An in vitro cell culture model simulating bruch membrane-choroid complex and a method for constructing the same

Abstract

This invention provides an in vitro cell culture model simulating the Bruch membrane-choroid complex (BMCC) and its construction method. The model is composed of polyacrylamide hydrogels of varying stiffness coated with type I collagen. It can be used to study the effects of BMCC stiffness changes on retinal pigment epithelial (RPE) cells, explore the pathogenesis of RPE layer structural dysfunction caused by changes in BMCC mechanical properties, and screen drugs for treating related diseases. Compared to traditional 2D RPE cell line culture models, this model better simulates the physiological and pathological processes of RPE cells in vivo under changes in the mechanical microenvironment, providing convenience for studying the role of mechanical signals in RPE structural dysfunction. The variable stiffness polyacrylamide hydrogels provided by this invention change stiffness by adjusting the ratio of monomers and crosslinking agents, and simulate the environment of RPE cells through type I collagen coating, exhibiting high simulation accuracy and high biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of cell culture model construction, and particularly relates to an in vitro cell culture model simulating the Bruch membrane-choroid complex and its construction method. Background Technology

[0002] Retinal-related diseases are a group of blinding eye diseases caused by structural and functional disorders of the retina, with a blindness rate second only to glaucoma and cataracts. The retina consists of the retinal neuroepithelial layer and the retinal pigment epithelium (RPE), and is connected to the choroid posteriorly by the Bruch's membrane. The choroid is a pigmented vascular membrane that primarily provides nutrition to the outer layer of the retina. The Bruch's membrane consists of five layers, including the basement membrane on the RPE and choroidal sides, the elastic layer in the middle, and two collagen layers. Various types of collagen are important components of the Bruch's membrane. Because the Bruch's membrane and choroid are structurally tightly bound and difficult to separate, they are often studied as a whole, called the Bruch's membrane-choroid complex (BMCC).

[0003] Studies have shown that the conductivity of the Bruch membrane gradually declines with age, indicating a reduction in its water content, a process particularly pronounced in the macula. This decrease in water content slows the transport of retinal metabolites, leading to lipid deposition and causing the Bruch membrane to fragment into a pastry-like layered structure, further affecting retinal metabolite transport. This decline in metabolite transport capacity is considered one of the main causes of age-related degenerative changes in the retina.

[0004] In recent years, with the deepening of research on biomechanics and cellular mechanical microenvironment perception, some researchers have proposed that changes in the retinal mechanical microenvironment may be a mechanism leading to retinal cell structural and functional disorders and age-related degenerative diseases. As mentioned earlier, the structure of the Bruch membrane changes with age, and this "crispy" structural alteration inevitably causes changes in its mechanical properties. A study on the stiffness of the Bruch membrane-choroidal complex (BMCC) showed that after age 21, the BMCC stiffness decreased by 1% annually with age, demonstrating the change in its mechanical properties.

[0005] However, the effects of changes in the mechanical properties of the Bruch's membrane-choroid complex on retinal pigment epithelial cells (RPE cells) and the retina remain unclear. Whether aging affects the normal function of RPE cells and the retina by altering the mechanical microenvironment requires further investigation. Because the BMCC is located deep within the fundus, in vivo studies of it are often challenging. Therefore, constructing an in vitro cell culture model that can simulate the mechanical microenvironment of the retina under aging conditions is of great significance for exploring changes in retinal cells and their mechanisms. To date, there are no reports on in vitro cell culture models that simulate the mechanical properties of the BMCC under aging conditions. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing an in vitro cell culture model that simulates the Bruch membrane-choroid complex and a method for constructing it.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a method for constructing an in vitro cell culture model simulating the Bruch membrane-choroid complex, comprising the following steps:

[0009] (1) A polyacrylamide hydrogel with a stiffness of 5-100 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0010] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a sulfotricyclic aromatic hydrocarbon crosslinking agent at a concentration of 0.5–5 mmol / L, and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1-2 hours; after sterilization, use a concentration of 1-5μg / cm³. 2 Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0011] (3) ARPE-19 complete culture medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete culture medium was replaced every 2 days. After 14 days of culture, the required cells were retained according to the inclusion criteria of the in vitro simulated Bruch membrane-choroid complex cell culture model as the in vitro simulated Bruch membrane-choroid complex cell culture model.

[0012] Furthermore, the ARPE-19 cells were derived from the first three generations after resuscitation.

[0013] Furthermore, the complete culture medium consists of 15 wt% fetal bovine serum, 1 wt% penicillin-streptomycin mixture, and 84 wt% F12 / DMEM basal medium.

[0014] Furthermore, the inclusion criteria for the in vitro simulated Bruch membrane-choroid complex cell culture model include: 1) ARPE-19 cells completely covering the bottom of the dish; 2) ARPE-19 cells being uncontaminated; and 3) ZO-1 cell immunofluorescence staining showing tight junctions between ARPE-19 cells.

[0015] Secondly, the present invention also provides a method for constructing an in vitro cell culture model simulating the Bruch membrane-choroid complex, comprising the following steps:

[0016] (1) A polyacrylamide hydrogel with a stiffness of 1-5 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0017] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a sulfotricyclic aromatic hydrocarbon crosslinking agent at a concentration of 0.5–5 mmol / L, and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1-2 hours; after sterilization, use a concentration of 1-5μg / cm³. 2 Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0018] (3) ARPE-19 complete medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete medium was replaced every 2 days. After 14 days of culture, the cells required for the BMCC in vitro cell culture model simulating the aging mechanical microenvironment were included as the in vitro cell culture model simulating the Bruch membrane-choroid complex.

[0019] Furthermore, the ARPE-19 cells were derived from the first three generations after resuscitation.

[0020] Furthermore, the complete culture medium consists of 15 wt% fetal bovine serum, 1 wt% penicillin-streptomycin mixture, and 84 wt% F12 / DMEM basal medium.

[0021] Furthermore, the inclusion criteria for the BMCC in vitro cell culture model simulating the aging mechanical microenvironment include: 1) ARPE-19 cells completely covering the bottom of the dish; 2) ARPE-19 cells being uncontaminated; 3) ZO-1 cell immunofluorescence staining showing tight junctions between ARPE-19 cells; 4) ARPE-19 cell senescence β-galactosidase staining showing deep green staining; and 5) WB and qPCR showing increased expression of cell senescence-related indicators.

[0022] Thirdly, the present invention also provides an in vitro cell culture model that simulates the Bruch membrane-choroid complex.

[0023] The beneficial effects of this invention are:

[0024] 1) This invention simulates the stiffness changes of the Bruch membrane-choroid complex during aging by preparing polyacrylamide hydrogels with different stiffnesses, and simulates the environment of retinal pigment epithelial cells by coating with collagen. This invention breaks through the limitation of previous 2D cell culture models that cannot simulate the stiffness changes of surrounding tissues, and obtains a cell culture model that can be used to simulate the stiffness changes of the Bruch membrane-choroid complex at different age stages in vitro.

[0025] 2) The low-stiffness Bruch membrane-choroid complex cell culture model provided by the above technical solutions can simulate the changes in the mechanical properties of the Bruch membrane-choroid complex under the aging conditions of the body in vitro, providing an in vitro research and drug treatment screening model for further exploring the effects of age-related changes in the mechanical microenvironment on the retina and its cells.

[0026] 3) This invention establishes a stable cell culture model of altered RPE cell mechanical microenvironment by constructing an in vitro Bruch membrane-choroid complex cell culture model; in addition, the low stiffness condition simulates the decrease in BMCC stiffness caused by aging, and ARPE-19 cells underwent similar cell aging changes as in vivo when cultured on the low stiffness substrate of this model. Attached Figure Description

[0027] Figure 1 A schematic diagram illustrating the construction of an in vitro cell culture model simulating the Bruch membrane-choroid complex;

[0028] Figure 2 Images of ARPE-19 cells cultured for 14 days on pretreated polyacrylamide hydrogels of varying stiffness under a 10x microscope.

[0029] Figure 3 β-gal staining image of cell senescence in an in vitro cell culture model simulating the Bruch membrane-choroid complex;

[0030] Figure 4 Figure showing the expression of aging-related mRNAs in ARPE-19 cells on day 14 of an in vitro cell culture model simulating the Bruch membrane-choroid complex.

[0031] Figure 5 The figure shows the expression of the aging-related protein p21 in ARPE-19 cells on day 14 of in vitro culture in a cell culture model simulating the Bruch membrane-choroid complex. Figure 5 (a) Image showing the expression of the aging-related protein p21 in ARPE-19 cells on day 14 of an in vitro cell culture model simulating the Bruch membrane-choroid complex. Figure 5 (b) is a graph showing the numerical expression of the aging-related protein p21 in ARPE-19 cells on day 14 of in vitro culture in a cell culture model simulating the Bruch membrane-choroid complex. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0033] Given the current lack of in vitro cell culture models that simulate changes in the biomechanical microenvironment of retinopathy of prematurity (RPE), this invention aims to provide an in vitro cell culture model simulating the Bruch's membrane-choroid complex and its construction method, and explores the application of this in vitro Bruch's membrane-choroid complex cell culture model in simulating the biomechanical microenvironment of RPE cells under aging conditions. This invention can simulate the stiffness changes of the Bruch's membrane-choroid complex in vitro, providing an effective research model for investigating the impact of changes in the biomechanical microenvironment on RPE cells. Furthermore, this invention can also simulate the age-related stiffness changes of the Bruch's membrane-choroid complex in vitro, providing a reliable research tool for further investigation into the effects and mechanisms of changes in the biomechanical microenvironment of RPE cells on age-related retinopathy.

[0034] In a first aspect, the present invention provides a method for constructing an in vitro cell culture model simulating the Bruch membrane-choroid complex, comprising the following steps:

[0035] (1) A polyacrylamide hydrogel with a stiffness of 5-100 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0036] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a sulfotricyclic aromatic hydrocarbon crosslinking agent (Sulfo-SANPAH) at a concentration of 0.5–5 mmol / L, and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1-2 hours; after sterilization, use a concentration of 1-5μg / cm³. 2 Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0037] (3) ARPE-19 complete culture medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete culture medium was replaced every 2 days. After 14 days of culture, the required cells were retained according to the inclusion criteria of the in vitro simulated Bruch membrane-choroid complex cell culture model as the in vitro simulated Bruch membrane-choroid complex cell culture model.

[0038] The ARPE-19 cells were derived from the first three generations after resuscitation.

[0039] The complete culture medium consists of 15 wt% fetal bovine serum, 1 wt% penicillin-streptomycin mixture, and 84 wt% F12 / DMEM basal medium.

[0040] The inclusion criteria for the cell culture model of the in vitro Bruch membrane-choroid complex included: 1) ARPE-19 cells completely covered the bottom of the dish; 2) ARPE-19 cells were free of contamination; and 3) immunofluorescence staining of ZO-1 cells showed that ARPE-19 cells formed tight junctions.

[0041] Secondly, the present invention also provides a method for constructing an in vitro cell culture model simulating the Bruch membrane-choroid complex, comprising the following steps:

[0042] (1) A polyacrylamide hydrogel with a stiffness of 1-5 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0043] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a sulfotricyclic aromatic hydrocarbon crosslinking agent at a concentration of 0.5–5 mmol / L, and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1-2 hours; after sterilization, use a concentration of 1-5μg / cm³. 2 Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0044] (3) ARPE-19 complete medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete medium was replaced every 2 days. After 14 days of culture, the cells required for the BMCC in vitro cell culture model simulating the aging mechanical microenvironment were included as the in vitro cell culture model simulating the Bruch membrane-choroid complex.

[0045] The ARPE-19 cells were derived from the first three generations after resuscitation.

[0046] The complete culture medium consists of 15 wt% fetal bovine serum, 1 wt% penicillin-streptomycin mixture, and 84 wt% F12 / DMEM basal medium.

[0047] The inclusion criteria for the BMCC in vitro cell culture model simulating the aging mechanical microenvironment also include: 1) ARPE-19 cells completely covering the bottom of the dish; 2) ARPE-19 cells being uncontaminated; 3) ZO-1 cell immunofluorescence staining showing tight junctions between ARPE-19 cells; 4) ARPE-19 cell senescence (β-gal) staining showing deep green staining; and 5) WB and qPCR showing increased expression of cell senescence-related indicators.

[0048] Example 1

[0049] (1) A polyacrylamide hydrogel with a stiffness of 5 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0050] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a 0.5 mmol / L sulfotricyclic aromatic hydrocarbon crosslinking agent (Sulfo-SANPAH), and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1 hour; after sterilization, use a concentration of 1μg / cm³. 2 Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0051] (3) ARPE-19 complete medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete medium was replaced every 2 days. After 14 days of culture, the cells required for the BMCC in vitro cell culture model simulating the aging mechanical microenvironment were included as the in vitro cell culture model simulating the Bruch membrane-choroid complex.

[0052] Example 2

[0053] (1) A polyacrylamide hydrogel with a stiffness of 20 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0054] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a 2 mmol / L sulfotricyclic aromatic hydrocarbon crosslinking agent (Sulfo-SANPAH), and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1 hour; after sterilization, use a concentration of 2μg / cm³. 2 Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0055] (3) ARPE-19 complete culture medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete culture medium was replaced every 2 days. After 14 days of culture, the required cells were retained according to the in vitro Bruch membrane-choroid complex cell culture model inclusion standard as the in vitro Bruch membrane-choroid complex cell culture model.

[0056] Example 3

[0057] (1) A polyacrylamide hydrogel with a stiffness of 100 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel.

[0058] (2) The surface of the non-biotoxic polyacrylamide hydrogel was covered with a 5 mmol / L sulfotricyclic aromatic hydrocarbon crosslinking agent (Sulfo-SANPAH), and crosslinked under ultraviolet light at a wavelength of 365 nm for 20 min, with an energy of 9999 mJ / cm². 2 After cross-linking, wash three times with 50mM HEPES buffer, 5 minutes each time, then sterilize in a biosafety cabinet under 254nm UV-C light for 1 hour; after sterilization, use a concentration of 5μg / cm³. 2Type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and stored at 4°C for later use to obtain pretreated polyacrylamide hydrogel.

[0059] (3) ARPE-19 complete culture medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete culture medium was replaced every 2 days. After 14 days of culture, the required cells were retained according to the in vitro Bruch membrane-choroid complex cell culture model inclusion standard as the in vitro Bruch membrane-choroid complex cell culture model.

[0060] The obtained in vitro cell culture model of the Bruch membrane-choroid complex

[0061] Figure 2 Images of ARPE-19 cells cultured for 14 days on pretreated polyacrylamide hydrogels of varying stiffness are shown under a 10x microscope. Figure 2 As shown, after 14 days of culture, ARPE-19 cells completely covered the bottom of the dish. Compared to ARPE-19 cells cultured on the bottom of the dish for 14 days, ARPE-19 cells cultured on a 5 kPa polyacrylamide hydrogel were smaller in size and had a more rounded shape. TCP: Normal cell culture plate, serving as a control group.

[0062] β-gal staining for cell senescence in an in vitro cell culture model simulating the Bruch membrane-choroid complex (see [link]). Figure 3 )

[0063] Aspirate the culture medium from the in vitro Bruch membrane-choroid complex cell culture model, wash gently once with PBS, and aspirate dry. Add cell senescence β-gal staining fixative (Beyotime) to the cell culture dish and fix at room temperature for 15 minutes. Aspirate the fixative, wash three times with PBS for 3 minutes each time, aspirate dry, and remove unbound fixative. Add 1 ml of pre-prepared cell senescence β-gal staining working solution (Beyotime) to each well and incubate overnight at 37°C. Observe under an optical microscope.

[0064] Figure 3 This image shows a β-gal staining pattern of ARPE-19 cells undergoing senescence in an in vitro cell culture model simulating the Bruch membrane-choroid complex, illustrating the senescence process in these cells under culture conditions with substrates of varying stiffness. Figure 3 As shown, β-gal staining for cell senescence revealed that ARPE-19 cells stained more prominently in an in vitro cell culture model with a softer RPE cell biomechanical microenvironment, suggesting that senescent cells accounted for a larger proportion of all cells.

[0065] Gene expression in an in vitro cell culture model simulating the Bruch membrane-choroid complex (see [reference]). Figure 4 , Figure 5 )

[0066] ARPE-19 cells were cultured for 14 days in an in vitro cell culture model simulating the Bruch membrane-choroid complex. After washing twice with preheated PBS (37°C) and aspirating, the cells were dried. RNA was extracted using Trizol and then processed using PrimeScript. TM Reverse transcription was performed using the RT Master Mix Kit (Takara, #RR036) and TB was used on the ABI Pirism 7500 rapid sequencing system (Thermo Fisher Scientific). Premix Ex Taq TM II (Takara, #RR420) was used to repeatedly amplify the synthesized cDNA. The sample was denatured at 95°C for 30 seconds, followed by 40 cycles of 3 seconds at 95°C and 30 seconds at 60°C.

[0067] Figure 4 Figure showing the expression of aging-related mRNAs in ARPE-19 cells on day 14 of in vitro culture in a cell culture model simulating the Bruch membrane-choroid complex.

[0068] like Figure 4 As shown, as the stiffness of the culture substrate decreased, the expression of p21 mRNA increased in the cell culture model simulating the Bruch membrane-choroid complex in vitro, suggesting that the expression of senescence-related factors in ARPE-19 cells increased when the stiffness of the substrate decreased.

[0069] After culturing ARPE-19 cells in an in vitro Bruch membrane-choroid complex-mimicking cell culture model for 14 days, the cells were washed twice with pre-warmed PBS (37°C) and aspirated. Cells were then digested with pre-warmed trypsin (37°C) for 3 minutes until floating, collected in 1.5 ml centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS and washed. The cells were centrifuged again at 1000 rpm for 5 minutes, and the supernatant was discarded. Protein was extracted using a protein extraction kit (Solepro), and protein samples from ARPE-19 cells were collected and quantified using a Pierce BCA protein assay kit (Thermo Fisher Scientific). The same amount of protein was electrophoresed on a 10% SDS-PAGE gel (Novozymes), and then transferred to a PVDF membrane (Millipore, NJ, USA). The membrane was then blocked with protein-free rapid blocking buffer (Novozymes). After blocking, the PVDF membrane was incubated overnight at 4°C with p21 primary antibody and then incubated at room temperature with secondary antibody for 1 hour. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference, and the relative expression of the protein was detected and evaluated by measuring the gray values ​​of the bands using ImageJ software (National Institutes of Health, DC, USA).

[0070] Figure 5 The figure shows the expression of the aging-related protein p21 in ARPE-19 cells on day 14 of in vitro culture in a cell culture model simulating the Bruch membrane-choroid complex. Figure 5 (a) Image showing the expression of the aging-related protein p21 in ARPE-19 cells on day 14 of an in vitro cell culture model simulating the Bruch membrane-choroid complex. Figure 5 (b) This figure shows the numerical expression of the aging-related protein p21 in ARPE-19 cells on day 14 of in vitro culture in a cell culture model simulating the Bruch membrane-choroid complex. Figure 5 (a) and Figure 5 As shown in (b), as the stiffness of the culture substrate decreased, the expression of p21 protein increased in the cell culture model simulating the Bruch membrane-choroid complex in vitro, suggesting that the expression of ARPE-19 cells related to senescence increased when the substrate stiffness decreased.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing an in vitro cell culture model simulating the aging Bruch membrane-choroid complex, characterized in that, Includes the following steps: (1) A polyacrylamide hydrogel with a stiffness of 5 kPa was prepared. After the polyacrylamide hydrogel solidified, it was soaked in PBS for 24 h and washed three times with 50 mM HEPES buffer for 5 minutes each time to remove the biotoxic monomer raw material and obtain a non-biotoxic polyacrylamide hydrogel. (2) the surface of the non-biologically toxic polyacrylamide hydrogel is covered with a sulfonated tricyclic aromatic hydrocarbon crosslinking agent with a concentration of 0.5-5 mmol / L, crosslinked under ultraviolet light with a wavelength of 365 nm for 20 min, and the energy is 9999 mJ / cm2; after crosslinking, the polyacrylamide hydrogel is washed with 50 mM HEPES buffer for 3 times, 5 min each time, and then placed in a biological safety cabinet for sterilization by 254 nm UV-C ultraviolet light for 1-2 h; after sterilization, the surface of the polyacrylamide hydrogel is coated with type I collagen with a concentration of 1-5 μg / cm 2 at room temperature for 1 h, washed with PBS buffer for 3 times, and placed in a 4°C refrigerator for standby, to obtain a pretreated polyacrylamide hydrogel; (3) ARPE-19 complete culture medium was then added to the pretreated polyacrylamide hydrogel and immersed. ARPE-19 cells were then seeded on the surface of the pretreated polyacrylamide hydrogel and cultured. The ARPE-19 complete culture medium was replaced every 2 days. After 14 days of culture, the cells required for the inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment were used as the cell culture model of the Bruch membrane-choroid complex simulating aging. The inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment included: 1) ARPE-19 cells covered the bottom of the dish; 2) ARPE-19 cells were free of contamination; 3) ZO-1 cell immunofluorescence staining showed that ARPE-19 cells formed tight junctions; 4) ARPE-19 cell senescence β-galactosidase staining showed deep green staining; 5) WB and qPCR showed increased expression of cell senescence-related indicators.

2. The method for constructing an in vitro cell culture model of the Bruch membrane-choroid complex simulating aging, as described in claim 1, is characterized in that... The ARPE-19 cells were derived from the first three generations after resuscitation.

3. The method for constructing an in vitro Bruch membrane-choroid complex cell culture model simulating aging according to claim 1, characterized in that, The complete culture medium consists of 15 wt% fetal bovine serum, 1 wt% penicillin-streptomycin mixture, and 84 wt% F12 / DMEM basal medium.

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