Preparation method and application of a retinal neurovascular unit in vitro 3D printing model

Abstract

The application provides a preparation method and application of a retinal nerve vascular unit in-vitro 3D printing model, and relates to the field of biomedical technology, and comprises the following steps: preparing a composite hydrogel precursor solution by using GelMA, HAMA and LAP; selecting RECs, RGCs and RMCs in an exponential growth phase, respectively resuspending after trypsin digestion, mixing the three single-cell suspensions to obtain a cell mixture; mixing the composite hydrogel precursor solution and the cell mixture to obtain a biological ink; printing and packaging the biological ink according to preset parameters to form a preliminary 3D-RNVU model; and culturing the preliminary 3D-RNVU model again. The 3D-RNVU model constructed in the application realizes the reproduction of the physical and chemical properties of ECM and the neural-glial-vascular three-way interaction in a three-dimensional structure, and provides an in-vitro platform with high physiological correlation for mechanism research and drug screening of diseases such as diabetic retinopathy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing and applying an in vitro 3D printed model of a retinal neurovascular unit. Background Technology

[0002] The retinal neurovascular unit (RNVU) is composed of neurons (bipolar cells, ganglion cells, amacrine cells, and horizontal cells), glial cells (Müller cells, astrocytes, and microglia), vascular cells (endothelial cells and pericytes), and the basement membrane, playing a crucial role in retinal metabolism and visual signal transmission. RNVU homeostasis imbalance is closely related to various major retinal diseases, including diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion. Given the difficulty of observing changes in the retinal microenvironment in real-time, non-invasively, and at the single-cell level in in vivo studies, in vitro models have become an important tool for simulating the pathological states of various retinal vascular diseases and elucidating the mechanisms of RNVU functional decompensation under various pathological conditions.

[0003] Currently, the main methods for in vitro research on RNVU involve two-dimensional culture of two cell types, which can be divided into direct or indirect co-culture methods. Cells derived from humans, rats, mice, and monkeys are commonly used in these studies. Depending on the research objective, the target cells are primarily retinal microvascular endothelial cells (RECs) and retinal ganglion cells (RGCs).

[0004] Direct co-culture involves directly seeding RECs and RGCs into the same culture system. Indirect co-culture, typically exemplified by the Transwell assay, involves taking RGCs and RECs in logarithmic growth phase and seeding them in the upper (RGC) and lower (REC) chambers of a Transwell cell line in a specific ratio. After 24 hours of culture, a "vascular-neural" binary system is formed. These methods have played a crucial role in early mechanistic exploration due to their simplicity and low cost. However, its inherent limitations significantly restrict the depth and reliability of research: First, when cells grow on rigid two-dimensional culture surfaces, their cell polarity, cytoskeleton arrangement, and expression patterns of signaling molecules (such as tight junction proteins and vascular endothelial growth factor receptors) differ significantly from those in the in vivo three-dimensional microenvironment, making it difficult to simulate real cell-cell interactions. Second, with the deepening research into the pathogenesis of retinal diseases (especially diabetic retinopathy), researchers have gradually realized that the functional decompensation of RNVUs is a complex dynamic process involving multiple cell types and pathways: for example, Müller cells (RMCs), as the main glial supporting cells of the retina, maintain neuronal survival by regulating potassium ion buffering and glutamate uptake, while simultaneously working with endothelial cells to maintain the blood-retinal barrier. Existing models, lacking functional integration of key components such as RMCs, are insufficient to meet the research needs of revealing the overall functional network and decompensation mechanisms of RNVUs. Therefore, establishing a novel, more physiologically accurate three-dimensional co-culture model is urgently needed.

[0005] In view of the limitations of current in vitro models, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical challenges in simulating retinal neurovascular units (RNVUs) in vitro, this invention provides a method for preparing and applying a 3D-printed in vitro model of the retinal neurovascular unit (3D-RNVU). The 3D-RNVU model constructed in this invention offers significant advantages over traditional 2D systems. In terms of cell interaction, this invention upgrades from planar contact between cells to a three-dimensional network of information and transportation. Regarding the breadth of research content, this invention expands from two-cell interactions to three cell systems: nerve, glial, and vascular. In terms of microenvironment simulation, this invention elevates the simulation from rigid substrates such as culture dishes to a biomimetic microenvironment of the retinal extracellular matrix (ECM).

[0007] The construction of this model provides an in vitro research platform that is closer to the physiological state for the study of the pathogenesis of diabetic retinopathy and other retinal vascular diseases and for drug screening, showing great application potential in the study of pathogenesis and drug development.

[0008] Technical solution:

[0009] This invention provides a method for preparing an in vitro 3D printed model of a retinal neurovascular unit, comprising the following steps:

[0010] S1. Prepare composite hydrogel precursor solutions using GelMA, HAMA and LAP;

[0011] S2. Select cells in the exponential growth phase, digest them with trypsin, and then resuspend them to obtain three single-cell suspensions. Then, mix the three single-cell suspensions to prepare a cell mixture.

[0012] S3. Mix the composite hydrogel precursor solution and the cell mixture to obtain bio-ink;

[0013] S4. The bio-ink is printed and packaged according to preset parameters, and the photocrosslinking reaction of GelMA and HAMA is initiated by ultraviolet light to form a preliminary 3D-RNVU model.

[0014] S5. The preliminary 3D-RNVU model is cultured again to obtain a 3D-RNVU model.

[0015] Furthermore, the specific steps for re-cultivating the initial 3D-RNVU model in step S5 are as follows:

[0016] S501. After rinsing the initial 3D-RNVU model with DPBS, aspirate and discard to remove excess and uncured bio-ink.

[0017] S502. Subsequently, DMEM complete medium containing 10% fetal bovine serum was added to the preliminarily rinsed 3D-RNVU model, and the model was cultured in a sterile cell culture incubator at 37°C and 5% CO2 for 72 hours to obtain the 3D-RNVU model.

[0018] Furthermore, in step S1, the mass concentration ratio of GelMA, HAMA and LAP is (1.5-4):(0.5-1):(0.1-0.2).

[0019] Furthermore, the mass concentration ratio of GelMA, HAMA, and LAP in the bio-ink in step S3 is 2:0.5:0.1.

[0020] Furthermore, in step S2, the volume ratio of REC single-cell suspension: RGC single-cell suspension: RMC single-cell suspension is 1:1:1.

[0021] Furthermore, in step S3, the composite hydrogel precursor solution and the cell mixture are mixed at a volume ratio of 1:1.

[0022] The cell concentrations of REC, RGC, and RMC in the bio-ink are all 5 × 10⁶ cells / mL.

[0023] Furthermore, the preset parameters are specifically as follows: using an 8mm diameter circular pattern, a 0.5mm thickness, a 20% ultraviolet light intensity, and a 20s exposure time in a single static printing mode.

[0024] Furthermore, the glucose concentration in the DMEM complete culture medium is 5.5 mM-125 mM.

[0025] Furthermore, the glucose concentration in the DMEM complete culture medium is 50 mM.

[0026] Furthermore, the mass concentration of trypsin used in step S1 is 0.25%.

[0027] The present invention also provides a method for preparing an in vitro 3D printed model of a retinal neurovascular unit to obtain a 3D-RNVU model.

[0028] The present invention also provides the application of a 3D-RNVU model in any of the following: preparing and developing drugs for the treatment and / or prevention of retinal vascular diseases, constructing a biological model of the pathogenesis of diabetic retinopathy, or screening drugs for diabetic retinopathy.

[0029] The present invention has the following technical effects:

[0030] This invention develops a biomimetic 3D model of the retinal neurovascular unit (3D-RNVU). This model utilizes bioactive scaffolds derived from natural polymers such as gelatin methacryloyl (GelMA) and hyaluronic acid methacryloyl (HAMA) to simulate the composition and mechanical properties of the retinal extracellular matrix (ECM), thus providing RNVU cells with a more closely in vivo three-dimensional microenvironment. Simultaneously, RGCs, RECs, and RMCs are mixed with biomaterials in physiological proportions to create a bio-ink, enabling co-culture of the three cells via 3D printing. This establishes a ternary interaction system of nerve-glial-vascular cells, supporting a more complete study of cellular synergistic mechanisms and providing an advanced in vitro research tool for in-depth analysis of the molecular mechanisms of RNVU functional decompensation, screening of targeted therapeutic drugs, and development of regenerative medicine strategies. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 : Schematic diagram of 3D-RNVU model construction;

[0033] Figure 2 Composition and mechanical properties of 3D-RNVU biomaterials;

[0034] Figure 3 The effects of 3D-RNVU biomaterial formulation and printing parameters on cell viability;

[0035] Figure 4 3D-RNVU model live / dead cell staining;

[0036] Figure 5 Comparison of growth morphology between 3D-RNVU multi-cell co-culture system and 3D-single-cell culture;

[0037] Figure 6 3D-RNVU cell-specific labeling and spatial localization imaging;

[0038] Figure 7 The effect of different glucose concentrations on cell viability in a 3D-RNVU model;

[0039] Figure 8 : Reactive oxygen species levels in an in vitro 3D-RNVU diabetic retinopathy model; (A) representative images of DCF fluorescence intensity detected by flow cytometry; (B) quantitative statistics of the average fluorescence intensity of DCF detected by flow cytometry;

[0040] Figure 9 : Changes in mitochondrial membrane potential in an in vitro 3D-RNVU diabetic retinopathy model; (A) Representative images of JC-1 fluorescence detected by flow cytometry; (B) Quantitative statistics of JC-1 monomers detected by flow cytometry. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] First, the present invention provides a method for preparing an in vitro 3D printed model of a retinal neurovascular unit.

[0043] S1. Prepare composite hydrogel precursor solutions using GelMA, HAMA and LAP;

[0044] S2. Select REC, RGC and RMC in the exponential growth phase, digest them with trypsin and resuspend them to obtain three single-cell suspensions. Then mix the three single-cell suspensions to prepare a cell mixture.

[0045] S3. Mix the composite hydrogel precursor solution and the cell mixture to obtain bio-ink;

[0046] S4. The bio-ink is printed and packaged according to preset parameters, and the photocrosslinking reaction of GelMA and HAMA is initiated by ultraviolet light to form a preliminary 3D-RNVU model.

[0047] S5. The preliminary 3D-RNVU model is cultured again to obtain a 3D-RNVU model.

[0048] In this invention, REC, RGC, and RMC are selected as the core components of RNVU. Single-cell suspensions are obtained through trypsin digestion to ensure cell viability and homogeneity. The cell mixture is then combined with a hydrogel precursor solution to form a bio-ink, which is then precisely spatially distributed via 3D printing. Ultraviolet light cross-linking and curing further solidifies the preliminary 3D structure. Subsequent cell culture allows for further adaptation, proliferation, and the establishment of intercellular connections in a three-dimensional environment, promoting functional maturation of the model.

[0049] This preparation method enables the three-dimensional construction of RNVU, breaking through the limitations of traditional 2D models and upgrading from planar interaction to three-dimensional network interaction. At the same time, the photocrosslinked hydrogel scaffold provides a tunable mechanical microenvironment to support cell migration, proliferation and functional expression. It can also simulate the spatial configuration of neuron-glial-vascular units in retinal tissue, which is closer to the physiological state, providing a reliable platform for studying the mechanisms of diseases such as diabetic retinopathy.

[0050] In some embodiments, the specific steps for re-cultivating the initial 3D-RNVU model in step S5 are as follows:

[0051] S501. After rinsing the initial 3D-RNVU model with DPBS, aspirate and discard to remove excess and uncured bio-ink.

[0052] S502. Subsequently, DMEM complete medium containing 10% fetal bovine serum was added to the preliminarily rinsed 3D-RNVU model, and the model was cultured in a sterile cell culture incubator at 37°C and 5% CO2 for 72 hours to obtain the 3D-RNVU model.

[0053] This step involves washing away uncrosslinked bio-ink and cell debris to reduce background interference and ensure model cleanliness. Simultaneously, reculturing the model provides nutrients and growth signals, promoting cell survival and functional maturation. This effectively improves model stability and cell survival rate, avoiding interference from uncrosslinked materials on cell behavior. Furthermore, prolonged culturing allows cells to form more mature cell-cell and cell-matrix interactions within the three-dimensional scaffold, enhancing the model's physiological relevance.

[0054] In some embodiments, the mass concentration ratio of GelMA, HAMA and LAP in step S1 is (1.5-4):(0.5-1):(0.1-0.2).

[0055] In some embodiments, the mass concentration ratio of GelMA, HAMA and LAP in the bio-ink in step S3 is 2:0.5:0.1.

[0056] GelMA mimics the collagen component in the retinal emulsion membrane (ECM) and provides cell adhesion sites, HAMA mimics the hyaluronic acid component in the ECM, and LAP controls the efficiency of photocrosslinking. This ratio balances the stiffness, porosity, and biodegradability of the hydrogel.

[0057] By optimizing the composition ratio of the bio-ink, the hydrogel scaffold is designed to mimic the natural softness and hardness of the inner retinal tissue, forming a three-dimensional network with appropriately sized pores. This ensures sufficient mechanical strength to support the printed structure while maintaining good biocompatibility to allow cell embedding and migration. Furthermore, by strictly controlling the amount of components such as LAP to avoid cytotoxicity, cell survival is guaranteed, and cell proliferation and functional expression are promoted.

[0058] In some embodiments, in step S2, the volume ratio of REC single-cell suspension:RGC single-cell suspension:RMC single-cell suspension is 1:1:1.

[0059] This proportion simulates the relative abundance of the three cell types in RNVU under physiological conditions, ensuring balanced intercellular interactions. Furthermore, RMCs, as glial cells, play a role in connecting neurons and blood vessels in RNVU, and their inclusion compensates for the shortcomings of traditional models that only focus on RECs and RGCs. This system can more comprehensively simulate RNVU function and avoid the over-dominance of any one cell type, improving the predictive accuracy of the model in drug screening.

[0060] In some embodiments, the composite hydrogel precursor solution and the cell mixture are mixed in a volume ratio of 1:1 in step S3;

[0061] The cell concentrations of REC, RGC, and RMC in the bio-ink are all 5 × 10⁻⁶. 6 per mL.

[0062] This formulation ensures uniform distribution of hydrogel and cells, and moderate viscosity of the bio-ink, thereby guaranteeing excellent printability and cell activity of the bio-ink, and maintaining a high level of cell survival rate after printing.

[0063] In some embodiments, the preset parameters are specifically: using an 8mm diameter circular pattern, a 0.5mm thickness, a 20% UV light intensity, and a 20s exposure time in a single static printing mode.

[0064] This thickness is suitable for standard culture dish operation and allows for sufficient light penetration and nutrient diffusion. The 20% UV light intensity and 20s exposure time balance crosslinking efficiency and cell protection, preventing insufficient crosslinking due to too low light intensity or potential DNA damage due to too high light intensity, thus ensuring the stability of the hydrogel scaffold.

[0065] The single-shot static printing mode ensures structural uniformity and repeatability, resulting in consistent 3D model structures with good reproducibility, making it easier for high-throughput experiments.

[0066] In some embodiments, the glucose concentration in the DMEM complete medium is 5.5 mM-125 mM.

[0067] In some embodiments, the glucose concentration in the DMEM complete medium is 50 mM.

[0068] In some embodiments, the mass concentration of trypsin used in step S1 is 0.25%.

[0069] Secondly, the present invention also provides a 3D-RNVU model prepared by the above-mentioned method for preparing an in vitro 3D printed model of a retinal neurovascular unit.

[0070] Thirdly, the present invention also provides the application of the above-mentioned 3D-RNVU model in any one of the following: preparing and developing drugs for the treatment and / or prevention of retinal vascular diseases, constructing a biological model of the pathogenesis of diabetic retinopathy, or screening drugs for diabetic retinopathy.

[0071] The following is a detailed explanation using specific embodiments:

[0072] Example 1: In vitro three-dimensional neurovascular unit culture model of the retina

[0073] The rat RECs used in the following examples were purchased from Shanghai Xuanya Biotechnology Co., Ltd.

[0074] RGCs from rats were purchased from Shanghai Xuanya Biotechnology Co., Ltd.

[0075] RMCs of rats were purchased from Shanghai Bosen Biotechnology Co., Ltd.

[0076] GelMA and HAMA were purchased from Shanghai Yuju Technology Co., Ltd., and LAP was purchased from Shanghai Huaxia Siyin Biotechnology Co., Ltd.

[0077] Dulbecco's Modified Eagle Medium (DMEM medium, with sugar contents of 1 g / L and 4.5 g / L, where the 1 g / L DMEM medium is the 5.5 mM low-sugar DMEM medium, and the 4.5 g / L DMEM medium is used to prepare the high-sugar DMEM medium) was purchased from Gibco.

[0078] Fetal bovine serum (FBS) was purchased from Gibco;

[0079] L-Glucose powder was purchased from AbMole;

[0080] 0.25% trypsin (% refers to mass ratio, i.e., g / 100mL) was purchased from Gibco;

[0081] CellTiter-Glo 3D (CTG) assay kits were purchased from Promega;

[0082] CytoTrace Red CMTPX and CellTracker Green CMFDA reagents were purchased from CYTOCH, and CellTracker Blue CMAC was purchased from AbMole.

[0083] The cell viability (live and dead cell staining) assay kit was purchased from MeilunBio;

[0084] 96-well and 24-well cell culture plates and culture dishes were purchased from Corning, and PBS and DPBS buffer were purchased from GibcoLife Technologies.

[0085] The complete culture medium used in the following examples is 5.5 mM low-sugar DMEM medium containing 10% FBS (% refers to volume ratio).

[0086] To prepare 100 mL of 1M (1000 mM) glucose stock solution: Weigh 18.016 g of glucose and add it to 80 mL of PBS. Stir magnetically until completely dissolved. Make up to 100 mL with PBS, mix well, and filter through a 0.22 μm filter membrane for sterilization. The following are detailed formulations (including serum) for preparing 10 mL target volumes of complete culture media with different glucose concentrations (5.5, 11.1, 25, 50, 75, 100, 125 mM) based on 1M glucose stock solution and DMEM low-glucose (5.5 mM) or high-glucose (25 mM) media, as shown in Tables 1 and 2.

[0087] Table 1: Based on DMEM low-sugar (5.5 mM) (10 mL system)

[0088]

[0089] Table 2: Based on DMEM high sugar (25 mM) (10 mL system)

[0090]

[0091] GelMA, HAMA, and LAP were mixed in proportions to form a photocrosslinkable composite hydrogel precursor solution with concentrations of 4%, 1%, and 0.2%, respectively, and temporarily stored at 37°C in the dark.

[0092] REC, RGC, and RMC cells in the exponential growth phase were selected, digested with 0.25% trypsin, and resuspended as single-cell suspensions. The three cell types were then mixed in a 1:1:1 volume ratio to form a cell mixture, ensuring that the concentration of each cell type was 1×10⁻⁶. 7 Cells / ml. The composite hydrogel precursor solution and the cell mixture were thoroughly mixed at a 1:1 volume ratio to form the bio-ink (the final concentration of each cell type in the final system was 5 × 10⁻⁶). 6 The bioprinting material contains 2% GelMA, 0.5% HAMA, and 0.1% LAP (per ml).

[0093] Using a digital light processing (DLP) bioprinter, the prepared bio-ink was printed and encapsulated in cell culture plates according to preset parameters (20% light intensity, 20s exposure time, 8mm diameter circular pattern, 0.5mm thickness, and single-shot static printing mode). The preliminarily printed 3D-RNVU model was rinsed with an appropriate amount of DPBS for 1-2 minutes and then aspirated to remove excess, uncured bio-ink. 1g / L DMEM complete medium containing 10% fetal bovine serum was added, and the cell culture plates were placed in a sterile cell culture incubator at 37℃ and 5% (v / v) CO2 for cell culture. After 72 hours of culture, the 3D-RNVU model was obtained (see [link to model construction procedure]). Figure 2 (This illustration was generated using the Figdraw scientific drawing tool.)

[0094] Example 2: Diabetic retinopathy model

[0095] Based on Example 1, the complete culture medium (i.e., 5.5 mM low-glucose DMEM medium containing 10% FBS) was replaced with 50 mM high-glucose DMEM medium to achieve high-glucose modeling and obtain a diabetic retinopathy model.

[0096] Comparative Example 1: Three-dimensional in vitro single-culture models of REC, RGC, and RMC

[0097] Based on Example 1, the cell mixture (a 1:1:1 volume ratio of REC, RGC, and RMC cells) was replaced with a REC (or RGC / RMC) single-cell suspension, which was then thoroughly mixed with the composite hydrogel precursor solution at a 1:1 volume ratio to form a bio-ink (with a final concentration of 5 × 10⁻⁶ for single-cell types). 6 The bioprinting material contains 2% GelMA, 0.5% HAMA, and 0.1% LAP per ml.

[0098] Experimental Example 1: Performance Validation of a Three-Dimensional Retinal Neurovascular Unit In Vitro Three-Cultural Model (3D-RNVU Model)

[0099] 1. Experimental Procedure

[0100] 1.1 Influence of the selection of biomaterials on the mechanical properties of the 3D-RNVU model

[0101] GelMA, HAMA, and LAP were mixed in the specified proportions (see Table 3 below). Using a digital light processing (DLP) bioprinter, the prepared biomaterials were printed onto 14mm cell slides according to the preset parameters (light intensity and exposure time as shown in Table 3). After washing with an appropriate amount of DPBS for 1-2 minutes, the sample was discarded. DPBS was added again to cover the sample, and the mixture was incubated overnight at 4°C. The sample was then removed and placed on a hydrogel biomechanical analyzer (EFL-MT-5600, EFL) for compression testing to measure its mechanical properties. The experimental results are shown in [Table 3]. Figure 2 .

[0102] Table 3: Composition ratio of biomaterials and 3D printing parameters

[0103]

[0104] Figure 2 The results showed that under the conditions of bioprinting material ratio of 2% GelMA, 0.5% HAMA, and 0.1% LAP, and bioprinter parameters of 20% light intensity, and bioprinting material ratio of 2% GelMA, 1% HAMA, and 0.1% LAP, and bioprinter parameters of 10% light intensity, the Young's modulus of the printed hydrogel was approximately 3 kPa, which is similar to the mechanical properties of the inner retinal layer.

[0105] Experimental Example 2: Effects of Biomaterial Formulation and Printing Parameters on Cell Viability of 3D-RNVU Models

[0106] Based on Example 1 and Comparative Example 1, the parameters of the bioprinting material and the bioprinter were set as follows:

[0107] Sample 2 contained 2% GelMA, 0.5% HAMA and 0.1% LAP, and was printed under conditions of 20% light intensity and 20s exposure time.

[0108] Sample 3 contained 2% GelMA, 1% HAMA, and 0.1% LAP, and was printed under 10% light intensity and 20s exposure time conditions. After 72 hours of incubation, the cell culture plate and 3D-RNVU model were removed, equilibrated to room temperature for 30 minutes, and an equal volume of CellTiter Glo was added. ® The 3D reagent was placed on a shaker at room temperature, horizontally shaken, mixed, and incubated for 30 minutes. The luminescence signal (emission wavelength range of 360 to 750 nm) was recorded using a microplate reader at room temperature, and relative cell viability was calculated. Experimental results are shown below. Figure 3 .

[0109] Figure 3 The results showed that in the 3D-RNVU model and the single-cell in vitro single-culture three-dimensional model, the cell growth trend was better under the conditions of bioprinting material ratio of 2% GelMA, 0.5% HAMA, 0.1% LAP and bioprinter parameter of 20% light intensity than under the conditions of bioprinting material ratio of 2% GelMA, 1% HAMA, 0.1% LAP and bioprinter parameter of 10% light intensity.

[0110] Experimental Example 3: Validation of Cell Viability in 3D-RNVU Model

[0111] A 3D-RNVU model was constructed based on Example 1. The selected bioprinting materials and parameters were: 2% GelMA, 0.5% HAMA, and 0.1% LAP, printed under conditions of 20% light intensity and 20s exposure time. Following the instructions of the cell viability (live / dead cell staining) assay kit, Calcein-AM Solution and PI Solution were first taken out and equilibrated at room temperature for 30 min. Then, 5µL of PI Solution and 5µL of Calcein-AM Solution were added to 10mL of Assay buffer, and the mixture was vortexed to prepare the working solution. At this point, the concentration of Calcein-AM was 2µM and the concentration of PI was 8µM. The 3D-RNVU model was gently washed 2-3 times with DPBS to remove active esterases from the culture medium. Then, sufficient Calcein-AM / PI staining working solution was added, ensuring that the 3D-RNVU model was completely submerged, and incubated at 37℃ for 15-30 min. Aspirate the staining working solution to terminate incubation. Gently wash cells 2-3 times with DPBS, add sufficient Assay buffer, and then observe live cells (green fluorescence) and dead cells (red fluorescence) using a fluorescence microscope at excitation wavelengths of 490±10 nm and 545 nm. Experimental results are shown below. Figure 4 .

[0112] Figure 4 The results showed that the 3D-RNVU model had extremely high cell viability, with a survival rate of over 90%, confirming the good biocompatibility of the selected materials and printing parameters.

[0113] Experimental Example 5: Cellular Morphological Identification of the Neuro-Glial-Vascular Ternary Interaction in a 3D-RNVU Model

[0114] Cell morphology identification under an optical microscope: Based on Example 1 and Comparative Example 1, the bioprinting materials and bioprinter parameters were selected as follows: 2% GelMA, 0.5% HAMA, and 0.1% LAP, printed under conditions of 20% light intensity and 20s exposure time. The preliminary 3D-RNVU model was cultured for 72 hours, and cell growth morphology was observed under an optical microscope. The experimental results are shown in [Figure number missing]. Figure 5 .

[0115] Cell-specific identification under fluorescence microscopy: The culture medium for three cell types (RGC, REC, and RMC) in the exponential growth phase was aspirated, and CellTracker Blue CMAC, CytoTraceRed CMTPX, and CellTracker Green CMFDA working solutions, prepared according to the manufacturer's instructions, were added respectively. The cells were incubated at 37°C for 45 minutes. The dye working solutions were discarded, and complete culture medium was added for further incubation for 2 hours. Then, based on Example 1, the bioprinting materials and bioprinter parameters were selected as follows: 2% GelMA, 0.5% HAMA, and 0.1% LAP, printed under 20% light intensity and 20s exposure time conditions. The preliminary 3D-RNVU model was cultured for 72 hours, and the growth morphology and relative positions of the three cell types were observed using a laser confocal microscope. The experimental results are shown in [Figure number missing]. Figure 6 .

[0116] Figure 5 and Figure 6 The results showed that the RGC, REC, and RMC in the 3D environment maintained a more similar three-dimensional morphology to those in vivo and formed a spatial mosaic, suggesting the initial formation of a retinal neuron-glial-vascular unit interaction network. In other words, the 3D-RNVU model successfully constructed a multi-cell three-dimensional co-culture system, forming a biomimetic retinal microstructure with physiological topology.

[0117] Experiment Example 6: Effects of different glucose concentrations on the viability of 3D-RNVU model cells

[0118] Based on Example 1, the parameters of the bioprinting material and bioprinter were set to 2% GelMA, 0.5% HAMA, 0.1% LAP, 20% light intensity, and 20s exposure time, respectively; the glucose concentrations of the DMEM complete culture medium were set to 5.5 mM, 11.1 mM, 25 mM, 50 mM, 75 mM, 100 mM, and 125 mM, respectively. After culturing for 72 hours, the cell culture plate and 3D-RNVU model were removed, equilibrated to room temperature for 30 minutes, and an equal volume of CellTiter Glo was added. ® The 3D reagent was placed on a shaker at room temperature and protected from light, horizontally shaken, mixed, and incubated for 30 minutes. The luminescence signal (emission wavelength range of 360 to 750 nm) was recorded using a microplate reader at room temperature, and relative cell viability was calculated. The experimental results are as follows: Figure 7 As shown.

[0119] Figure 7 The results showed that, compared with normal glucose concentration (5.5 mM), the cell viability of the 3D-RNVU model began to decrease when the glucose concentration was increased to 50 mM, suggesting that the 50 mM glucose concentration condition can be used to construct a diabetic retinopathy model.

[0120] Experimental Example 7: Evaluation of a Diabetic Retinopathy Model Prepared Using In Vitro 3D-RNVU

[0121] Based on Examples 1 and 2, the preliminary 3D-RNVU models were cultured for 72 hours. Digestive enzyme working solutions were prepared: containing 2 mg / mL collagenase (Yisheng, Cat No: 40507ES60), 1 mg / mL hyaluronidase (Yisheng, Cat No: 20426ES80), and 0.1 mg / mL DNASE I (Yisheng, Cat No: 10608ES60). Fixation and permeabilization working solutions were prepared: Fixation / Permeabilization Concentrate (eBioscience, Cat No: 00-5123-43) and Fixation / Permeabilization Diluent (eBioscience, Cat No: 00-5223-56) were diluted 1:3 and used immediately; 1X buffer: Permeabilization Buffer (eBioscience, Cat No: 00-8333) was diluted 1:10 with experimental pure water and used immediately. Discard the culture medium from the cell culture plate, rinse three times with DPBS, add 20 times the volume of the 3D-RNVU model digestive enzyme working solution, digest in a 37°C incubator for 10-15 minutes, add an equal volume of complete culture medium to stop digestion, centrifuge to collect cells, discard the supernatant, and perform reactive oxygen species detection or mitochondrial membrane potential detection experiments.

[0122] Reactive oxygen species (ROS) detection assay: Following the instructions of the ROS detection kit (KeyGEN BioTECH, catalog number KGA7308), a 10 µM DCFH-DA loading working solution was prepared using DPBS at a 1:1000 dilution. Cells were resuspended in the DCFH-DA loading working solution and incubated at 37°C for 20 min, inverting every 3-5 min to ensure adequate contact with the cells. The cells were centrifuged at 200-300 × g for 3 min to remove the DCFH-DA working solution. Cells were washed three times with serum-free culture medium to remove any unadulterated DCFH-DA. Flow cytometry was used to detect the ROS at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Experimental results are shown below. Figure 8 .

[0123] Mitochondrial membrane potential detection experiment: Following the instructions of the mitochondrial membrane potential detection kit (KeyGEN BioTECH, catalog number KGA1904), prepare 1×Incubation Buffer: Dilute 100 µL of 10×Incubation Buffer with 900 µL of sterile deionized water to make 1×Incubation Buffer, mix well, and preheat to 37℃. Prepare JC-1 working solution: Take 2 µL of JC-1 (500×), add it to 898 µL of sterile deionized water, vortex vigorously to fully dissolve and mix JC-1, then add 100 µL of 10×Incubation Buffer, mix well, and this is 1 mL of JC-1 working solution. Resuspend the cells evenly in 500 µL of JC-1 working solution, and incubate at 37℃ in a 5% CO2 incubator in the dark for 15-20 min. Cells were collected by centrifugation at room temperature (300×g, 5 min), washed twice with 1× Incubation Buffer, resuspended in 1 mL of 1× Incubation Buffer, centrifuged again, and collected. The cells were then resuspended again by centrifugation with 1 mL of 1× Incubation Buffer. Flow cytometry was performed within 1 h to detect the cells: green fluorescence (Ex = 488 nm, Em = 530 nm); red fluorescence (Ex = 488 nm, Em ≥ 630 nm). Results are shown below. Figure 9 .

[0124] from Figure 8 and Figure 9 The results show that, compared with the control group at normal glucose concentration (5.5 mM), the in vitro 3D-RNVU diabetic retinopathy model exhibited significantly enhanced oxidative stress and mitochondrial dysfunction. Specifically, this was manifested by elevated levels of reactive oxygen species (ROS) accompanied by a decrease in mitochondrial membrane potential (ΔΨm), the latter being an early marker event of apoptosis. These results collectively indicate that the model successfully reproduces key pathological phenotypes in diabetic retinopathy, including oxidative stress, mitochondrial damage, and early apoptosis events.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an in vitro 3D printed model of a retinal neurovascular unit, characterized in that, Includes the following steps: S1. A composite hydrogel precursor solution was prepared using gelatin methacrylate, hyaluronic acid methacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphonate. S2. Select REC, RGC and RMC in the exponential growth phase, digest them with trypsin and resuspend them to obtain three single-cell suspensions. Then mix the three single-cell suspensions to prepare a cell mixture. S3. Mix the composite hydrogel precursor solution and the cell mixture to obtain bio-ink; S4. The bio-ink is printed and packaged according to preset parameters, and a photocrosslinking reaction between gelatin methacrylate and lithium phenyl-2,4,6-trimethylbenzoylphosphonate is initiated by ultraviolet light to form a preliminary 3D-RNVU model. S5. The preliminary 3D-RNVU model is cultured again to obtain a 3D-RNVU model; In step S2, the volume ratio of REC single-cell suspension: RGC single-cell suspension: RMC single-cell suspension is 1:1:

1. The specific steps for re-cultivating the initial 3D-RNVU model in step S5 are as follows: S501. After rinsing the initial 3D-RNVU model with DPBS, aspirate and discard to remove excess and uncured bio-ink. S502. Subsequently, DMEM complete medium containing 10% fetal bovine serum was added to the preliminarily rinsed 3D-RNVU model and cultured in a sterile cell culture incubator at 37°C and 5% CO2 for 72 hours to obtain the 3D-RNVU model. In step S1, the mass concentration ratio of gelatin methacrylate, hyaluronic acid methacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphonate is (1.5-4):(0.5-1):(0.1-0.2). In step S3, the mass concentration ratio of gelatin methacrylate, hyaluronic acid methacrylate, and lithium phenyl-2,4,6-trimethylbenzoylphosphonate in the bio-ink is 2:0.5:0.

1. In step S3, the composite hydrogel precursor solution and the cell mixture are mixed at a volume ratio of 1:

1. The cell concentrations of REC, RGC, and RMC in the bio-ink are all 5 × 10⁻⁶. 6 per mL.

2. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, The preset parameters are as follows: using an 8mm diameter circular pattern, a 0.5mm thickness, 20% UV light intensity, and a 20s exposure time in a single static printing mode.

3. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, The glucose concentration in the DMEM complete medium is 5.5 mM-125 mM.

4. A 3D-RNVU model prepared by a method for preparing an in vitro 3D printed model of a retinal neurovascular unit as described in any one of claims 1-3.

5. The application of the 3D-RNVU model as described in claim 4 in constructing a biological model of the pathogenesis of diabetic retinopathy.

6. The application of the 3D-RNVU model as described in claim 4 in screening drugs for diabetic retinopathy.

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