Preparation method of cochlear organ chip integrated with blood labyrinth barrier

Abstract

The invention discloses a preparation method of a cochlear organ chip integrated with a blood labyrinth barrier, and belongs to the field of biomedical engineering. The preparation method of the simulated blood labyrinth barrier comprises the following steps: constructing an endothelial cell culture cavity and a pericyte culture cavity, and separating the endothelial cell culture cavity and the pericyte culture cavity through a simulated basement membrane; and respectively inoculating percutaneous cells and endothelial cells into a percutaneous cell culture cavity and an endothelial cell culture cavity, and culturing to obtain the simulated blood lost barrier. The chip can effectively simulate the structure and the function of the BLB, and the barrier integrity of the BLB is evaluated through TEER (trans-epithelial electrical resistance) measurement and an apparent permeability coefficient. The invention also relates to application of the chip in NIHL drug screening, in particular to drug evaluation for oxidative stress induced inner ear organ injury. Through the platform, the protection effect of the candidate drugs on the TBHP-induced oxidative stress injury of the inner ear organs can be evaluated, and a new tool and thought are provided for developing novel NIHL treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and in particular to a preparation method of a cochlea organ chip integrating a blood-labyrinth barrier. BACKGROUND

[0002] Noise-induced hearing loss (NIHL) is the most common form of sensorineural hearing loss worldwide, and its pathological features mainly include cochlear hair cell damage or loss, spiral ganglion neuron degeneration, and stria vascularis dysfunction. This damage often leads to permanent hearing loss, severely affecting the quality of life of patients. Although there are currently some treatment strategies, such as the use of glucocorticoids, neurotrophic factors, etc., the efficacy of these methods is still limited and often cannot completely reverse hearing loss.

[0003] The blood-labyrinth barrier (BLB) is a key structure of the inner ear, which is composed of a layer of tightly connected endothelial cells, strictly controlling the composition of endolymph and maintaining the homeostasis of the cochlear environment. However, the BLB also becomes a major obstacle to drug delivery, greatly limiting the entry of most systemically administered compounds into the inner ear, thereby reducing the efficacy of drugs. Therefore, how to effectively deliver drugs to the target of the inner ear is a key problem in the field of NIHL treatment that needs to be solved.

[0004] Currently, the methods for evaluating the permeability of drugs to the BLB mainly rely on in vitro cell models and in vivo animal models. However, in vitro cell models are difficult to simulate the complex cochlear microenvironment, while in vivo animal models have problems such as high cost, long cycle, ethical controversy, and the results have certain limitations in relevance to the human body. Therefore, developing an in vitro model that can better simulate the cochlear microenvironment and integrate the function of the BLB is of great significance for the drug screening and development of NIHL.

[0005] In recent years, microfluidic technology has provided new possibilities for constructing in vitro tissue models. Microfluidic chips can simulate the in vivo microenvironment by precisely controlling the flow of microfluids, providing cells with culture conditions closer to physiological conditions. Based on this, the present application proposes a preparation method of a cochlea organoid chip (COC) with a BLB for NIHL drug screening, aiming to solve the shortcomings of the prior art and provide a new tool for developing more effective NIHL treatment drugs. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application proposes a preparation method of a cochlea organ chip integrating a blood-labyrinth barrier.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] The first aspect of the application relates to a preparation method of a blood labyrinth barrier simulation, comprising the following steps:

[0009] The endothelial cell culture cavity and the pericyte culture cavity are constructed and separated by a simulated basement membrane;

[0010] The pericyte and the endothelial cell are inoculated into the pericyte culture cavity and the endothelial cell culture cavity respectively,

[0011] After culture, the blood labyrinth barrier simulation is obtained.

[0012] The second aspect of the application relates to a preparation method of an organoid chip, comprising the following steps:

[0013] The chip system is constructed to form an organoid culture cavity, an endothelial cell culture cavity and a pericyte culture cavity;

[0014] The auditory cell culture is inoculated into the organoid culture cavity to obtain an organoid;

[0015] The pericyte and the endothelial cell are inoculated into the pericyte culture cavity and the endothelial cell culture cavity respectively, and the blood labyrinth barrier simulation is obtained after culture;

[0016] Optionally, the ratio of the PDMS prepolymer of the chip to the curing agent is 10:1, the curing temperature is 60 DEG C, and the curing time is 40 minutes.

[0017] Optionally, each layer of the chip is cleaned using a 300W plasma cleaner for 60 seconds before assembly.

[0018] Optionally, after assembly, the chip is subjected to overnight thermal bonding in an oven at 75 DEG C.

[0019] Optionally, the bottom ultra-thin high-definition glass of the chip is pre-coated with 0.1 mg / mL of polylysine (PDL).

[0020] Optionally, the perfusion flow rate of the chip is set to 30 muL / min.

[0021] Optionally, the size of the chip is the same as that of a standard microscope slide, the length is 75 mm, the width is 25 mm, the total height is less than 10 mm, the height of the top liquid storage chamber is 8 mm, the height of the organoid culture chamber is 300 mu m, and the height of the pericyte (PC) and endothelial cell (EC) culture chambers is 375 mu m.

[0022] Optionally, the culture method of ECs and PCs comprises: isolating the vascular stripe tissue from 10-15 day old mouse cochlea and tearing it into small pieces, inoculating these tissue pieces into collagen I coated culture dishes and culturing them in the corresponding culture medium; EC culture is based on endothelial cell culture medium, supplemented with 5% FBS, 1% P / S and 1% ECGF; PC culture is based on low-sugar DMEM culture medium, supplemented with 10% FBS, 1% penicillin-streptomycin and 100 nM pigment epithelium-derived factor (PEDF); culture at 37°C in a 5% CO2 atmosphere, usually forming cell clones within 1-2 days; after the cells reach 90% confluence, digest with trypsin-EDTA and inoculate at a seeding density of 1.5x10 4 cells / cm 2 (ECs) and 7.5x10 3 cells / cm 2 (PCs) into new collagen I coated culture dishes; replace the culture medium every 2 days and monitor cell growth regularly; evaluate cell purity by immunofluorescence staining, with von Willebrand factor (vWF) and platelet-derived growth factor receptor beta (PDGFRbeta) as specific markers for ECs and PCs, respectively; finally, the cells are cryopreserved in a culture medium containing 10% DMSO and 20% FBS, placed overnight at -80°C and then transferred to liquid nitrogen for long-term storage; the entire procedure is carried out under sterile conditions, using new, sharp ophthalmic forceps for tissue isolation and avoiding moving the culture dishes or changing the culture medium within 24 hours after cell attachment; strictly control the glucose and PEDF concentrations in the PC culture medium and ensure that the cell viability is over 90% before cryopreservation.

[0023] Optionally, the organoid culture method comprises: collecting the auditory sensory epithelium from the cochlea of a P0-1 neonatal mouse, first digesting with 0.3 mg / mL collagenase type I at 37°C for 10 minutes, then adding an equal volume of 0.25% trypsin and continuing to digest for 15 minutes; terminating digestion by adding complete DMEM medium containing 10% FBS; blowing the mixture to obtain a single cell suspension, then centrifuging and washing again with complete medium; counting the obtained single cells and mixing with Matrigel hydrogel, then inoculating into an organoid culture chamber; the hydrogel is solidified at 37°C for 20 minutes, then fresh advanced DMEM / F12 medium containing 1% N2, 2% B27, 1% P / S, 50 ng / mL EGF, 50 ng / mL FGF, 50 ng / mL IGF, 3 μM CHIR99021, 500 μM VPA, 100 μg / mL p-Vc and 2 μM 616452 is added, and the amplification culture is carried out for 10 days; after the amplification stage, the medium is replaced with a differentiation medium, i.e. advanced DMEM / F12 added with 1% N2, 2% B27, 1% PS, 3 μM CHIR99021 and 5 μM LY411575, and cultured for an additional 18 days; the growth state of the organoids is monitored using a bright field microscope or immunofluorescence staining.

[0024] Optionally, the BLB assembly method comprises: seeding high-purity PCs into a designated PCs culture chamber at a density of 1.2 x 10 5 cells, then inverting the chip and culturing for 6 hours to ensure that the cells are fully attached; thereafter, 2 x 10 5 ECs are gently introduced into the ECs culture chamber, and after 18 hours of culture, the chip is connected to the microfluidic system; in order to simulate the basement membrane of the BLB, a polyethylene terephthalate (PET) film pre-coated with collagen type I is placed between the ECs and PCs culture chambers.

[0025] In a third aspect, the present application relates to an organoid chip system, comprising:

[0026] a chip;

[0027] an organoid culture cavity for accommodating an organoid and its culture solution;

[0028] a pericyte culture cavity for accommodating pericytes and their culture solution;

[0029] an endothelial cell culture cavity for accommodating endothelial cells and their culture solution;

[0030] the organoid culture cavity, the pericyte culture cavity and the endothelial cell culture cavity are arranged in sequence and separated by a porous membrane;

[0031] In a fourth aspect, the application relates to the use of the blood-labyrinth barrier mimicking system prepared by the method, the organoid-on-a-chip, and the organoid-on-a-chip system in evaluating or screening drugs for treating noise-induced hearing loss.

[0032] Advantages of the application:

[0033] The application provides a COC platform-based NIHL drug screening method, which can simulate BLB and evaluate drug permeability, and is particularly suitable for evaluating the protective effect of drugs on oxidative stress-induced inner ear organoid damage.

[0034] 1. Simulate BLB: The COC platform can better simulate the BLB structure of the inner ear, providing a model closer to the physiological state for drug screening.

[0035] 2. Evaluate drug permeability: The ability of candidate drugs to penetrate the BLB can be evaluated to screen drugs with potential therapeutic effects.

[0036] 3. Target oxidative stress: Targeting oxidative stress, a key pathological mechanism of NIHL, establishes a corresponding damage model, improving the specificity of drug screening.

[0037] 4. Guide clinical application: By evaluating the BLB permeability of drugs, the dosage form and administration method of drugs can be guided to improve the bioavailability and clinical efficacy of drugs.

[0038] In summary, the application provides an effective NIHL drug screening method, providing a new tool and approach for developing new therapeutic drugs. BRIEF DESCRIPTION OF DRAWINGS

[0039] The application will be further described below with reference to the accompanying drawings.

[0040] Figure 1 Figure 1 is a schematic diagram and a photograph of the COC system, wherein a is a schematic diagram of the layers of the chip, b is a photograph of the assembled chip integrating the microfluid perfusion system, c is a schematic diagram of the microfluid channels in the chip, and d is a schematic diagram of the organoid culture chamber, including size parameters and microchannel configuration.

[0041] Figure 2 Figure 3 is a characteristic characterization diagram of the organoid culture chamber and BLB assembly in the chip, wherein a is an immunofluorescence image of the cochlear organoid at different time points, b is a side view and immunofluorescence staining of vascular stripe endothelial cells and pericytes, c is the transepithelial electrical resistance of the BLB assembly, and d is the apparent permeability coefficient of the BLB assembly.

[0042] Figure 3Figure 4 is a dose-dependent effect diagram of TBHP on the inner ear organoids, wherein a is a bright field image of the inner ear organoids contacted with different concentrations of TBHP, b is the survival rate of the inner ear organoids after being treated with different concentrations of TBHP, and c is the survival rate of the blood-labyrinth barrier components after being treated with different concentrations of TBHP.

[0043] Figure 4 Figure 5 is a diagram of the effects of various compounds on the survival rate of the inner ear organoids treated with TBHP, wherein a is the relative survival rate of the organoids treated with ebselen at different concentrations, b is the relative survival rate of the organoids treated with curcumin at different concentrations, and c is the relative survival rate of the organoids treated with Gypenoside at different concentrations. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the present application.

[0045] In some embodiments of the present application, a COC chip system is provided, as shown in Figure 1 which includes a plurality of PDMS chips.

[0046] The PDMS chip is configured with, from top to bottom, an endothelial cell culture cavity, a pericyte culture cavity, and an organoid culture cavity, and the endothelial cell culture cavity and the pericyte culture cavity are separated by a porous membrane B, and the pericyte culture cavity and the organoid culture cavity are separated by a porous membrane A.

[0047] The PDMS chip needs to be bonded with another PDMS, glass sheet or silicon sheet to form a closed microchannel.

[0048] In some embodiments, in order to achieve permanent bonding, the surface of the PDMS is subjected to plasma treatment (such as oxygen plasma treatment) to produce hydrophilic groups on the surface, and then the surface-treated substrate (such as a glass sheet) is tightly contacted and heated to form irreversible chemical bonding.

[0049] The PDMS chip can be configured with a plurality of input and output ports, which can specifically include:

[0050] The input port i is an organoid culture medium input port;

[0051] The input port ii is an endothelial cell inoculation port, an endothelial cell culture medium input port, and a test drug administration port;

[0052] Input port iii as organoid progenitor cell seeding port;

[0053] Input port iv as pericyte seeding port and pericyte medium input port;

[0054] Output port v as organoid culture medium waste output port;

[0055] Output port vi as endothelial cell culture medium waste output port;

[0056] Output port vii as organoid culture medium waste output port;

[0057] Output port viii as pericyte culture medium waste output port.

[0058] The PDMS chip where the endothelial cell culture cavity is located can be configured with a flow channel that sequentially connects the input port ii, the endothelial cell culture cavity and the output port vi.

[0059] The PDMS chip where the pericyte culture cavity is located can be configured with a flow channel that sequentially connects the input port iv, the pericyte culture cavity and the output port viii.

[0060] The PDMS chip where the organoid culture cavity is located can be configured with a flow channel that respectively connects the input port i, the input port iii, the output port v and the output port vii.

[0061] The test drug is input from the input port ii into the endothelial cell culture cavity, penetrates into the pericyte culture cavity through the porous membrane B, penetrates into the organoid culture cavity through the porous membrane A, and finally acts on the organoid.

[0062] The porous membrane A and the porous membrane B are polyethylene terephthalate (PET) membranes with a pore size of 0.4 μm, and are pre-coated with 10 μg / cm 2 of type I collagen.

[0063] Example 1

[0064] The present embodiment provides a preparation method of a cochlear organoid chip with blood labyrinth barrier (BLB) for noise-induced hearing loss (NIHL) drug screening, which has the structure as shown in Figure 1 , and the specific steps are as follows:

[0065] Step S1, chip preparation and assembly:

[0066] Mold preparation and PDMS curing were performed using soft lithography. PDMS pre-polymer and curing agent were mixed at a ratio of 10:1 and cured at 60 °C for 40 min. Before chip cleaning and assembly, each layer of chips was cleaned using a 300 W plasma cleaner for 60 s, and then assembled sequentially and tightly wrapped with low-adhesion tape. Thermal bonding and sterilization. The assembled chips were placed in a disposable sterilization bag and subjected to overnight thermal bonding in an oven at 75 °C. The bottom of the chip was pre-coated with 0.1 mg / mL poly-lysine (PDL). Chip perfusion and washing. After bonding and sterilization were completed, the chip was perfused with ddH2O for 5 min and then PBS for 5 min in a sterile environment. The perfusion flow rate was set to 30 pL / min. Chip size and structure. The final prepared chip had the same size as a standard microscope slide, with a length of 75 mm, a width of 25 mm, and a total height of less than 10 mm. The height of the top reservoir was 8 mm, the height of the organoid culture chamber was 300 pm, and the height of the pericyte (PCs) and endothelial cell (ECs) culture chambers was 375 pm. Microchannel design verification. Computational fluid dynamics simulation of COC was performed by COMSOL Multiphysics to verify and optimize the microchannel design and determine the perfusion rate.

[0067] Step S2, cell culture:

[0068] Culture of ECs and PCs. First, the vascular stripe tissue was isolated from 10-15-day-old mouse cochlea as the cell source. Then, the tissue pieces were seeded into collagen type I-coated culture dishes and cultured in the corresponding medium. ECs culture was based on endothelial cell medium with the addition of 5% FBS, 1% P / S, and 1% ECGF; while PCs culture was based on low-glucose DMEM medium with the addition of 10% FBS, 1% penicillin-streptomycin, and 100 nM pigment epithelium-derived factor (PEDF). The culture was carried out at 37 °C in a 5% CO2 atmosphere, and cell clones were usually formed within 1-2 days. When the cells reached 90% confluence, they were digested with trypsin-EDTA and seeded into new collagen type I-coated culture dishes at a seeding density of 1.5 x 10 4 cells / cm 2 (ECs) and 7.5 x 10 3 cells / cm 2 (PCs). Cell purity was evaluated by immunofluorescence staining, in which ECs and PCs showed specific markers of von Willebrand factor (vWF) and platelet-derived growth factor receptor beta (PDGFRβ), respectively. Figure 2b). Cells were cryopreserved in culture medium containing 10% DMSO and 20% FBS, placed overnight at -80°C, and then transferred to liquid nitrogen for long-term storage. The entire procedure was performed under sterile conditions, tissue isolation was performed using new, sharp ophthalmic forceps, and the culture dishes were not moved or the medium was not changed within 24 hours after cell attachment, and cell viability was ensured to be over 90% before cryopreservation.

[0069] Organoid culture: First, the auditory sensory epithelium was collected from the cochlea of P0-1 neonatal mice as the cell source. Next, it was digested with 0.3 mg / mL collagenase type I for 10 minutes at 37°C, followed by the addition of an equal volume of 0.25% trypsin and continued digestion for 15 minutes. The digestion was terminated by the addition of complete DMEM medium containing 10% FBS, the mixture was blown to obtain a single cell suspension, and then centrifuged and washed again with complete medium. The obtained single cells were counted and mixed with Matrigel hydrogel, which was then inoculated into the organoid culture chamber, and the hydrogel was solidified at 37°C for 20 minutes. Fresh advanced DMEM / F12 medium containing 1% N2, 2% B27, 1% P / S, 50 ng / mL EGF, 50 ng / mL FGF, 50 ng / mL IGF, 3 mM CHIR99021, 500 mM VPA, 100 pg / mL p-Vc, and 2 mM 616452 was added for 10 days of expansion culture. After the expansion stage, the medium was replaced with differentiation medium, i.e., advanced DMEM / F12 supplemented with 1% N2, 2% B27, 1% PS, 3 mM CHIR99021, and 5 mM LY411575, and cultured for an additional 18 days. Immunofluorescence staining was used to monitor the growth and differentiation state of the organoids Figure 2 a).

[0070] Step S3, BLB assembly:

[0071] At day 13 of organoid culture, 1.2 x 10 5 High-purity PCs of cells were seeded into the designated PC culture chamber. The chip was then inverted and cultured for 6 hours to ensure sufficient cell attachment. Subsequently, 2 x 10 5 ECs were gently introduced into the EC culture chamber. After an additional 18 hours of culture, the chip was integrated with the microfluidic system. To simulate the basement membrane of the BLB, a PET membrane precoated with collagen type I was placed between the EC and PC culture chambers (e.g., porous membrane B shown in Figure 1 Cell positioning of the BLB was evaluated using immunofluorescence imaging methods, showing successful positioning of the two types of cells at their respective locations Figure 2b); the barrier integrity of the BLB was assessed using the TEER (Trans-epithelial electrical resistance) measurement method, showing stable and constant resistance values, indicative of the effectiveness of the tight junctions Figure 2 c); the barrier permeability of the BLB was assessed using FITC-dextran, the COC showing a lower permeability, further confirming the stability of the tight junctions Figure 2 d).

[0072] In the above steps, the measurement of the trans-endothelial electrical resistance (TEER) and the apparent permeability (Papp) can be carried out using the following steps:

[0073] 1.2 x 10 5 Purified PC cells were seeded into the PC chamber. Then, the chip was inverted and the cells were cultured for 6 hours to ensure full adhesion. Subsequently, 2 x 10 5 Purified EC cells were gently seeded into the EC chamber and the co-culture was continued for 18 hours before the chip was connected to the microfluidic system.

[0074] TEER values were measured daily using a cell resistance meter and recorded. During the measurements, the microfluidic system was disconnected and the upper reservoir of the chip was filled with culture medium and the volatile chamber was filled with PBS. The separated electrode tips were inserted into the corresponding ports (inlet ii and iv) of the PC and EC chambers and the average of three measurements was calculated. After four days of BLB (blood brain barrier) formation, the microfluidic system was disconnected and the culture medium in the chip was replaced with D-Hank’s solution to gently rinse each chamber. Then, a working solution of FITC-dextran with a molecular weight of 4 kDa or 40 kDa (1000 pg / mL) was added to the upper chamber of the BLB through inlet ii. After 3 hours, the solution in the organ chamber was aspirated through outlet vii and placed in a 384-well plate. The fluorescence intensity of the FITC-dextran leakage was measured using a microplate reader and subsequently the Papp value was calculated. The calculation of Papp followed the following formula: Papp (cm / s) = AQ / (At x A x C), where Papp represents the apparent permeability, AQ is the total amount of drug or substance that has accumulated through the biological membrane in the time interval At, A is the surface area of the membrane (0.33 cm 2 ), and C is the concentration difference of the drug.

[0075] Step S4, drug application and TBHP treatment:

[0076] On day 16, the organoids were subjected to the drug to be evaluated for 24 hours through the ECs chamber. After 24 hours, the organoids were subjected to the TBHP attack and the drug to be evaluated was continuously applied.

[0077] Step S5, evaluation of the status of the organoids:

[0078] On day 18, the status of the organoids is evaluated, with Ebselen selected as the positive control drug for COC chip evaluation. Through the above steps, the preparation of the cochlear organoid chip with blood-labyrinth barrier (BLB) can be completed, and the platform can be used for NIHL drug screening, especially for drug evaluation for oxidative stress-induced inner ear organoid damage.

[0079] Example 2

[0080] Application of COC platform in NIHL drug screening:

[0081] Step S1, COC platform preparation:

[0082] According to steps S1-S5 in Example 1, the COC platform is constructed, and the integrity of the BLB and the normal growth of the organoids are ensured.

[0083] Step S2, drug evaluation:

[0084] A series of candidate drugs, such as Ebselen, curcumin and gypenoside, are selected, and different concentrations of drugs are applied to the organoids through the ECs culture chamber. After 24 hours of drug application, the organoids are subjected to TBHP attack to simulate oxidative stress environment. On day 18, the status of the organoids is evaluated, including the survival rate, morphology and function of the organoids.

[0085] Step S3, data analysis:

[0086] The survival rate, morphology and function data of the organoids under different drug treatments are collected, and statistical analysis is performed to compare the protective effects of different drugs on TBHP-induced inner ear organoid damage.

[0087] Step S4, result verification:

[0088] Ebselen is used as a positive control to verify the effectiveness and reliability of the COC platform in NIHL drug screening, and the therapeutic potential of the candidate drugs is evaluated according to the evaluation results of the organoid status.

[0089] Through the above steps, the COC platform can be used for NIHL drug screening, especially for drug evaluation for oxidative stress-induced inner ear organoid damage.

[0090] In the description of the present specification, the description of the terms "one embodiment", "example", "specific example" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0091] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and practice described. Accordingly, all such variations and modifications are intended to be included within the scope of the application as defined in the following claims.

Claims

1. A method for preparing a blood-labyrinth barrier analog, comprising the following steps: constructing an endothelial cell culture chamber and a pericyte culture chamber, and separating them by a basement membrane analog; separately inoculating pericytes and endothelial cells into the pericyte culture chamber and the endothelial cell culture chamber, and obtaining the blood-labyrinth barrier analog after culture.

2. The method of claim 1, wherein the blood-brain barrier mimicking preparation is prepared by the steps of: The basement membrane analog is a PET membrane coated with collagen type I. 3.A method for preparing an organoid chip, comprising the following steps: constructing a chip system to form an organoid culture chamber, an endothelial cell culture chamber and a pericyte culture chamber; taking auditory cell culture and inoculating it into the organoid culture chamber to obtain an organoid; separately inoculating pericytes and endothelial cells into the pericyte culture chamber and the endothelial cell culture chamber, and obtaining the blood-labyrinth barrier analog after culture.

4. The method for preparing an organoid-on-a-chip according to claim 3, wherein The method for constructing the organoid comprises the following steps: taking auditory sensory epithelium, digesting it with collagenase type I, then adding an equal volume of trypsin and continuing to digest; stopping the digestion by adding complete DMEM medium containing FBS, blowing the mixture to obtain a single cell suspension, then centrifuging and washing with complete medium again; counting the obtained single cells and mixing them with Matrigel hydrogel, then inoculating into the organoid culture chamber; adding DMEM / F12 medium containing 1% N2, 2% B27, 1% P / S, 50 ng / mL EGF, 50 ng / mL FGF, 50 ng / mL IGF, 3 μM CHIR99021, 500 μM VPA, 100 μg / mL p-Vc and 2 μM 616452 for expansion culture; after expansion culture, the medium is replaced with differentiation medium, Advanced DMEM / F12 containing 1% N2, 2% B27, 1% PS, 3 μM CHIR99021 and 5 μM LY411575, and the organoid is obtained after additional culture.

5. The method for preparing an organoid-on-a-chip according to claim 3, wherein The method for culturing the pericytes and endothelial cells comprises the following steps: taking vascular stria tissue and inoculating it into a collagen type I coated culture dish, and culturing it in the corresponding medium; taking endothelial cell culture and basing it on endothelial cell culture medium, adding 5% FBS, 1% P / S and 1% ECGF; taking pericyte culture and basing it on low-sugar DMEM medium, adding 10% FBS, 1% penicillin-streptomycin and 100 nM pigment epithelium-derived factor; when the cells reach 90% confluence, digest them with trypsin-EDTA and inoculate them into new collagen type I coated culture dishes.

6. The method for preparing an organoid-on-a-chip according to claim 3, wherein The method for preparing the chip system comprises the following steps: mold preparation and PDMS curing using soft lithography technology, mixing PDMS prepolymer with curing agent and curing; chip cleaning and assembly, each layer of chip is cleaned using a plasma cleaner before assembly, and then each layer of chip is bonded; chip bottom packaging glass, and the glass surface is pre-coated with polylysine; after chip bonding, use PBS to perfuse the chip. 7.An organoid chip system, comprising: a chip; an organoid culture chamber for containing an organoid and its culture solution; a pericyte culture chamber for containing pericytes and their culture solution; an endothelial cell culture cavity for accommodating endothelial cells and a culture solution thereof; The organoid culture cavity, the pericyte culture cavity and the endothelial cell culture cavity are arranged in sequence and are separated by a porous membrane; the organoid culture cavity, the pericyte culture cavity and the endothelial cell culture cavity are each provided with an output port and an input port which are connected by a flow channel.

8. The organoid chip system of claim 7, wherein, The porous membrane is a PET membrane coated with collagen type I.

9. The organoid chip system of claim 7, wherein, The chip on which the endothelial cell culture cavity is located is provided with a flow channel which is in communication with the input port ii, the endothelial cell culture cavity and the output port vi in sequence; The chip on which the pericyte culture cavity is located is provided with a flow channel which is in communication with the input port iv, the pericyte culture cavity and the output port viii in sequence; The chip on which the organoid culture cavity is located is provided with a flow channel which is connected to the input port i, the input port iii, the output port v and the output port vii respectively; The input port i is an organoid culture medium input port; The input port ii is an endothelial cell inoculation port, an endothelial cell culture medium input port and a test drug administration port; The input port iii is an organoid progenitor cell inoculation port; The input port iv is a pericyte inoculation port and a pericyte culture medium input port; The output port v is an organoid culture medium waste liquid output port; The output port vi is an endothelial cell culture medium waste liquid output port; The output port vii is an organoid culture medium waste liquid output port; The output port viii is a pericyte culture medium waste liquid output port.

10. Use of the blood-brain barrier simulation prepared by the method of claim 1 or 2, the organoid chip prepared by the method of any one of claims 3 to 6, and the organoid chip system of claims 7 to 9 in evaluating or screening drugs for treating noise-induced hearing loss.

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