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

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a hydrogel for evaluating functions of corneal endothelial seed cells, and a preparation method and application thereof. The hydrogel is a three-dimensional cross-linked network structure formed by free radical polymerization of acrylamide in the presence of N,N'-methylene bisacrylamide. Meanwhile, extracellular matrix components are fixed on the surface of the three-dimensional cross-linked network structure, and the hydrogel is obtained. The hydrogel prepared by the application improves the problems of mismatching of mechanical properties of traditional materials, large batch difference and the like, and provides a more reliable standardized tool for HCE cell behavior research. This breakthrough technology solves the problem that traditional materials cannot simultaneously provide physical support and function evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a hydrogel for evaluating the function of corneal endothelial seed cells, its preparation method, and its application. Background Technology

[0002] The cornea is an avascular, transparent tissue located at the front of the eyeball. Its refractive power, combined with that of the lens, allows light to be accurately focused onto the retina, thus forming vision. The corneal endothelium (HCE), located at the innermost layer of the cornea, is composed of a single layer of regularly spaced, hexagonal, flattened cells. A complete monolayer of HCE cells functions as an endothelial pump and a corneal-aqueous humor barrier, playing an irreplaceable role in maintaining the cornea's semi-dehydrated state, normal thickness, transparency, and the acquisition of nutrients and oxygen from the aqueous humor. A sufficient number of corneal endothelial cells is a major factor in ensuring the function of the corneal endothelium. The death of local cells 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 the physiological function of the corneal endothelium, irreversible damage occurs, namely corneal endothelial decompensation, leading to corneal edema and opacity, and in severe cases, blindness. Currently, the only treatment for corneal endothelial blindness is corneal endothelial transplantation. However, due to a severe shortage of donated corneas and corneal aging, the vast majority of patients with corneal endothelial blindness remain blind because they cannot obtain usable donated corneas for transplantation and treatment. The development and production of tissue-engineered human corneal endothelium (TE-HCE), an equivalent substitute for donated corneal endothelium, has become a source of hope for many patients with corneal endothelial blindness and is key to fundamentally solving the problems of insufficient corneal transplant donor materials and postoperative immune rejection.

[0003] In the construction of TE-HCE, the mechanical properties of the carrier scaffold, especially its thickness and stiffness, are the core parameters determining the effective functionalization of seed cells. The cellular functions of corneal endothelial seed cells mainly include cell proliferation and cell metabolism. Under normal physiological conditions, the Descemet's membrane, to which HCE cells attach, has precise mechanical characteristics, with a thickness of 10μm~13μm and a stiffness of 50±17.8kPa. Its microenvironmental parameters directly affect key biological behaviors such as the formation of tight junctions, nutrient penetration, and mechanical signal transduction. 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, there is a significant deviation between the mechanical properties and physiological values; for example, the stiffness of amniotic membrane and collagen may not be matched or may not cover the stiffness environment that HCE cells may encounter under physiological conditions, or the thickness may be inappropriate; second, batch-to-batch variations in materials interfere with experimental reproducibility. These reasons prevent amniotic membrane and collagen from evaluating the cell proliferation and metabolic capacity of human corneal endothelial seed cells during the construction of TE-HCE.

[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 (HCEs). By simulating the natural mechanical microenvironment of HCEs and utilizing hydrogel materials with adjustable stiffness and ultrathin properties, this invention provides a standardized tool for HCE cell behavior research, thereby providing theoretical support and experimental basis for the development of TE-HCEs.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a hydrogel for evaluating the function of corneal endothelial seed cells. The hydrogel is formed by the free radical polymerization of acrylamide in the presence of N,N'-methylenebisacrylamide to form a three-dimensional cross-linked network structure. At the same time, extracellular matrix components are fixed on the surface of the three-dimensional cross-linked network structure to obtain the hydrogel.

[0008] Preferably, the extracellular matrix components include at least one of type IV collagen, type VIII collagen, fibronectin, and laminin.

[0009] Preferably, the mass percentages of type IV collagen, type VIII collagen, fibronectin, and laminin in the extracellular matrix components are as follows:

[0010] Type VIII collagen accounts for 23.5% to 33.3%;

[0011] Fibronectin accounted for 11.7% to 16.7%;

[0012] Laminin accounts for 5.9% to 8.3%.

[0013] Type IV collagen supplementation reaches 100%.

[0014] Preferably, cellulose nanocrystals and sodium alginate are also added to the hydrogel.

[0015] A second aspect of the present invention provides a method for preparing the hydrogel, comprising the following steps:

[0016] Acrylamide and N,N'-methylenebisacrylamide were dissolved in water to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, which were then mixed to obtain a polyacrylamide premix.

[0017] A coagulant and a stiffening agent are added to a polyacrylamide premix to obtain a hydrogel solution; the stiffening agent is cellulose nanocrystals and sodium alginate.

[0018] Unmodified hydrogels with a thickness of 10 μm to 13 μm were prepared using hydrogel solutions;

[0019] A crosslinking agent is added to an unmodified hydrogel, causing the crosslinking agent to crosslink onto the unmodified hydrogel, resulting in a crosslinked hydrogel.

[0020] Extracellular matrix components are added to the cross-linked hydrogel, and the N-hydroxysuccinimide ester groups activated by the cross-linking agent undergo an 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, thus obtaining the hydrogel.

[0021] Preferably, the coagulant is ammonium persulfate and tetramethylethylenediamine;

[0022] Ammonium persulfate is added to the polyacrylamide premix as a 20%~25% mass fraction ammonium persulfate solution, and the amount of ammonium persulfate solution added is 0.2%~0.6% of the volume of the polyacrylamide premix.

[0023] The amount of tetramethylethylenediamine added is 0.1% to 0.2% of the volume of the polyacrylamide premix.

[0024] Preferably, the amount of cellulose nanocrystals added is 1%~5% wt; the amount of sodium alginate added is 0.1%~0.2% wt.

[0025] Preferably, the crosslinking agent is a sulfotricyclic aromatic hydrocarbon.

[0026] A third aspect of the present invention provides an application of the hydrogel for evaluating the cellular function of human corneal endothelial seed cells.

[0027] Preferably, the cell function is cell proliferation and cell metabolism.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention provides a hydrogel for evaluating the function of corneal endothelial seed cells. The hydrogel is formed by the free radical polymerization of acrylamide in the presence of N,N'-methylenebisacrylamide to create a three-dimensional cross-linked network structure. Simultaneously, extracellular matrix components are immobilized on the surface of this three-dimensional cross-linked network structure, thus obtaining the hydrogel. The core of this invention lies in: first forming a polyacrylamide-based hydrogel framework with controllable mechanical properties, and then covalently anchoring the extracellular matrix to the framework surface using a cross-linking agent, providing a specifically recognizable adhesion microenvironment for corneal endothelial cells.

[0030] In this invention, acrylamide and N,N'-methylenebisacrylamide first undergo free radical chain polymerization under the action of ammonium persulfate and tetramethylethylenediamine as coagulants. The double bonds of N,N'-methylenebisacrylamide simultaneously participate in both main chains, forming a three-dimensional cross-linked network. Subsequently, cellulose nanocrystals are embedded into the network through hydrogen bonding and physical entanglement, improving rigidity; the carboxyl groups of sodium alginate form secondary hydrogen bonds with the amide / hydroxyl groups in the network, synergistically strengthening the gel. Then, the N-hydroxysuccinimide ester in the cross-linking agent undergoes an amidation reaction with the -NH2 in the polyacrylamide network, covalently grafting the cross-linking agent onto the gel surface; ultraviolet light triggers the azide group at the other end of the cross-linking agent to generate reactive nitric oxide radicals, which can covalently couple with the primary amino groups in the ECM protein to form stable amide bonds; the covalently fixed ECM protein exposes its integrin binding sites such as the RGD sequence, which are recognized by corneal endothelial cells, triggering the formation of adhesion spots, achieving directional cell spreading and functional maintenance.

[0031] The hydrogel described in this invention can be used to evaluate the proliferation, metabolism, and barrier function of HCE cells under different mechanical conditions. This includes, but is not limited to: observing the effect of stiffness on cell adhesion and proliferation, analyzing glucose metabolism and ATP levels to assess cellular metabolic capacity, and detecting the pump and barrier functions of cells through corneal endothelial cell functional proteins and cell junction proteins. This invention improves upon the problems of mismatched mechanical properties and large batch-to-batch variations in traditional materials, providing a more reliable standardized tool for HCE cell behavior research. This breakthrough technology solves the problem that traditional materials cannot simultaneously provide physical support and functional evaluation, providing novel research tools and treatment strategies for corneal endothelial tissue engineering and clinical transplantation.

[0032] The innovation of this invention lies in the fact that it is the first time that stiffness and thickness have been simultaneously matched to the physiological microenvironment of HCE cells, overcoming the shortcomings of traditional materials in terms of standardization and balancing mechanical support and functional evaluation. Furthermore, the high-precision controllability of the hydrogel described in this invention makes it not only suitable for corneal endothelial research, but also provides a theoretical reference for other cell behavior studies that rely on stiffness regulation, such as cardiomyocytes or osteoblasts.

[0033] 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 the behavior of HCE cells, laying a theoretical foundation for optimizing the preparation of tissue-engineered corneal endothelium. Simultaneously, its standardized design can guide the precise development of carrier scaffolds in clinical transplantation, promoting the development of functional TE-HCEs and thus alleviating the shortage of corneal donors. Furthermore, the design concept and methods of this technology can be extended to other tissue engineering fields such as stem cell differentiation, providing a referable technical solution for related research. In summary, this invention has broad application prospects in the fields of 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. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of a double-layer glass interlayer adhesive forming device. The attached figures are labeled as follows: 1 is the first glass plate; 2 is the second glass plate; 3 is the magnetic micro-pressure strip; 4 is the PET film; 5 is the miniature pressure sensor; 6 is the spring plunger; and 7 is the medical three-way valve.

[0035] Figure 2 The stiffness of the hydrogels prepared in Examples 1 to 3 was tested. A: Stress-strain curve of 20 kPa hydrogel; B: Stress-strain curve of 50 kPa hydrogel; C: Stress-strain curve of 10 MPa hydrogel.

[0036] Figure 3 The appearance and transparency of the hydrogels prepared in Examples 1 to 3 were tested. A: Appearance of the hydrogel; B: Statistical results of the transmittance of the hydrogel.

[0037] Figure 4 MTT assay of HCE cells on hydrogels prepared in Examples 1 to 3.

[0038] Figure 5 For the detection of HCE cell proliferation rate on hydrogels prepared in Examples 1 to 3, A: EdU staining; B: Proliferation rate statistics.

[0039] Figure 6 The ATP levels of HCE cells on hydrogels prepared in Examples 1 to 3 were detected. Detailed Implementation

[0040] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0041] The inventive concept of this invention is as follows:

[0042] Polyacrylamide hydrogel, as an important polymer material, has attracted much attention due to its adjustable mechanical properties, chemical properties, and batch stability. However, its application in HCE cell behavior evaluation faces two shortcomings: (1) insufficient upper limit of stiffness, making it difficult to reach the high stiffness range of 0.1MPa~10MPa required by tissues such as the cornea; (2) biological function defects, the inherent biological inertness forces reliance on surface modification, such as using a single layer of adhesion proteins to support cell adhesion, but it cannot reproduce the complex extracellular matrix component network in the natural elastic layer and its synergistic regulation of cell behavior.

[0043] This invention provides a hydrogel for evaluating the proliferation and metabolic behavior of hematopoietic stem cell (HCE) cells. By mimicking the natural mechanical microenvironment of HCE cells and utilizing a hydrogel material with adjustable stiffness and ultrathin properties, this invention provides a standardized tool for HCE cell behavior research, thereby offering theoretical support and experimental basis for the development of TE-HCE. The composition and construction method of the hydrogel described in this invention include:

[0044] 1) Adjustable stiffness: Using polyacrylamide solution and N,N'-methylenebisacrylamide as the base, low-stiffness ultrathin hydrogels of 0.1kPa~100kPa were prepared by adjusting the ratio of monomeric acrylamide and crosslinking agent bisacrylamide; on this basis, cellulose nanocrystals and sodium alginate were added to construct high-stiffness ultrathin hydrogels of 0.1MPa~10MPa, covering the mechanical microenvironment that HCE cells may encounter under physiological conditions.

[0045] 2) Ultra-thin design: The double-layer glass sandwich gel-making device controls the thickness of the hydrogel to 10μm~13μm, which is highly matched with the thickness of the posterior elastic layer, the natural carrier of the human corneal endothelium. This provides sufficient mechanical support without hindering the penetration of nutrients.

[0046] 3) Functional modification: Through chemical coupling, extracellular matrix components such as type IV collagen, type VIII collagen, fibronectin and laminin are immobilized on the surface of hydrogel for functional modification to simulate the natural microenvironment of HCE cells and promote cell attachment and functional expression.

[0047] The above-mentioned method for preparing low-stiffness ultrathin hydrogel solution is as follows: Weigh 30g~40g of acrylamide monomer powder, dissolve it in 60mL~70mL of ultrapure water to prepare an acrylamide monomer solution with a mass fraction of 30%~40%; then weigh 1g~2g of... N,N'-methylenebisacrylamide powder was dissolved in 98 mL to 99 mL of ultrapure water to prepare a 1% to 2% N,N'-methylenebisacrylamide solution. 20 g to 25 g of ammonium persulfate powder was weighed and dissolved in 75 mL to 80 mL of ultrapure water to prepare a 20% to 25% ammonium persulfate solution. The acrylamide monomer solution and the N,N'-methylenebisacrylamide solution were mixed at a volume ratio of 1.1:1 to 33.3:1. Then, 0.2% to 0.6% (v / v) of ammonium persulfate solution and 0.1% to 0.2% (v / v) of TEMED were added to obtain the hydrogel solution.

[0048] The preparation method of the above-mentioned high-rigidity ultrathin hydrogel solution is as follows: Weigh 30g-40g of acrylamide monomer powder and dissolve it in 60mL-70mL of ultrapure water to prepare an acrylamide monomer solution with a mass fraction of 30%-40%; weigh 1g-2g of N,N'-methylenebisacrylamide powder and dissolve it in 98mL-99mL of ultrapure water to prepare an N,N'-methylenebisacrylamide solution with a mass fraction of 1%-2%; mix the acrylamide monomer solution and the N,N'-methylenebisacrylamide solution in a ratio of 1.1:1 to 33.3:1, then weigh CNC powder with a mass-to-volume ratio of 1g:99mL to 5g:95mL and dissolve it in the above mixed solution to prepare a CNC mixed solution with a mass fraction of 1%-5%; add... Sodium alginate powder with a mass-to-volume ratio of 1g:99mL to 3g:97mL is used to prepare a sodium alginate mixed solution with a mass fraction of 1% to 3%. Finally, 20g to 25g of ammonium persulfate powder is weighed and dissolved in 75mL to 80mL of ultrapure water to prepare an ammonium persulfate solution with a mass fraction of 20% to 25%. Add 0.2% to 0.6% (v / v) of ammonium persulfate solution and 0.1% to 0.2% (v / v) of TEMED to the above mixed solution to obtain a high-rigidity hydrogel solution.

[0049] See the structure of the double-glass interlayer adhesive forming device. Figure 1 The system includes a frame, which comprises a base plate and an n-shaped frame mounted on the base plate. The n-shaped frame includes a top rod and two vertical rods connected to the top rod. A spring plunger 6 passes through the top rod, with one part of the spring plunger 6 located above the top rod and the other part below it. A second glass plate 2 is fixedly connected to the bottom of the spring plunger 6. A magnetic micro-pressure strip 3 is provided on the second glass plate 2. The second glass plate 2 is connected to a micro-pressure sensor 5, which can test the pressure of the second glass plate 2. A 1mm diameter circular hole is provided on the edge of the second glass plate 2. 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. A first glass plate 1 is fixed to the base plate and is located directly below the second glass plate 2. The magnetic micro-pressure strip 3 is fixed to the back of the first glass plate 1. A PET film 4 is placed between the first glass plate 1 and the second glass plate 2.

[0050] Assembly of the double-layer glass interlayer adhesive forming device:

[0051] A 5%~10% v / v glycerol aqueous solution and a 1%~2% v / v PVA mixed solution are pre-coated on the surfaces of the first glass plate 1 and the second glass plate 2, which are equipped with magnetic micro-pressure strips 3 on both sides. Then, the first glass plate 1 and the second glass plate 2 are placed in an oven to dry. A PET film 4 with a thickness of 10μm~13μm is cut into a U-shape slightly smaller than the size of the glass plate and placed tightly against the first glass plate 1. The first glass plate 1 and the second glass plate 2 are aligned and covered, and the initial pre-pressure is completed by magnetic attraction to avoid misalignment. The plate is placed horizontally in the locking device, and the spring plunger 6 is adjusted to a moderate pressure of 0N~10N. This completes the assembly of the double-layer glass interlayer adhesive device. The first glass plate 1 has dimensions of 10cm × 7.5cm × 1mm; the second glass plate 2 has dimensions of 10cm × 7.3cm × 0.75mm; the magnetic micro-pressure strip 3 has a thickness of 0.5mm and is model MC-YN001; the PET film 4 has dimensions of 9.8cm × 7.2cm and a thickness of 12μm; the miniature pressure sensor 5 is UNEO and model GD25; the spring plunger 6 is WIXROYD and model 3215.W105; and the medical three-way valve 7 is YY 0585.2-3SM-P-II. Examples 1 to 3 below all use this double-layer glass sandwich gel-forming device to prepare hydrogels for evaluating the function of corneal endothelial seed cells.

[0052] The method for preparing the hydrogel using the double-glass sandwich gel-forming apparatus is as follows:

[0053] A 5%~10% glycerol aqueous solution and a 1%~2% glycerol aqueous solution were pre-coated onto the surfaces of the first glass plate 1 and the second glass plate 2, which were equipped with magnetic micro-pressure strips 3 on both sides. The PVA mixture solution was then dried in an oven to form a micro-lubricating layer, reducing demolding damage. Simultaneously, the short edge of the second glass plate 2 had a 1mm diameter circular hole connected to a medical three-way valve 7, allowing for the injection of 0.1mL~0.2mL of air after curing, creating a peeling initiation point between the gel and the glass, achieving low-stress demolding of the ultra-thin hydrogel. A 10μm~13μm thick PET film 4 was cut into a U-shape slightly smaller than the glass plate size, 9.8cm×7.2cm, with micro-notches pre-cut at the edges to facilitate the release of excess air and prevent film bulging caused by the glass plates clamping. The first glass plate 1 and the second glass plate 2 were aligned and covered, with initial pre-pressing achieved through magnetic attraction. This prevented misalignment leading to glue leakage and excessive localized stress on the glass plates caused by manual pressure, which could deform the PET film 4 and result in uneven gel thickness. The first glass plate 1 and the second glass plate 2, which have been covered, are placed horizontally in the clamping device to avoid uneven glue formation caused by gravity drooping due to verticality; the spring plunger 6 is adjusted 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.

[0054] The preparation method of the above-mentioned low stiffness ultrathin hydrogel is as follows: the above hydrogel solution is gently stirred and mixed with a glass rod, and the mixed solution is quickly added to the double-layer glass sandwich gel-making device using a pipette to ensure uniform distribution of the solution and avoid the generation of air bubbles. The solution is placed in an oven at 25℃~50℃ for 2h~12h to obtain the hydrogel.

[0055] The above-mentioned high-rigidity ultrathin hydrogel preparation method is as follows: The high-rigidity hydrogel solution is placed in an ultrasonic instrument and ultrasonically dispersed at 50W~80W for 10min~15min to avoid gelation and clumping; after ultrasonication, 0.2%~0.6% v / v APS solution and 0.1%~0.2% v / v TEMED are added to the above mixed solution. After stirring evenly with a glass rod, the mixed solution is quickly added to a double-layer glass sandwich gel-forming device using a pipette to ensure uniform distribution of the solution and avoid the generation of bubbles. The solution is then placed at room temperature for 2h~4h; the solidified hydrogel is immersed in 1%~5% w / v CaCl2 solution for 30min~60min; the hydroxyl-OH groups on the CNC surface form hydrogen bonds with the amide groups -CONH2 in the polyacrylamide hydrogel to restrict chain segment movement, while the carboxyl groups -COOH of sodium alginate form ionic crosslinks in the presence of Ca²⁺, increasing the crosslinking density, thereby obtaining a high-rigidity hydrogel.

[0056] The above ultrathin hydrogel was sterilized as follows: the hydrogel was completely immersed in phosphate buffer solution; it was placed in a constant temperature shaker at 4°C, and the shaker speed was set to 50 rpm to 100 rpm, and the hydrogel was slowly shaken and soaked for 5 min to 10 min; the above soaking and washing steps were repeated 3 to 5 times, and fresh PBS solution was replaced each time; the washed ultrathin hydrogel was placed in a low temperature steam sterilizer, and the formaldehyde concentration was set to 1.5 g / m³ to 2 g / m³, the temperature to 55°C to 60°C, the humidity to 75% to 80%, and the hydrogel was sterilized by low temperature steam for 20 min to 30 min to obtain sterile ultrathin hydrogel.

[0057] Surface functionalization modification method: Using a 10mm diameter corneal trephine, the above-mentioned sterile ultrathin hydrogel was ringed into independent scaffold discs and laid flat in a 48-well cell culture plate; the surface of the sterile ultrathin hydrogel was gently rinsed with sterile PBS to remove any possible residual impurities; using a sterile pipette, 0.01%~0.02% w / v poly-L-lysine solution was aspirated and soaked for 5min~10min, followed by washing three times with sterile PBS, 5min~10min each time; 100mg~200mg of [the solution was then used]. Sulfo-SANPAH powder was dissolved in 50-100 mL of sterile PBS to prepare a sulfo-SANPAH solution with a concentration of 1-2 mg / mL. Using a sterile pipette, 30-50 μL of the sulfo-SANPAH solution was evenly added to the surface of the hydrogel and gently spread with a sterile spatula until it completely covered the hydrogel surface. The solution was allowed to stand for 3-5 minutes. Subsequently, the 48-well cell culture plate containing the hydrogel was placed under a 320-420 nm UV lamp for UV cross-linking at a distance of 10-15 cm for 5-10 minutes. Wash three times with sterile PBS for 5-10 minutes each time. Poly-L-lysine enhances the crosslinking efficiency of sulfo-SANPAH by providing additional amino groups, solving the problems of poor coating stability and low functionalization efficiency in existing modification methods. Then, remove the PBS and use a sterile pipette to draw 200-300 μL of the special modification solution A, and place it in a 37°C incubator for 8-12 hours. Remove the modified hydrogel from the 37°C incubator, remove the special modification solution A, and wash three times with sterile PBS, replacing the PBS every 3-5 minutes. The functionalized ultrathin hydrogel is then obtained.

[0058] The formulation of the above-mentioned special modification solution A is as follows: Weigh 5 mg~10 mg of type IV collagen lyophilized powder, 2 mg~4 mg of type VIII collagen lyophilized powder, 1 mg~2 mg of fibronectin lyophilized powder, and 0.5 mg~1 mg of laminin lyophilized powder, and dissolve them in 50 mL~100 mL of sterile PBS; prepare a mixed solution containing 0.05 mg / mL~0.1 mg / mL type IV collagen, 0.02~0.08 mg / mL type VIII collagen, 0.01~0.04 mg / mL fibronectin, and 0.005~0.02 mg / mL laminin. The mass percentages of each substance in the extracellular matrix components 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 is supplemented to 100%.

[0059] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0060] The list of abbreviations for this invention is shown in Table 1.

[0061] Table 1 List of Abbreviations

[0062]

[0063] The collagen freeze-dried powder involved in this invention originates from the following sources:

[0064] Type IV collagen freeze-dried powder: Sigma, Beijing Innocare Technology Co., Ltd., C5533.

[0065] Fibronectin freeze-dried powder: Sigma, Beijing Innocare Technology Co., Ltd., F2006-2MG.

[0066] Adhesion protein lyophilized powder: Sigma, Beijing Innocare Technology Co., Ltd., L6274.

[0067] Type VIII collagen lyophilized powder was obtained by purifying porcine corneal posterior elastic lamina. The preparation method is as follows:

[0068] Pig eyes were obtained from the slaughterhouse. The cornea was peeled off the eyeball, and then the posterior lamellar membrane was peeled off with forceps. The membrane was digested with 0.5 mg / ml pepsin in 0.5 mol / L acetic acid for 12 h and centrifuged. The supernatant was lyophilized and then dissolved in 1 mol / L NaCl and 50 mmol / L Tris at pH 7.5. Collagen was separated from other proteins by gradient precipitation in 4 mol / L, 0.7 mol / L, and 1.5 mol / L NaCl, with each precipitation followed by dialyzing with 0.5 mol / L acetic acid. The final separation of type VIII collagen from contaminated type V collagen was achieved by chromatography on an agarose A1.5-m column.

[0069] Example 1

[0070] The preparation method of the hydrogel for evaluating the function of corneal endothelial seed cells is as follows:

[0071] 1.1 Preparation of unmodified 50 kPa hydrogel: Acrylamide and N,N'-methylenebisacrylamide were dissolved in water to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, respectively. After mixing, a polyacrylamide premix was obtained. A coagulant was added to the polyacrylamide premix to obtain a hydrogel solution. An unmodified 50 kPa hydrogel was prepared using the hydrogel solution.

[0072] Preparation of reaction solutions: Measure 70 mL of ultrapure water, add 30 g of acrylamide monomer powder, and stir slowly until completely dissolved to obtain an acrylamide monomer solution; measure 98 mL of ultrapure water, add 2 g of N,N'-methylenebisacrylamide powder, and stir slowly until completely dissolved to obtain an N,N'-methylenebisacrylamide solution; measure 75 mL of ultrapure water, add 25 g of ammonium persulfate powder, and stir slowly until completely dissolved to obtain an ammonium persulfate solution.

[0073] Pretreatment adhesive preparation device: Use a pipette to draw a v / v 10% glycerol aqueous solution and a v / v 2% PVA mixed solution, and evenly drop it onto the surface of the first glass plate 1 and the second glass plate 2. Then spread it evenly with a coater. After standing for 7 minutes, place the first glass plate 1 and the second glass plate 2 in a 55℃ oven to dry. After drying, take out the first glass plate 1 and the second glass plate 2. Place a 12μm thick U-shaped PET film 4 tightly against the first glass plate 1 coated with glycerol and PVA. Then align and cover it with the second glass plate 2. Initial pre-pressing is completed 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. The assembly of the double-layer glass interlayer adhesive preparation device is completed.

[0074] Preparation of unmodified 50 kPa hydrogel: The acrylamide monomer solution prepared above was mixed with N,N'-methylenebisacrylamide solution at a volume ratio of 14.3:1 to obtain a polyacrylamide premix. Then, 0.4% v / v ammonium persulfate solution and 0.1% v / v TEMED were added. The mixture was gently stirred with a glass rod and then quickly added to the double-layer glass sandwich gel casting device to ensure uniform distribution and avoid air bubble formation. After gel casting, the mixture was placed in a 37°C oven for 4 hours. After curing, 0.15 mL of air was injected through a valve to form a peeling initiation point between the gel and the glass. The hydrogel was gently lifted from both sides with tweezers and slowly demolded to avoid damaging the hydrogel structure. The stress-strain curve of the obtained ultrathin hydrogel was measured by a universal testing machine to confirm its stiffness of 50 kPa.

[0075] 1.2 Sterilization and surface functionalization modification.

[0076] 1.2.1 Low-temperature steam sterilization.

[0077] Using tweezers, gently remove the unmodified 50 kPa hydrogel from the gel casting apparatus to avoid damaging the hydrogel structure. Gently spread the hydrogel evenly in a culture dish, ensuring its surface is smooth. Add phosphate-buffered saline (PBS) to the culture dish, ensuring the hydrogel is completely submerged. Transfer the culture dish to a 4°C constant-temperature shaker, set the shaker speed to 100 rpm, and slowly shake for 5 minutes. Repeat the above soaking and washing steps three times, replacing the PBS solution each time, to ensure thorough removal of any remaining unreacted monomers and catalysts from the hydrogel. Place the washed hydrogel in a low-temperature steam sterilizer, set the formaldehyde concentration to 2 g / m³, the temperature to 60°C, and the humidity to 80%, and sterilize with low-temperature steam for 20 minutes to obtain a 50 kPa sterile ultrathin hydrogel.

[0078] 1.2.2 Preparation of crosslinking agent.

[0079] Weigh 100 mg of sulfo-SANPAH powder into a sterile centrifuge tube, add 100 mL of pH 7.4 sterile PBS solution, and vortex at low speed for 5 min to fully dissolve the powder, thus obtaining 1 mg / mL sulfo-SANPAH.

[0080] 1.2.3. Prepare a special modification solution A.

[0081] Weigh 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, and dissolve them in 100 mL of sterile PBS at pH 7.4. After mixing by inverting the container, place it in a constant temperature incubator at 37°C for 4 h until the flocculent precipitate is completely dissolved, thus obtaining 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.

[0082] 1.2.4. Add poly-L-lysine as an anchoring point for the cross-linking agent.

[0083] Subsequently, using a 10mm diameter corneal trephine, the aforementioned 50kPa sterile ultrathin hydrogel was ringed into independent scaffold discs. These discs were then gently lifted with forceps and laid flat in 48-well cell culture plates, ensuring a smooth surface. The surface was gently rinsed with 300μL of sterile PBS using a sterile pipette to remove any remaining impurities. A 0.02% w / v poly-L-lysine solution was then used for immersion in the solution for 10 min, followed by washing three times with sterile PBS for 10 min each time. The poly-L-lysine solution serves as an anchoring site for subsequent sulfo-SANPAH, ensuring uniform distribution of the cross-linking agent on the substrate, thereby more efficiently capturing target molecules such as fibronectin and adhesion proteins.

[0084] 1.2.5. Sulfo-SANPAH is crosslinked onto the unmodified hydrogel via ultraviolet crosslinking to obtain a crosslinked hydrogel. Extracellular matrix components are added to the crosslinked hydrogel, and the N-hydroxysuccinimide ester groups activated by the crosslinking agent 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.

[0085] Remove the PBS, and use a sterile pipette to draw 50 μL of sulfo-SANPAH solution and evenly drop it onto the surface of the hydrogel. Gently spread the solution with a sterile spatula to ensure even coverage of the hydrogel surface, avoiding excessive or insufficient application in any area. After spreading evenly, let it stand for 5 minutes to allow the solution to fully wet the hydrogel surface. Then, place the 48-well cell culture plate containing the hydrogel under a 365 nm UV lamp for UV cross-linking at a distance of 10 cm for 10 minutes. Avoid moving the hydrogel during irradiation to ensure uniform illumination. Afterward, wash three times with sterile PBS for 5 minutes each time, replacing the PBS solution each time to remove unreacted cross-linking solution. After washing, remove the PBS and use a sterile pipette to add 200 μL of the special modification solution A to the crosslinked hydrogel. Incubate at 37°C for 12 h. After the chemical crosslinking is complete, remove the modified hydrogel from the 37°C incubator, remove the special modification solution A, and wash it three times with sterile PBS, replacing the PBS every 3 min. The functionalized ultrathin hydrogel is then obtained. Transfer the treated hydrogel to a sterile container and store it at 4°C.

[0086] Example 2

[0087] The preparation method of the hydrogel for evaluating the function of corneal endothelial seed cells is as follows:

[0088] 1.1 Preparation of unmodified 20 kPa hydrogel: Acrylamide and N,N'-methylenebisacrylamide were dissolved in water to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, respectively. After mixing, a polyacrylamide premix was obtained. A coagulant was added to the polyacrylamide premix to obtain a hydrogel solution. An unmodified 20 kPa hydrogel was prepared using the hydrogel solution.

[0089] Preparation of reaction solutions: Measure 70 mL of ultrapure water, add 30 g of acrylamide monomer powder, and stir slowly until completely dissolved to obtain an acrylamide monomer solution; measure 99 mL of ultrapure water, add 1 g of N,N'-methylenebisacrylamide powder, and stir slowly until completely dissolved to obtain an N,N'-methylenebisacrylamide solution; measure 80 mL of ultrapure water, add 20 g of ammonium persulfate powder, and stir slowly until completely dissolved to obtain an ammonium persulfate solution.

[0090] Pretreatment adhesive preparation device: Use a pipette to draw a 10% v / v glycerol aqueous solution and a 2% v / v PVA mixed solution, and evenly drop it onto the surface of the first glass plate 1 and the second glass plate 2. Then spread it evenly with a coater. After standing for 10 minutes, place the first glass plate 1 and the second glass plate 2 in a 60℃ oven to dry. After drying, take out the first glass plate 1 and the second glass plate 2. Place a 10μm thick U-shaped PET film 4 tightly against the first glass plate 1 coated with glycerol and PVA. Then align and cover it with the second glass plate 2. Initial pre-pressing is completed 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. The assembly of the double-layer glass interlayer adhesive preparation device is completed.

[0091] Preparation of unmodified 20 kPa hydrogel: The acrylamide monomer solution obtained above was mixed with N,N'-methylenebisacrylamide solution at a volume ratio of 33.3:1 to obtain a polyacrylamide premix. Then, 0.2% v / v ammonium persulfate solution and 0.1% v / v TEMED were added. The mixture was stirred with a glass rod and then quickly added to the double-layer glass sandwich gel casting device to ensure uniform distribution and avoid bubble generation. After gel casting, the mixture was placed in a 25°C oven for 12 hours. After curing, 0.1 mL of air was injected through a valve to form a peeling initiation point between the gel and the glass. The hydrogel was gently lifted from both sides with tweezers and slowly demolded to avoid damaging the hydrogel structure. The stress-strain curve of the obtained ultrathin hydrogel was measured by a universal testing machine to confirm its stiffness of 20 kPa.

[0092] 1.2 Sterilization and surface functionalization modification.

[0093] The method is exactly the same as that in Example 1.

[0094] Example 3

[0095] The preparation method of the hydrogel for evaluating the function of corneal endothelial seed cells is as follows:

[0096] 1.1 Preparation of unmodified 10MPa hydrogel: Acrylamide and N,N'-methylenebisacrylamide were dissolved in water to obtain an acrylamide monomer solution and an N,N'-methylenebisacrylamide solution, respectively. After mixing, a polyacrylamide premix was obtained. A coagulant and a stiffening agent were added to the polyacrylamide premix to obtain a hydrogel solution. An unmodified 10MPa hydrogel was prepared using the hydrogel solution.

[0097] Preparation of the reaction solution: Weigh 40g of acrylamide monomer powder and dissolve it in 60mL of ultrapure water to prepare a 40% (w / w) acrylamide monomer solution; weigh 2g of N,N'-methylenebisacrylamide powder and dissolve it in 98mL of ultrapure water to prepare a 2% (w / w) N,N'-methylenebisacrylamide solution; mix the acrylamide monomer solution and the N,N'-methylenebisacrylamide solution at a volume ratio of 1.1:1. Then, weigh CNC powder at a mass-to-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% (w / w) CNC mixed solution. Finally, add sodium alginate powder to the CNC mixed solution at a mass-to-volume ratio of 2g:97mL to obtain a final mixed solution containing CNC and sodium alginate.

[0098] Pretreatment adhesive preparation device: Use a pipette to draw a 5% v / v glycerol aqueous solution and a 1% v / v PVA mixed solution, and evenly drop it onto the surface of the first glass plate 1 and the second glass plate 2. Then spread it evenly with a coater. After standing for 5 minutes, place the first glass plate 1 and the second glass plate 2 in a 50℃ oven to dry. After drying, take out the first glass plate 1 and the second glass plate 2. Place a 13μm thick U-shaped PET film 4 tightly against the first glass plate 1 coated with glycerol and PVA. Then align and cover it with the second glass plate 2. Initial pre-pressing is completed 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, thus completing the assembly of the double-layer glass interlayer adhesive preparation device.

[0099] Preparation of unmodified 10MPa hydrogel: A mixed solution containing CNC and sodium alginate was ultrasonically dispersed at 80W for 10 min in an ultrasonic instrument. After ultrasonication, 0.2% v / v ammonium persulfate solution and 0.2% v / v TEMED were added to the mixed solution. After stirring evenly with a glass rod, the mixed solution was quickly added to a double-layer glass sandwich gel casting device using a pipette to ensure uniform distribution of the solution and avoid the generation of air bubbles. The solution was left at room temperature for 2 h. The cured hydrogel was then immersed in 5% w / v CaCl2 solution for 60 min. After curing, 0.2 mL of air was injected through a valve to form a peeling initiation point between the gel and the glass. The hydrogel was gently lifted from both sides with tweezers and slowly demolded to avoid damaging the hydrogel structure. The stress-strain curve of the obtained ultrathin hydrogel was measured by a universal testing machine to confirm its stiffness of 10 MPa.

[0100] 1.2 Sterilization and surface functionalization modification.

[0101] The method is exactly the same as that in Example 1.

[0102] Example 4

[0103] The application of hydrogels for evaluating the function of corneal endothelial seed cells was verified by examining the hydrogels prepared in Examples 1-3, as follows:

[0104] Using a BOSE ElectroForce 3100 testing system, with a thickness of 10 μm to 13 μm and a stiffness range of 0.1 MPa to 10 MPa required for corneal tissue as a benchmark, the elastic modulus of the prepared hydrogel scaffold was measured, and stress-strain curves were plotted. The results showed that an ultrathin hydrogel with an elastic modulus of 50 kPa could be obtained using the preparation method and solution ratio in Example 1; an ultrathin hydrogel with an elastic modulus of 20 kPa could be obtained using the preparation method and solution ratio in Example 2; and an ultrathin hydrogel with an elastic modulus of 10 MPa could be obtained using the preparation method and solution ratio in Example 3. The prepared ultrathin hydrogel scaffold not only conforms to 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. See [see details]. Figure 2 .

[0105] Hydrogel scaffolds of varying hardness were placed on white paper printed with the letter "A," and then observed and photographed. The results showed that these hydrogels were uniformly transparent, with smooth surfaces and high transparency. Transmittance analysis revealed no significant difference in transmittance among the hydrogel scaffolds of different hardnesses within the visible light range of 400 nm to 800 nm, and all exhibited transmittance exceeding 90%, demonstrating excellent light transmission performance. (See results below.) Figure 3 .

[0106] The viability of HCE cells on hydrogel scaffolds with different stiffnesses (20 kPa, 50 kPa, and 10 MPa) was detected using the MTT assay kit. The results showed that the viability of HCE cells was regulated by the stiffness of the scaffolds, and gradually increased with increasing scaffold stiffness. (See attached table). Figure 4 .

[0107] HCE cells were seeded onto hydrogel scaffolds with different stiffnesses (20 kPa, 50 kPa, and 10 MPa), and the effect of different scaffold stiffnesses on HCE cell proliferation was observed using EdU staining. The results showed that the cell proliferation rate gradually increased with increasing scaffold stiffness, exhibiting a stiffness-dependent relationship. (See attached table for details.) Figure 5 .

[0108] The energy production levels of HCE cells cultured on hydrogel scaffolds of different stiffness were assessed. The results showed that the ATP content of the cells significantly increased with increasing scaffold stiffness, indicating that the energy production level of the cells was significantly enhanced with increasing scaffold stiffness. See below for details. Figure 6 .

[0109] Furthermore, it should be noted that the preparation methods for various solutions of this invention are as follows:

[0110] 1. Preparation of acrylamide monomer solution: Place a beaker on a magnetic stirrer, add a magnetic stir bar, measure 60mL~70mL of ultrapure water with a graduated cylinder, add 30g~40g of acrylamide monomer powder, turn on the stirrer, and stir slowly until the acrylamide monomer powder is completely dissolved. This is the acrylamide monomer solution.

[0111] 2. Preparation of N,N'-methylenebisacrylamide solution: Place a beaker on a magnetic stirrer, add a magnetic stir bar, measure 98mL~99mL of ultrapure water with a graduated cylinder, add 1g~2g of N,N'-methylenebisacrylamide powder, turn on the stirrer, and stir slowly until the N,N'-methylenebisacrylamide powder is completely dissolved. This is the N,N'-methylenebisacrylamide solution.

[0112] 3. Preparation of ammonium persulfate solution: Place the beaker on a magnetic stirrer, add the magnetic stir bar, measure 75mL~80mL of ultrapure water with a graduated cylinder, add 20g~25g of ammonium persulfate powder, turn on the stirrer, and stir slowly until the ammonium persulfate powder is completely dissolved. This is the ammonium persulfate solution.

[0113] 4. Preparation of sulfo-SANPAH solution: Weigh 50mg~100mg of sulfo-SANPAH powder, transfer the weighed sulfo-SANPAH to a sterile centrifuge tube, add 50mL~100mL of sterile PBS solution, and vortex at low speed for 3min~5min to fully dissolve the sulfo-SANPAH, which is the sulfo-SANPAH solution.

[0114] 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; after mixing by inverting, place in a 37℃ constant temperature incubator for 2h~4h until the flocculent precipitate is completely dissolved, which is the special modification solution A.

[0115] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0116] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A hydrogel for evaluating the function of corneal endothelial seed cells, characterized by, The hydrogel is prepared by the following method: Acrylamide and N,N'-methylene bisacrylamide are dissolved in water respectively to obtain an acrylamide monomer solution and an N,N'-methylene bisacrylamide solution, and a polyacrylamide premix is obtained after mixing; A coagulant and a rigidity enhancer are added to the polyacrylamide premix to obtain a hydrogel solution; The rigidity enhancer is cellulose nanocrystals and sodium alginate; An unmodified hydrogel with a thickness of 10-13 μm is prepared using the hydrogel solution; A crosslinking agent is added to the unmodified hydrogel, so that the crosslinking agent is crosslinked to the unmodified hydrogel to obtain a crosslinked hydrogel; An extracellular matrix component is added to the crosslinked hydrogel, so that the N-hydroxysuccinimide ester group of the activated crosslinking agent reacts with the primary amino group of the protein in the extracellular matrix component to form an amide, and then the extracellular matrix component is covalently coupled to provide specific adhesion sites for corneal endothelial seed cells, thereby obtaining the hydrogel.

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

3. The hydrogel of claim 2, wherein, The mass fraction of type IV collagen, type VIII collagen, fibronectin, and laminin in the extracellular matrix component is as follows: The mass fraction of type VIII collagen is 23.5%-33.3%; The mass fraction of fibronectin is 11.7%-16.7%; The mass fraction of laminin is 5.9%-8.3%, Type IV collagen makes up the remaining 100%.

4. The hydrogel of claim 1, wherein, The coagulant is ammonium persulfate and tetramethyl ethylenediamine; The ammonium persulfate is added to the polyacrylamide premix in the form of a 20%-25% ammonium persulfate solution, and the addition amount of the ammonium persulfate solution is 0.2%-0.6% of the volume of the polyacrylamide premix; The addition amount of tetramethyl ethylenediamine is 0.1%-0.2% of the volume of the polyacrylamide premix.

5. The hydrogel of claim 1, wherein, The addition amount of cellulose nanocrystals is 1%-5% wt; The addition amount of sodium alginate is 0.1%-0.2% wt.

6. The hydrogel of claim 1, wherein, The crosslinking agent is a sulfonated tricyclic aromatic hydrocarbon.

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

8. Use according to claim 7, wherein the compound is ###0002### The cell function is cell proliferation and cell metabolic capacity.

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