Cell culture model for simulating Bruch membrane-choroid complex in vitro and construction method thereof

Abstract

The invention provides a cell culture model for simulating a Bruch membrane-choroid complex in vitro and a construction method of the cell culture model. The model is composed of polyacrylamide hydrogel coated by type I collagen and having different rigidities, can be used for researching the influence of BMCC rigidity change on retinal pigment epithelium (RPE) cells, explores the pathogenesis of RPE layer structure dysfunction caused by BMCC mechanical property change, and can be used for screening drugs for treating related diseases. Compared with a traditional 2D RPE cell line culture model, the model can better simulate the physiological and pathological process of in-vivo RPE cells when the mechanical microenvironment is changed, and convenience is provided for studying the effect of mechanical signals in RPE structure dysfunction. The rigidity of the variable-rigidity polyacrylamide hydrogel provided by the invention is changed by adjusting the ratio of the monomer to the cross-linking agent, and the environment in which RPE cells are located is simulated through I-type collagen coating, so that the variable-rigidity polyacrylamide hydrogel has the characteristics of high simulation property, high biological safety and the like.

Description

Technical Field

[0001] The invention belongs to the field of cell culture model construction, and particularly relates to a cell culture model for simulating a Bruch's membrane-choroid complex in vitro and a construction method thereof. Background Art

[0002] Retinal diseases are a type of blinding eye disease caused by retinal structural and functional disorders, and their blindness rate is second only to glaucoma and cataracts. The retina is composed of the retinal neuroepithelium and the retinal pigment epithelium (RPE), which are connected to the choroid through Bruch's membrane at the back. The choroid is a vascular membrane containing pigments, which mainly provides nutrition for the outer layer of the retina. Bruch's membrane is composed of 5 layers, including the basement membrane on the RPE side and the choroid side, the elastic layer in the middle, and two collagen layers. Various types of collagen are important components of Bruch's membrane. Because Bruch's membrane and choroid are tightly combined in structure and difficult to separate, they are often studied as a whole, called Bruch's membrane-choroid complex (BMCC).

[0003] Studies have shown that with age, the conductivity of Bruch's membrane gradually decreases, indicating a decrease in its water content, which is particularly evident in the macular area; the decrease in water content slows down the transport of retinal-related metabolites, leading to lipid deposition, which causes Bruch's membrane to fragment into a layered structure similar to puff pastry, further affecting the transport of metabolites in the retina. The decline in metabolite transport capacity is considered to be 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 cell mechanical microenvironment perception, some researchers have proposed that changes in the retinal mechanical microenvironment may be a mechanism that causes structural and functional disorders of retinal cells and leads to age-related degenerative diseases. As mentioned earlier, with age, the structure of Bruch's membrane changes, and the "puff pastry-like" structural changes will inevitably cause changes in its mechanical properties. A study on the stiffness of the Bruch's membrane-choroid complex showed that after the age of 21, the stiffness of BMCC decreases by 1% each year with age, proving the change in its mechanical properties.

[0005] However, the effect of changes in the mechanical properties of the Bruch's membrane-choroid complex on retinal pigment epithelial cells and the retina is still unclear. Whether aging affects the normal function of RPE cells and the retina by changing the mechanical microenvironment still needs further study. Since BMCC is located in a deeper part of the fundus, it is often difficult to study it in vivo. Therefore, it is of great significance to construct an in vitro cell culture model that can simulate the retinal mechanical microenvironment under the condition of aging in order to explore the changes in retinal cells and their mechanisms. So far, there has been no report on an in vitro cell culture model that simulates the mechanical properties of BMCC under aging. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies of the prior art and provide a cell culture model for simulating Bruch's membrane-choroid complex in vitro and a construction method thereof.

[0007] The objective of the present invention is achieved through the following technical solutions:

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

[0009] (1) preparing a polyacrylamide hydrogel with a stiffness of 5 to 100 kPa; after the polyacrylamide hydrogel solidifies, soaking it in PBS for 24 hours, and washing it three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw material with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel;

[0010] (2) using a sulfotricyclic aromatic hydrocarbon crosslinker with a concentration of 0.5 to 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 minutes under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, wash with 50 mM HEPES buffer for 3 times, 5 minutes each time, and then sterilize with 254 nm UV-C light in a biosafety cabinet for 1-2 hours. After sterilization, 2 The type I collagen was coated at room temperature for 1 hour, washed with PBS buffer for 3 times, and placed in a refrigerator at 4°C for later use to obtain the pretreated polyacrylamide hydrogel;

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

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

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

[0014] Furthermore, the inclusion criteria of the in vitro simulated Bruch's membrane-choroid complex cell culture model include: 1) ARPE-19 cells cover the bottom of the dish; 2) ARPE-19 cells are free of contamination; 3) ZO-1 cell immunofluorescence staining shows tight junctions formed between ARPE-19 cells.

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

[0016] (1) preparing a polyacrylamide hydrogel with a stiffness of 1 to 5 kPa; after the polyacrylamide hydrogel solidifies, soaking it in PBS for 24 hours, and washing it three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw material with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel;

[0017] (2) using a sulfotricyclic aromatic hydrocarbon crosslinker with a concentration of 0.5 to 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 minutes under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, wash with 50 mM HEPES buffer for 3 times, 5 minutes each time, and then sterilize with 254 nm UV-C light in a biosafety cabinet for 1-2 hours. After sterilization, 2 The type I collagen was coated at room temperature for 1 hour, washed with PBS buffer for 3 times, and placed in a refrigerator at 4°C for later use to obtain the pretreated polyacrylamide hydrogel;

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

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

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

[0021] Furthermore, the inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment include: 1) ARPE-19 cells cover the bottom of the dish; 2) ARPE-19 cells are free of contamination; 3) ZO-1 cell immunofluorescence staining shows tight junctions between ARPE-19 cells; 4) ARPE-19 cell senescence β-galactosidase staining shows dark green staining; 5) WB and qPCR show increased expression of cell senescence-related indicators.

[0022] The third invention, the present invention also provides a cell culture model for simulating Bruch's membrane-choroid complex in vitro.

[0023] The beneficial effects of the present invention are:

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

[0025] 2) The low-rigidity Bruch's membrane-choroid complex cell culture model provided by the above technical solution can simulate the changes in the mechanical properties of the Bruch's membrane-choroid complex under the condition of body aging in vitro, and provide an in vitro research and drug treatment screening model for further exploring the effects of mechanical microenvironment changes caused by aging on the retina and its cells;

[0026] 3) The present invention established a stable cell culture model of RPE cell mechanical microenvironment changes by constructing a Bruch's membrane-choroid complex cell culture model in vitro; in addition, the low-stiffness condition simulated the decrease in BMCC stiffness caused by aging, and ARPE-19 cells underwent cell aging changes similar to those in vivo when cultured on the low-stiffness substrate of this model. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of the construction of a cell culture model for simulating Bruch's membrane-choroid complex in vitro;

[0028] Figure 2 The images of ARPE-19 cells cultured on pre-treated polyacrylamide hydrogels with different stiffnesses after 14 days under a 10x microscope;

[0029] Figure 3 This is a β-gal staining image of cell senescence in a cell culture model that simulates Bruch's membrane-choroid complex in vitro;

[0030] Figure 4 This is a graph showing the expression of senescence-related mRNA in ARPE-19 cells on the 14th day of culture in a cell culture model that simulates Bruch's membrane-choroid complex in vitro;

[0031] Figure 5 This is a diagram showing the expression of senescence-related protein p21 in ARPE-19 cells on the 14th day of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro. Figure 5 (a) is the image expression of the senescence-related protein p21 in ARPE-19 cells on the 14th day of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro. Figure 5 (b) is a graph showing the numerical expression of the senescence-related protein p21 in ARPE-19 cells on the 14th day of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0033] In view of the lack of in vitro cell culture models that simulate changes in the mechanical microenvironment of RPE cells, the present invention aims to provide a cell culture model and a construction method for simulating Bruch's membrane-choroid complex in vitro, and explores the application of the in vitro Bruch's membrane-choroid complex cell culture model in the mechanical microenvironment of RPE cells under simulated aging conditions. The present invention can simulate the stiffness changes of Bruch's membrane-choroid complex in vitro, and provide an effective research model for exploring the effects of mechanical microenvironment changes on RPE cells. In addition, the present invention can also simulate the hardness changes of Bruch's membrane-choroid complex with age in vitro, and provide a reliable research tool for further studying the effects of changes in the mechanical microenvironment of RPE cells on age-related retinopathy and its mechanism.

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

[0035] (1) preparing a polyacrylamide hydrogel with a stiffness of 5 to 100 kPa; after the polyacrylamide hydrogel solidifies, soaking it in PBS for 24 hours, and washing it three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw material with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel;

[0036] (2) using a sulfo-tricyclic aromatic hydrocarbon crosslinking agent (Sulfo-SANPAH) with a concentration of 0.5 to 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 minutes under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, wash with 50 mM HEPES buffer for 3 times, 5 minutes each time, and then sterilize with 254 nm UV-C light in a biosafety cabinet for 1-2 hours. After sterilization, 2 The type I collagen was coated at room temperature for 1 hour, washed with PBS buffer for 3 times, and placed in a refrigerator at 4°C for later use to obtain the pretreated polyacrylamide hydrogel;

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

[0038] The ARPE-19 cells were derived from the first 3 passages after recovery.

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

[0040] The inclusion criteria of the cell culture model simulating Bruch's membrane-choroid complex in vitro include: 1) ARPE-19 cells cover the bottom of the dish; 2) ARPE-19 cells are free of contamination; 3) ZO-1 cell immunofluorescence staining shows tight junctions formed between ARPE-19 cells.

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

[0042] (1) preparing a polyacrylamide hydrogel with a stiffness of 1 to 5 kPa; after the polyacrylamide hydrogel solidifies, soaking it in PBS for 24 hours, and washing it three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw material with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel;

[0043] (2) using a sulfotricyclic aromatic hydrocarbon crosslinker with a concentration of 0.5 to 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 minutes under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, wash with 50 mM HEPES buffer for 3 times, 5 minutes each time, and then sterilize with 254 nm UV-C light in a biosafety cabinet for 1-2 hours. After sterilization, 2 The type I collagen was coated at room temperature for 1 hour, washed with PBS buffer for 3 times, and placed in a refrigerator at 4°C for later use to obtain the pretreated polyacrylamide hydrogel;

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

[0045] The ARPE-19 cells were derived from the first 3 passages after recovery.

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

[0047] The inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment also include: 1) ARPE-19 cells cover the bottom of the dish; 2) ARPE-19 cells are free of contamination; 3) ZO-1 cell immunofluorescence staining shows tight junctions between ARPE-19 cells; 4) ARPE-19 cell senescence (β-gal) staining shows dark green staining; 5) WB and qPCR show 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 was solidified, it was soaked in PBS for 24 hours and washed three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw materials with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel.

[0050] (2) using a 0.5 mmol / L sulfo-tricyclic aromatic hydrocarbon crosslinker (Sulfo-SANPAH) to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 min under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, the cells were washed three times with 50 mM HEPES buffer for 5 minutes each time, and then sterilized with 254 nm UV-C light in a biosafety cabinet for 1 hour. After sterilization, the cells were sterilized with a concentration of 1 μg / cm 2 The type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and placed in a 4°C refrigerator for later use to obtain the pretreated polyacrylamide hydrogel.

[0051] (3) ARPE-19 complete medium was then added to the pretreated polyacrylamide hydrogel and immersed, and then ARPE-19 cells were inoculated on the surface of the pretreated polyacrylamide hydrogel for culture. The ARPE-19 complete medium was replaced every 2 days. After 14 days of culture, the cells required by the inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment were used as an in vitro cell culture model simulating Bruch's 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 hours and washed three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw materials with biological toxicity, thereby obtaining a non-biologically toxic polyacrylamide hydrogel.

[0054] (2) using a sulfo-tricyclic aromatic hydrocarbon crosslinker (Sulfo-SANPAH) with a concentration of 2 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 min under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, the cells were washed three times with 50 mM HEPES buffer for 5 minutes each time, and then sterilized with 254 nm UV-C light in a biosafety cabinet for 1 hour. After sterilization, the cells were sterilized with a concentration of 2 μg / cm 2 The type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and placed in a 4°C refrigerator for later use to obtain the pretreated polyacrylamide hydrogel.

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

[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 hours and washed three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw materials with biological toxicity, thereby obtaining a non-biologically toxic polyacrylamide hydrogel.

[0058] (2) using a sulfo-tricyclic aromatic hydrocarbon crosslinker (Sulfo-SANPAH) with a concentration of 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 min under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, the cells were washed three times with 50 mM HEPES buffer for 5 minutes each time, and then sterilized with 254 nm UV-C light in a biosafety cabinet for 1 hour. After sterilization, the cells were sterilized with a concentration of 5 μg / cm 2The type I collagen was coated at room temperature for 1 hour, washed three times with PBS buffer, and placed in a 4°C refrigerator for later use to obtain the pretreated polyacrylamide hydrogel.

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

[0060] Obtained in vitro cell culture model simulating Bruch's membrane-choroid complex

[0061] Figure 2 The image of ARPE-19 cells cultured on pre-treated polyacrylamide hydrogels with different stiffnesses after 14 days under a 10x microscope is shown. Figure 2 As shown, after 14 days of culture, ARPE-19 cells covered the bottom of the dish. Compared with ARPE-19 cells cultured on the bottom of the dish for 14 days, the size of ARPE-19 cells cultured on 5kPa polyacrylamide hydrogels became smaller and the cell shape was round. TCP: normal cell culture plate, used as the control group.

[0062] β-gal staining of cellular senescence in a cell culture model that simulates Bruch's membrane-choroid complex in vitro (see Figure 3 )

[0063] Aspirate the culture medium of the cell culture model simulating Bruch's membrane-choroid complex in vitro, wash gently once with PBS, and aspirate dry. Add the cell senescence β-gal staining fixative (Biyuntian) 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 the unbound fixative. Add 1 ml of the pre-prepared cell senescence β-gal staining working solution (Biyuntian) to each well and incubate overnight at 37°C. Observe under an optical microscope.

[0064] Figure 3 This is a β-gal staining image of cell senescence in a cell culture model simulating the Bruch's membrane-choroid complex in vitro, showing the cell senescence of ARPE-19 cells under different substrate stiffness culture conditions. Figure 3 As shown, cell senescence β-gal staining showed that in the in vitro cell culture model of RPE cell mechanical microenvironment with a softer substrate, ARPE-19 cells were more obviously stained, suggesting that senescent cells accounted for a larger proportion of all cells.

[0065] Gene expression in a cell culture model that mimics the Bruch's membrane-choroid complex in vitro (see Figure 4 , Figure 5 )

[0066] After 14 days of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro, ARPE-19 cells were washed twice with PBS preheated at 37°C and dried. After RNA was extracted using Trizol, PrimeScript TM Reverse transcription was performed using the RT Master Mix Kit (Takara, #RR036) and the DNA was analyzed using the TB DNA sequencing system (Thermo Fisher Scientific). Premix Ex Taq TM The synthesized cDNA was amplified repeatedly using ELISA II (Takara, #RR420). The samples were denatured at 95°C for 30 seconds and then subjected to 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds.

[0067] Figure 4 This is a graph showing the expression of senescence-related mRNA in ARPE-19 cells on the 14th day of culture in a cell culture model that simulates Bruch's membrane-choroid complex in vitro.

[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 Bruch's membrane-choroid complex in vitro, suggesting that when the stiffness of the substrate decreased, the senescence-related expression of ARPE-19 cells increased.

[0069] After 14 days of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro, ARPE-19 cells were washed twice with PBS preheated at 37°C and dried. Use 37°C preheated trypsin digestion for 3 minutes until the cells float, collect them in a 1.5ml centrifuge tube, centrifuge at 1000r / min for 5 minutes, discard the supernatant, add PBS to resuspend and wash. Centrifuge again at 1000r / min for 5 minutes, discard the supernatant. Use a protein extraction kit (Solerbo) to extract proteins, collect and use the Pierce BCA protein assay kit (Thermo Fisher Scientific) to quantify the protein samples of ARPE-19 cells. On a 10% SDS PAGE gel (Novizen), the same amount of protein was electrophoresed and then transferred to a PVDF membrane (Millipore, NJ, USA). The membrane was then blocked with a protein-free fast blocking buffer (Novizen). After blocking, the PVDF membrane was incubated with p21 primary antibody overnight at 4°C and incubated with secondary antibody for 1 hour at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference, and the relative expression of proteins was detected and evaluated by measuring the gray value of the bands using Image J software (National Institutes of Health, DC, USA).

[0070] Figure 5 This is a diagram showing the expression of senescence-related protein p21 in ARPE-19 cells on the 14th day of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro. Figure 5 (a) is the image expression of the senescence-related protein p21 in ARPE-19 cells on the 14th day of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro. Figure 5 (b) is the numerical expression of the senescence-related protein p21 in ARPE-19 cells on the 14th day of culture in a cell culture model simulating Bruch's membrane-choroid complex in vitro. 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 Bruch's membrane-choroid complex in vitro, suggesting that when the substrate stiffness decreased, the senescence-related expression of ARPE-19 cells increased.

[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 in the scope of protection of the present invention.

Claims

1. A method for constructing a cell culture model for simulating Bruch's membrane-choroid complex in vitro, characterized in that: The following steps are involved: (1) preparing a polyacrylamide hydrogel with a stiffness of 5 to 100 kPa; after the polyacrylamide hydrogel solidifies, soaking it in PBS for 24 hours, and washing it three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw material with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel; (2) using a sulfotricyclic aromatic hydrocarbon crosslinker with a concentration of 0.5 to 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 minutes under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, wash with 50 mM HEPES buffer for 3 times, 5 minutes each time, and then sterilize with 254 nm UV-C light in a biosafety cabinet for 1-2 hours. After sterilization, 2 The type I collagen was coated at room temperature for 1 hour, washed with PBS buffer for 3 times, and placed in a refrigerator at 4°C for later use to obtain the pretreated polyacrylamide hydrogel; (3) ARPE-19 complete medium was then added to the pretreated polyacrylamide hydrogel and immersed, and then ARPE-19 cells were inoculated on the surface of the pretreated polyacrylamide hydrogel for culture. The ARPE-19 complete medium was replaced every 2 days. After 14 days of culture, the required cells were retained as the in vitro simulated Bruch's membrane-choroid complex cell culture model according to the inclusion criteria.

2. A method for constructing a cell culture model for simulating Bruch's membrane-choroid complex in vitro, characterized in that: The following steps are involved: (1) preparing a polyacrylamide hydrogel with a stiffness of 1 to 5 kPa; after the polyacrylamide hydrogel solidifies, soaking it in PBS for 24 hours, and washing it three times with 50 mM HEPES buffer, each time for 5 minutes, to remove the monomer raw material with biological toxicity, and obtain a non-biologically toxic polyacrylamide hydrogel; (2) using a sulfotricyclic aromatic hydrocarbon crosslinker with a concentration of 0.5 to 5 mmol / L to cover the surface of the non-biotoxic polyacrylamide hydrogel, and crosslinking for 20 minutes under ultraviolet light with a wavelength of 365 nm and an energy of 9999 mJ / cm 2 After cross-linking, wash with 50 mM HEPES buffer for 3 times, 5 minutes each time, and then sterilize with 254 nm UV-C light in a biosafety cabinet for 1-2 hours. After sterilization, 2 The type I collagen was coated at room temperature for 1 hour, washed with PBS buffer for 3 times, and placed in a refrigerator at 4°C for later use to obtain the pretreated polyacrylamide hydrogel; (3) ARPE-19 complete medium was then added to the pretreated polyacrylamide hydrogel and immersed, and then ARPE-19 cells were inoculated on the surface of the pretreated polyacrylamide hydrogel for culture. The ARPE-19 complete medium was replaced every 2 days. After 14 days of culture, the cells required by the inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment were used as an in vitro cell culture model simulating Bruch's membrane-choroid complex.

3. A method for constructing a cell culture model for simulating Bruch's membrane-choroid complex in vitro according to claim 1 or 2, characterized in that: The ARPE-19 cells were derived from the first 3 passages after recovery.

4. The method for constructing a cell culture model for simulating Bruch's membrane-choroid complex in vitro according to claim 1 or 2, characterized in that: The complete culture medium consists of 15wt% fetal bovine serum, 1wt% penicillin-streptomycin mixed solution and 84wt% F12 / DMEM basic culture medium.

5. The method for constructing a cell culture model for simulating Bruch's membrane-choroid complex in vitro according to claim 1, characterized in that: The inclusion criteria of the in vitro simulated Bruch's membrane-choroid complex cell culture model include: 1) ARPE-19 cells cover the bottom of the dish; 2) ARPE-19 cells are free of contamination; 3) ZO-1 cell immunofluorescence staining shows tight junctions formed between ARPE-19 cells.

6. The method for constructing a cell culture model for simulating Bruch's membrane-choroid complex in vitro according to claim 2, characterized in that: The inclusion criteria of the BMCC in vitro cell culture model simulating the aging mechanical microenvironment include: 1) ARPE-19 cells cover the bottom of the dish; 2) ARPE-19 cells are free of contamination; 3) ZO-1 cell immunofluorescence staining shows tight junctions between ARPE-19 cells; 4) ARPE-19 cell senescence β-galactosidase staining shows dark green staining; 5) WB and qPCR show increased expression of cell senescence-related indicators.

7. A cell culture model for simulating Bruch's membrane-choroid complex in vitro prepared by any method of claims 1-6.

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