Hydrogel for evaluating corneal endothelial seed cell function and preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to hydrogel for evaluating corneal endothelium seed cell functions and a preparation method and application thereof.The hydrogel is of a three-dimensional cross-linked network structure formed by acrylamide in the presence of N, N '-methylene bisacrylamide through free radical polymerization; meanwhile, an extracellular matrix component is fixed on the surface of the three-dimensional cross-linked network structure, and the hydrogel is obtained. According to the hydrogel prepared by the method, the problems of mismatching of mechanical properties, large batch difference and the like of a traditional material are solved, and a more reliable standardized tool is provided for HCE cell behavior research. The breakthrough technology solves the problem that a traditional material cannot give consideration to both physical support and function evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a hydrogel for evaluating the function of corneal endothelial seed cells, and a preparation method and application thereof. Background Art

[0002] The cornea is a transparent, avascular tissue located at the front of the eyeball. Its refractive power, combined with that of the lens, allows light to accurately focus on the retina, thus enabling vision. The human corneal endothelium (HCE), located at the innermost layer of the cornea, is composed of a mosaic of regularly arranged, hexagonal, flat cells. This intact monolayer of HCE cells functions as an endothelial pump and a barrier between the cornea and the aqueous humor. They play an irreplaceable role in maintaining the cornea's semi-dehydrated state, normal thickness, and transparency, and in sourcing nutrients and oxygen from the aqueous humor. A sufficient number of corneal endothelial cells is crucial for maintaining corneal endothelial function. Localized cell death can only be repaired by the expansion and migration of neighboring cells. Once the density of human corneal endothelial cells falls below the critical density required to maintain physiological corneal endothelial function, irreversible changes, known as corneal endothelial decompensation, can occur, leading to corneal edema and opacity, and severe blindness. Currently, the only treatment for corneal endothelial blindness is corneal endothelial transplantation. However, due to the severe shortage of donated corneas and the aging of donated corneas, the vast majority of corneal endothelial blindness patients cannot regain their sight due to the lack of available donor corneas for transplantation and treatment. As an equivalent substitute for donated corneal endothelium, the development and production of tissue-engineered human corneal endothelium (TE-HCE) offers hope for many corneal endothelial blindness patients to regain their sight. It is also the key to fundamentally solving the problems of corneal transplant donor material shortage and postoperative immune rejection.

[0003] During the TE-HCE construction process, the mechanical properties of the scaffold, particularly its thickness and stiffness, are key parameters determining the effective functionalization of seed cells. The cellular functions of corneal endothelial seed cells primarily include cell proliferation and metabolism. Under normal physiological conditions, the Descemet's membrane, to which HCE cells attach, exhibits precise mechanical properties, with a thickness of 10μm–13μm and a stiffness of 50±17.8kPa. These microenvironmental parameters directly influence key biological behaviors such as intercellular tight junction formation, nutrient penetration, and mechanical signaling. Existing materials used to evaluate the function of human corneal endothelial seed cells, such as amniotic membrane and collagen, generally suffer from two major technical bottlenecks: first, significant deviations from physiological values ​​in mechanical properties, such as incompatibility between the stiffness of amniotic membrane and collagen, or inability to encompass the stiffness environment that HCE cells may encounter under physiological conditions or inappropriate thickness; and second, batch-to-batch variability in materials interferes with experimental reproducibility. These factors make it difficult to evaluate the cell proliferation and metabolic capacity of human corneal endothelial seed cells using amniotic membrane and collagen during the TE-HCE construction process.

[0004] Therefore, there is an urgent need to develop a carrier scaffold for evaluating the function of human corneal endothelial seed cells. Summary of the Invention

[0005] The purpose of this invention is to provide a hydrogel for evaluating the function of corneal endothelial seed cells. By simulating the natural mechanical microenvironment of HCE cells and utilizing hydrogel materials with adjustable stiffness and ultra-thin properties, a standardized tool is provided for studying HCE cell behavior, thereby providing theoretical support and experimental basis for the development of TE-HCE.

[0006] The technical solution adopted in the present invention is: The present invention provides a hydrogel for evaluating the function of corneal endothelial seed cells. The hydrogel is formed by acrylamide in the presence of N,N'-methylenebisacrylamide through free radical polymerization to form a three-dimensional cross-linked network structure; and extracellular matrix components are simultaneously fixed on the surface of the three-dimensional cross-linked network structure to obtain the hydrogel.

[0007] Preferably, the extracellular matrix component includes at least one of type IV collagen, type VIII collagen, fibronectin and laminin.

[0008] Preferably, the mass proportions of type IV collagen, type VIII collagen, fibronectin and laminin in the extracellular matrix components are as follows: Type VIII collagen accounts for 23.5% to 33.3%; Fibronectin accounts for 11.7% to 16.7%; Laminin accounts for 5.9% to 8.3%. Type IV collagen is replenished 100%.

[0009] Preferably, cellulose nanocrystals and sodium alginate are also added to the hydrogel.

[0010] The second aspect of the present invention provides a method for preparing the hydrogel, comprising the following steps: dissolving acrylamide and N,N'-methylenebisacrylamide in water respectively to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, and mixing them to obtain a polyacrylamide premix solution; Adding a coagulant and a stiffness enhancer to a polyacrylamide premix to obtain a hydrogel solution; the stiffness enhancer is cellulose nanocrystals and sodium alginate; Using the hydrogel solution, an unmodified hydrogel with a thickness of 10 μm to 13 μm was prepared; adding a cross-linking agent to the unmodified hydrogel, so that the cross-linking agent is cross-linked to the unmodified hydrogel to obtain a cross-linked hydrogel; The extracellular matrix components are added to the cross-linked hydrogel, and the N-hydroxysuccinimide ester groups activated by the cross-linker undergo amidation reaction with the primary amino groups of the proteins in the extracellular matrix components, thereby covalently coupling the extracellular matrix components and providing specific adhesion sites for corneal endothelial seed cells to obtain the hydrogel.

[0011] Preferably, the coagulant is ammonium persulfate and tetramethylethylenediamine; Ammonium persulfate is added to the polyacrylamide premix solution in a 20% to 25% mass fraction of ammonium persulfate solution. The amount of ammonium persulfate solution added is 0.2% to 0.6% of the volume of the polyacrylamide premix solution. The amount of tetramethylethylenediamine added is 0.1%~0.2% of the volume of the polyacrylamide premix.

[0012] Preferably, the added amount of the cellulose nanocrystals is 1% to 5% wt; and the added amount of the sodium alginate is 0.1% to 0.2% wt.

[0013] Preferably, the cross-linking agent is a sulfotricyclic aromatic hydrocarbon.

[0014] A third aspect of the present invention provides a use of the hydrogel, wherein the hydrogel is used to evaluate the cell function of human corneal endothelial seed cells.

[0015] Preferably, the cell functions are cell proliferation and cell metabolic capacity.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a hydrogel for evaluating the function of corneal endothelial seed cells. The hydrogel is formed by free radical polymerization of acrylamide in the presence of N,N'-methylenebisacrylamide to form a three-dimensional cross-linked network structure. Extracellular matrix components are simultaneously fixed to the surface of the three-dimensional cross-linked network structure to obtain the hydrogel. The core of the invention lies in first forming a polyacrylamide-based hydrogel skeleton with adjustable mechanical properties, then covalently anchoring the extracellular matrix to the skeleton surface using a cross-linking agent, thereby providing a specific, recognizable adhesion microenvironment for corneal endothelial cells.

[0017] In the present invention, acrylamide and N,N'-methylenebisacrylamide first undergo free radical chain polymerization under the action of coagulants ammonium persulfate and tetramethylethylenediamine. The double bond of N,N'-methylenebisacrylamide simultaneously participates in both main chains, forming a three-dimensional cross-linked network. Cellulose nanocrystals are then embedded in the network through hydrogen bonds and physical entanglement, enhancing rigidity. The carboxyl groups of sodium alginate form secondary hydrogen bonds with the amide / hydroxyl groups in the network, providing synergistic toughness. The N-hydroxysuccinimide ester in the cross-linker then undergoes an amidation reaction with the -NH2 in the polyacrylamide network to covalently graft the cross-linker to the gel surface. Ultraviolet light triggers the azide group at the other end of the cross-linker to generate an active nitrene radical, which can undergo CN covalent coupling with the primary amino group in the ECM protein to form a stable amide bond. The covalently fixed ECM protein exposes its integrin binding sites, such as the RGD sequence, which is recognized by corneal endothelial cells, triggering focal adhesion formation, achieving directional cell spreading and maintaining function.

[0018] The hydrogels described in this invention can be used to evaluate the proliferation, metabolism, and barrier function of HCE cells under different mechanical conditions. These include, but are not limited to, observing the effects of stiffness on cell adhesion and proliferation, analyzing glucose metabolism and ATP levels to assess cellular metabolic capacity, and measuring the pump and barrier functions of cells by measuring corneal endothelial cell functional proteins and cell junction proteins. This invention addresses the issues of mechanical property mismatch and large batch variability in traditional materials, providing a more reliable, standardized tool for studying HCE cell behavior. This breakthrough technology addresses the inability of traditional materials to balance physical support and functional evaluation, providing a novel research tool and therapeutic strategy for corneal endothelial tissue engineering and clinical transplantation.

[0019] The innovation of this invention lies in its first simultaneous matching of stiffness and thickness to the physiological microenvironment of HCE cells, overcoming the limitations of traditional materials, which are difficult to standardize and balance mechanical support and functional evaluation. Furthermore, the high-precision controllable properties of the hydrogels described in this invention make them suitable not only for corneal endothelial research but also for the study of other cell behaviors that rely on stiffness regulation, such as cardiomyocytes and osteoblasts.

[0020] In terms of application prospects, the hydrogel described in this invention provides a tool for basic research to reveal the mechanism by which the mechanical microenvironment affects HCE cell behavior, laying a theoretical foundation for optimizing the preparation of tissue-engineered corneal endothelium. At the same time, its standardized design can guide the precise development of carrier scaffolds in clinical transplantation, promote the development of functional TE-HCE, and thus alleviate the shortage of corneal donors. In addition, the design concepts and methods of this technology can also be extended to other tissue engineering fields such as stem cell differentiation, providing a technical solution that can be used as a reference for related research. In summary, the present invention has broad application prospects in fields such as cell mechanical response research, tissue engineering, drug screening, and regenerative medicine, providing an important theoretical research platform for scientific research and technological development in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of a double-layer glass interlayer glue-making device, with reference numerals: 1 is the first glass plate; 2 is the second glass plate; 3 is a magnetic micro-pressing strip; 4 is a PET film; 5 is a micro pressure sensor; 6 is a spring plunger; and 7 is a medical three-way valve.

[0022] Figure 2 Stiffness test of the hydrogels prepared in Examples 1 to 3, A: stress-strain curve of 20 kPa hydrogel; B: stress-strain curve of 50 kPa hydrogel; C: stress-strain curve of 10 MPa hydrogel.

[0023] Figure 3 Appearance and transparency test of the hydrogels prepared in Examples 1 to 3, A: hydrogel appearance; B: statistical results of hydrogel transmittance.

[0024] Figure 4 This is the MTT assay of HCE cells on the hydrogels prepared in Examples 1 to 3.

[0025] Figure 5 Detection of the proliferation rate of HCE cells on the hydrogels prepared in Examples 1 to 3. A: EdU staining; B: proliferation rate statistics.

[0026] Figure 6 Detection of ATP levels of HCE cells on the hydrogels prepared in Examples 1 to 3. DETAILED DESCRIPTION

[0027] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0028] The inventive concept of the present invention is as follows: As an important polymer material, polyacrylamide hydrogel has attracted much attention due to its tunable mechanical properties, chemical and batch stability. However, its application in evaluating HCE cell behavior faces two shortcomings: (1) insufficient upper limit of stiffness, which makes it difficult to achieve the high stiffness range of 0.1MPa to 10MPa required for tissues such as the cornea; (2) biological function defects. Its inherent biological inertness forces reliance on surface modification, such as using a single layer of adhesion protein to support cell adhesion, which cannot reproduce the complex network of extracellular matrix components in the natural elastic layer and its coordinated regulation of cell behavior.

[0029] The present invention provides a hydrogel for evaluating the proliferation and metabolic behavior of HCE cells. By simulating the natural mechanical microenvironment of HCE cells and utilizing hydrogel materials with controllable stiffness and ultrathin properties, the present invention provides a standardized tool for studying HCE cell behavior, thereby providing theoretical support and experimental basis for the development of TE-HCE. The composition and construction method of the hydrogel described in the present invention include:

[0030] 1) Adjustable stiffness: Using polyacrylamide solution and N,N'-methylenebisacrylamide as the substrate, by adjusting the ratio of monomer acrylamide and crosslinker bisacrylamide, low-stiffness ultrathin hydrogels with a range of 0.1kPa to 100kPa are prepared. On this basis, cellulose nanocrystals and sodium alginate are added to construct high-stiffness ultrathin hydrogels with a range of 0.1MPa to 10MPa, covering the mechanical microenvironment that HCE cells may encounter under physiological conditions.

[0031] 2) Ultra-thin design: Using a double-layer glass sandwich gel-making device, the hydrogel thickness is controlled to 10μm~13μm, which is highly matched with the thickness of the natural carrier of the human corneal endothelium, the posterior elastic layer, which can provide sufficient mechanical support without hindering the penetration of nutrients.

[0032] 3) Functional modification: Through chemical coupling, extracellular matrix components such as type IV collagen, type VIII collagen, fibronectin, and laminin are fixed on the hydrogel surface for functional modification to simulate the natural microenvironment of HCE cells and promote cell attachment and functional expression.

[0033] The preparation method of the low-rigidity ultrathin hydrogel solution is as follows: 30g~40g of acrylamide monomer powder is weighed and dissolved in 60mL~70mL of ultrapure water to prepare an acrylamide monomer solution with a mass fraction of 30%~40%; then 1g~2g of N,N'-methylenebisacrylamide powder is dissolved in 98mL~99mL of ultrapure water to prepare an N,N'-methylenebisacrylamide solution with a mass fraction of 1%~2%. 20g~25g of ammonium persulfate powder is weighed again and dissolved in 75mL~80mL of ultrapure water to prepare an ammonium persulfate solution with a mass fraction of 20%~25%. The acrylamide monomer solution and the N,N'-methylenebisacrylamide solution are mixed in a volume ratio of 1.1:1~33.3:1, and then 0.2%~0.6% v / v ammonium persulfate solution and 0.1%~0.2% v / v TEMED are added to obtain a hydrogel solution.

[0034] The configuration method of the above high-rigidity ultra-thin hydrogel solution is as follows: 30-40 g of acrylamide monomer powder is dissolved in 60-70 mL of ultrapure water to prepare a 30-40% acrylamide monomer solution; 1-2 g of N,N'-methylenebisacrylamide powder is dissolved in 98-99 mL of ultrapure water to prepare a 1-2% N,N'-methylenebisacrylamide solution; the acrylamide monomer solution and the N,N'-methylenebisacrylamide solution are mixed in a ratio of 1.1:1-33.3:1, then 1 g:99 mL-5 g:95 mL of CNC powder is added to the mixed solution to prepare a 1-5% CNC mixed solution; 1 g:99 mL-3 g:97 mL of sodium alginate powder is added to the mixed solution to prepare a 1-3% sodium alginate mixed solution; finally, 20-25 g of ammonium persulfate powder is dissolved in 75-80 mL of ultrapure water to prepare a 20-25% ammonium persulfate solution; 0.2-0.6% of the ammonium persulfate solution and 0.1-0.2% of TEMED are added to the mixed solution, and a high-rigidity hydrogel solution is obtained.

[0035] The structure of the double-layer glass interlayer glue making device is shown in Figure 1 , which comprises a frame including a bottom plate and an n-shaped frame arranged on the bottom plate. The n-shaped frame comprises a top rod and two vertical rods connected with the top rod. A spring plunger 6 is arranged on the top rod, and a part of the spring plunger 6 is located above the top rod and another part is located below the top rod. The bottom of the spring plunger 6 is fixedly connected with the second glass plate 2. The second glass plate 2 is provided with a magnetic micro-pressure strip 3, and the second glass plate 2 is connected with a micro-pressure sensor 5. The micro-pressure sensor 5 can test the pressure of the second glass plate 2. The edge of the second glass plate 2 is provided with a circular hole with a diameter of 1 mm. One port of a medical three-way valve 7 is connected to the circular hole, and the other two ports are connected to a sterile air generator. The first glass plate 1 is fixed on the bottom plate, and the first glass plate 1 is located directly below the second glass plate 2. The magnetic micro-pressure strip 3 is fixed on the back of the first glass plate 1. The PET film 4 is used to be placed between the first glass plate 1 and the second glass plate 2.

[0036] Assembly of the double-layer glass interlayer glue making device: A mixed solution of 5% to 10% v / v glycerol aqueous solution and 1% to 2% v / v PVA is pre-coated on the surfaces of the first and second glass plates 1 and 2, both of which are loaded with magnetic micro-beads 3 on both sides. The first and second glass plates 1 and 2 are then placed in an oven for drying. A PET film 4 with a thickness of 10 μm to 13 μm is cut into a U-shaped shape slightly smaller than the size of the glass plates and placed close to the first glass plate 1. The first and second glass plates 1 and 2 are aligned and covered, and initial pre-compression is performed by magnetic attraction to avoid misalignment. The plates are placed horizontally in a locking device, and the spring plunger 6 is adjusted to a moderate pressure of 0 N to 10 N. This completes the assembly of the double-layer glass interlayer glue making device. The first glass plate 1 measures 10 cm x 7.5 cm x 1 mm; the second glass plate 2 measures 10 cm x 7.3 cm x 0.75 mm; the magnetic micro-bead 3 is 0.5 mm thick and is model MC-YN001; the PET film 4 measures 9.8 cm x 7.2 cm in length and width and is 12 μm thick; the micro pressure sensor 5 is UNEO, model GD25; the spring plunger 6 is WIXROYD, model 3215.W105; and the medical three-way valve 7 is YY 0585.2-3SM-P-II. Examples 1 to 3 below all used this double-glass sandwich gel-making apparatus to prepare hydrogels for evaluating the function of corneal endothelial seed cells.

[0037] The method for preparing the hydrogel using the double-layer glass interlayer glue making device is as follows: The surfaces of the first glass plate 1 and the second glass plate 2 with magnetic micro-beads 3 on both sides are pre-coated with a 5% to 10% glycerol aqueous solution and a 1% to 2% A PVA mixed solution is prepared, and then the first glass plate 1 and the second glass plate 2 are placed in an oven for drying to form a micro-lubricating layer to reduce demolding damage; at the same time, a circular hole with a diameter of 1 mm is provided on the short edge of the second glass plate 2, and a medical three-way valve 7 is connected to facilitate the injection of a trace amount of air of 0.1 mL to 0.2 mL through the valve after solidification to form a peeling starting point between the gel and the glass, thereby achieving low-stress demolding of the ultra-thin hydrogel; a PET film with a thickness of 10 μm to 13 μm is cut 4 into a circular shape slightly smaller than the size of the glass plate, 9.8 cm × 7.2 cm, and a micro-notch is pre-cut on the edge to facilitate the discharge of excess air and prevent the film from bulging due to the clamping of the glass plates; the first glass plate 1 and the second glass plate 2 are aligned and covered, and the initial pre-compression is completed by magnetic attraction to avoid glue leakage caused by misalignment and excessive local force on the glass plate caused by manual pressure, which squeezes the PET film 4 and deforms it, resulting in uneven gel thickness. Place the covered first glass plate 1 and the second glass plate 2 horizontally in the clamping device to avoid uneven glue formation caused by gravity sagging of the glue liquid due to vertical movement; adjust the spring plunger 6 until the pressure reading displayed by the micro pressure sensor 5 is 0N~10N to avoid deformation of the PET film 4 caused by squeezing; thus, the assembly of the double-layer glass interlayer glue making device is completed.

[0038] The preparation method of the above-mentioned low-rigidity ultra-thin hydrogel is as follows: the above-mentioned hydrogel solution is gently stirred and mixed with a glass rod, and the mixed solution is quickly added to a double-layer glass sandwich glue-making device using a pipette to ensure that the solution is evenly distributed and to avoid the generation of bubbles. The hydrogel is placed in an oven at 25°C~50°C for 2h~12h to obtain the hydrogel.

[0039] The preparation method of the above-mentioned high-stiffness ultra-thin hydrogel is as follows: the above-mentioned high-stiffness hydrogel solution is placed in an ultrasonic instrument at 50W~80W for 10min~15min to avoid gelation and agglomeration; after the ultrasonication is completed, v / v 0.2%~0.6% APS solution and v / v 0.1%~0.2% TEMED are added to the above-mentioned mixed solution, and after stirring evenly with a glass rod, the mixed solution is quickly added to a double-layer glass sandwich glue-making device using a pipette to ensure that the solution is evenly distributed and avoid the generation of bubbles, and placed at room temperature for 2h~4h; the solidified hydrogel is immersed in w / v 1%~5% CaCl2 solution for 30min~60min; the hydroxyl -OH rich on the CNC surface forms hydrogen bonds with the amide group -CONH2 in the polyacrylamide hydrogel to restrict the movement of the chain segments, and at the same time, the carboxyl -COOH of sodium alginate forms ionic crosslinking in the presence of Ca²⁺ to increase the crosslinking density, thereby obtaining a high-stiffness hydrogel.

[0040] Sterilization treatment of the above-mentioned ultra-thin hydrogel: completely immerse the above-mentioned hydrogel in phosphate buffer; place it in a constant temperature shaker at 4°C, set the shaker speed to 50rpm~100rpm, and slowly shake and immerse for 5min~10min; repeat the above-mentioned immersion and cleaning steps 3~5 times, replacing fresh PBS solution each time; place the cleaned ultra-thin hydrogel in a low-temperature steam machine, set the formaldehyde concentration to 1.5g / m³~2g / m³, the temperature to 55°C~60°C, and the humidity to 75%~80%, and low-temperature steam sterilize for 20min~30min to obtain a sterile ultra-thin hydrogel.

[0041] Surface functionalization modification method: Use a 10mm diameter corneal ring drill to form the above sterile ultrathin hydrogel into an independent scaffold disc, and spread it flat in a 48-well cell culture plate; gently rinse the surface of the sterile ultrathin hydrogel with sterile PBS to remove any remaining impurities; use a sterile pipette to draw up a w / v 0.01%~0.02% poly-lysine solution, soak it for 5min~10min, and then wash it with sterile PBS three times, each time for 5min~10min; weigh 100mg~200mg Dissolve sulfo-SANPAH powder in 50 mL to 100 mL of sterile PBS to prepare a sulfo-SANPAH solution with a concentration of 1 mg / mL to 2 mg / mL. Use a sterile pipette to draw 30 μL to 50 μL of sulfo-SANPAH solution, evenly drop it onto the surface of the hydrogel, and gently spread it with a sterile spatula until the surface of the hydrogel is evenly covered. Let it stand for 3 to 5 minutes. Then, place the 48-well cell culture plate containing the hydrogel under a 320 nm to 420 nm UV lamp for UV crosslinking at a distance of 10 cm to 15 cm for 5 to 10 minutes. Wash with sterile PBS three times, each time for 5 minutes to 10 minutes; polylysine enhances the cross-linking efficiency of sulfo-SANPAH by providing additional amino groups, thereby solving the problems of poor coating stability and low functionalization efficiency in existing modification methods; then remove PBS, use a sterile pipette to draw 200μL~300μL of special modification solution A, and place it in a 37℃ constant temperature incubator for 8h~12h; take the modified hydrogel out of the 37℃ constant temperature incubator, draw out the special modification solution A, and wash with sterile PBS three times, replacing PBS every 3min~5min; thus, a functionally modified ultrathin hydrogel is obtained.

[0042] The formula for the above-mentioned special modification solution A is as follows: Weigh 5mg-10mg of type IV collagen lyophilized powder, 2mg-4mg of type VIII collagen lyophilized powder, 1mg-2mg of fibronectin lyophilized powder, and 0.5mg-1mg of laminin lyophilized powder and dissolve them in 50mL-100mL of sterile PBS. A mixed solution containing 0.05mg / mL-0.1mg / mL type IV collagen, 0.02-0.08mg / mL type VIII collagen, 0.01-0.04mg / mL fibronectin, and 0.005-0.02mg / mL laminin is prepared. The weight proportions of each substance in the extracellular matrix component are as follows: type VIII collagen accounts for 23.5%-33.3%; fibronectin accounts for 11.7%-16.7%; laminin accounts for 5.9%-8.3%, and type IV collagen accounts for 100%.

[0043] In order to make the skilled in the art better understand the technical solutions of the present application can be implemented, the following specific examples of the present application is further described. In the description of the present application, if not special, the reagents used are commercially available, the method used is the conventional technology in the art.

[0044] The present application abbreviations table is shown in Table 1.

[0045] Table 1 Abbreviations table The collagen lyophilized powder involved in the present application is as follows: IV collagen lyophilized powder: Sigma, Beijing Inokai Technology Co., Ltd., C5533.

[0046] Fibronectin lyophilized powder: Sigma, Beijing Inokai Technology Co., Ltd., F2006-2MG.

[0047] Nectin lyophilized powder: Sigma, Beijing Inokai Technology Co., Ltd., L6274.

[0048] VIII collagen lyophilized powder, VIII collagen is obtained after the posterior elastic layer of pig cornea is purified, and the preparation method is as follows: Pig eyes are obtained from slaughterhouse, corneas are peeled off from eyeballs, and then the posterior lamina membrane is peeled off with tweezers. The membrane is digested with 0.5 mg / ml pepsin in 0.5 mol / L acetic acid for 12 h and centrifuged, and the supernatant is lyophilized and then dissolved in 1 mol / L NaCl and 50 mmol / L Tris at pH 7.5. The collagen is separated from other proteins by gradient precipitation in 4 mol / L, 0.7 mol / L and 1.5 mol / L NaCl, and each precipitation is then dialyzed with 0.5 mol / L acetic acid. The final separation of VIII collagen from contaminated V collagen is achieved by chromatography on a Sepharose A1.5-m column.

[0049] Example 1 The preparation method of the hydrogel for evaluating the function of corneal endothelial seed cells is as follows: 1.1, Preparation of unmodified 50kPa hydrogel: acrylamide and N,N'-methylene bisacrylamide are dissolved in water respectively to obtain acrylamide monomer solution and N,N'-methylene bisacrylamide solution, and then mixed to obtain a polyacrylamide premix solution; a coagulant is added to the polyacrylamide premix solution to obtain a hydrogel solution; and the unmodified 50kPa hydrogel is prepared by using the hydrogel solution.

[0050] Prepare a reaction solution: add 30 g of acrylamide monomer powder to 70 mL of ultrapure water and slowly stir until completely dissolved to prepare an acrylamide monomer solution; add 2 g of N,N'-methylenebisacrylamide powder to 98 mL of ultrapure water and slowly stir until completely dissolved to prepare an N,N'-methylenebisacrylamide solution; and add 25 g of ammonium persulfate powder to 75 mL of ultrapure water and slowly stir until completely dissolved to prepare an ammonium persulfate solution.

[0051] Pretreatment glue-making device: Use a pipette to suck up a v / v 10% glycerol aqueous solution and a v / v 2% PVA mixed solution, and evenly add it to the surface of the first glass plate 1 and the second glass plate 2, then spread it evenly with an applicator. After standing for 7 minutes, place the first glass plate 1 and the second glass plate 2 in a 55°C oven to dry; after drying, take out the first glass plate 1 and the second glass plate 2, and place a 12μm thick U-shaped PET film 4 close to the first glass plate 1 coated with glycerol and PVA, and then align and cover it with the second glass plate 2, complete the initial pre-pressing by magnetic attraction, place the covered first glass plate 1 and the second glass plate 2 horizontally in the locking device, adjust the spring plunger 6 until the pressure sensor displays 7N, and the assembly of the double-layer glass interlayer glue-making device is completed.

[0052] Preparation of unmodified 50kPa hydrogels: The acrylamide monomer solution prepared above was mixed with the N,N'-methylenebisacrylamide solution in a volume ratio of 14.3:1 to obtain a polyacrylamide premix. 0.4% (v / v) ammonium persulfate solution and 0.1% (v / v) TEMED were then added. Gently stir the mixture with a glass rod and quickly add the mixed solution to the double-layer glass sandwich gel-making apparatus described above using a pipette to ensure even distribution of the solution and avoid air bubbles. After the gel was injected, it was placed in a 37°C oven for 4 hours. After curing, 0.15mL of air was injected through the valve to form a peeling starting point between the gel and the glass. The hydrogel was gently lifted on both sides with tweezers and slowly demolded to avoid damaging the hydrogel structure. The resulting ultrathin hydrogel was tested for stress-strain curves using a universal materials testing machine, confirming a stiffness of 50kPa.

[0053] 1.2. Sterilization and surface functionalization modification.

[0054] 1.2.1. Low-temperature steam sterilization.

[0055] Use tweezers to gently remove the unmodified 50kPa hydrogel from the gel-making device to avoid damaging the hydrogel structure; gently spread the hydrogel flat on the culture dish to ensure its surface is flat; add phosphate buffer saline (PBS) to the culture dish to ensure that the hydrogel is completely immersed; transfer the culture dish to a 4°C constant temperature shaker, set the shaker speed to 100 rpm, and slowly shake and soak for 5 minutes; repeat the above soaking and washing steps three times, replacing fresh PBS solution each time to ensure that the residual unreacted monomers and catalysts in the hydrogel are completely removed; place the cleaned hydrogel in a low-temperature steam machine, set the formaldehyde concentration to 2g / m³, the temperature to 60°C, and the humidity to 80%, and low-temperature steam sterilize for 20 minutes to obtain a 50kPa sterile ultrathin hydrogel.

[0056] 1.2.2. Prepare the cross-linking agent.

[0057] Weigh 100 mg of sulfo-SANPAH powder into a sterile centrifuge tube, add 100 mL of pH 7.4 sterile PBS solution, and use a vortex oscillator to oscillate at low speed for 5 minutes to fully dissolve, thereby preparing 1 mg / mL of sulfo-SANPAH.

[0058] 1.2.3. Prepare special modification solution A.

[0059] 10 mg of type IV collagen lyophilized powder, 4 mg of type VIII collagen lyophilized powder, 2 mg of fibronectin lyophilized powder, and 1 mg of laminin lyophilized powder were weighed and dissolved in 100 mL of sterile PBS, pH 7.4. After inversion to mix, the mixture was placed in a 37°C constant temperature incubator for 4 h until the flocculent precipitate was completely dissolved, thereby preparing a special modification solution A containing 0.1 mg / mL type IV collagen, 0.04 mg / mL type VIII collagen, 0.02 mg / mL fibronectin, and 0.01 mg / mL laminin.

[0060] 1.2.4. Add poly-lysine as the anchoring point of the cross-linking agent.

[0061] A 10mm diameter corneal trephine was then used to form the 50kPa sterile ultrathin hydrogel into individual scaffold discs. These discs were gently grasped with tweezers and spread flatly across a 48-well cell culture plate, ensuring a smooth surface. A sterile pipette was used to gently rinse the surface with 300μL of sterile PBS to remove any remaining impurities. A sterile pipette was then used to soak the discs in a 0.02% w / v poly-lysine solution for 10 minutes, followed by three 10-minute washes with sterile PBS. The poly-lysine solution served as an anchoring site for the subsequent sulfo-SANPAH crosslinker, ensuring uniform distribution across the substrate and enabling more efficient capture of target molecules such as fibronectin and laminin.

[0062] 1.2.5. Cross-link sulfo-SANPAH to the unmodified hydrogel via UV cross-linking to obtain a cross-linked hydrogel. Then, add extracellular matrix components to the cross-linked hydrogel to allow the N-hydroxysuccinimide ester groups activated by the cross-linker to undergo amidation reaction with the primary amino groups of proteins in the extracellular matrix components, thereby covalently coupling the extracellular matrix components and providing specific adhesion sites for corneal endothelial seed cells.

[0063] Aspirate the PBS, use a sterile pipette to draw up 50 μL of sulfo-SANPAH solution, and evenly add it to the surface of the hydrogel; gently spread it with a sterile spatula to evenly cover the surface of the hydrogel with the solution, avoiding local excess or deficiency; after evenly spreading, let it stand for 5 minutes to allow the solution to fully infiltrate the surface of the hydrogel; then place the 48-well cell culture plate containing the hydrogel under a 365nm UV lamp for UV crosslinking at a distance of 10 cm and irradiate for 10 minutes; avoid moving the hydrogel during irradiation to ensure uniform illumination; then wash with sterile PBS three times, 5 minutes each time, replacing new PBS solution each time to remove unreacted special cross-linking solution. After washing, remove PBS, use a sterile pipette to draw 200 μL of special modification solution A and add it to the cross-linked hydrogel, and place it in a 37°C constant temperature incubator for 12 hours; after the chemical cross-linking is completed, take out the modified hydrogel from the 37°C constant temperature incubator, remove the special modification solution A, and wash it with sterile PBS three times, replacing PBS every 3 minutes; thus, a functionally modified ultrathin hydrogel is obtained; the treated hydrogel is transferred to a sterile container and stored in a 4°C refrigerator.

[0064] Example 2 The preparation method of the hydrogel for evaluating the function of corneal endothelial seed cells is as follows: 1.1. Preparation of unmodified 20 kPa hydrogel: Acrylamide and N,N'-methylenebisacrylamide were separately dissolved in water to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, which were then mixed to obtain a polyacrylamide premix solution; a coagulant was added to the polyacrylamide premix solution to obtain a hydrogel solution; and an unmodified 20 kPa hydrogel was prepared using the hydrogel solution.

[0065] Prepare a reaction solution: add 30 g of acrylamide monomer powder to 70 mL of ultrapure water and slowly stir until completely dissolved to prepare an acrylamide monomer solution; add 1 g of N,N'-methylenebisacrylamide powder to 99 mL of ultrapure water and slowly stir until completely dissolved to prepare an N,N'-methylenebisacrylamide solution; and add 20 g of ammonium persulfate powder to 80 mL of ultrapure water and slowly stir until completely dissolved to prepare an ammonium persulfate solution.

[0066] Pretreatment glue-making device: Use a pipette to suck up a 10% v / v glycerol aqueous solution and a 2% PVA v / v mixed solution, and evenly add it to the surface of the first glass plate 1 and the second glass plate 2, then spread it evenly with an applicator, let it stand for 10 minutes, and then place the first glass plate 1 and the second glass plate 2 in a 60°C oven to dry; after drying, take out the first glass plate 1 and the second glass plate 2, and place a 10μm thick U-shaped PET film 4 close to the first glass plate 1 coated with glycerol and PVA, and then align and cover it with the second glass plate 2, complete the initial pre-pressing by magnetic attraction, place the covered first glass plate 1 and the second glass plate 2 horizontally in the locking device, adjust the spring plunger 6 until the pressure sensor displays 10N, and the assembly of the double-layer glass interlayer glue-making device is completed.

[0067] Preparation of unmodified 20kPa hydrogel: The acrylamide monomer solution prepared above was mixed with N,N'-methylenebisacrylamide solution in a volume ratio of 33.3:1 to obtain a polyacrylamide premix, and then v / v 0.2% ammonium persulfate solution and v / v 0.1% TEMED were added; stirred and mixed with a glass rod, and the mixed solution was quickly added to the above-mentioned double-layer glass sandwich glue-making device using a pipette to ensure that the solution was evenly distributed and to avoid the generation of bubbles; after the glue injection was completed, it was placed in a 25°C oven for 12 hours; after the curing was completed, 0.1mL of air was injected through the valve to form a peeling starting point between the gel and the glass, and the two sides of the hydrogel were gently lifted with tweezers to slowly demold to avoid damaging the hydrogel structure; the obtained ultrathin hydrogel was measured for stress-strain curve using a universal material testing machine to confirm that its stiffness was 20kPa.

[0068] 1.2. Sterilization and surface functionalization modification.

[0069] The method is exactly the same as in Example 1.

[0070] Example 3 The preparation method of the hydrogel for evaluating the function of corneal endothelial seed cells is as follows: 1.1. Preparation of unmodified 10 MPa hydrogel: Acrylamide and N,N'-methylenebisacrylamide were separately dissolved in water to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, which were then mixed to obtain a polyacrylamide premix solution; a coagulant and a stiffness enhancer were added to the polyacrylamide premix solution to obtain a hydrogel solution; and an unmodified 10 MPa hydrogel was prepared using the hydrogel solution.

[0071] Prepare the reaction solution: Weigh 40g of acrylamide monomer powder and dissolve it in 60mL of ultrapure water to prepare a 40% acrylamide monomer solution. Weigh 2g of N,N'-methylenebisacrylamide powder and dissolve it in 98mL of ultrapure water to prepare a 2% N,N'-methylenebisacrylamide solution. Mix the acrylamide monomer solution and the N,N'-methylenebisacrylamide solution in a volume ratio of 1.1:1. Then, weigh carbon nanotubes (CNC) powder in a mass-volume ratio of 5g:95mL and dissolve it in the mixture of the acrylamide monomer solution and the N,N'-methylenebisacrylamide solution to prepare a 5% CNC mixed solution. Add sodium alginate powder to the CNC mixed solution in a mass-volume ratio of 2g:97mL to obtain a mixed solution containing CNC and sodium alginate.

[0072] Pretreatment glue making device: Use a pipette to suck up a 5% v / v glycerol aqueous solution and a 1% PVA v / v mixed solution, and evenly add it to the surface of the first glass plate 1 and the second glass plate 2, then spread it evenly with an applicator, let it stand for 5 minutes, and then place the first glass plate 1 and the second glass plate 2 in a 50°C oven to dry; after drying, take out the first glass plate 1 and the second glass plate 2, and place a 13μm thick U-shaped PET film 4 close to the first glass plate 1 coated with glycerol and PVA, and then align and cover it with the second glass plate 2, complete the initial pre-pressing by magnetic attraction, place the covered first glass plate 1 and the second glass plate 2 horizontally in the locking device, adjust the spring plunger 6 until the pressure sensor displays 3N, and the assembly of the double-layer glass interlayer glue making device is completed.

[0073] Preparation of unmodified 10MPa hydrogel: Place the mixed solution containing CNC and sodium alginate in an ultrasonic instrument and ultrasonically disperse it at 80W for 10 minutes; after the ultrasonication is completed, add v / v 0.2% ammonium persulfate solution and v / v 0.2% TEMED to the above mixed solution, stir evenly with a glass rod, and then use a pipette to quickly add the mixed solution to a double-layer glass sandwich glue-making device to ensure that the solution is evenly distributed and avoid bubble generation. Place it at room temperature for 2 hours; immerse the solidified hydrogel in 5% w / v CaCl2 solution for 60 minutes; after the solidification is completed, inject 0.2mL of air through the valve to form a peeling starting point between the gel and the glass, and use tweezers to gently lift both sides of the hydrogel and slowly demold it to avoid damaging the hydrogel structure; the obtained ultra-thin hydrogel is measured by a universal material testing machine The stress-strain curve of the hydrogel confirms that its stiffness is 10MPa.

[0074] 1.2. Sterilization and surface functionalization modification.

[0075] The method is exactly the same as in Example 1.

[0076] Example 4 The application of the hydrogel to evaluate the function of corneal endothelial seed cells was verified by testing the hydrogels prepared in Examples 1 to 3 as follows: Using the BOSE ElectroForce3100 test system, the elastic modulus of the prepared hydrogel scaffold was tested and the stress-strain curve was drawn based on the thickness of 10μm~13μm and the stiffness range of 0.1MPa~10MPa required for corneal tissue. The results showed that by adopting the preparation method and solution ratio in Example 1, an ultra-thin hydrogel with an elastic modulus of 50kPa can be obtained; by adopting the preparation method and solution ratio in Example 2, an ultra-thin hydrogel with an elastic modulus of 20kPa can be obtained; by adopting the preparation method and solution ratio in Example 3, an ultra-thin hydrogel with an elastic modulus of 10MPa can be obtained. The prepared ultra-thin hydrogel scaffold not only matches the thickness of the elastic layer after the natural attachment carrier of HCE cells, but also covers the stiffness microenvironment that HCE cells may encounter under physiological conditions. Results are shown in Figure 2 .

[0077] Hydrogel scaffolds of different hardness were placed on a white paper printed with the letter "A" and then observed and photographed. The results showed that these hydrogels were uniform and transparent, with a smooth surface and high transparency. Transmittance analysis showed that within the visible light range of 400nm~800nm, there was no significant difference in the transmittance of hydrogel scaffolds of different hardness, and the transmittance was over 90%, showing good light transmission performance. Figure 3 .

[0078] The MTT cell viability assay kit was used to detect the cell viability of HCE cells on hydrogel scaffolds with three different stiffnesses: 20kPa, 50kPa, and 10MPa. The results showed that the viability level of HCE cells was regulated by the stiffness of the scaffold and gradually increased with the increase of the stiffness of the scaffold. Figure 4 .

[0079] HCE cells were seeded on hydrogel scaffolds with three different stiffnesses: 20kPa, 50kPa, and 10MPa. The effect of different stiffness scaffolds on HCE cell proliferation was observed by EdU staining. The results showed that the cell proliferation rate gradually increased with the increase of scaffold stiffness, showing stiffness dependence. Figure 5 .

[0080] The energy production level of HCE cells cultured on hydrogel scaffolds with different stiffness was tested. The results showed that as the stiffness of the scaffold gradually increased, the ATP content of the cells increased significantly. This indicates that the energy production level of the cells increased significantly with the increase of the scaffold stiffness. Figure 6 .

[0081] In addition, it should be noted that the preparation methods of the various solutions of the present invention are: 1. Preparation of acrylamide monomer solution: Place a beaker on a magnetic stirrer, add a magnetic stirrer, measure 60mL~70mL of ultrapure water in a measuring cylinder, add 30g~40g of acrylamide monomer powder, start stirring, and slowly stir until the acrylamide monomer powder is completely dissolved. This is the acrylamide monomer solution.

[0082] 2. Preparation of N,N'-methylenebisacrylamide solution: Place a beaker on a magnetic stirrer, add a magnetic stirrer, measure 98mL~99mL of ultrapure water in a measuring cylinder, add 1g~2g of N,N'-methylenebisacrylamide powder, start stirring, and slowly stir until the N,N'-methylenebisacrylamide powder is completely dissolved. This is the N,N'-methylenebisacrylamide solution.

[0083] 3. Preparation of ammonium persulfate solution: Place a beaker on a magnetic stirrer, add a magnetic stirrer, measure 75mL~80mL of ultrapure water with a measuring cylinder, add 20g~25g of ammonium persulfate powder, start stirring, and slowly stir until the ammonium persulfate powder is completely dissolved. This is the ammonium persulfate solution.

[0084] 4. Preparation of sulfo-SANPAH solution: Weigh 50 mg to 100 mg of sulfo-SANPAH powder and transfer it to a sterile centrifuge tube. Add 50 mL to 100 mL of sterile PBS solution and vortex at low speed for 3 to 5 minutes to fully dissolve the sulfo-SANPAH. This is the sulfo-SANPAH solution.

[0085] 5. Preparation of Special Modification Solution A: Weigh 5mg-10mg of type IV collagen lyophilized powder, 2mg-4mg of type VIII collagen lyophilized powder, 1mg-2mg of fibronectin lyophilized powder, and 0.5-1mg of laminin lyophilized powder, and dissolve them in 50mL-100mL of sterile PBS; mix thoroughly by inverting the tube, and place in a 37°C constant temperature incubator for 2h-4h until the flocculent precipitate is completely dissolved. This is Special Modification Solution A.

[0086] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A hydrogel for evaluating the function of corneal endothelial seed cells, characterized in that: The hydrogel is formed by acrylamide forming a three-dimensional cross-linked network structure through free radical polymerization in the presence of N,N'-methylenebisacrylamide; and the extracellular matrix components are fixed on the surface of the three-dimensional cross-linked network structure to obtain the hydrogel.

2. The hydrogel according to claim 1, wherein The extracellular matrix component includes at least one of type IV collagen, type VIII collagen, fibronectin and laminin.

3. The hydrogel according to claim 2, wherein The mass proportions of type IV collagen, type VIII collagen, fibronectin, and laminin in the extracellular matrix are as follows: Type VIII collagen accounts for 23.5% to 33.3%; Fibronectin accounts for 11.7% to 16.7%; Laminin accounts for 5.9% to 8.3%. Type IV collagen is replenished 100%.

4. The hydrogel according to claim 1, wherein Cellulose nanocrystals and sodium alginate are also added into the hydrogel.

5. The method for preparing the hydrogel according to claim 1, wherein: The following steps are involved: dissolving acrylamide and N,N'-methylenebisacrylamide in water respectively to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, and mixing them to obtain a polyacrylamide premix solution; adding a coagulant and a stiffness enhancer to a polyacrylamide premix to obtain a hydrogel solution; The stiffness enhancers are cellulose nanocrystals and sodium alginate; Using the hydrogel solution, an unmodified hydrogel with a thickness of 10 μm to 13 μm was prepared; adding a cross-linking agent to the unmodified hydrogel, so that the cross-linking agent is cross-linked to the unmodified hydrogel to obtain a cross-linked hydrogel; The extracellular matrix components are added to the cross-linked hydrogel, and the N-hydroxysuccinimide ester groups activated by the cross-linker undergo amidation reaction with the primary amino groups of the proteins in the extracellular matrix components, thereby covalently coupling the extracellular matrix components and providing specific adhesion sites for corneal endothelial seed cells to obtain the hydrogel.

6. The preparation method according to claim 5, wherein The coagulant is ammonium persulfate and tetramethylethylenediamine; Ammonium persulfate is added to the polyacrylamide premix solution in a 20% to 25% mass fraction of ammonium persulfate solution. The amount of ammonium persulfate solution added is 0.2% to 0.6% of the volume of the polyacrylamide premix solution. The amount of tetramethylethylenediamine added is 0.1%~0.2% of the volume of the polyacrylamide premix.

7. The preparation method according to claim 5, wherein The amount of cellulose nanocrystals added is 1% to 5% wt; The added amount of the sodium alginate is 0.1%~0.2%wt.

8. The preparation method according to claim 5, wherein The cross-linking agent is sulfotricyclic aromatic hydrocarbon.

9. The use of the hydrogel according to claim 1, characterized in that: The hydrogel was used to evaluate the cell function of human corneal endothelial seed cells.

10. The use according to claim 9, characterized in that The cell functions include cell proliferation and cell metabolic capacity.

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