An ultra-high cell density organ module based on click chemistry photocured hydrogel and construction method and application thereof

Abstract

The application relates to an organ module with super-high cell density based on a photo-cured hydrogel of click chemistry and a construction method and application thereof. The construction method comprises the following steps: providing high-grafting-rate norbornene-modified high-molecular-weight hyaluronic acid and thiol-modified high-molecular-weight hyaluronic acid; mixing a photo initiator, cells, a HANb solution and a HASH solution; and through click chemistry reaction, the organ module is quickly cross-linked and solidified through very short-time light irradiation. The application utilizes the synergistic effect of the high-molecular-weight hyaluronic acid main body material and the high-grafting-rate modification, realizes the construction of the organ module with super-high cell density under the super-mild conditions of very low photo initiator concentration and very short light irradiation time. The liver module constructed based on the method has very high cell density, good mechanical performance and excellent liver function.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering, biomedical materials, tissue engineering and regenerative medicine, and particularly relates to an ultra-high cell density organ module based on click chemistry photocurable hydrogel, its construction method and application. Background Technology

[0002] An organ module refers to a functional unit of tissue or organ constructed from cells, biological materials, and a specific microenvironment, possessing spatial structure and functional characteristics. Natural tissues and organs typically exhibit extremely high cell density and complex spatial organizational features. Examples include the liver (hepatocyte density approximately 1.0-1.5 × 10^8 cells / mL), skin (epidermal cell density reaching 10^8-10^9 cells / mL), heart (10^8-10^9 cells / mL), pancreas (10^8 cells / mL), and kidney (10^8 cells / mL), all of which consist of a large number of functional cells and supporting cells arranged in an orderly manner within a limited space. High cell density is a crucial prerequisite for maintaining tissue structural integrity and for achieving cell-cell interactions, cell-matrix interactions, metabolic coupling, signal transduction, and organ-specific functions.

[0003] Compared to traditional two-dimensional culture, organ modules can better simulate cell density, cell interactions, and nutrient and oxygen transport processes in vivo in three-dimensional space. Therefore, they have significant value in tissue engineering, regenerative medicine, disease models, drug screening, toxicology evaluation, and personalized medicine. Larger organ modules with higher cell loads can be used to construct functional units of tissues and organs such as the liver, skin, heart, pancreas, and kidneys. They can also serve as in vitro organ models, transplantable tissue building blocks, or biomanufacturing intermediates, providing new pathways for organ function reconstruction and tissue defect repair.

[0004] Currently, organ module construction typically relies on methods such as three-dimensional cell culture, bio-3D printing, micromolding, microfluidic assembly, cell cluster aggregation, and hydrogel embedding. Among these, bio-3D printing enables the spatially controlled deposition of cells, biomaterials, and bioactive factors. To achieve printability, molding stability, and a certain mechanical strength, existing technologies often employ crosslinkable hydrogels as bio-inks or scaffold materials. Common crosslinking systems include: 1) free radical polymerization reactions based on acrylamide or methacrylamide groups, with representative materials including gelatin methacrylamide (GelMA), polyethylene glycol diacrylate (PEGDA), and hyaluronic acid methacrylamide (HAMA). These materials have mature processes and easily controllable mechanical properties, but they usually require photoinitiators (such as LAP) and strong light, which can generate free radicals, affecting the activity, functional maintenance, and long-term culture of high-density embedded cells. They are also susceptible to oxygen interference, leading to reduced curing efficiency. 2) Hydrogel systems based on click chemistry reactions, such as hyaluronic acid or gelatin derivatives containing thiol (SH) and norbornene (Nb) groups, with representative materials including HASH, HANb, and GelNb. These systems exhibit good selectivity and high cross-linking efficiency; however, the light-mediated thiol-ene click reaction still requires LAP and light irradiation, leading to issues such as free radical damage, phototoxicity, and initiator residue. 3) Hydrogel systems based on bioorthogonal chemistry reactions, with representative materials including gelatin or other natural polymer derivatives containing functional groups such as norbornene (Nb) and tetrazine (Tz), such as GelNb and GelTz. These reactions offer good selectivity and biocompatibility, enabling specific cross-linking in complex biological environments; however, the reaction of Nb and Tz produces nitrogen gas as a byproduct. In high-viscosity, high-cell-density, or rapidly gelling systems, if the gas cannot escape in time, it easily forms bubbles, causing pores, interfacial discontinuities, or local structural defects, affecting molding accuracy, mechanical stability, and structural uniformity.

[0005] However, existing organ module construction still faces the challenge of reconciling biocompatibility, cell loading capacity, and mechanical properties. Taking free radical polymerized hydrogel systems as an example, in ultra-high cell density systems, such as with a cell loading capacity of approximately 1 × 10^8 cells / mL, the aggregation of a large number of cells within a limited volume not only significantly increases the system viscosity, affecting material mixing, printing, and molding, but also enhances the absorption and scattering of light by the cell population, leading to uneven light distribution within the gel and a decrease in photoinitiation efficiency. To induce rapid gelation, existing technologies typically require increasing the concentration of photoinitiator, extending the illumination time, or enhancing the light intensity. However, this increases the risk of damage to cells from free radicals, reactive oxygen species, and light. Especially in ultra-high cell density environments, the damage to cell membranes, proteins, and DNA caused by photoinitiation may be amplified, thereby reducing cell viability and affecting functional maintenance and long-term culture. On the other hand, to ensure the stable morphology of centimeter-scale organ modules during printing, transfer, culture, and application, the material needs a certain cross-linking density and mechanical strength. However, a high cross-linking density reduces the pore size and porosity of the hydrogel network, limiting cell spreading, migration, proliferation, cell interaction, and the transport of nutrients, oxygen, and metabolic waste. Conversely, reducing material concentration, photoinitiator dosage, or shortening exposure time to improve biocompatibility and material transport efficiency may lead to insufficient cross-linking, decreased mechanical strength, and affect structural integrity during long-term culture. Traditional chain-based free radical polymerization systems may also suffer from uneven internal cross-linking due to light attenuation, oxygen inhibition, and uneven local reactions, resulting in localized weak areas or structural defects. Therefore, in constructing centimeter-scale, ultra-high cell density organ modules, achieving uniform, efficient, and gentle cross-linking with lower photoinitiator dosage and shorter exposure time, while simultaneously ensuring high cell load, good cell viability, suitable pore structure, and sufficient mechanical stability, remains a key technical challenge. This directly relates to its subsequent large-scale preparation, standardized quality control, and clinical translational applications. Summary of the Invention

[0006] The technical problem solved by this invention is: how to achieve uniform, efficient and mild crosslinking with a low amount of photoinitiator and a short exposure time when constructing centimeter-scale, ultra-high cell density organ modules, while taking into account high cell load, good cell activity, suitable pore structure and sufficient mechanical stability.

[0007] In view of the technical problems existing in the prior art, this invention designs an ultra-high cell density organ module based on click chemistry photocurable hydrogel, its construction method, and its applications. This invention relates to the preparation of photocurable hydrogels using click chemistry reactions under extremely low photoinitiator conditions, and combines this with high-density cell integration, three-dimensional structure construction, and in vitro culture regulation to construct organ modules with ultra-high cell loading, tissue-like spatial structures, and certain organ functional characteristics. These organ modules can be used in centimeter-scale organ construction, organoid construction, organ functional unit reconstruction, tissue defect repair, drug evaluation, disease model establishment, and regenerative medicine research.

[0008] It should be noted that, in this invention, unless otherwise specified, the specific meaning of "comprising" in relation to composition definition and description includes both open-ended meanings such as "comprising," "including," etc., and closed-ended meanings such as "composed of," etc., and similar meanings.

[0009] To solve the aforementioned technical problems, the present invention adopts the following solution:

[0010] [First technical solution]

[0011] A method for constructing ultra-high cell density organ modules based on click chemistry photocurable hydrogels includes the following steps:

[0012] Step 1: Synthesis of norbornene-modified high molecular weight hyaluronic acid;

[0013] Step 2: Synthesis of thiol-modified high molecular weight hyaluronic acid;

[0014] Step 3: Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels;

[0015] In step 1, sodium hyaluronate powder with a weight-average molecular weight of 2.03 MDa is used as a raw material to synthesize norbornene-modified high molecular weight hyaluronic acid.

[0016] In step 2, sodium hyaluronate powder with a weight-average molecular weight of 2.03 MDa is used as a raw material to synthesize thiol-modified high molecular weight hyaluronic acid.

[0017] Step 3 involves mixing a photoinitiator and a solution containing cells and / or bioactive substances with the norbornene-modified hyaluronic acid solution described in step 1 and the thiol-modified hyaluronic acid solution described in step 2 to obtain a prepolymer-cell mixture; irradiating the prepolymer-cell mixture with light to allow it to crosslink and solidify through a click chemical reaction to form the ultra-high cell density organ module.

[0018] Furthermore, the method for synthesizing norbornene-modified high molecular weight hyaluronic acid in step 1 is as follows:

[0019] Step 1.1 Add sodium hyaluronate powder to the buffer solution and stir until dissolved. The final concentration of the hyaluronic acid (HA) solution is 0.5-1.0 wt%.

[0020] Step 1.2 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 5-norbornene-2-methylamine (Nb-NH2) to the hyaluronic acid (HA) solution from Step 1.1, adjust the pH of the reaction solution, and stir the reaction.

[0021] Step 1.3 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 5-norbornene-2-methylamine to the reaction solution in Step 1.2 again, adjust the pH of the reaction solution, and continue stirring the reaction.

[0022] Step 1.4 Repeat the operation of Step 1.3, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 5-norbornene-2-methylamine to the reaction solution of Step 1.3 again, adjust the pH of the reaction solution, and continue to stir the reaction.

[0023] Step 1.5 Place the reaction solution from Step 1.4 into a dialysis bag, dialyze it in pure water, and freeze-dry it to obtain norbornene-modified hyaluronic acid, denoted as HANb.

[0024] Furthermore, the method for synthesizing the thiol-modified high molecular weight hyaluronic acid in step 2 is as follows:

[0025] Step 2.1 Add sodium hyaluronate powder to the buffer solution and stir until dissolved. The final concentration of the hyaluronic acid (HA) solution is 0.5-1.0 wt%.

[0026] Step 2.2 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the hyaluronic acid HA solution from Step 2.1 and stir the mixture initially. Then add cystamine dihydrochloride (CysA·2HCl) to the reaction solution after the initial stirring in Step 2.2 and stir the mixture in the dark.

[0027] Step 2.3 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the reaction solution of step 2.2 and stir the mixture initially. Then add cystamine dihydrochloride to the reaction solution after the initial stirring in step 2.3 and stir the mixture in the dark.

[0028] Step 2.4 Place the reaction solution from Step 2.3 into a dialysis bag and dialyze it in pure water. Then adjust the pH of the reaction solution and add a reducing agent to carry out the reaction of reducing disulfide bonds.

[0029] Step 2.5 Adjust the pH of the reaction solution from Step 2.4 and continue to place it in a dialysis bag for gradient dialysis. Then freeze-dry the dialysis solution to obtain thiol-modified hyaluronic acid, denoted as HASH.

[0030] Further, step 3 involves a method for constructing ultra-high cell density organ modules based on click chemistry photocurable hydrogels:

[0031] Step 3.1 Dissolve the HANb synthesized in Step 1 and the HASH synthesized in Step 2 in sterile PBS solution to prepare HANb solution and HASH solution respectively;

[0032] Short peptides were dissolved in cell culture medium to prepare short peptide solutions;

[0033] The photoinitiator was dissolved in sterile PBS solution to prepare a photoinitiator solution;

[0034] Step 3.2 Take the HANb solution, HASH solution, short peptide solution and initiator solution from step 3.1 and mix them evenly to prepare a prepolymer-cell mixture;

[0035] Step 3.3 After digesting and centrifuging mouse primary hepatocytes, the supernatant was discarded to obtain hepatocyte precipitate. The prepolymer-cell mixture from step 3.2 was added to the hepatocyte precipitate and mixed thoroughly. The precipitate was then exposed to light at a wavelength of 405 nm with an intensity of 15-50 mW / cm² for 5-17 s to obtain an organ module with ultra-high cell density, denoted as HANS.

[0036] Further, in step 1.1, the buffer solution is 2-(N-morpholine)ethanesulfonic acid buffer (MES), and the pH of the buffer solution is 5.5-6.5;

[0037] The cumulative addition of steps 1.2, 1.3, and 1.4 results in a total reaction molar ratio of the carboxyl group of high molecular weight hyaluronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and 5-norbornene-2-methylamine of 1:(6-10):(6-10):(6-10), with a total reaction time of 72-108 h.

[0038] Further, in step 2.1, the buffer solution is 2-(N-morpholine)ethanesulfonic acid buffer (MES), and the pH of the buffer solution is 5.5-6.5;

[0039] The cumulative addition amounts in steps 2.2 and 2.3 resulted in a total reaction molar ratio of carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine dihydrochloride in high molecular weight hyaluronic acid of 1:(6-10):(6-10):(6-10), with a total reaction time of 48 h.

[0040] In step 2.4, the molar ratio of the carboxyl group in the high molecular weight hyaluronic acid to the reducing agent is 1:5; the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP).

[0041] Furthermore, in step 3.1, the concentrations of both the HANb solution and the HASH solution are 4.0 wt%.

[0042] The concentration of the short peptide solution was 22.0-44.0 wt%;

[0043] The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and the concentration of the photoinitiator solution is 1-3 mg / mL;

[0044] In step 3.2, the final concentration of HANb is 1.2-2.0 wt%; the final concentration of HASH is 1.2-2.0 wt%; the final concentration of the short peptide solution is 2.2-4.4 wt%; and the final concentration of the photoinitiator solution is 0.05 mg / mL.

[0045] In step 3.3, the prepolymer-cell mixture from step 3.2 is used, and the cell density is controlled to be 1.5 × 10^8 cells / mL.

[0046] The abbreviations for each substance are as follows:

[0047] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC);

[0048] N-hydroxysuccinimide (NHS);

[0049] 5-Norbornene-2-methylamine (Nb-NH2);

[0050] Cystamine dihydrochloride (CysA·2HCl);

[0051] Hyaluronic acid (HA)

[0052] Lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP);

[0053] 2-(N-morpholine)ethanesulfonic acid buffer (MES);

[0054] Dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

[0055] In this invention, the reaction temperature can be controlled according to the specific needs of the reaction. Generally, in step 1, the reaction temperature is 37°C; in step 2, the reaction temperature is room temperature.

[0056] In step 1.5 of this invention, dialysis continues until the infrared characteristic peaks of EDC and NHS are no longer detectable in the dialysate, wherein the positions of the infrared characteristic peaks of each substance are: EDC: 2110–2140 cm⁻¹ -1 NHS: 1810–1820 cm -1 .

[0057] In steps 2.2 and 2.3 of this invention, after adding EDC and NHS, a preliminary carboxyl activation reaction is carried out, and the activation time is generally 30-60 minutes.

[0058] In steps 2.4 and 2.5 of this invention, dialysis continues until the infrared characteristic peaks of EDC and NHS are no longer detectable in the dialysate. Specifically, EDC: 2110–2140 cm⁻¹ -1 NHS: 1810–1820 cm -1 In step 2.4, the reduction reaction time is generally determined by allowing the substances in the solution to react completely, which is typically 6-8 hours in this invention. In the above steps, the pH value is adjusted to allow the reactants to participate normally in the reaction; a typical pH range is 1.5-9.0. Gradient dialysis is generally performed sequentially in 5mM, 3mM, and 1mM hydrochloric acid solutions, followed by freeze-drying of the dialysate to obtain thiol-modified hyaluronic acid.

[0059] In this invention, short peptides can be selected as needed. In the embodiments of this invention, the short peptides are prepared by dissolving the cysteine-containing short peptide Cys-Arg-Gly-Asp-Ser, synthesized in solid state by Hefei Guotai Company, in cell culture medium to form a short peptide solution, namely CRGDS short peptide solution.

[0060] This invention uses a weight-average molecular weight (Mw) of 2.03 × 10⁻⁶. 6 Using high molecular weight hyaluronic acid (g / mol) as a raw material to prepare norbornene-modified hyaluronic acid (HANB) and thiolated hyaluronic acid (HASH) is of great significance.

[0061] On the one hand, due to the long molecular chain and high degree of chain entanglement of hyaluronic acid, the solution viscosity is significantly increased, posing certain technical challenges in the processes of dissolution, mixing, reaction, purification, and aseptic filtration. Specifically, these challenges manifest as slow dissolution rates, easy formation of aggregates, limited mass transfer in the reaction system, uneven local grafting, easy stringing and foaming of the solution, and difficulties in pipetting and filtration under high concentration conditions. Therefore, in the synthesis of HANB and HASH, it is necessary to improve reaction uniformity and product stability by extending the dissolution time, using gentle stirring, adding reaction reagents stepwise, controlling the concentration of the reaction system, and optimizing purification conditions.

[0062] On the other hand, HANB and HASH formed from high molecular weight hyaluronic acid possess stronger molecular chain entanglement and network support capabilities. After click chemical crosslinking to form hydrogels, they can achieve better structural integrity, deformation resistance, and cell encapsulation stability at lower polymer concentrations. This property is particularly suitable for constructing high cell density systems, maintaining the continuity and mechanical support of the gel network when encapsulating large numbers of cells, reducing cell sedimentation, gel rupture, and cell leakage, which is beneficial for forming three-dimensional hydrogel organ modules with high cell density, high cell retention rate, and good maneuverability. Therefore, a molecular weight of 2.03 × 10⁻⁶ was used. 6 The preparation of HANb and HASH with g / mol hyaluronic acid not only solved the problem of insufficient mechanical support of hydrogels during high cell density encapsulation, but also improved the uniform fixation and long-term retention of cells in three-dimensional space.

[0063] [Second Technical Solution]

[0064] An ultra-high cell density organ module based on click chemistry photocurable hydrogel is constructed according to the above-described method for constructing an ultra-high cell density organ module based on click chemistry photocurable hydrogel.

[0065] Furthermore, the organ module is a gel with a three-dimensional structure, with an internal cell density of ≥1×10^8 cells / mL, and the cumulative encapsulation rate of the encapsulated cells is ≥95% after 48 hours of culture, and the cell viability is ≥90%. The organ module has clampable mechanical strength and structural stability, and can maintain its overall shape under forceps clamping conditions to meet the needs of surgical transfer and implantation operations.

[0066] [Third technical solution]

[0067] The aforementioned ultra-high cell density organ module based on click chemistry photocurable hydrogel has applications in products or models for centimeter-scale organ construction, organoid construction, organ functional unit reconstruction, tissue defect repair, drug evaluation, disease model establishment, regenerative medicine research, tissue engineering culture, in vitro organ function simulation, organ-on-a-chip construction, cell therapy delivery, vascularized tissue construction, and high-density three-dimensional cell culture evaluation.

[0068] Applications of this invention include, but are not limited to:

[0069] 1. Used to construct centimeter-scale three-dimensional tissue or organ-like structures with physiologically relevant cell densities;

[0070] 2. Used to prepare functional units for organs such as liver, heart, pancreas, kidney, skin, cartilage, bone, muscle, blood vessels, or nerves;

[0071] 3. Used for constructing functional tissue modules in organoids, assemblies, tissue-engineered grafts, or organ-on-a-chip;

[0072] 4. Used for tissue defect repair, wound repair, organ damage repair, or pre-transplant tissue engineering culture;

[0073] 5. Used for drug screening, efficacy evaluation, toxicity evaluation, metabolic evaluation, and personalized drug response testing;

[0074] 6. Used to establish in vitro disease models such as tumors, fibrosis, inflammation, ischemic injury, metabolic abnormalities, genetic diseases, or degenerative diseases;

[0075] 7. Used to study cell survival, proliferation, differentiation, maturation, function maintenance, cell-cell interactions, cell-matrix interactions, and tissue remodeling processes in high-density three-dimensional cellular microenvironments;

[0076] 8. Used in regenerative medicine, tissue engineering, biomanufacturing, cell therapy, precision medicine and translational medicine research.

[0077] The mechanism by which this invention can successfully construct organ modules lies in:

[0078] Firstly, this invention possesses an excellent material basis: the role of the high molecular weight host material: using high molecular weight hyaluronic acid (HA) as the backbone, its longer molecular chains, after cross-linking, can form a more loose, continuous, and larger-pore three-dimensional network structure. This structure has dual advantages: first, it significantly improves the material transport efficiency within the gel, allowing oxygen and nutrients to penetrate deep into the center of the module, while metabolic waste can be promptly discharged, thus supporting the long-term survival of ultra-high-density cells within the module; second, it provides cells with more ample space for growth and remodeling, which is conducive to cell spreading, migration, and the formation of cell-cell interactions, thereby promoting the formation of tissue-like spatial structures.

[0079] Secondly, the high grafting rate modification significantly improves the grafting rate of norbornene and thiol groups on the HA molecular chain through an optimized multi-step synthesis process. This results in extremely high local effective concentrations of reactive groups at the molecular scale. When activated by a photoinitiator, these densely packed groups can rapidly undergo click chemistry reactions. Its advantages are: first, it allows the reaction to be initiated at extremely low photoinitiator concentrations, reducing the generation of free radicals at the source; second, it allows cross-linking to be completed within a very short light exposure time, minimizing the time cells are exposed to light and free radical environments. These two factors combined greatly improve the cell biocompatibility of the photocuring process, solving the core problem of cell damage under high-density embedding.

[0080] The mechanism by which the organ module constructed in this invention exhibits unique and superior effects lies in:

[0081] The unique effects of this invention stem from the synergistic effect of the high molecular weight host material and the high grafting rate modification. On the one hand, the three-dimensional network structure formed by the crosslinking of high molecular weight HA is relatively loose, with larger pore sizes and higher porosity. This not only facilitates the efficient transport of nutrients, oxygen, and metabolic waste, but also provides the necessary physical space for the survival, spread, migration, and intercellular interactions of ultra-high density cells. On the other hand, the high grafting rate results in extremely high local concentrations of reactive groups on the polymer chains, greatly improving the microscopic efficiency of the thiol-alkene click chemistry reaction. This allows the hydrogel to rapidly and uniformly complete crosslinking at extremely low photoinitiator concentrations and extremely short exposure times, thereby minimizing the damage to high-density embedded cells caused by free radicals and phototoxicity.

[0082] This invention provides an ultra-high cell density organ module based on click chemistry photocurable hydrogel, its construction method, and its application, which have the following beneficial effects:

[0083] 1. This invention uses a high molecular weight host material, which enables the long polymer chain segments to form a relatively loose and continuous three-dimensional network during the cross-linking process. While ensuring the overall mechanical stability and structural integrity of the gel, it increases the pore size and porosity inside the module, thereby improving the transport of nutrients, oxygen and metabolites, and providing space for ultra-high density cell survival, cell interaction and tissue-like structure formation.

[0084] 2. This invention improves the grafting rate of chemically reactive groups in the main material, resulting in a higher local effective concentration of thiol and norbornene groups on the molecular chain, thereby improving the efficiency of micro-reactions. This allows the hydrogel to crosslink rapidly and uniformly with lower photoinitiator dosage and shorter exposure time, reducing free radical damage and phototoxicity caused by high concentrations of photoinitiators or prolonged light exposure.

[0085] 3. The construction method of the present invention is based on the synergistic effect of high molecular weight host material and high grafting rate, which can induce HS-Nb click crosslinking reaction to form a gel rapidly with extremely low LAP dosage and extremely short exposure time, thereby obtaining good cell compatibility, high cell load, suitable pore structure and sufficient mechanical strength at the same time under ultra-high cell load.

[0086] 4. The organ modules constructed by the method of the present invention promote the formation and maintenance of organ-specific functions; in the constructed liver module, hepatocyte markers are clearly expressed, the expression levels of related functional genes are significantly higher than those in 2D culture, and a relatively complete cytoskeleton structure is formed.

[0087] 5. This invention is expected to overcome the technical bottlenecks in the construction of existing organ modules, such as the difficulty in achieving compatibility in terms of biocompatibility, cell load, and mechanical properties, and to provide a technical basis for constructing organ modules with centimeter-scale, ultra-high cell density, and tissue-like structural and functional characteristics. Attached Figure Description

[0088] Figure 1 : These are the GPC characterization results of the high molecular weight hyaluronic acid in all embodiments and comparative examples of this invention;

[0089] Figure 2 : A schematic diagram of the chemical structure and its NMR spectrum of norbornene-modified hyaluronic acid HANb synthesized in Example 1;

[0090] Figure 3 : A schematic diagram of the chemical structure and its NMR spectrum of the thiol-modified hyaluronic acid HASH synthesized in Example 1;

[0091] Figure 4 : This is a schematic diagram of the construction of an ultra-high cell density organ module based on click chemistry hydrogel in this invention;

[0092] Figure 5: A schematic diagram of the chemical structure and its NMR spectrum of the methacryloyl hyaluronic acid HAMA synthesized in Comparative Example 1;

[0093] Figure 6 : This is a viscosity comparison chart of HANb and HASH in Example 1, HAMA and unmodified HA in Comparative Example 1;

[0094] Figure 7 : This is a diagram of the ultra-high cell density organ module based on HANS constructed in Example 1;

[0095] Figure 8 : This represents the cumulative cell encapsulation rate of the HANS-based ultra-high cell density organ module at different time points in Example 1;

[0096] Figure 9 : Microscopic images and quantitative diagrams of cell encapsulation efficiency of ultra-high cell density organ modules constructed based on HANS in Example 1 and HAMA in Comparative Example 1;

[0097] Figure 10 : Encapsulation efficiency of HANS in Example 1 and HAMA in Comparative Example 1 at different cell densities;

[0098] Figure 11 : NMR spectrum of norbornene-modified hyaluronic acid HANb in Example 2;

[0099] Figure 12 : NMR spectrum of thiol-modified hyaluronic acid HASH in Example 2;

[0100] Figure 13 : This is a diagram of the ultra-high cell density organ module constructed in Example 2;

[0101] Figure 14 : A confocal image of the ultra-high cell density organ module constructed in Example 2;

[0102] Figure 15 : This is a comparison of the cell compatibility of HANS in Example 2 and HAMA in Comparative Example 1 with mouse hepatocytes;

[0103] Figure 16 : This is a comparison of the cell compatibility of HANS in Example 2 and HAMA in Comparative Example 1 with human umbilical vein endothelial cells (HUVECs).

[0104] Figure 17 : NMR spectrum of norbornene-modified hyaluronic acid HANb in Example 3;

[0105] Figure 18 : NMR spectrum of thiol-modified hyaluronic acid HASH in Example 3;

[0106] Figure 19 : This is a diagram of the ultra-high cell density organ module constructed in Example 3;

[0107] Figure 20 : The results of live / dead staining based on the HANS organ module constructed in Example 3;

[0108] Figure 21 This is a graph showing the comparison of the mechanical properties of the HANS hydrogel in Example 3 and the HAMA hydrogel in Comparative Example 1.

[0109] Figure 22 : Immunofluorescence staining image of the HANS-based ultra-high cell density organ module in Example 3;

[0110] Figure 23 Example 3 shows the expression of hepatocyte-related genes after different culture times based on the HANS organ module. Detailed Implementation

[0111] The present invention will be further described below with reference to specific embodiments and accompanying drawings:

[0112] Regarding cell acquisition and culture in the embodiments of the present invention

[0113] The specific steps for the extraction and culture of mouse primary hepatocytes (mH) in this invention are as follows:

[0114] C57BL6 mice were anesthetized by intraperitoneal injection of sodium pentobarbital. The skin was disinfected with alcohol, and the abdominal cavity was opened to expose the portal vein and inferior vena cava. A small incision was made distal to the portal vein, and an indwelling needle was inserted after no blood flow was observed. A small incision was made in the inferior vena cava as the outlet for perfusion fluid. Approximately 30 mL of physiological saline was perfused until the liver turned grayish-white. Perfusion was continued with 0.05% collagenase IV (the inferior vena cava was ligated before perfusion; after the liver swelled, it was held for one minute, then the inferior vena cava was opened to allow the collagenase to flow out; this process was repeated 4-5 times, lasting approximately 10 minutes). The liver lobe was removed, the liver was cut into a chyme-like shape, filtered, and centrifuged in 40% Percoll separation medium for 5 minutes to obtain primary mouse hepatocytes. These cells were cultured, with fresh hepatocyte culture medium changed every two days. When using mTmG-positive C57BL6 mice, mTmG-positive primary mouse hepatocytes (mTmG...) were obtained. + mH).

[0115] Regarding the culture of human umbilical vein endothelial cells (HUVECs) in this embodiment of the invention:

[0116] The HUVECs used in this invention were purchased from the cell resource platform of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. After cell resuscitation, the cells were seeded into sterile cell culture containers and cultured in commercially available endothelial cell culture medium (ECM) at 37°C, 5% CO2, and saturated humidity. During culture, the culture medium was changed periodically according to the cell growth status. When the cell confluence reached approximately 80%-90%, conventional digestion and passage methods were used for expansion. Before use, HUVECs with good growth status, uniform morphology, and in the logarithmic growth phase were selected, digested, centrifuged, resuspended, and counted before use.

[0117] In this embodiment of the invention, the short peptide in the short peptide solution is prepared by dissolving the cysteine-containing short peptide Cys-Arg-Gly-Asp-Ser (batch number GTB30723-0801) synthesized in cell culture medium, which is called CRGDS short peptide solution.

[0118] The raw materials used in other applications in this invention can all be obtained through commercially available channels.

[0119] Example 1

[0120] Step 1. The method for synthesizing norbornene-modified high molecular weight hyaluronic acid (HANb) is as follows:

[0121] Step 1.1 Add high molecular weight sodium hyaluronate powder to a MES buffer solution with pH=5.5 and stir at 37°C until dissolved. The final HA concentration is 1.0 wt%.

[0122] Step 1.2 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 5-norbornene-2-methylamine (Nb-NH2) to the HA solution from Step 1.1, wherein the molar ratio of the carboxyl group of HA to EDC, NHS, and Nb-NH2 is 1:4:4:4. Adjust the pH of the reaction solution to 5.5-6.5 and stir the reaction at 37°C for 24 hours.

[0123] Step 1.3 Add EDC, NHS and Nb-NH2 to the reaction solution in Step 1.2 again, wherein the molar ratio of the carboxyl group of HA to EDC, NHS and Nb-NH2 is 1:3:3:3. Adjust the pH of the reaction solution to 5.5-6.5 and continue to stir the reaction at 37°C for 24 hours.

[0124] Step 1.4: Add EDC, NHS, and Nb-NH2 to the reaction solution from Step 1.3 again, with the molar ratio of HA carboxyl groups to EDC, NHS, and Nb-NH2 being 1:3:3:3. Adjust the pH of the reaction solution to 5.5-6.5 and continue stirring at 37°C for 24 hours. The total molar ratio of HA carboxyl groups to EDC, NHS, and Nb-NH2 is 1:10:10:10, and the total reaction time is 72 hours.

[0125] Step 1.5: Place the reaction solution in a dialysis bag, dialyze in pure water for 3 days, and freeze-dry to obtain norbornene-modified high molecular weight hyaluronic acid (HANb).

[0126] Step 2. The synthesis method of thiol-modified high molecular weight hyaluronic acid (HASH) is as follows:

[0127] Step 2.1 Add high molecular weight sodium hyaluronate powder to a MES buffer solution with a pH of 5.5 and stir at room temperature until dissolved, with a final HA concentration of 1.0 wt%.

[0128] Step 2.2 Add EDC and NHS to the HA solution from Step 2.1 and stir for 45 minutes at room temperature. Then add cystamine dihydrochloride (CysA·2HCl) to the above reaction solution. The molar ratio of carboxyl group in HA, EDC, NHS and CysA·2HCl is 1:5:5:5. Adjust the pH of the reaction solution to 5.5-6.5 and stir for 24 hours at room temperature in the dark.

[0129] Step 2.3 Repeat step 2.2 once to make the total molar ratio of carboxyl group, EDC, NHS and CysA·2HCl in HA 1:10:10:10, and the total reaction time 48h.

[0130] Step 2.4 Place the reaction solution from Step 2.3 into a dialysis bag and dialyze it in pure water for 2 days. Then adjust the pH of the reaction solution to 8.5 and add DTT to reduce the disulfide bond. The molar ratio of the carboxyl group in HA to DTT is 1:5. React at room temperature for 6 hours.

[0131] Step 2.5: Adjust the pH of the reaction solution from Step 2.4 to 3.5 and continue to place it in a dialysis bag. Perform gradient dialysis in 5mM, 3mM, and 1mM hydrochloric acid solutions for a total of 3 days. Afterward, freeze-dry the dialysate to obtain thiol-modified high molecular weight hyaluronic acid (HASH).

[0132] Step 3. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels:

[0133] Step 3.1 Dissolve HANb synthesized in Step 1 and HASH synthesized in Step 2 in sterile PBS solution, each with a concentration of 4.0 wt%; dissolve the solid-state synthesized short peptide Cys-Arg-Gly-Asp-Ser (CRGDS) in cell culture medium, with a concentration of 22.0 wt%; dissolve the photoinitiator LAP in sterile PBS solution, with a concentration of 2 mg / mL.

[0134] Step 3.2 Take a certain volume of HANb, HASH, CRGDS and LAP solution from step 3.1 and mix them evenly so that the final concentration of HANb is 1.6wt%, the final concentration of HASH is 1.8wt%, the final concentration of CRGDS is 2.2wt% and the final concentration of LAP is 0.05mg / mL.

[0135] Step 3.3: Primary mouse hepatocytes were digested, centrifuged, and the supernatant was discarded to obtain a hepatocyte precipitate. A certain volume of the mixed solution from step 3.2 was added to the hepatocyte precipitate, and the mixture was gently pipetted to mix thoroughly. The cell density was controlled at 1.5 × 10^8 cells / mL. The mixture was then heated at 405 nm wavelength with 25 mW / cm². 2 With light intensity and irradiation for 10 seconds, organ modules with ultra-high cell density (HANS) were obtained.

[0136] Figure 1 The molecular weight GPC characterization results of high molecular weight hyaluronic acid in the embodiments of the present invention are shown below.

[0137] from Figure 1 As can be seen from A, the weight-average molecular weight (Mw) of hyaluronic acid is 2.03 × 10⁻⁶. 6 g / mol, which belongs to ultra-high molecular weight hyaluronic acid. Figure 1 B is the molecular weight distribution diagram.

[0138] Figure 2 This is a schematic diagram of the chemical structure and NMR spectrum of norbornene-modified hyaluronic acid (HANb) synthesized in Example 1. Figure 2 It can be concluded that the grafting rate of norbornene in the product HANb is 66.4%. The grafting rate of HANb synthesized in this invention can reach as high as 66.4%, which is significantly higher than the grafting rates reported in the literature (14.5-45%).

[0139] Figure 3 Here is a schematic diagram of the chemical structure and NMR spectrum of the thiol-modified hyaluronic acid HASH synthesized in Example 1. Figure 3 It can be concluded that the grafting rate of thiol groups in the product HASH is 52.5%. The grafting rate of HASH synthesized in this invention can reach as high as 52.5%, which is comparable to the highest grafting rate reported in the literature (10-60%).

[0140] Figure 4 This is a schematic diagram illustrating the construction of an ultra-high cell density organ module based on click chemistry hydrogel in this invention. Figure 4 The entire organ module construction process is clearly displayed. Furthermore, the photocuring conditions of this invention—LAP concentration of 0.05 mg / mL and light irradiation for 10 s—are significantly lower than those reported in the literature.

[0141] Comparative Example 1

[0142] Step 1. The synthesis method for methacrylamide-modified high molecular weight hyaluronic acid (HAMA) is as follows:

[0143] Step 1.1 Add high molecular weight sodium hyaluronate powder to pure water and stir at 4°C until dissolved, where the final HA concentration is 0.8 wt%.

[0144] Step 1.2 Add 1M sodium hydroxide solution (NaOH) and methacrylic anhydride (MA) to the HA solution from Step 1.1, so that the molar ratio of the carboxyl group of HA to NaOH and MA is 1:3:3, and stir the reaction at 4°C for 1 hour.

[0145] Step 1.3 Add NaOH and MA to the reaction solution from Step 1.2 again, wherein the molar ratio of the carboxyl group of HA to NaOH and MA is 1:3:3, and continue stirring the reaction at 4°C for 1 hour.

[0146] Step 1.4: Add NaOH and MA to the reaction solution from Step 1.3 again, with the molar ratio of the carboxyl group of HA to NaOH and MA being 1:2:2. Continue stirring the reaction at 4°C for 1 hour. The total molar ratio of the carboxyl group of HA to NaOH and MA is 1:8:8, and the total reaction time is 3 hours.

[0147] Step 1.5: Place the reaction solution in a dialysis bag, dialyze in pure water for 3 days, and freeze-dry to obtain methacrylamide high molecular weight hyaluronic acid (HAMA).

[0148] Step 2: Construction of organ modules based on HAMA photocurable hydrogels and ultra-high cell density:

[0149] Step 2.1 Dissolve the HAMA synthesized in Step 1 in sterile PBS solution at a concentration of 1.8 wt%; dissolve the photoinitiator LAP in sterile PBS solution at a concentration of 2 mg / mL.

[0150] Step 2.2 Take the HAMA and LAP solutions from Step 2.1 and mix them thoroughly to make the final concentration of HAMA 1.6 wt% and the final concentration of LAP 0.2 mg / mL.

[0151] Step 2.3 After digesting and centrifuging the primary mouse hepatocytes, the supernatant was discarded to obtain a hepatocyte precipitate. A certain volume of the mixed solution from Step 2.2 was added to the hepatocyte precipitate and gently blown to mix evenly, so that the cell density was 1.5×10^8 cells / mL. The cells were then irradiated at a wavelength of 405nm for 60s to obtain an organ module with ultra-high cell density.

[0152] Figure 5 The chemical structure diagram and NMR spectrum of the methacrylamide hyaluronic acid (HAMA) synthesized in Comparative Example 1 are shown. The grafting rate of norbornene in the HAMA product was calculated to be 65.4%. Currently, the grafting rate of commercially available HAMA typically ranges from 10% to 65%. Sigma-Aldrich's high molecular weight HAMA has a substitution degree of 10%–30%, while CELLINK PhotoHAStiff and some supplier products can reach 45%–65%, but their molecular weights are relatively low. Therefore, the grafting rate of the high molecular weight HAMA synthesized in this study is higher than that of commercially available products, achieving a good comparative result.

[0153] Figure 6 This is a comparison chart of the different viscosities of HANb and HASH in Example 1, HAMA in Comparative Example 1, and unmodified HA. HANb and HASH synthesized in Example 1, HAMA synthesized in Comparative Example 1, and unmodified HA were dissolved in sterile PBS solution. Four concentrations were set for each material: 1wt%, 2wt%, 3wt%, and 4wt%. After complete dissolution, their viscosity was measured.

[0154] Depend on Figure 6 It can be seen that the viscosities of HA and HAMA are similar and significantly higher than those of HANb and HASH. The limiting solubility concentrations of HANb and HASH are much higher than those of HAMA, which is beneficial for constructing ultra-high cell density organ modules by increasing the concentration in a very low photoinitiator system.

[0155] Figure 7 This is a diagram of the ultra-high cell density organ module based on HANS constructed in Example 1. Figure 7 It can be seen that the organ module has a diameter of 7mm and a thickness of 2mm.

[0156] Figure 8 The cumulative cell encapsulation efficiency of the HANS-based ultra-high cell density organ module in Example 1 is shown at different time points. After the construction of the ultra-high cell density organ module in Example 1, cells leaked into the culture medium were collected at 6h, 12h, 24h, and 48h, respectively, and the cumulative encapsulation efficiency of the ultra-high cell density organ module was calculated. After 48h, the cell encapsulation efficiency of the HANS-based ultra-high cell density organ module remained above 90%, demonstrating its excellent cell encapsulation ability.

[0157] Figure 9 Microscopic images and quantitative cell encapsulation rates of the ultra-high cell density organ modules constructed based on HANS in Example 1 and HAMA in Comparative Example 1 are shown. Under a microscope, when the photocuring conditions were the same (LAP concentration 0.05 mg / mL, 10 s of light exposure), significant cell leakage was observed in the HAMA-based ultra-high cell density organ module, which could not completely encapsulate the cells. Only when the photocuring conditions of HAMA were changed to LAP concentration 0.2 mg / mL and 60 s of light exposure could HAMA encapsulate a cell density of 1.5 × 10^8 cells / mL. Figure 9 A). The ultra-high cell density organ module constructed based on HANS exhibits excellent cell encapsulation ability at a LAP concentration of 0.2 mg / mL and 60 s of light irradiation. Figure 9 B). Figure 9 The quantitative analysis results of C further confirmed that HANS can achieve an ultra-high cell encapsulation rate under extremely low photocuring conditions.

[0158] Figure 10 The encapsulation efficiency of HANS in Example 1 and HAMA in Comparative Example 1 was compared at different cell densities. To compare the cell encapsulation efficiency of HANS and HAMA under the same photocuring conditions, a final HA concentration of 0.8 wt%, a LAP concentration of 0.05 mg / mL, and a light irradiation time of 10 s were used. The encapsulation efficiency of HANS and HAMA was compared at different cell densities (0.5 × 10^6, 1 × 10^6, 2 × 10^6 cells / mL). HANS showed better cell encapsulation efficiency than HAMA, and the difference increased with increasing cell density.

[0159] Example 2

[0160] Step 1. The method for synthesizing norbornene-modified high molecular weight hyaluronic acid (HANb) is as follows:

[0161] Step 1.1 Add high molecular weight sodium hyaluronate powder to a MES buffer solution with pH=5.5 and stir at 37°C until dissolved, with a final HA concentration of 0.6wt%.

[0162] Step 1.2 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 5-norbornene-2-methylamine (Nb-NH2) to the HA solution from Step 1.1, wherein the molar ratio of the carboxyl group of HA to EDC, NHS, and Nb-NH2 is 1:3:3:3. Adjust the pH of the reaction solution to 5.5-6.5 and stir the reaction at 37°C for 36 hours.

[0163] Step 1.3 Add EDC, NHS and Nb-NH2 to the reaction solution from Step 1.2 again, wherein the molar ratio of the carboxyl group of HA to EDC, NHS and Nb-NH2 is 1:3:3:3. Adjust the pH of the reaction solution to 5.5-6.5 and continue to stir the reaction at 37°C for 36 hours.

[0164] Step 1.4: Add EDC, NHS, and Nb-NH2 to the reaction solution from Step 1.3 again, with the molar ratio of HA carboxyl groups to EDC, NHS, and Nb-NH2 being 1:2:2:2. Adjust the pH of the reaction solution to 5.5-6.5 and continue stirring at 37°C for 36 hours. The total molar ratio of HA carboxyl groups to EDC, NHS, and Nb-NH2 is 1:8:8:8, and the total reaction time is 108 hours.

[0165] Step 1.5: Place the reaction solution in a dialysis bag, dialyze in pure water for 3 days, and freeze-dry to obtain norbornene-modified high molecular weight hyaluronic acid (HANb).

[0166] Step 2. The synthesis method of thiol-modified high molecular weight hyaluronic acid (HASH) is as follows:

[0167] Step 2.1 Add high molecular weight sodium hyaluronate powder to a MES buffer solution with a pH of 5.5 and stir at room temperature until dissolved, with a final HA concentration of 0.8 wt%.

[0168] Step 2.2 Add EDC and NHS to the HA solution from Step 2.1 and stir for 30 minutes at room temperature. Then add cystamine dihydrochloride (CysA·2HCl) to the above reaction solution. The molar ratio of the carboxyl group in HA, EDC, NHS and CysA·2HCl is 1:4:4:4. Adjust the pH of the reaction solution to 5.5-6.5 and stir for 24 hours at room temperature in the dark.

[0169] Step 2.3 Repeat step 2.2 once to make the total molar ratio of carboxyl group, EDC, NHS and CysA·2HCl in HA 1:8:8:8, and the total reaction time 48h.

[0170] Step 2.4 Place the reaction solution from Step 2.3 in a dialysis bag and dialyze it in pure water for 3 days. Then adjust the pH of the reaction solution to 8.5 and add TCEP to reduce disulfide bonds. The molar ratio of the carboxyl group in HA to TCEP is 1:5, and the concentration of TCEP is 10mM. React at room temperature for 6 hours.

[0171] Step 2.5 Place the reaction solution from Step 2.4 into an ultrafiltration tube, centrifuge and concentrate it at 4°C, and repeatedly replenish the solution with deionized water and change the solution through ultrafiltration 6 times to remove residual TCEP, salt and other small molecule byproducts. Then freeze-dry the dialysate to obtain thiol-modified high molecular weight hyaluronic acid HASH.

[0172] Step 3. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels:

[0173] Step 3.1 Dissolve the HANb synthesized in Step 1 and the HASH synthesized in Step 2 in sterile PBS solution, with a concentration of 4.0 wt% for each.

[0174] The solid-state synthesized short peptide Cys-Arg-Gly-Asp-Ser (CRGDS) was dissolved in cell culture medium at a concentration of 44.0 wt%.

[0175] The photoinitiator LAP was dissolved in sterile PBS solution at a concentration of 3 mg / mL.

[0176] Step 3.2 Take a certain volume of HANb, HASH, CRGDS and LAP solution from step 3.1 and mix them evenly so that the final concentration of HANb is 1.2 wt%, the final concentration of HASH is 2.0 wt%, the final concentration of CRGDS is 2.7 wt% and the final concentration of LAP is 0.05 mg / mL.

[0177] Step 3.3 After digesting and centrifuging mouse primary hepatocytes, the supernatant was discarded to obtain hepatocyte precipitate. A certain volume of the mixed solution from step 3.2 was added to the hepatocyte precipitate and gently mixed by pipetting. The cell density was controlled at 1.5 × 10^8 cells / mL. The precipitate was then exposed to light at a wavelength of 405 nm with an intensity of 15 mW / cm² for 17 s to obtain organoids with ultra-high cell density (HANS).

[0178] Figure 11 The image shows the NMR spectrum of norbornene-modified hyaluronic acid HANb in Example 2. Based on this, the grafting rate of norbornene in the product HANb was calculated to be 61.5%.

[0179] Figure 12 The image shows the NMR spectrum of the thiol-modified hyaluronic acid HASH in Example 2. Based on this, the grafting rate of thiol groups in the product HASH was calculated to be 46%.

[0180] Figure 13 The image shows the ultra-high cell density organ module constructed in Example 2. As can be seen from the image, the organ module has a diameter of approximately 20 mm.

[0181] Figure 14This is a confocal image of the ultra-high cell density organ module constructed in Example 2. Ultra-high cell density organ modules were obtained using mTmG-positive hepatocytes. After fixation in paraformaldehyde at 4°C, the modules were stained with DAPI and observed under a confocal microscope. The cells were tightly packed, highlighting extremely high cell density.

[0182] Figure 15 This section compares the cell compatibility of HANS (Example 2) and HAMA (Comparative Example 1) with mouse hepatocytes. Under the same photocuring conditions, primary mouse hepatocytes (mH) were encapsulated using HANS (Example 2) and HAMA (Comparative Example 1), respectively. After 1, 3, and 5 days of culture, cell viability was quantified using a PI / Calcein AM live / dead staining kit. Figure 15 A and Figure 15 As shown in Figure B, at different time points, the survival rate of mouse hepatocytes in HANS was better than that in HAMA. With the extension of culture time, the cell survival rate in HANS was greater than 90%, while the cell survival rate in HAMA gradually decreased, proving that HANS has better cell compatibility with mouse hepatocytes.

[0183] Figure 16 This section compares the cell compatibility of HANS (Example 2) and HAMA (Comparative Example 1) with human umbilical vein endothelial cells (HUVECs). Under the same photopolymerization conditions, HUVECs were encapsulated using HANS (Example 2) and HAMA (Comparative Example 1), respectively. After 1, 3, and 5 days of culture, cell viability was quantified using a PI / Calcein AM live / dead staining kit. Figure 16 A and Figure 16 As shown in Figure B, at different time points, the survival rate of HUVECs in HANS was better than that in HAMA. With the extension of culture time, the cell survival rate in HANS was greater than 90%, while the cell survival rate in HAMA gradually decreased, proving that HANS has better cell compatibility with HUVECs.

[0184] Example 3

[0185] Step 1. The method for synthesizing norbornene-modified high molecular weight hyaluronic acid (HANb) is as follows:

[0186] Step 1.1 Add high molecular weight sodium hyaluronate powder to a MES buffer solution with pH=5.5 and stir at 37°C until dissolved, with a final HA concentration of 0.5wt%.

[0187] Step 1.2 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 5-norbornene-2-methylamine (Nb-NH2) to the HA solution from Step 1.1, wherein the molar ratio of the carboxyl group of HA to EDC, NHS and Nb-NH2 is 1:3:3:3. Adjust the pH of the reaction solution to 5.5-6.5 and stir the reaction at 37°C for 24 hours.

[0188] Step 1.3 Add EDC, NHS and Nb-NH2 to the reaction solution in Step 1.2 again, wherein the molar ratio of the carboxyl group of HA to EDC, NHS and Nb-NH2 is 1:2:2:2. Adjust the pH of the reaction solution to 5.5-6.5 and continue to stir the reaction at 37°C for 24 hours.

[0189] Step 1.4: Add EDC, NHS, and Nb-NH2 to the reaction solution from Step 1.3 again, with the molar ratio of the carboxyl group of HA to EDC, NHS, and Nb-NH2 being 1:1:1:1. Adjust the pH of the reaction solution to 5.5-6.5 and continue stirring at 37°C for 24 hours. The total molar ratio of the carboxyl group of HA to EDC, NHS, and Nb-NH2 is 1:6:6:6, and the total reaction time is 72 hours.

[0190] Step 1.5: Place the reaction solution in a dialysis bag, dialyze in pure water for 3 days, and freeze-dry to obtain norbornene-modified high molecular weight hyaluronic acid (HANb).

[0191] Step 2. The synthesis method of thiol-modified high molecular weight hyaluronic acid (HASH) is as follows:

[0192] Step 2.1 Add high molecular weight sodium hyaluronate powder to a MES buffer solution with a pH of 5.5 and stir at room temperature until dissolved, with a final HA concentration of 0.5 wt%.

[0193] Step 2.2 Add EDC and NHS to the HA solution from Step 2.1 and stir for 60 minutes at room temperature. Then add cystamine dihydrochloride (CysA·2HCl) to the above reaction solution. The molar ratio of carboxyl group in HA, EDC, NHS and CysA·2HCl is 1:3:3:3. Adjust the pH of the reaction solution to 5.5-6.5 and stir for 24 hours at room temperature in the dark.

[0194] Step 2.3 repeats step 2.2 once, so that the total molar ratio of carboxyl group, EDC, NHS and CysA·2HCl in HA is 1:6:6:6, and the total reaction time is 48h.

[0195] Step 2.4 Place the reaction solution from Step 2.3 into a dialysis bag and dialyze it in pure water for 3 days. Then adjust the pH of the reaction solution to 8.5 and add TCEP to reduce disulfide bonds. The molar ratio of the carboxyl group in HA to TCEP is 1:5, and the concentration of TCEP is 10mM. React at room temperature for 6 hours.

[0196] Step 2.5 The reaction solution from step 2.4 was placed in an ultrafiltration tube, centrifuged and concentrated at 4°C, and repeatedly replenished with deionized water and ultrafiltration solution was performed 6 times to remove residual TCEP, salt and other small molecule byproducts. Then the dialysate was freeze-dried to obtain thiol-modified high molecular weight hyaluronic acid HASH.

[0197] Step 3. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels:

[0198] Step 3.1 Dissolve the HANb synthesized in Step 1 and the HASH synthesized in Step 2 in sterile PBS solution, with a concentration of 4.0 wt% for each.

[0199] The solid-state synthesized short peptide Cys-Arg-Gly-Asp-Ser (CRGDS) was dissolved in cell culture medium at a concentration of 33.0 wt%.

[0200] The photoinitiator LAP was dissolved in sterile PBS solution at a concentration of 1 mg / mL.

[0201] Step 3.2 Take a certain volume of HANb, HASH, CRGDS and LAP solution from step 3.1 and mix them evenly so that the final concentration of HANb is 2.0 wt%, the final concentration of HASH is 1.2 wt%, the final concentration of CRGDS is 4.4 wt% and the final concentration of LAP is 0.05 mg / mL.

[0202] Step 3.3 After digesting and centrifuging mouse primary hepatocytes, the supernatant was discarded to obtain hepatocyte precipitate. A certain volume of the mixed solution from step 3.2 was added to the hepatocyte precipitate and gently mixed by pipetting. The cell density was controlled at 1.0 × 10^8 cells / mL. At a wavelength of 405 nm and a light intensity of 50 mW / cm², the illumination time was 5 s to obtain organoid modules (HANS) with ultra-high cell density.

[0203] Figure 17 The image shows the NMR spectrum of norbornene-modified hyaluronic acid HANb in Example 3. Based on this, the grafting rate of norbornene in the product HANb was calculated to be 57.5%.

[0204] Figure 18 The image shows the NMR spectrum of the thiol-modified hyaluronic acid HASH in Example 3. The calculated grafting rate of thiol groups in the product HASH is 40.5%.

[0205] Figure 19 This is a diagram of the ultra-high cell density organ module constructed in Example 3.

[0206] Figure 20 The results show the viability staining of the HANS-based ultra-high cell density organ module constructed in Example 3. After the ultra-high cell density organ module was constructed, it was cultured for 5 days and then stained using the PI / Calcein AM viability staining kit. The results showed that it still had good cell viability.

[0207] To compare the mechanical properties of hydrogels formed based on two chemical reactions, the same final HA concentration was used to study the mechanical properties of HANS and HAMA with different photoinitiator concentrations and different light exposure times. The preparation conditions are shown in Table 1.

[0208] Figure 21 This is a comparison of the mechanical properties of the HANS hydrogel in Example 3 and the HAMA hydrogel in Comparative Example 1. Figure 21 It can be seen that, under the same photocuring conditions, HANS has a higher energy storage modulus and a higher photocuring efficiency.

[0209] Table 1. Preparation conditions of HANS and HAMA hydrogels

[0210]

[0211] Figure 22 This image shows the immunofluorescence staining of the HANS-based ultra-high cell density organ module from Example 3. The HANS-based ultra-high cell density organ module from Example 3 was cultured in vitro for 24 h, then fixed with paraformaldehyde, dehydrated in 30% sucrose solution, embedded with OCT embedding medium, frozen, sectioned, and blocked. Specific primary antibodies against hepatocyte markers (ALB, HNF4a) and cytoskeleton markers (F-actin) were then added and incubated overnight at 4°C. The sections were then washed three times with PBS for 5 min each time, followed by incubation with fluorescently labeled secondary antibodies at room temperature in the dark for 1 h. After three repeated washes with PBS, the cell nuclei were counterstained with DAPI for 5 min. Finally, the sections were washed three times with PBS and observed and images were acquired under a fluorescence microscope.

[0212] Depend on Figure 22 Immunofluorescence staining showed that ALB and HNF-4α were significantly expressed in the liver organoids, suggesting that the organ modules constructed based on HANS have hepatocyte-like differentiation and functional phenotypes. At the same time, F-actin staining showed that a relatively complete cytoskeleton structure was formed inside the organ modules, indicating that the culture system can support the formation of a relatively stable three-dimensional tissue structure of the liver organ modules.

[0213] Figure 23 This section describes the expression of hepatocyte-related genes in the HANS-based organ module after different culture times in Example 3. After 1, 3, and 7 days of in vitro culture of the organ module, the expression of hepatocyte-related genes in the organ module was detected. Two-dimensionally cultured mouse hepatocytes were selected as a control. The expression levels of the corresponding genes were detected by real-time quantitative PCR. The reaction system for real-time quantitative PCR is shown in Table 2, the reaction conditions are shown in Table 3, and the primer sequences are shown in Table 4.

[0214] Depend on Figure 23 It can be seen that, compared with two-dimensional culture, the expression levels of hepatocyte-related genes in the HANS organ module are significantly increased, and the expression levels increase with the extension of culture time, indicating that hepatocytes in the HANS organ module have good function.

[0215] Table 2. Figure 23 The reaction system used in real-time quantitative PCR

[0216]

[0217] Table 3. Figure 23 The reaction conditions used in real-time quantitative PCR

[0218]

[0219] Table 4. Figure 23 Primer sequences used in real-time quantitative PCR

[0220]

[0221] The present invention has been described above by way of example with reference to the embodiments and accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A method for constructing ultra-high cell density organ modules based on click chemistry photocurable hydrogels, characterized in that... Therefore, the following steps are included: Step 1: Synthesis of norbornene-modified high molecular weight hyaluronic acid; Step 2: Synthesis of thiol-modified high molecular weight hyaluronic acid; Step 3: Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels; In step 1, sodium hyaluronate powder with a weight-average molecular weight of 2.03 MDa is used as a raw material to synthesize dehydrogenase. High molecular weight hyaluronic acid modified with laminarin; In step 2, sodium hyaluronate powder with a weight-average molecular weight of 2.03 MDa is used as a raw material to synthesize thiol-modified high-molecular-weight hyaluronic acid. Hyaluronic acid with a molecular weight of [missing information] Step 3 involves mixing the photoinitiator and the solution containing cells and / or bioactive substances with the solution described in step 1. The norbornene-modified hyaluronic acid solution and the thiol-modified hyaluronic acid solution from step 2 are mixed to obtain a prepolymer-cell mixture; the prepolymer-cell mixture is then irradiated with light to crosslink and solidify through click chemistry, forming... The aforementioned ultra-high cell density organ module; Step 3: Method for constructing ultra-high cell density organ modules based on click chemistry photocurable hydrogels: Step 3.1 Dissolve the HANb synthesized in Step 1 and the HASH synthesized in Step 2 in sterile PBS solution to prepare separate formulations. HANb solution and HASH solution; Short peptides were dissolved in cell culture medium to prepare short peptide solutions; The photoinitiator was dissolved in sterile PBS solution to prepare a photoinitiator solution; Step 3.2 Take the HANb solution, HASH solution, short peptide solution and initiator solution from step 3.1 and mix them evenly to prepare a prepolymer-cell mixture; Step 3.3: Primary mouse hepatocytes were digested, centrifuged, and the supernatant was discarded to obtain a hepatocyte precipitate. The prepolymer-cell mixture from Step 3.2 was added to the hepatocyte precipitate and mixed thoroughly. The mixture was then irradiated at a wavelength of 405 nm. With an illumination of 15-50 mW / cm² and an illumination time of 5-17 s, organ modules with ultra-high cell density were obtained, denoted as HANS. In step 3.1, the concentrations of both the HANb solution and the HASH solution are 4.0 wt%. The concentration of the short peptide solution was 22.0-44.0 wt%; The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and the concentration of the photoinitiator solution is 1-3 mg / mL; In step 3.2, the final concentration of HANb is 1.2-2.0 wt%; the final concentration of HASH is 1.2-2.0 wt%; the final concentration of the short peptide solution is 2.2-4.4 wt%; and the final concentration of the photoinitiator solution is 0.05 mg / mL. In step 3.3, the prepolymer-cell mixture from step 3.2 is used, and the cell density is controlled to be 1.5 × 10^8. cells / mL; The norbornene grafting rate in the norbornene-modified high molecular weight hyaluronic acid prepared in step 1 was 57.5%, 61.5%, or 66.4%. The thiol-modified high molecular weight hyaluronic acid prepared in step 2 has a thiol grafting rate of 40.5%, 46%, or 52.5%.

2. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels according to claim 1. The method is characterized by: The method for synthesizing norbornene-modified high molecular weight hyaluronic acid in step 1 is as follows: Step 1.1 Add sodium hyaluronate powder to the buffer solution and stir until dissolved. The final concentration of the hyaluronic acid (HA) solution is... It is 0.5-1.0 wt% Step 1.2 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and 5-hydroxylamine hydrochloride are added. Bornene-2-methylamine was added to the hyaluronic acid (HA) solution from step 1.1, the pH of the reaction solution was adjusted, and the reaction was stirred. Step 1.3 Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloric acid to the reaction solution from Step 1.2 again. Salt, N-hydroxysuccinimide, and 5-norbornene-2-methylamine were added to adjust the pH of the reaction solution, and the reaction was continued with stirring. Step 1.4 Repeat the operation of Step 1.3, adding 1-ethyl-(3-dimethylamino) to the reaction solution of Step 1.3 again. Add propyl carbodiimide hydrochloride, N-hydroxysuccinimide and 5-norbornene-2-methylamine, adjust the pH of the reaction solution and continue stirring the reaction; Step 1.5 Place the reaction solution from Step 1.4 in a dialysis bag, dialyze it in pure water, and freeze-dry to obtain norbornene-modified solution. Hyaluronic acid is denoted as HANb.

3. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels according to claim 1. The method is characterized by: The method for synthesizing the thiol-modified high molecular weight hyaluronic acid in step 2 is as follows: Step 2.1 Add sodium hyaluronate powder to the buffer solution and stir until dissolved. The final concentration of the hyaluronic acid (HA) solution is 0.5-1.0 wt%. Step 2.2: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the solution. In step 2.1, the hyaluronic acid (HA) solution was initially stirred and reacted. Then, cystamine dihydrochloride was added to the reaction solution after the initial stirring in step 2.2, and the reaction was stirred in the dark. Step 2.3: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the solution. In the reaction solution of step 2.2, the reaction was initially stirred. Then, cystamine dihydrochloride was added to the reaction solution after the initial stirring in step 2.3, and the reaction was stirred in the dark. Step 2.4 Place the reaction solution from Step 2.3 into a dialysis bag and dialyze it in pure water. Then adjust the pH of the reaction solution. A reducing agent is added to initiate a reaction that reduces disulfide bonds; Step 2.5 Adjust the pH of the reaction solution from Step 2.4, and continue to place it in the dialysis bag for gradient dialysis. Then... The dialysate was freeze-dried to obtain thiol-modified hyaluronic acid, denoted as HASH.

4. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels according to claim 2. The method is characterized by: In step 1.1, the buffer solution is a 2-(N-morpholine)ethanesulfonic acid buffer solution with a pH of 5.5-6.

5. The cumulative addition amounts in steps 1.2, 1.3, and 1.4 cause the carboxyl group of the high molecular weight hyaluronic acid to react with the 1-ethyl group. The total trans-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and 5-norbornene-2-methylamine The molar ratio should be 1:(6-10):(6-10):(6-10), and the total reaction time is 72-108h.

5. Construction of ultra-high cell density organ modules based on click chemistry photocurable hydrogels according to claim 3. The method is characterized by: In step 2.1, the buffer solution is 2-(N-morpholine)ethanesulfonic acid buffer solution with a pH of 5.5-6.

5. The cumulative addition of steps 2.2 and 2.3 results in the presence of carboxyl groups and 1-ethyl-(3-diethyl-2 ... The total molar ratio of (methylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine dihydrochloride is 1: (6-10):(6-10):(6-10), the total reaction time is 48h; In step 2.4, the molar ratio of the carboxyl group to the reducing agent in the high molecular weight hyaluronic acid is 1:5; the further... The original agent is dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride.

6. A high-cell-density organ module based on click chemistry photocurable hydrogel, characterized in that: It is according to Construction method of ultra-high cell density organ module based on click chemistry photocurable hydrogel as described in any one of claims 1-5 It was constructed using the method.

7. The ultra-high cell density organ module based on click chemistry photocurable hydrogel according to claim 6, its characteristics are... The evidence lies in: The organ module is a gel with a three-dimensional structure, and its internal cell density is ≥1×10^8 cells / mL, and it is encapsulated. The cells exhibited a cumulative encapsulation rate of ≥95% and a cell viability of ≥90% after 48 hours of culture; the organ module was clampable. Its mechanical strength and structural stability enable it to maintain its overall shape under forceps holding conditions, thus meeting the requirements for surgical transfer and implantation. Operational requirements.

8. A high-cell-density organ module based on click chemistry photocurable hydrogel as described in claim 6, at a density of centimeter... Meter-scale organ construction, organoid construction, organ functional unit reconstruction, tissue defect repair, drug evaluation, disease model establishment, Applications in products or models for regenerative medicine research, tissue engineering culture, in vitro organ function simulation, organ-on-a-chip construction, cell therapy delivery, vascularized tissue construction, and high-density three-dimensional cell culture evaluation.

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