm, x-axis row number is 10, y-axis interval is 9mm, y-axis row number is 6), needle moving speed is 30-50mm / s, dot interval needle lifting 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 CaCl2(s) and dissolve it in 50 mL PBS (pH = 7.0) to make a 1.5% solution. Shake to dissolve and filter using a bacterial filter to sterilize.

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

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

[0121] Note: The height of the printing needle from the substrate is 20-100 μm. Prepare 100 μL of CaCl2 solution, cellulase solution, and complete culture medium solution per well. Prepare 200 μL of separation solution per well.

[0122] Example 6 Printing and cell / organoid culture using CMC-NaA-HAMA hydrogel

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

[0124] 2. After printing, irradiate with a blue light (405nm) for 30s, add 100μL of 1.5% CaCl2 solution to each well using a spray gun, and aspirate the solution after curing for 5-10min.

[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 adding and aspirating liquid.

[0130] Example 7 Printing using CMC-HAMA hydrogel

[0131] Similar to Example 6, except that: the step of "adding 100 μL of 1.5% CaCl2 solution to each well using a spray gun and aspirating the solution after the curing reaction for 5-10 minutes" is 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 solidification 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 replacement operations 10-20 times to check the adhesion of the ink droplets to the substrate.

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

[0135] Table 2 Ink dot fall 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 of 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 without any falling off problem, which met the requirements of long-term cell experiments.

[0141] Experimental Example 2 Cell / Organoid Drug Testing

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

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

[0144] (1) Preparation of dye solution: Use DMEM culture 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 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, add 100 μL 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 well plate under a fluorescence confocal microscope, use 488 nm / 561 nm excitation light for multi-channel imaging and record. The images are processed using Image J, and the cell / organoid live / death rate is the green fluorescence area / (red fluorescence area + green fluorescence area) in the same image.

[0149] (6) Data were statistically analyzed and graphed 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 live cells in hydrogel-mixed cells was greater than 90%, which is close to the effect of matrix gel. It can be seen that the activity of cells 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 a bright field image of cells cultured in CMC-NaA-HAMA hydrogel on the fourth day. Figure 2 b is a bright field image of cells cultured in matrix gel on the fourth day. Figure 2 c is a 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 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 in the same period of time.

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

[0161] Multiple sets of repeated experiments were performed 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 organoid area statistics of cells cultured in hydrogel and matrix gel on the fourth day (normalized using the area on day 0), c is the staining fluorescence image of cells cultured in hydrogel on the fourth day (AM: live cells; EthD-1: dead cells), and d is the staining fluorescence image of cells cultured in matrix gel on the fourth day.

[0163] Conclusion: After 4 days of culture with hydrogel mixed cells, the proportion of live cells was greater than 90%, which is close to the effect of matrix gel, indicating that the cells were more active during the culture process. After 4 days of culture with hydrogel mixed cells, the organoid area (cell density) increased by about 0.83-0.87 times that of matrix gel.

[0164] Experimental Example 3 Drug sensitivity test of hydrogel cultured organoids

[0165] The hydrogel prepared in experimental group 2 was used as an example for drug sensitivity testing. The statistical chart of cell survival rate in the hydrogel after adding different concentrations of pentafluorouracil (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 follows the trend of decreasing with increasing drug concentration.

[0167] Test Example 4: Mechanical properties test of hydrogel

[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 a compression performance test was performed after curing and degrading 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 hydrolyzed, the main component is Ca 2+The cross-linked network of cross-linked NaA molecules has dynamic bonds and is easily broken by external forces. After adding PDA to the 5% NaA (very Low)-2% CMC hydrogel (Control Group 2), the phenomenon of non-elastic deformation of the material under external force cannot be changed. 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). The material strength is too low to meet the requirements of cell culture for up to 10 days. Figure 7 b is a comparison chart of the compression modulus of four different hydrogels. The compression modulus of human tissue is low, so using a hydrogel with a lower compression modulus is beneficial to cell culture. The compression modulus of 0.75% HAMA (150K)-2% CMC hydrogel (control group 11) is low and its stability in an aqueous environment is poor. Therefore, the compression modulus of 1% HAMA (150K)-2% CMC (experimental group 2) is the minimum value to ensure that the ink droplets are stable in an aqueous environment for a long time (more than 10 days).

[0170] 10 μg / mL of fluorescent nanospheres (10 nm) were added to the hydrogel, and the ink droplets were 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) will be deformed and broken during the separation process, and more residues will remain on the substrate; the ink droplets of 1% HAMA (150K)-2% CMC hydrogel (experimental group 2) can be completely separated from the substrate, and less residues will remain on the substrate.

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

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. 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 solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should 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, methacryloyl hyaluronic acid (HAMA), a photoinitiator and a solvent. The ionic compound comprises one or two of carboxymethyl cellulose and sodium alginate.

2. A composite hydrogel composition according to claim 1, characterized in that: 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.

3. The composite hydrogel composition according to claim 1, characterized in that: The composition includes carboxymethyl cellulose, methacryloyl hyaluronic acid (HAMA), and a photoinitiator; Preferably, the composition comprises the following ingredients in percentage: 2% carboxymethyl cellulose (CMC), 0.5-3% methacryloyl hyaluronic acid (HAMA) and 0.05-0.25% photoinitiator; Preferably, the molecular weight of the methacryloyl hyaluronic acid is 100KDa-1,000K Da; more preferably, the molecular weight of the methacryloyl hyaluronic acid is 150K Da-400K Da; Preferably, the composition further comprises sodium alginate; Preferably, the percentage content of sodium alginate in the composition is 3-5%; Preferably, the sodium alginate comprises a combination of low-viscosity sodium alginate and medium-viscosity sodium alginate; Preferably, the mass ratio of the low viscosity sodium alginate to the medium viscosity sodium alginate is 2:1; Preferably, 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.

4. A composite hydrogel, characterized in that: The raw material is the composition described in any one of claims 1 to 3.

5. A method for preparing the composite hydrogel according to claim 4, characterized in that: The method comprises mixing an ionic compound, methacryloyl hyaluronic acid (HAMA), a photoinitiator and a solvent to obtain the composite hydrogel.

6. A biological ink, characterized in that: The composite hydrogel according to claim 4 also includes cells, tissues or organoids.

7. A cell chip, characterized in that: Comprising the biological ink as claimed in claim 6; Preferably, the bio-ink is printed on a substrate to obtain the cell chip.

8. Use of the composite hydrogel composition according to any one of claims 1 to 3, or the composite hydrogel according to claim 4, in improving the adhesion of biological ink to a substrate, improving the stability of biological ink, and / or improving the elasticity of biological ink.

9. Use of the composite hydrogel composition according to any one of claims 1 to 3, the composite hydrogel according to claim 4, the bio-ink according to claim 6, or the cell chip according to claim 7 in drug screening, drug toxicity and efficacy testing, or organ model construction or tissue engineering.

10. 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 described in claim 4 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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