Preparation method and application of retina nerve and blood vessel unit in-vitro 3D printing model

Abstract

The invention provides a preparation method and application of a retinal nerve and blood vessel unit in-vitro 3D printing model, and relates to the technical field of biomedicines.The preparation method comprises the steps that GelMA, HAMA and LAP are used for preparing a composite hydrogel precursor solution; the method comprises the following steps: selecting REC, RGC and RMC in an exponential growth phase, digesting the REC, RGC and RMC with trypsin respectively, resuspending, and mixing three single-cell suspensions to obtain a cell mixture; mixing the composite hydrogel precursor solution with the cell mixture to obtain biological ink; the bio-ink is printed and packaged according to preset parameters, and a preliminary 3D-RNVU model is formed; and the preliminary 3D-RNVU model is cultured again. According to the constructed 3D-RNVU model, the physical and chemical characteristics of ECM and nerve-colloid-blood vessel ternary interaction are represented in a three-dimensional structure, and an in-vitro platform with high physiological correlation is provided for mechanism research and drug screening of diseases such as diabetic retinopathy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a preparation method and application of a retinal neurovascular unit in vitro 3D printing model. BACKGROUND

[0002] The retinal neurovascular unit (RNVU) is composed of neurons (bipolar cells, ganglion cells, amacrine cells, horizontal cells), glial cells (Muller cells, astrocytes, microglia cells), vascular cells (endothelial cells, pericytes) and basement membrane, and plays an important role in retinal metabolism and visual signal transmission. The steady-state imbalance of RNVU is closely related to various major retinal diseases such as diabetic retinopathy, age-related macular degeneration and retinal vein occlusion. Since in vivo studies are difficult to observe the changes in the retinal microenvironment in real time, non-invasively and at the single cell level, in vitro models have become an important means to simulate the pathological state of various retinal vascular diseases and analyze the functional compensation mechanism of RNVU under various pathological states.

[0003] At present, the method for in vitro research of RNVU is mainly two-dimensional culture of two cells, which can be divided into direct or indirect co-culture method. Cells from humans, rats, mice, monkeys and other sources are commonly used in research. According to different research purposes, the target cells are mainly retinal vascular endothelial cells (Retinal Microvascular Endothelial Cells, REC) and retinal ganglion cells (Retinal Ganglion Cells, RGC).

[0004] Direct co-culture method is to directly inoculate REC and RGC in the same culture system; indirect co-culture method, typically such as Transwell chamber experiment - the specific operation is generally: taking the logarithmic growth period of RGC and REC cells, inoculating in the Transwell upper chamber (RGC) and lower chamber (REC) in proportion, and forming a "blood vessel-neural" binary system after 24 hours of culture. These methods play an important role in early mechanism exploration because of simple operation and low cost. However, their inherent defects significantly limit the depth and reliability of the research: first, when cells grow on the hard two-dimensional culture surface, their cell polarity, cytoskeleton arrangement and expression pattern of signal molecules (such as tight junction proteins, vascular endothelial growth factor receptors, etc.) are obviously different from the three-dimensional microenvironment in vivo, and it is difficult to simulate the real intercellular interaction; second, with the deepening of the research on the pathogenesis of retinal diseases (especially diabetic retinopathy), researchers gradually realize that the functional failure of RNVU is a complex dynamic process involving multiple cell types and multiple pathway interactions: for example, Müller cells (Retinal Müller Cells, RMC) as the main glial support cells of the retina, maintain neuronal survival by regulating potassium ion buffering and glutamate uptake, and cooperate with endothelial cells to maintain the blood-retinal barrier. However, the existing model is difficult to meet the research needs of revealing the overall functional network and failure mechanism of RNVU due to the lack of functional integration of key components such as RMC. Therefore, it is urgent to establish a new three-dimensional co-culture model that is closer to the physiological state.

[0005] In view of the limitations of various in vitro models, the present application is proposed. SUMMARY

[0006] In order to solve the above technical problems of simulating RNVU in vitro, the present application provides a preparation method and application of a retinal neurovascular unit in vitro 3D printing model (3D-RNVU). The 3D-RNVU model constructed by the present application has significant advantages compared to traditional 2D systems. In terms of cell interaction dimension, the present application upgrades from planar contact between cells to three-dimensional network information traffic; in terms of research content, the present application expands from the interaction of two cells to a neural-glial-vascular three-cell system; in terms of microenvironment simulation, the present application improves from rigid substrates such as culture dishes to extracellular matrix (ECM) biomimetic microenvironment.

[0007] The construction of this model provides a more physiological in vitro research platform for the pathogenesis research and drug screening of retinal vascular diseases such as diabetic retinopathy, and shows great application potential in pathogenesis research and drug development.

[0008] Technical scheme: The application provides a preparation method of a retinal nerve vascular unit in vitro 3D printing model, comprising the following steps: S1, preparing a composite hydrogel precursor solution by using GelMA, HAMA and LAP; S2, selecting an exponential growth period, respectively using trypsin digestion and resuspension to obtain three single cell suspensions, and then mixing the three single cell suspensions to prepare a cell mixture; S3, mixing the composite hydrogel precursor solution and the cell mixture to obtain a biological ink; S4, printing and packaging the biological ink according to preset parameters, initiating a photo-crosslinking reaction of GelMA and HAMA by ultraviolet light to form a preliminary 3D-RNVU model; S5, culturing the preliminary 3D-RNVU model again to obtain a 3D-RNVU model.

[0009] Further, the specific steps of culturing the preliminary 3D-RNVU model again in the S5 step are as follows: S501, rinsing the preliminary 3D-RNVU model with DPBS and then discarding the excess and un-solidified biological ink; S502, then adding DMEM complete medium containing 10% fetal bovine serum to the rinsed preliminary 3D-RNVU model, and culturing in a sterile cell incubator at 37°C and 5% volume concentration of CO2 for 72h to obtain a 3D-RNVU model.

[0010] Further, the mass concentration ratio of GelMA, HAMA and LAP in the S1 step is (1.5-4):(0.5-1):(0.1-0.2).

[0011] Further, the mass concentration ratio of GelMA, HAMA and LAP in the biological ink in the S3 step is 2:0.5:0.1.

[0012] Further, in the S2 step, the volume ratio of the REC single cell suspension, the RGC single cell suspension and the RMC single cell suspension is 1:1:1.

[0013] Further, the composite hydrogel precursor solution and the cell mixture are mixed in a volume ratio of 1:1 in the S3 step. The cell concentration of REC, RGC and RMC in the biological ink is 5x106 / mL.

[0014] Further, the preset parameter is specifically: using 8mm diameter circular pattern, 0.5mm thickness, using 20% ultraviolet light intensity, 20s exposure time single static printing mode.

[0015] Further, the concentration of glucose in the DMEM complete culture medium is 5.5mM-125mM.

[0016] Further, the concentration of glucose in the DMEM complete culture medium is 50mM.

[0017] Further, the mass concentration of trypsin used in the S1 step is 0.25%.

[0018] The application further provides a 3D-RNVU model prepared by the preparation method of the retinal nerve vascular unit in vitro 3D printing model.

[0019] The application further provides application of the 3D-RNVU model in any one of the following: preparation of a drug for treating and / or preventing a retinal vascular disease, construction of a biological model of pathogenesis of diabetic retinopathy or screening of a drug for diabetic retinopathy.

[0020] The application has the following technical effects: The application develops a bionic in-vitro retinal nerve vascular unit 3D model (3D-RNVU). The model uses a bioactive scaffold derived from natural high polymer materials such as gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA), simulates the composition and mechanical properties of the retinal ECM, and thus provides a three-dimensional microenvironment support closer to the in-vivo for RNVU cells. Meanwhile, the RGC, REC and RMC are mixed with the biomaterials at a physiological ratio to form a biological ink, and through 3D printing, the three-cell co-culture is realized, a nerve-glial-vascular ternary interaction system is established, and thus a more complete cell coordination mechanism research is supported, which provides an advanced in-vitro research tool for in-depth analysis of the molecular mechanism of RNVU functional decompensation, screening of targeted therapeutic drugs and development of regenerative medicine strategies. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0022] Figure 1 : 3D-RNVU model construction schematic diagram; Figure 23D-RNVU biomaterial composition and mechanical properties Figure 3 3D-RNVU biomaterial ratio and printing parameters on cell activity Figure 4 3D-RNVU model live / dead cell staining Figure 5 3D-RNVU multi-cell co-culture system and 3D-single cell culture growth morphology comparison Figure 6 3D-RNVU cell-specific marker and spatial localization imaging Figure 7 3D-RNVU model, different glucose concentrations on cell activity Figure 8 3D-RNVU diabetic retinopathy model in vitro reactive oxygen species level; wherein (A) representative image of flow cytometry detection of DCF fluorescence intensity; (B) quantification statistics of flow cytometry detection of DCF average fluorescence intensity; Figure 9 3D-RNVU diabetic retinopathy model in vitro mitochondrial membrane potential changes; wherein (A) representative image of flow cytometry detection of JC-1 fluorescence; (B) quantification statistics of flow cytometry detection of JC-1 monomer. DETAILED DESCRIPTION

[0023] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0024] Firstly, the present application provides a preparation method of a retinal neurovascular unit 3D printing model in vitro, S1, using GelMA, HAMA and LAP to prepare a composite hydrogel precursor solution; S2, selecting REC, RGC and RMC in the exponential growth phase, respectively resuspending after trypsin digestion, to obtain three single cell suspensions, and then mixing the three single cell suspensions to prepare a cell mixture; S3, mixing the composite hydrogel precursor solution and the cell mixture to obtain a biological ink; S4, printing and packaging the biological ink according to the preset parameters, initiating the photocrosslinking reaction of GelMA and HAMA by ultraviolet light to form a preliminary 3D-RNVU model. S5, re-culturing the preliminary 3D-RNVU model to obtain a 3D-RNVU model.

[0025] In the present application, REC, RGC and RMC are selected as the core components of RNVU, and single-cell suspensions are obtained by trypsin digestion to ensure cell activity and uniformity. The cell mixture is mixed with the hydrogel precursor solution to form a bio-ink, and precise spatial distribution is achieved by 3D printing. The preliminary 3D structure is formed by UV cross-linking and solidification. The post-cultured cells further adapt, proliferate and establish cell-cell connections in the three-dimensional environment, promoting the functional maturation of the model.

[0026] The preparation method realizes the three-dimensional construction of RNVU, breaks through the limitations of traditional 2D models, and upgrades from planar interaction to three-dimensional network interaction. At the same time, through the light cross-linking hydrogel scaffold, a controllable mechanical microenvironment is provided to support cell migration, proliferation and functional expression. It can also simulate the spatial configuration of the neuron-glia-vascular unit in the retina tissue, which is closer to the physiological state, and provides a reliable platform for studying the mechanisms of diseases such as diabetic retinopathy.

[0027] In some embodiments, the specific steps of re-culturing the preliminary 3D-RNVU model in the S5 step are as follows: S501, using DPBS to rinse the preliminary 3D-RNVU model and then discard it, to remove excess and un-solidified bio-ink; S502, then add DMEM complete medium containing 10% fetal bovine serum to the rinsed preliminary 3D-RNVU model, and place it in a sterile cell incubator at 37°C and 5% CO2 by volume for 72h to obtain a 3D-RNVU model.

[0028] In this step, un-crosslinked bio-ink and cell debris are removed to reduce background interference and ensure model cleanliness. At the same time, the model is re-cultured to provide nutritional factors and growth signals to promote cell survival and functional maturation. This effectively improves the stability of the model and the survival rate of the cells, avoiding interference with cell behavior by un-crosslinked materials. At the same time, by culturing for a long time, the cells form more mature cell-cell and cell-matrix interactions in the three-dimensional scaffold, enhancing the physiological relevance of the model.

[0029] In some embodiments, the mass concentration ratio of GelMA, HAMA and LAP in the S1 step is (1.5-4):(0.5-1):(0.1-0.2).

[0030] In some embodiments, the mass concentration ratio of GelMA, HAMA and LAP in the bio-ink of the S3 step is 2:0.5:0.1.

[0031] GelMA mimics the collagen component in the retinal ECM, HAMA mimics the hyaluronic acid component in the ECM, and LAP controls the efficiency of photocrosslinking. This proportion balances the stiffness, porosity, and biodegradability of the hydrogel.

[0032] By optimizing the proportion of the components of the bio-ink, the hydrogel scaffold can mimic the natural softness and hardness of the inner retinal tissue, and form a three-dimensional network with moderate pore size, so that it has sufficient mechanical strength to support the printed structure, and good biocompatibility to allow cell embedding and migration. In addition, by strictly controlling the amount of LAP and other components to avoid cytotoxicity, the survival rate of cells is ensured, and cell proliferation and functional expression are promoted.

[0033] In some embodiments, in the S2 step, the volume ratio of the REC single-cell suspension: RGC single-cell suspension: RMC single-cell suspension is 1:1:1.

[0034] This proportion simulates the relative abundance of the three types of cells in the RNVU under physiological conditions, ensuring balanced cell-cell interactions; in addition, RMC, as a glial cell, plays a role in connecting neurons and blood vessels in the RNVU, and its addition makes up for the shortcomings of traditional models that only focus on REC and RGC. This system can more comprehensively simulate the function of the RNVU and avoid the dominance of a certain cell type, improving the prediction accuracy of the model in drug screening.

[0035] In some embodiments, in the S3 step, the composite hydrogel precursor solution and the cell mixture are mixed at a volume ratio of 1:1. The cell concentration of REC, RGC, and RMC in the bio-ink is 5x10 6 6 / mL.

[0036] This proportion ensures uniform distribution of the hydrogel and cells, and moderate viscosity of the bio-ink, thereby ensuring excellent printing performance and cell activity of the bio-ink, and maintaining a high level of cell survival rate after printing.

[0037] In some embodiments, the preset parameters are specifically: using an 8mm diameter circular pattern, 0.5mm thickness, using 20% UV light intensity, 20s exposure time, and single static printing mode.

[0038] This thickness is suitable for standard culture dish operation and allows sufficient light penetration and nutrient diffusion, 20% UV light intensity and 20s exposure time balance the crosslinking efficiency and cell protection, preventing insufficient crosslinking caused by too low light intensity, or possible DNA damage to cells caused by too high light intensity, ensuring the stability of the hydrogel scaffold.

[0039] The single static printing mode can ensure structural uniformity and repeatability, so that the generated 3D model structure is consistent and has good reproducibility, and is more convenient for high-throughput experiments.

[0040] In some embodiments, the concentration of glucose in the DMEM complete medium is 5.5 mM-125 mM.

[0041] In some embodiments, the concentration of glucose in the DMEM complete medium is 50 mM.

[0042] In some embodiments, the mass concentration of trypsin used in the S1 step is 0.25%.

[0043] In a second aspect, the present application also provides a 3D-RNVU model prepared by the preparation method of the above-mentioned retinal neurovascular unit in vitro 3D printing model.

[0044] In a third aspect, the present application also provides a use of the above-mentioned 3D-RNVU model in any one of the following: preparation of a drug for treating and / or preventing a retinal vascular disease, construction of a biological model of the onset of diabetic retinopathy, or screening of a drug for diabetic retinopathy.

[0045] The following will be described in conjunction with specific embodiments: Embodiment 1: Retinal three-dimensional neurovascular unit in vitro culture model The rat REC involved in the following embodiments is purchased from Shanghai Xiyaji Biological Company; The rat RGC is purchased from Shanghai Xiyaji Biological Company; The rat RMC is purchased from Shanghai Bolson Biological Technology Co., Ltd.; GelMA and HAMA are purchased from Shanghai Yujia Technology Co., Ltd., and LAP is purchased from Shanghai Huaxia Sijing Biological Technology Co., Ltd.; Dulbecco's Modified Eagle Medium (DMEM medium, containing 1 g / L and 4.5 g / L of sugar, wherein the DMEM medium containing 1 g / L of sugar is a 5.5 mM low-sugar DMEM medium, and the DMEM medium containing 4.5 g / L of sugar is used for the preparation of a high-sugar DMEM medium) is purchased from Gibco; Fetal bovine serum (FBS) is purchased from Gibco; Glucose powder (L-Glucose) is purchased from AbMole; 0.25% trypsin (% refers to mass ratio, i.e., g / 100 mL) is purchased from Gibco; CellTiter-Glo 3D (CTG) detection reagent is purchased from Promega; CytoTrace Red CMTPX and CellTracker Green CMFDA reagents were purchased from CYTOCH, and CellTracker Blue CMAC was purchased from AbMole. The cell viability (live and dead cell staining) assay kit was purchased from MeilunBio; 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. The complete culture medium used in the following examples is 5.5 mM low-sugar DMEM medium containing 10% FBS (% refers to volume ratio).

[0046] 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.

[0047] Table 1: Based on DMEM low-sugar (5.5 mM) (10 mL system)

[0048] Table 2: Based on DMEM high sugar (25 mM) (10 mL system)

[0049] 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.

[0050] 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).

[0051] The prepared bio-ink was printed in a cell culture plate using a digital light processing (DLP) bioprinter according to the preset parameters (20% light intensity, 20s exposure time, 8mm diameter circular pattern, 0.5mm thickness, and single static printing mode). The preliminarily printed 3D-RNVU model was washed with an appropriate amount of DPBS for 1-2 minutes and then removed to remove the excess and un-solidified bio-ink. The cell culture plate was added with 1g / L DMEM complete medium containing 10% fetal bovine serum and placed in a sterile cell incubator at 37°C and 5% (v / v) CO2 for cell culture. After 72h of culture, a 3D-RNVU model was obtained (see the model construction process in Figure 2 , and the diagram was generated using Figdraw scientific drawing tool).

[0052] Example 2: Diabetic retinopathy model On the basis of Example 1, the complete medium used (i.e., 5.5mM low-sugar DMEM medium containing 10% FBS) was replaced with 50mM high-sugar DMEM medium to achieve high-sugar modeling, thereby obtaining a diabetic retinopathy model.

[0053] Comparative Example 1: REC, RGC, and RMC in-vitro single-culture three-dimensional model On the basis of Example 1, the cell mixture (mixed by REC, RGC, and RMC at a volume ratio of 1:1:1) was replaced with a single-cell suspension of REC (or RGC / RMC), which was completely mixed with the composite hydrogel precursor solution at a volume ratio of 1:1 to form a bio-ink (the final concentration of single-cell type was 5x10 6 6 / ml, and the bio-printing material contained 2% GelMA, 0.5% HAMA, and 0.1% LAP).

[0054] Experimental Example 1: Performance verification of the retinal three-dimensional neurovascular unit in-vitro three-culture model (3D-RNVU model) 1. Experimental process 1.1 Effect of selection of biomaterials on mechanical properties of 3D-RNVU model GelMA, HAMA, and LAP were mixed at a certain ratio (see Table 3 below). The prepared biomaterials were printed on a 14mm cell climbing sheet using a digital light processing (DLP) bioprinter according to the preset parameters (light intensity and exposure time as shown in Table 3). An appropriate amount of DPBS was added to wash for 1-2 minutes and then removed. DPBS was added again to cover the sample, and after 4 degrees overnight, the sample was taken out and placed on a hydrogel bioforce instrument (EFL-MT-5600, EFL) for compression test to measure the mechanical properties. The experimental results are shown inFigure 2 .

[0055] Table 3: Biomaterial composition and 3D printing parameters

[0056] Figure 2 The results show that the Young's modulus of the printed hydrogel is about 3 Kpa, which is similar to the mechanical properties of the inner layer of the retina, under the conditions of a biomaterial ratio of 2% GelMA, 0.5% HAMA, and 0.1% LAP, and a biomaterial ratio of 2% GelMA, 1% HAMA, and 0.1% LAP, and a 3D printer parameter of 10% light intensity.

[0057] Experimental Example 2: Effect of biomaterial formulation and printing parameters on cell activity of 3D-RNVU model On the basis of Example 1 and Comparative Example 1, the biomaterial and the 3D printer parameters are set as follows: Sample 2 contains 2% GelMA, 0.5% HAMA, and 0.1% LAP, and is printed under the conditions of 20% light intensity and 20s exposure time; Sample 3 contains 2% GelMA, 1% HAMA, and 0.1% LAP, and is printed under the conditions of 10% light intensity and 20s exposure time. After 72h of culture, the cell culture plate and 3D-RNVU model are taken out, equilibrated to room temperature for 30 minutes, and an equal volume of CellTiter Glo ® 3D reagent is added to the shaker at room temperature, mixed well, incubated for 30 minutes, and the luminescence signal (emission wavelength range of 360 to 750 nm) is recorded using a microplate reader at room temperature. The relative cell activity is calculated. The experimental results are shown in Figure 3 .

[0058] Figure 3 The results show that the cell growth trend in the 3D-RNVU model and the single cell in vitro single culture three-dimensional model is better under the conditions of a biomaterial ratio of 2% GelMA, 0.5% HAMA, and 0.1% LAP, and a 3D printer parameter of 20% light intensity, than under the conditions of a biomaterial ratio of 2% GelMA, 1% HAMA, and 0.1% LAP, and a 3D printer parameter of 10% light intensity.

[0059] Experimental Example 3: Verification of cell survival rate of 3D-RNVU model A 3D-RNVU model was constructed based on Example 1, and the selected bioprinting materials and parameters were: 2% GelMA, 0.5% HAMA and 0.1% LAP, and the printing was performed at a light intensity of 20% and an exposure time of 20 s. According to the steps 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 5 μL of PI Solution and 5 μL of Calcein-AM Solution were added to 10 mL of Assay buffer, and vortexed to mix well to prepare the working solution. At this time, the concentration of Calcein-AM was 2 μM, and the concentration of PI was 8 μM. The 3D-RNVU model was gently washed with DPBS for 2-3 times to remove the active esterase in the culture medium, and then sufficient Calcein-AM / PI staining working solution was added to ensure that the 3D-RNVU model was covered, and was placed in a 37°C incubator for 15-30 min. The staining working solution was aspirated to terminate incubation, and the cells were gently washed with DPBS for 2-3 times, and sufficient Assay buffer was added. Then a fluorescence microscope was used to observe the live cells (green fluorescence) and dead cells (red fluorescence) at an excitation wavelength of 490±10 nm and 545 nm, and the experimental results are shown in Figure 4 .

[0060] Figure 4 The results show that the 3D-RNVU model has very high cell activity, with a survival rate of more than 90%, which confirms the good biocompatibility of the selected materials and printing parameters.

[0061] Experimental Example 5: Cell morphological identification of neural-glial-vascular three-way interaction in 3D-RNVU model Cell morphological identification under an optical microscope: Based on Example 1 and Comparative Example 1, the selected bioprinting materials and bioprinter parameters were: 2% GelMA, 0.5% HAMA and 0.1% LAP, and the printing was performed at a light intensity of 20% and an exposure time of 20 s. The preliminary 3D-RNVU model was cultured for 72 h, and the cell growth morphology was observed by an optical microscope, and the experimental results are shown in Figure 5 .

[0062] Cell-specific identification under fluorescence microscope: discard the culture medium of the three kinds of cells RGC, REC and RMC in exponential growth phase, and add the working solution of live cell tracer CellTracker Blue CMAC, CytoTrace Red CMTPX and CellTracker Green CMFDA configured according to the instructions, respectively, and incubate in a 37°C incubator for 45 minutes. Discard the dye working solution, add complete culture medium and continue to incubate for 2 hours. Then, on the basis of Example 1, the parameters of the bioprinting material and the bioprinter are selected as follows: 2% GelMA, 0.5% HAMA and 0.1% LAP, and the printing is performed under the conditions of 20% light intensity and 20s exposure time. The preliminary 3D-RNVU model is cultured for 72h, and the growth morphology and relative position of the three kinds of cells are observed by laser confocal microscope. The experimental results are shown in Figure 6 .

[0063] Figure 5 and Figure 6 The results show that RGC, REC and RMC in the 3D environment maintain a more in-vivo three-dimensional morphology and form a spatial chimeric, indicating that the retinal neuron-glia-vascular unit interaction network is preliminarily formed. That is, the 3D-RNVU model successfully constructs a multi-cell three-dimensional co-culture system and forms a biomimetic retinal microstructure with a physiological topology.

[0064] Example 6: Effect of different glucose concentrations on the cell activity of 3D-RNVU model On the basis of Example 1, the parameters of the bioprinting material and the bioprinter are set as follows: 2% GelMA, 0.5% HAMA, 0.1% LAP, and 20% light intensity and 20s exposure time; and the glucose concentration of the DMEM complete culture medium is set as 5.5 mM, 11.1 mM, 25 mM, 50 mM, 75 mM, 100 mM and 125 mM, respectively. After 72h of culture, the cell culture plate and the 3D-RNVU model are taken out, equilibrated to room temperature for 30 minutes, and an equal volume of CellTiter Glo ® 3D reagent is added, and the mixture is shaken, mixed, incubated at room temperature for 30 minutes in the dark, and the luminescence signal (emission wavelength range: 360-750 nm) is recorded at room temperature using a microplate reader, and the relative cell activity is calculated. The experimental results are shown in Figure 7 .

[0065] Figure 7 The results show that compared with the normal sugar concentration (5.5 mM), the cell activity of the 3D-RNVU model starts to decrease when the glucose concentration is increased to 50 mM, indicating that a glucose concentration of 50 mM can be used for the construction of a diabetic retinopathy model.

[0066] Experimental Example 7: Evaluation of the diabetic retinopathy model prepared using in vitro 3D-RNVU On the basis of Examples 1 and 2, the preliminary 3D-RNVU model was cultured for 72 h, respectively. Digestive enzyme working solution was prepared: containing 2 mg / mL collagenase (Yoxin, Cat No: 40507ES60), 1 mg / ml hyaluronidase (Yoxin, Cat No: 20426ES80), 0.1 mg / mL DNASE I (Yoxin, Cat No: 10608ES60). Fixation and membrane permeation working solution was prepared: Fixation / Permeabilization Concentrate (eBioscience, Cat No: 00-5123-43) and Fixation / Permeabilization Diluent (eBioscience, Cat No: 00-5223-56) were diluted at a ratio of 1:3 and used immediately; 1X buffer: Permeabilization Buffer (eBioscience, Cat No: 00-8333) and pure water for experiments were diluted at a ratio of 1:10 and used immediately. The culture medium of the cell culture plate was aspirated, washed with DPBS for 3 times, then the digestive enzyme working solution was added at 20 times the volume of the 3D-RNVU model, and was digested in a 37-degree incubator for 10-15 minutes. After adding an equal volume of complete medium to terminate digestion, the cells were collected by centrifugation, the supernatant was discarded, and the reactive oxygen species detection or mitochondrial membrane potential detection experiment was performed.

[0067] Reactive oxygen species detection experiment: according to the instructions of the reactive oxygen species detection kit (KeyGEN BioTECH, Cat No: KGA7308), the DCFH-DA loading working solution was prepared at a dilution ratio of 1:1000 with DPBS to 10 µM. The DCFH-DA loading working solution was added to uniformly resuspend the cells, and was incubated in a 37°C cell incubator for 20 min, and was inverted and mixed every 3-5 min to ensure sufficient contact with the cells. The DCFH-DA working solution was removed by centrifugation at 200-300 x g for 3 min, and the cells were washed with serum-free medium for 3 times to completely remove the DCFH-DA that did not enter the cells. The flow cytometer was used for detection at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The experimental results are shown in Figure 8 .

[0068] Mitochondrial membrane potential detection experiment: according to the mitochondrial membrane potential detection kit (KeyGEN BioTECH, item number KGA1904) instruction book steps, 1x Incubation Buffer is configured: take 100 μL 10x Incubation Buffer and dilute 900 μL sterilized deionized water into 1x Incubation Buffer, mix well and preheat to 37℃ for standby. Configure JC-1 working solution: take 2 μL JC-1 (500x), add 898 μL sterilized deionized water, vortex thoroughly to dissolve and mix well JC-1, then add 100 μL 10x Incubation Buffer, mix well to get 1 mL of JC-1 working solution. Take 500 μL JC-1 working solution to uniformly resuspend the cells, 37℃, 5% CO2 incubator, avoid light for 15-20 min. Centrifuge (300xg, 5 min) to collect cells at room temperature, wash twice with 1x Incubation Buffer: add 1 mL 1x Incubation Buffer to resuspend the cells, centrifuge to collect the cells, add 1 mL 1x Incubation Buffer to resuspend the cells again, centrifuge to collect the cells. Take 500 μL 1x Incubation Buffer to resuspend the cells, and detect by flow cytometry within 1 h: green fluorescence (Ex = 488 nm, Em = 530 nm); red fluorescence (Ex = 488 nm, Em ≥ 630 nm). The experimental results are shown in Figure 9 .

[0069] From Figure 8 and Figure 9 , it can be seen that compared with the normal sugar concentration (5.5 mM) control group, the in vitro 3D-RNVU diabetic retinopathy model showed significant enhancement of oxidative stress and mitochondrial dysfunction. Specifically, the level of reactive oxygen species (ROS) was elevated, accompanied by a decrease in mitochondrial membrane potential (ΔΨm), which is an early marker event of apoptosis. These results collectively indicate that the model successfully reproduces key pathological phenotypes such as oxidative stress, mitochondrial damage and early events of apoptosis in diabetic retinopathy.

[0070] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a retinal neurovascular unit in vitro 3D printed model, characterized in that, It comprises the following steps: S1, using gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA) and lithium phenyl-2, 4, 6-trimethylbenzoyl phosphonate (LAP) to prepare a composite hydrogel precursor solution; S2, selecting REC, RGC and RMC in the exponential growth phase, respectively using trypsin digestion and resuspension to obtain three single cell suspensions, and then mixing the three single cell suspensions to prepare a cell mixture; S3, mixing the composite hydrogel precursor solution and the cell mixture to obtain a bio-ink; S4, printing and packaging the bio-ink according to the preset parameters, initiating the photo-crosslinking reaction of GelMA and HAMA by ultraviolet light to form a preliminary 3D-RNVU model; S5, re-culturing the preliminary 3D-RNVU model to obtain a 3D-RNVU model.

2. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, The specific steps of re-culturing the preliminary 3D-RNVU model in the S5 step are: S501, rinsing the preliminary 3D-RNVU model with DPBS and then discarding the excess and un-solidified bio-ink; S502, then adding DMEM complete medium containing 10% fetal bovine serum to the rinsed preliminary 3D-RNVU model, and culturing in a sterile cell incubator at 37℃ and 5% volume concentration of CO2 for 72h to obtain a 3D-RNVU model.

3. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, The mass concentration ratio of GelMA, HAMA and LAP in the S1 step is (1.5-4):(0.5-1):(0.1-0.2).

4. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, The mass concentration ratio of GelMA, HAMA and LAP in the bio-ink of the S3 step is 2:0.5:0.

1.

5. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, In the S2 step, the volume ratio of REC single cell suspension: RGC single cell suspension: RMC single cell suspension is 1:1:

1.

6. The method for preparing an in vitro 3D printed model of a retinal neurovascular unit according to claim 1, characterized in that, The composite hydrogel precursor solution and the cell mixture are mixed in a volume ratio of 1:1 in the S3 step. The cell concentration of REC, RGC and RMC in the bio-ink is 5 x 10 6 cells / mL.

7. The method of claim 1, wherein the method further comprises: culturing the 3D-printed retinal neurovascular unit model in a culture medium for a period of time sufficient to allow the 3D-printed retinal neurovascular unit model to develop into a functional 3D-printed retinal neurovascular unit model. The preset parameters are specifically: using an 8mm diameter circular pattern, 0.5mm thickness, using 20% ultraviolet light intensity, 20s exposure time single static printing mode.

8. The method of claim 2, wherein the method further comprises: culturing the 3D-printed retinal neurovascular unit model in a culture medium for a period of time sufficient to allow the 3D-printed retinal neurovascular unit model to develop into a functional 3D-printed retinal neurovascular unit model. The concentration of glucose in the DMEM complete medium is 5.5mM-125mM.

9. A 3D-RNVU model prepared by the method of any one of claims 1-8.

10. The use of the 3D-RNVU model of claim 9 in any one of the preparation of diabetic retinopathy, the development of a biological model of the onset of retinal vascular disease, or the screening of drugs for diabetic retinopathy.

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