In-vitro three-culture model of retina nerve and blood vessel unit and preparation method thereof

Abstract

The invention relates to a retina nerve and blood vessel unit in-vitro three-culture model and a preparation method thereof, and belongs to the technical field of biology. The invention provides an in-vitro three-culture model of a retina nerve and blood vessel unit. The in-vitro three-culture model is obtained by carrying out in-vitro Transwell indirect co-culture on retina microvascular endothelial cells, retina ganglion cells and retina Muller cells. The in-vitro three-culture model of the retina nerve and blood vessel unit is based on a three-cell indirect co-culture method, retina microvascular endothelial cells, retina ganglion cells and retina Muller cells are simultaneously incorporated by utilizing Transwell, and the three cells are in indirect contact. The in-vitro three-culture model of the retina nerve and blood vessel unit can be used for more objectively simulating pathophysiological states of various cells of the retina nerve and blood vessel unit, so that common pathophysiological changes of mutual dependence and interaction of the three cells under in-vitro conditions can be observed.

Description

TECHNICAL FIELD

[0001] The present application relates to a retinal neurovascular unit in vitro three-culture model and a preparation method thereof, and belongs to the field of biotechnology. BACKGROUND

[0002] Retinal vascular diseases refer to a general term of diseases caused by damage to the retinal vascular unit, and are one of the main causes of decreased vision, low vision and blindness. The retinal neurovascular unit is composed of nerve cells (bipolar cells, ganglion cells, amacrine cells and horizontal cells), glial cells (Muller cells, astrocytes and microglia cells) and vascular cells (endothelial cells, pericytes) and other cells, and basement membrane and the like. Various cells in the retinal neurovascular unit depend on each other, and play an important role in the maintenance of normal physiological functions of the retina and the occurrence and development of retinal vascular diseases. The current mainstream theory believes that the functional decompensation of the retinal neurovascular unit is the earliest and most important pathological change leading to retinal vascular diseases, and in recent years, a number of studies have also shown that the homeostasis imbalance of the retinal neurovascular unit is involved in the occurrence and development of various retinal diseases such as diabetic retinopathy (DR), etc. Therefore, the study of the functional decompensation of the retinal neurovascular unit in retinal diseases is helpful to analyze the pathogenesis of retinal vascular diseases and explore the treatment mechanism of retinal vascular diseases. However, due to the difficulty in studying the microenvironment of cell survival in vivo, the use of in vitro models to simulate the physiological state and pathological changes of the retinal neurovascular unit is currently the main method for studying the functional decompensation of the retinal neurovascular unit in retinal diseases.

[0003] At present, the in vitro research model of the retinal neurovascular unit mainly includes a two-culture model obtained by two-dimensional co-culture of retinal neurovascular unit cells. The construction method of such a model mainly includes direct co-culture method and indirect co-culture method (see Figure 1). Among them, the direct co-culture method first separates the cells of the two retinal neurovascular units (including retinal microvascular endothelial cells and retinal ganglion cells) in vitro, and then the two cells in the exponential growth phase are selected and inoculated in the culture hole plate at an appropriate density and ratio for direct co-culture. After the co-culture is completed, different disease models can be constructed according to the research content through physical or chemical means, so as to explore the changes in biological behavior in the pathological process, and the molecular phenotype of different cells under co-culture conditions can also be explored through gene expression sequence analysis and proteomics analysis. The indirect co-culture method needs to use Transwell. First, the cells of the two retinal neurovascular units are separated in vitro, and then the two cells in the exponential growth phase are selected and inoculated in the Transwell chamber and the lower chamber at an appropriate density and ratio for indirect co-culture. After the co-culture is completed, different disease models can be constructed according to the research content, or genomic and proteomic studies can be carried out.

[0004] Compared with the indirect co-culture method, the direct co-culture method has the advantages of increasing the types of inoculated cells, allowing the interaction between multiple cells, and more objectively simulating the pathological and physiological state of multiple cells of the retinal neurovascular unit. However, due to the need to mix different cells and inoculate them into the same culture container, the direct co-culture method is not convenient for the classification and collection of multiple cells and the next step of genomic and proteomic research. While the indirect co-culture method can more simply achieve the classification and collection of two cells and the next step of research, the two-culture model based on the indirect co-culture method can only include two cells, and cannot objectively simulate the pathological and physiological state of multiple cells of the retinal neurovascular unit. This makes most of the current retinal neurovascular unit two-culture models only be used to study the interaction between retinal microvascular endothelial cells and ganglion cells, the two most important cells in the retinal neurovascular unit, while ignoring other cell types that may play an important role in the retinal neurovascular unit. Therefore, it is urgent to find a retinal neurovascular unit in vitro model based on the indirect co-culture method that can include more cell types to overcome the defects of the existing retinal neurovascular unit in vitro model. SUMMARY

[0005] To solve the above-mentioned defects, the present application provides a retinal neurovascular unit in vitro three-culture model, which is obtained by indirect co-culture of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells in vitro.

[0006] In an embodiment of the present application, the preparation method of the in vitro tri-culture model comprises: culturing retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells on the inner side of the membrane of the Transwell chamber, the outer side of the membrane of the Transwell chamber and the lower chamber respectively to obtain the in vitro tri-culture model of retinal neurovascular unit.

[0007] In an embodiment of the present application, the preparation method of the in vitro tri-culture model comprises the following steps:

[0008] Step one: using the culture medium to prepare cell suspension A, and obtaining cell suspension A; inverting the Transwell chamber, inoculating cell suspension A to the outer side of the membrane of the inverted Transwell chamber, and culturing cell A on the outer side of the membrane of the Transwell chamber;

[0009] Step two: discarding the culture medium on the outer side of the membrane of the Transwell chamber, placing the Transwell chamber in the No. 1 lower chamber added with the culture medium, and continuing to culture cell A on the outer side of the membrane of the Transwell chamber;

[0010] Step three: using the culture medium to prepare cell suspension B, and obtaining cell suspension B; using the culture medium to prepare cell suspension C, and obtaining cell suspension C; inoculating cell suspension B to the inner side of the membrane of the Transwell chamber, and inoculating cell suspension C to the No. 2 lower chamber, and culturing the three kinds of cells on the two sides of the membrane of the Transwell chamber and in the No. 2 lower chamber respectively;

[0011] Step four: placing the Transwell chamber with cell A and cell B growing on the two sides of the membrane in the No. 2 lower chamber, and continuing to culture the three kinds of cells on the two sides of the membrane of the Transwell chamber and in the No. 2 lower chamber; obtaining the in vitro tri-culture model of retinal neurovascular unit;

[0012] The cell A is a retinal microvascular endothelial cell, a retinal ganglion cell or a retinal Muller cell; when the cell A is a retinal microvascular endothelial cell, the cell B and the cell C are a retinal ganglion cell and a retinal Muller cell respectively, or the cell B and the cell C are a retinal Muller cell and a retinal ganglion cell respectively; when the cell A is a retinal ganglion cell, the cell B and the cell C are a retinal microvascular endothelial cell and a retinal Muller cell respectively, or the cell B and the cell C are a retinal Muller cell and a retinal microvascular endothelial cell respectively; when the cell A is a retinal Muller cell, the cell B and the cell C are a retinal microvascular endothelial cell and a retinal ganglion cell respectively, or the cell B and the cell C are a retinal ganglion cell and a retinal microvascular endothelial cell respectively.

[0013] In an embodiment of the present application, the cell A, the cell B and the cell C are a retinal microvascular endothelial cell, a retinal ganglion cell and a retinal Muller cell respectively.

[0014] In an embodiment of the present application, the membrane material of the Transwell chamber is PET (polyethylene terephthalate).

[0015] In an embodiment of the present application, the seeding density of the cell A on the outside of the membrane of the Transwell chamber is 8000-12000 cells / well; the seeding density of the cell B on the inside of the membrane of the Transwell chamber is 8000-12000 cells / well; and the seeding density of the cell C in the lower chamber is 25000-35000 cells / well.

[0016] In an embodiment of the present application, the step one comprises: using a culture medium to prepare a cell suspension of the cell A to obtain a cell suspension A; inverting a Transwell chamber, inoculating the cell suspension A to the outside of the membrane of the inverted Transwell chamber, and then placing the inverted Transwell chamber in a culture well plate added with a buffer, taking another culture well plate as a cover to cover the culture well plate added with the buffer, and then culturing the cell A on the outside of the membrane of the Transwell chamber.

[0017] In an embodiment of the present application, the culture medium is a DMEM culture medium containing 8-12% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin mixture.

[0018] The application further provides a construction method of a retinal nerve blood vessel unit in-vitro three-culture model, which comprises: performing in-vitro indirect co-culture of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells to obtain the retinal nerve blood vessel unit in-vitro three-culture model.

[0019] In an embodiment of the application, the construction method comprises: performing cell culture of the retinal microvascular endothelial cells, the retinal ganglion cells and the retinal Muller cells on the inner side of the membrane of the Transwell chamber, the outer side of the membrane of the Transwell chamber and the lower chamber, respectively, to obtain the retinal nerve blood vessel unit in-vitro three-culture model.

[0020] In an embodiment of the application, the construction method comprises the following steps:

[0021] Step one: using a culture medium to prepare cell A into a cell suspension to obtain cell suspension A; inverting a Transwell chamber, inoculating cell suspension A to the outer side of the membrane of the inverted Transwell chamber, and performing cell culture of cell A on the outer side of the membrane of the Transwell chamber;

[0022] Step two: discarding the culture medium on the outer side of the membrane of the Transwell chamber, placing the Transwell chamber in 1# lower chamber added with a culture medium, and continuing to perform cell culture of cell A on the outer side of the membrane of the Transwell chamber;

[0023] Step three: using a culture medium to prepare cell B into a cell suspension to obtain cell suspension B; using a culture medium to prepare cell C into a cell suspension to obtain cell suspension C; inoculating cell suspension B to the inner side of the membrane of the Transwell chamber, and inoculating cell suspension C to 2# lower chamber, and performing cell culture of the three kinds of cells in the Transwell chamber and 2# lower chamber, respectively;

[0024] Step four: placing the Transwell chamber with cell A and cell B growing on both sides of the membrane in 2# lower chamber, and continuing to perform cell culture of the three kinds of cells in the Transwell chamber and 2# lower chamber; obtaining the retinal nerve blood vessel unit in-vitro three-culture model;

[0025] The cell A is a retinal microvascular endothelial cell, a retinal ganglion cell or a retinal Muller cell; when the cell A is a retinal microvascular endothelial cell, the cell B and the cell C are a retinal ganglion cell and a retinal Muller cell respectively, or the cell B and the cell C are a retinal Muller cell and a retinal ganglion cell respectively; when the cell A is a retinal ganglion cell, the cell B and the cell C are a retinal microvascular endothelial cell and a retinal Muller cell respectively, or the cell B and the cell C are a retinal Muller cell and a retinal microvascular endothelial cell respectively; when the cell A is a retinal Muller cell, the cell B and the cell C are a retinal microvascular endothelial cell and a retinal ganglion cell respectively, or the cell B and the cell C are a retinal ganglion cell and a retinal microvascular endothelial cell respectively.

[0026] In an embodiment of the present application, the cell A, the cell B and the cell C are a retinal microvascular endothelial cell, a retinal ganglion cell and a retinal Muller cell respectively.

[0027] In an embodiment of the present application, the membrane material of the Transwell chamber is PET.

[0028] In an embodiment of the present application, the seeding density of the cell A on the outside of the membrane of the Transwell chamber is 8000-12000 cells / well; the seeding density of the cell B on the inside of the membrane of the Transwell chamber is 8000-12000 cells / well; and the seeding density of the cell C in the lower chamber is 25000-35000 cells / well.

[0029] In an embodiment of the present application, the step one comprises: using a culture medium to prepare a cell suspension of the cell A to obtain a cell suspension A; inverting a Transwell chamber, inoculating the cell suspension A to the outside of the membrane of the inverted Transwell chamber, and then placing the inverted Transwell chamber in a culture well plate added with a buffer, taking another culture well plate as a cover to cover the culture well plate added with the buffer, and then culturing the cell A on the outside of the membrane of the Transwell chamber.

[0030] In an embodiment of the present application, the culture medium is a DMEM culture medium containing 8-12% fetal bovine serum and 1% penicillin-streptomycin mixture.

[0031] The present application also provides a retinal vascular disease in vitro model, which is obtained by modeling the above-mentioned retinal neurovascular unit in vitro three-culture model.

[0032] In an embodiment of the present application, the retinal vascular disease in vitro model comprises a diabetic retinopathy (DR) in vitro model.

[0033] In an embodiment of the present application, when the retinal vascular disease in vitro model is a diabetic retinopathy in vitro model, the modeling method comprises: using a high-sugar medium in the in vitro Transwell indirect co-culture process of the in vitro three-culture model.

[0034] In an embodiment of the present application, the sugar concentration of the high-sugar medium is 70-80 mM.

[0035] The present application also provides a method for constructing a retinal vascular disease in vitro model, which comprises: modeling the above-mentioned retinal neurovascular unit in vitro three-culture model to obtain a retinal vascular disease in vitro model.

[0036] In an embodiment of the present application, the retinal vascular disease in vitro model comprises a diabetic retinopathy in vitro model.

[0037] In an embodiment of the present application, when the retinal vascular disease in vitro model is a diabetic retinopathy in vitro model, the modeling method comprises: using a high-sugar medium in the in vitro Transwell indirect co-culture process of the in vitro three-culture model.

[0038] In an embodiment of the present application, the sugar concentration of the high-sugar medium is 70-80 mM.

[0039] The present application also provides a method for screening drugs, which comprises: using the above-mentioned retinal neurovascular unit in vitro three-culture model or the above-mentioned retinal vascular disease in vitro model to screen drugs.

[0040] In an embodiment of the present application, the drug is a drug for treating retinal vascular diseases.

[0041] In an embodiment of the present application, the retinal vascular disease comprises diabetic retinopathy.

[0042] The present application also provides the above-mentioned retinal neurovascular unit in vitro three-culture model or the above-mentioned retinal vascular disease in vitro model for use in drug screening.

[0043] In an embodiment of the present application, the drug is a drug for treating retinal vascular diseases.

[0044] In an embodiment of the present application, the retinal vascular disease comprises diabetic retinopathy.

[0045] The technical scheme of the present application has the following advantages:

[0046] The present application provides a retinal nerve blood vessel unit in vitro three culture model, which is obtained by indirect co-culturing retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells in vitro through a Transwell.

[0047] Muller cells only exist in the retinal tissue of vertebrates, and are the largest and most important glial cells in the retina. The cell body penetrates the entire retinal tissue. Muller cells play a structural supporting role in the retinal nerve blood vessel unit, and can regulate the activity of retinal neurons, maintain neurotransmitter transmission, and help material transport and information transmission between neurons and retinal microvascular endothelial cells. In addition, Muller cells also have the functions of regulating the water and electrolyte balance of the retina and participating in the immune and inflammatory response of the retina.

[0048] At present, studies have shown that Muller cells, retinal ganglion cells and retinal microvascular endothelial cells are interdependent in physiological function and jointly maintain the stability of the microenvironment (see document "Zhang X, Zeng H, Bao S, Wang N, Gillies MC. Diabetic macular edema: new concepts in patho-physiology and treatment. Cell Biosci. 2014; 4: 27. Published 2014 May 14."). And studies have also confirmed that diabetic macular edema (DME) is the main cause of visual loss in patients with diabetic retinopathy (DR), and Muller cells in the retina are directly involved in the pathogenesis of diabetic macular edema (DME) (see document "Lai D, Wu Y, Shao C, et al. The role of Muller cells in diabetic macular edema [J]. Investigative Ophthalmology & Visual Science, 2023, 64(10): 8-8."). Therefore, Muller cells not only maintain the normal physiological function of the retina, but also participate in the pathological process of retinal vascular diseases.

[0049] On this basis, the present application further found through research that in a high glucose environment, Muller cells have a supporting effect on retinal ganglion cells and retinal microvascular endothelial cells, and the interaction of these three cells helps to simulate the true state of diabetic retinopathy and other retinal vascular diseases involving high glucose environment. Based on the above research, considering the development of imaging technology and the like, especially the development of adaptive optics (AO), it has been possible to observe Muller cells in vivo (see document "Kadomoto S, Muraoka Y, Uji A, et al. Human foveal cone and Muller cells examined by adaptive optics optical coherence tomography [J]. Translational Vision Science & Technology, 2021, 10 (11): 17-17.") Therefore, if a retinal neurovascular unit in vitro model that can simultaneously incorporate the three cells of Muller cells, retinal ganglion cells and retinal microvascular endothelial cells can be found, the pathological and physiological state of the retinal neurovascular unit can be more objectively simulated, and a research basis can be provided for the analysis of the pathogenesis of retinal vascular diseases and the exploration of the treatment mechanism of retinal vascular diseases.

[0050] The retinal neurovascular unit in vitro three-culture model of the present application is based on the indirect co-culture method of three cells, and simultaneously incorporates the three cells of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells using Transwell, and makes the three cells indirectly contact, which not only ensures the interaction between the three cells, but also ensures the classified observation of the three cells. And compared with the physiological and pathological changes of two cells observed in vitro, the pathological response of the retinal neurovascular unit in vitro three-culture model of the present application participated by the three cells is clearer for explaining the interaction of the retinal neurovascular unit. Therefore, using the retinal neurovascular unit in vitro three-culture model of the present application can more objectively simulate the pathological and physiological state of the retinal neurovascular unit, and then observe the common pathological and physiological changes of the three cells under the condition of mutual dependence and interaction in vitro. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 : Direct co-culture method and indirect co-culture method of in vitro co-culture model.

[0052] Figure 2 : Construction process of retinal neurovascular unit in vitro three-culture model.

[0053] Figure 3: Differences of cell proliferation function of Müller cells in different sugar concentration environment under single culture and three culture conditions. Figure 3 In the figure, (A) CCK8 experiment results of 24h after low sugar (5.5mM) or high sugar (75mM) modeling; (B) CCK8 experiment results of 48h after low sugar (5.5mM) or high sugar (75mM) modeling; (C) CCK8 experiment results of 72h after low sugar (5.5mM) or high sugar (75mM) modeling.

[0054] Figure 4 : Culture state of different cells in the three-cell co-culture model of retinal neurovascular unit in vitro. Figure 4 In the figure, ① Growth state of the ganglion cells after adhering to the outside of the membrane of inverted Transwell chamber for 3h; ② Growth state of the ganglion cells after adhering to the outside of the membrane of inverted Transwell chamber for 24h; ③ Growth state of the Müller cells in the lower chamber after adhering to the membrane for 24h; ④ Growth state of the microvascular endothelial cells in the inside of the membrane of Transwell chamber after adhering to the membrane for 24h.

[0055] Figure 5 : Growth morphology of the Müller cells in the lower chamber in the three-cell co-culture model of retinal neurovascular unit in vitro.

[0056] Figure 6 : Differences of cell apoptosis of rat retinal ganglion cells (RGC) in different sugar concentration environment under three culture conditions. Figure 6 In the figure, (A) Model schematic diagram; (B) Apoptosis experiment results of 24h after high sugar (5.5mM, 50mM, 75mM or 125mM) modeling; (C) Apoptosis experiment results of 48h after high sugar (5.5mM, 50mM, 75mM or 125mM) modeling; (D) Apoptosis experiment results of 72h after high sugar (5.5mM, 50mM, 75mM or 125mM) modeling.

[0057] Figure 7 : Effect of inverted culture difference of different materials chambers on the three-cell co-culture model of retinal neurovascular unit in vitro.

[0058] Figure 8 : Effect of different inverted chamber inoculation densities on the three-cell co-culture model of retinal neurovascular unit in vitro.

[0059] Figure 9 : Effect of different chamber inoculation densities on the three-cell co-culture model of retinal neurovascular unit in vitro.

[0060] Figure 10 : Effect of different lower chamber inoculation densities on the three-cell co-culture model of retinal neurovascular unit in vitro.

[0061] Figure 11 : Effect of PBS infiltration on the difference between inverted culture and three-cell co-culture model of retinal neurovascular unit in vitro. DETAILED DESCRIPTION

[0062] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the scope of the application or the contents thereof, and do not constitute a limitation on the scope of the application and the scope of protection, and any person who obtains the same or similar products under the inspiration of the application or combines the application with other prior art features falls within the scope of protection of the application.

[0063] The specific experimental steps or conditions are not indicated in the following examples, and can be carried out according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0064] In the following examples, rat retinal microvascular endothelial cells (rRMECs) were purchased from Shanghai Xiaoya Biological Company, rat retinal ganglion cells (rRGCs) were purchased from Shanghai Xiaoya Biological Company, rat retinal Müller cells were purchased from Guangzhou Jinieuo 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 5.5 mM low-sugar DMEM medium, and the DMEM medium containing 4.5 g / L of sugar is used for the preparation of high-sugar DMEM medium) was purchased from Gibco, fetal bovine serum (FBS) was purchased from Gibco, L-glucose was purchased from AbMole, 0.5% trypsin (% refers to the mass-volume ratio, i.e. g / 100 mL) was purchased from Gibco, CCK-8 detection reagent (Cell Counting Kit) was purchased from AbMole, 24-well cell culture plates and suitable Transwell chambers (0.4 μm, 3470, PET film material) were purchased from Corning, 12-well cell culture plates and culture dishes were purchased from Corning, PBS buffer was purchased from Gibco Life Technologies, and penicillin-streptomycin mixture was purchased from Gibco. The complete culture medium in the following examples is 5.5 mM low-sugar DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixture (% refers to the volume ratio).

[0065] The preparation method of high-sugar medium with different sugar concentrations involved in the following examples is as follows:

[0066] 50mM high-sugar DMEM medium (50mL as an example): according to the formula of 5mL FBS (10% of the total volume) + 0.5mL penicillin-streptomycin mixture (1% of the total volume) + 44.5mL 4.5g / L DMEM medium, a basic compound solution is prepared; 225mg glucose powder is weighed and dissolved in the compound solution to obtain 50mM high-sugar DMEM medium.

[0067] 75mM high-sugar DMEM medium (50mL as an example): according to the formula of 5mL FBS (10% of the total volume) + 0.5mL penicillin-streptomycin mixture (1% of the total volume) + 44.5mL 4.5g / L DMEM medium, a basic compound solution is prepared; 450mg glucose powder is weighed and dissolved in the compound solution to obtain 75mM high-sugar DMEM medium.

[0068] 125mM high-sugar DMEM medium (50mL as an example): according to the formula of 5mL FBS (10% of the total volume) + 0.5mL penicillin-streptomycin mixture (1% of the total volume) + 44.5mL 4.5g / L DMEM medium, a basic compound solution is prepared; 900mg glucose powder is weighed and dissolved in the compound solution to obtain 125mM high-sugar DMEM medium.

[0069] Example 1: A retinal neurovascular unit in vitro three-culture model

[0070] The present embodiment provides a retinal neurovascular unit in vitro three-culture model, which is obtained by in vitro indirect co-culture of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells.

[0071] The preparation method of the retinal neurovascular unit in vitro three-culture model is as follows:

[0072] The rat retinal ganglion cells are used as target cells 1, the rat retinal microvascular endothelial cells are used as target cells 2, and the rat retinal Muller cells are used as target cells 3; the Transwell chamber is inverted, and 1×10 5 mL of cell suspension containing 1×10 4After the transwell chamber is placed in the 12-well plate containing 0.3 mL of PBS buffer and another 12-well plate is used as a cover to prevent liquid evaporation and contamination (infiltration operation), the cells are cultured in a sterile cell incubator at 37°C and 5% (v / v) CO2. After 3 hours of culture, the target cells 1 adhere to the outer side of the membrane of the transwell chamber. At this time, the culture medium on the outer side of the membrane of the transwell chamber is discarded, and 0.5 mL of complete medium is added to the first 24-well cell culture plate (lower chamber) at a volume of 0.5 mL per well. The transwell chamber is turned upside down and placed in the first 24-well cell culture plate, and the first 24-well cell culture plate is placed in a sterile cell incubator at 37°C and 5% (v / v) CO2 for cell culture. After 24 hours of culture, the target cells 1 grow adherently on the outer side of the membrane of the transwell chamber. At this time, a cell suspension containing 1 x 10 5 cells / mL of target cells 2 (the cell suspension is obtained by resuspending the target cells in complete medium) is inoculated into the inner side of the membrane of the transwell chamber (1 x 10 4 cells per well) at a volume of 0.1 mL per well, and a cell suspension containing 6 x 10 4 cells / mL of target cells 3 (the cell suspension is obtained by resuspending the target cells in complete medium, and the percentage refers to the volume ratio) is inoculated into the second 24-well cell culture plate (lower chamber) (3 x 10 4 cells per well) at a volume of 0.5 mL per well, and the two 24-well cell culture plates are placed in a sterile cell incubator at 37°C and 5% (v / v) CO2 for cell culture. After 24 hours of culture, the target cells 2 grow adherently on the inner side of the membrane of the transwell chamber, and the target cells 3 grow adherently on the inner wall of the second 24-well cell culture plate. At this time, the transwell chamber with target cells growing on both sides of the membrane is placed in the second 24-well cell culture plate, and the second 24-well cell culture plate is placed in a sterile cell incubator at 37°C and 5% (v / v) CO2 for cell culture. After 24 hours of culture, the retinal neurovascular unit in vitro three-culture model is obtained (see the construction process of the retinal neurovascular unit in vitro three-culture model in Figure 2 , which is generated by Figdraw scientific drawing tool).

[0073] Comparative Example 1: A Müller cell in vitro single-culture model

[0074] This comparative example provides a Müller cell in vitro single-culture model, which is obtained by in vitro culture of retinal Müller cells.

[0075] The preparation method of the Müller cell in vitro single-culture model is as follows:

[0076] Take the cell suspension containing 6 x 10 4 The cell suspension containing 6 x 10

[0077] Example 2: A diabetic retinopathy (DR) model

[0078] This example provides a diabetic retinopathy (DR) model, which is obtained by in vitro indirect co-culturing retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells under a high-sugar environment.

[0079] The preparation method of the diabetic retinopathy model is: on the basis of Example 1, the complete culture medium used (i.e. 5.5 mM low-sugar DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixture) is replaced with 75 mM high-sugar DMEM medium to achieve high-sugar modeling, thereby obtaining the diabetic retinopathy model.

[0080] Comparative Example 2: A high-sugar intervention Muller cell in vitro single culture model

[0081] This comparative example provides a high-sugar intervention Muller cell in vitro single culture model, which is obtained by in vitro culturing retinal Muller cells under a high-sugar environment.

[0082] The preparation method of the high-sugar intervention Muller cell in vitro single culture model is: on the basis of Comparative Example 1, the complete culture medium used (i.e. 5.5 mM low-sugar DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixture) is replaced with 75 mM high-sugar DMEM medium to achieve high-sugar modeling, thereby obtaining the high-sugar intervention Muller cell in vitro single culture model.

[0083] Experimental Example 1: Performance verification of the retinal neurovascular unit in vitro three-culture model and optimization of its construction conditions

[0084] This experimental example verifies the performance of the retinal neurovascular unit in vitro three-culture model and optimizes its construction conditions, and the experimental process is as follows:

[0085] 1. Experimental process

[0086] 1.1. Performance verification of retinal neurovascular unit in vitro three-culture model and optimization of culture system (single culture, three-culture)

[0087] Experiment 1: Cell proliferation function difference of Müller cells in different sugar concentration environments under single culture and three-culture conditions

[0088] Using rat retinal microvascular endothelial cells, rat retinal ganglion cells and rat retinal Müller cells, low-sugar single-cultured Müller cells (prepared by Comparative Example 1), high-sugar single-cultured Müller cells (prepared by Comparative Example 2), low-sugar three-cultured retinal neurovascular unit in vitro three-culture model (prepared by Example 1) and high-sugar three-cultured diabetic retinopathy model (prepared by Example 2) were prepared by the methods of Comparative Examples 1-2 and Examples 1-2, respectively. Cell proliferation function detection was performed on Müller cells at 24h, 48h and 72h after single culture and three-culture high-sugar modeling, respectively, and the experimental results are shown in Table 1. Figure 3 During the preparation of the low-sugar three-cultured retinal neurovascular unit in vitro three-culture model, the cell state in the Transwell chamber and the 24-well cell culture plate was observed under a microscope, and the observation results are shown in Table 2. Figures 4-5 The cell proliferation function detection method was as follows: the Transwell chamber was discarded, 600μL of 5.5mM low-sugar DMEM medium containing 10%(v / v) CCK-8 detection reagent (Cell Counting Kit, purchased from AbMole) was added to the lower chamber, and then the Multiskan FC enzyme marker (Thermo Fisher Scientific) was used to detect the optical density (OD) value of the absorbance of each well at 450nm.

[0089] Experiment 2: Cell apoptosis difference of rat retinal ganglion cells (RGC) in different sugar concentration environments under three-culture conditions

[0090] The rat retinal Muller cells were used as target cells 1, the rat retinal microvascular endothelial cells were used as target cells 2, and the rat retinal ganglion cells were used as target cells 3. The low-sugar three-culture retinal neurovascular unit in vitro three-culture model was prepared by using the method of Example 1, and on the basis of Example 1, the complete culture medium (i.e. 5.5 mM low-sugar DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin mixture) used was replaced with 50 mM high-sugar DMEM culture medium, 75 mM high-sugar DMEM culture medium, and 125 mM high-sugar DMEM culture medium, respectively, to achieve high-sugar modeling at different concentrations, thereby obtaining a three-culture diabetic retinopathy model in different sugar concentration environments. PI / Hoechst cell apoptosis staining (PI solution, Hoechst 33342 solution, purchased from AbMole) was performed on the Muller cells at 24 h, 48 h, and 72 h after three-culture high-sugar modeling, respectively, the number of PI-stained cells / the number of Hoechst-stained cells was calculated, and the apoptosis index was obtained. The experimental results are shown in Figure 6 .

[0091] 1.2, Effect of selection of Transwell chamber membrane material on the in vitro three-culture model of retinal neurovascular unit

[0092] The PET membrane material Transwell chamber and the PC membrane material Transwell chamber (purchased from Corning) in Example 1 were taken. After the two Transwell chambers were inverted, 0.5 mL of complete culture medium was added to the bottom of each Transwell chamber, and the Transwell chambers were placed in a sterile cell culture incubator at 37°C and 5% (v / v) CO2. After the addition was completed, the leakage of the two Transwell chambers was observed to evaluate the effect of the Transwell chamber membrane material on the in vitro three-culture model of retinal neurovascular unit. The experimental results are shown in Figure 7 .

[0093] 1.3, Effect of selection of cell seeding density on the outside of the chamber membrane on the in vitro three-culture model of retinal neurovascular unit

[0094] On the basis of Example 1, the seeding density of rat retinal ganglion cells on the outside of the chamber membrane was replaced from 1×10 5 cells / mL to 3×10 5 cells / mL. The cell morphology and density were observed by inverted microscope at 24 h and 72 h of three-culture, and the results were compared with those of Example 1 to explore the effect of cell seeding density on the outside of the chamber membrane on the in vitro three-culture model of retinal neurovascular unit. The experimental results are shown in Figure 8 .

[0095] 1.4, Effect of the selection of the seeding density of the cells on the inner side of the chamber membrane on the in vitro tri-culture model of retinal neurovascular unit

[0096] On the basis of Example 1, the seeding density of the rat retinal microvascular endothelial cells on the inner side of the chamber membrane was replaced from 1 x 10 5 cells / mL to 3 x 10 5 cells / mL. The cell morphology and density were observed by inverted microscope at 24h and 72h of tri-culture, and the results were compared with those of Example 1 to explore the effect of the seeding density of the cells on the inner side of the chamber membrane on the in vitro tri-culture model of retinal neurovascular unit, and the experimental results are shown in Figure 9 .

[0097] 1.5, Effect of the selection of the seeding density of the cells in the lower chamber (i.e. 24-well cell culture plate) on the in vitro tri-culture model of retinal neurovascular unit

[0098] On the basis of Example 1, the seeding density of the Müller cells in the lower chamber (i.e. 24-well cell culture plate) was replaced from 6 x 10 4 cells / mL to 1.2 x 10 5 cells / mL. The cell morphology and density were observed by inverted microscope at 24h and 72h of tri-culture, and the results were compared with those of Example 1 to explore the effect of the seeding density of the cells in the lower chamber on the in vitro tri-culture model of retinal neurovascular unit, and the experimental results are shown in Figure 10 .

[0099] 1.6, Effect of the implementation or not of the infiltration operation on the in vitro tri-culture model of retinal neurovascular unit

[0100] On the basis of Example 1, the infiltration operation in the inverted culture of the Transwell chamber was removed. The liquid evaporation in the Transwell chamber was observed by naked eye at 3h of tri-culture, and the results were compared with those of Example 1 to explore the effect of the infiltration operation on the in vitro tri-culture model of retinal neurovascular unit, and the experimental results are shown in Figure 11 .

[0101] 2, Experimental results

[0102] 2.1, Performance verification of the in vitro tri-culture model of retinal neurovascular unit and optimization of the culture system (single culture, tri-culture)

[0103] Figure 3The cell proliferation function results show that the proliferation function of the Muller cells in the low-sugar single culture and the high-sugar three culture has a significant statistical difference at 24 h, 48 h and 72 h. Among them, after 48 h and 72 h of establishing the disease model, the Muller cells in the high-sugar three culture system present an abnormal proliferation feature, and the proliferation degree is significantly higher than that of the in-vitro single culture group. Notably, in either the single culture or the three culture system, the high-sugar environment can promote the proliferation activity of the Muller cells, which is manifested as the enhanced proliferation function relative to the low-sugar control group. Further analysis finds that under the same sugar concentration condition, the proliferation level of the Muller cells in the three culture system is obviously lower than that in the single culture group, which indicates that the interaction of the ganglion cells, the Muller cells and the microvascular endothelial cells in the retinal microenvironment can effectively inhibit the pathological proliferation reaction. It can be seen that, compared with the single culture system, the interaction of the three kinds of cells in the three culture system can more objectively simulate the microenvironment of the retinal nerve blood vessel unit in the body. In addition, it is found through the experimental results that, in this three culture system, due to the influence of high sugar and the interaction of the three kinds of cells, the proliferation function of the Muller cells is enhanced compared with the in-vitro low-sugar single culture group, which can completely simulate the pathological and physiological process of the interaction among the three kinds of cells in DR in the body. Therefore, the use of the three-cell co-culture system is helpful for the construction of the retinal vascular disease model such as diabetic retinopathy, and can lay a foundation for subsequent drug screening in the disease model.

[0104] Figure 4 The microscope observation results show that in the retinal nerve blood vessel unit in-vitro three-cell co-culture model, the three kinds of cells of the ganglion cells, the Muller cells and the microvascular endothelial cells have no abnormal morphology, and all grow adherently. Figure 5 The microscope observation results show that in the retinal nerve blood vessel unit in-vitro three-cell co-culture model, the Muller cells have stable morphology, and the whole cell grows in a flat polygonal fusion state, the cell body can be long or short, and has several long protrusions. It can be seen that the use of the system of embodiment 1 for co-culturing the three kinds of cells not only ensures the culture conditions of the rRMECs and the rRGCs, but also meets the growth needs of the Muller cells, and the retinal nerve blood vessel unit in-vitro three-cell co-culture model is successfully constructed.

[0105] Figure 6 The cell apoptosis detection results show that after 48 h and 72 h of high-sugar modeling, the apoptosis of the ganglion cells is reduced in the high-sugar environment with different concentrations compared with the low-sugar 5.5 mM group, which proves that in the high-sugar environment, the mutual support of the three kinds of cells of the ganglion cells, the Muller cells and the microvascular endothelial cells inhibits the apoptosis and necrosis of the ganglion cells. This further proves the conclusion of Figure 3 .

[0106] 2.2 The Influence of Transwell Chamber Membrane Material Selection on the In Vitro Three-Culture Model of Retinal Neurovascular Unit

[0107] Figure 7 The results showed that the PC membrane Transwell chambers began to leak after 15 minutes of inversion, while the PET membrane Transwell chambers retained liquid even after 3 hours of standing in the incubator. This indicates that the PET membrane Transwell chambers (0.4 μm, 3470, Corning, USA) can maintain liquid levels during inverted cell culture without leakage, while the PC membrane Transwell chambers have high permeability, making it difficult to maintain liquid inversion and thus failing to meet the minimum cell adhesion time of 3 hours.

[0108] 2.3 The effect of the choice of cell seeding density on the lateral ventricular membrane of the retinal neurovascular unit in an in vitro three-culture model

[0109] Figure 8 The results showed that 1×10 4 A cell / well density of 3 × 10⁻⁶ cells / well is suitable for growth at 24-72 hours, allowing for experiments at multiple time points. 4 The density of cells / well filled the field of view after 24 hours, and after 72 hours, the cells died in patches due to excessive density.

[0110] 2.4 The effect of cell seeding density on the inner side of the ventricular membrane on the in vitro three-culture model of retinal neurovascular unit

[0111] Figure 9 The results showed that 1×10 4 A cell / well density of 3 × 10⁻⁶ cells / well is suitable for growth at 24-72 hours, allowing for experiments at multiple time points. 4 The density of cells / well filled the field of view after 24 hours, and after 72 hours, the cells died in patches due to excessive density.

[0112] 2.5 The effect of cell seeding density selection in the lower chamber (i.e., 24-well cell culture plate) on the in vitro three-culture model of retinal neurovascular unit.

[0113] Figure 10 The results showed that 3×10 4 A cell / well density of 100 cells per ... wells is suitable for growth at 24-72 hours, allowing for experiments at multiple time points. Meanwhile, a density of 6 × 100 cells per 100 wells is also suitable. 4 The density of cells / well filled the field of view after 24 hours, and after 72 hours, the cells died in patches due to excessive density.

[0114] 2.6 The effect of whether or not the infiltration operation is performed on the in vitro three-culture model of retinal neurovascular unit

[0115] Figure 11 The results show that the liquid in the inverted chamber remains after 3 hours in the incubator with PBS, while the liquid in the inverted chamber without PBS evaporates after 3 hours in the incubator. This result indicates that placing the chamber in a 12 well plate with PBS prevents liquid evaporation during incubation and ensures that the liquid does not evaporate too much during the cell's adherence period in the incubator.

[0116] Obviously, the above-mentioned embodiment is only an example for clearly illustrating, but not a limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. An in vitro tri-culture model of retinal neurovascular units, characterized in that, The in-vitro three-culture model is obtained by in-vitro indirect co-culturing of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells through a Transwell.

2. The retinal neurovascular unit in vitro tri-culture model of claim 1, wherein, The preparation method of the in-vitro three-culture model comprises: culturing the retinal microvascular endothelial cells, the retinal ganglion cells and the retinal Muller cells on the inner side of the membrane of a Transwell, the outer side of the membrane of the Transwell and the lower chamber respectively to obtain the in-vitro three-culture model of the retinal neurovascular unit.

3. The retinal neurovascular unit in vitro tri-culture model of claim 2, wherein the retinal pigment epithelial cells are ARPE-19 cells. The preparation method of the in-vitro three-culture model comprises the following steps: Step one: using a culture medium to prepare a cell suspension of cell A to obtain cell suspension A; inverting a Transwell, inoculating the cell suspension A to the outer side of the membrane of the inverted Transwell, and culturing the cell A on the outer side of the membrane of the Transwell; Step two: discarding the culture medium on the outer side of the membrane of the Transwell, inverting the Transwell and placing it in a No. 1 lower chamber added with a culture medium, and continuing to culture the cell A on the outer side of the membrane of the Transwell; Step three: using a culture medium to prepare a cell suspension of cell B to obtain cell suspension B; using a culture medium to prepare a cell suspension of cell C to obtain cell suspension C; inoculating the cell suspension B to the inner side of the membrane of the Transwell, and inoculating the cell suspension C to a No. 2 lower chamber, and culturing the three kinds of cells in the Transwell and the No. 2 lower chamber respectively; Step four: placing the Transwell with cell A and cell B growing on both sides of the membrane in the No. 2 lower chamber, and continuing to culture the three kinds of cells in the Transwell and the No. 2 lower chamber; obtaining the in-vitro three-culture model of the retinal neurovascular unit; The cell A is retinal microvascular endothelial cells, retinal ganglion cells or retinal Muller cells; when the cell A is retinal microvascular endothelial cells, the cell B and the cell C are retinal ganglion cells and retinal Muller cells respectively, or the cell B and the cell C are retinal Muller cells and retinal ganglion cells respectively; when the cell A is retinal ganglion cells, the cell B and the cell C are retinal microvascular endothelial cells and retinal Muller cells respectively, or the cell B and the cell C are retinal Muller cells and retinal microvascular endothelial cells respectively; when the cell A is retinal Muller cells, the cell B and the cell C are retinal microvascular endothelial cells and retinal ganglion cells respectively, or the cell B and the cell C are retinal ganglion cells and retinal microvascular endothelial cells respectively.

4. The retinal neurovascular unit in vitro tri-culture model of claim 3, wherein the retinal pigment epithelial cells are ARPE-19 cells. The membrane material of the Transwell is PET.

5. The retinal neurovascular unit in vitro tri-culture model of claim 3, wherein the retinal pigment epithelial cells are ARPE-19 cells. The seeding density of the cell A on the outside of the membrane of the Transwell chamber is 8000-12000 cells per well; the seeding density of the cell B on the inside of the membrane of the Transwell chamber is 8000-12000 cells per well; and the seeding density of the cell C in the lower chamber is 25000-35000 cells per well.

6. A method for constructing an in vitro tri-culture model of retinal neurovascular unit, characterized in that, The construction method comprises: in-vitro indirect co-culturing retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells in a Transwell to obtain an in-vitro three-culture model of retinal neurovascular units.

7. An in vitro model of retinal vascular disease, characterized in that, The in-vitro model of retinal vascular diseases is obtained by modeling the in-vitro three-culture model of retinal neurovascular units according to any one of claims 1-5.

8. A method for constructing an in vitro model of retinal vascular disease, comprising, The construction method comprises: modeling the in-vitro three-culture model of retinal neurovascular units according to any one of claims 1-5 to obtain an in-vitro model of retinal vascular diseases.

9. A method of screening for a drug, characterized by, The method comprises: using the in-vitro three-culture model of retinal neurovascular units according to any one of claims 1-5 or the in-vitro model of retinal vascular diseases according to claim 7 for drug screening.

10. Use of the in-vitro three-culture model of retinal neurovascular units according to any one of claims 1-5 or the in-vitro model of retinal vascular diseases according to claim 7 in drug screening.

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