A method for prolonging the in vitro culture time of cochlear hair cells

Abstract

The present invention provides a method for prolonging the in vitro culture time of cochlear hair cells, belonging to the field of cell culture, and the method steps are as follows: (1) dissecting the temporal bone: peeling off the mouse forebrain, cerebellum and brainstem, and separating and obtaining the cochlea; (2) extracting the cochlea: separating the spiral ligament on the Corti organ from the rest of the cochlea, peeling off the Reissner's membrane, and removing the tectorial membrane above the Corti organ; (3) culturing the cochlea in vitro: placing the cochlear hair cells upward on a cell culture plate containing a cell slide and a culture medium, wherein the cochlear hair cell culture medium is a DMEM high-glucose culture medium containing 0.6-0.7% glucose (including glucose in the basal culture medium), 1% N2 supplement and 100U / mL penicillin G sodium, and culturing the hair cells in a cell culture incubator, and replacing the culture medium every 72 hours. The in vitro culture method of cochlear hair cells provided by the present invention is the first reported method at home and abroad, and can culture hair cells in vitro for 30 days, which can meet the needs of exploring the mechanism of hair cell development and differentiation and collecting data on in vitro gene therapy for hearing impairment.

Description

Technical Field

[0001] The invention belongs to the field of cell culture, and in particular relates to a method for prolonging the in vitro culture time of cochlear hair cells. Technical Background

[0002] The cochlea is an important part of the inner ear. Together with the vestibular labyrinth, it forms the bony labyrinth of the inner ear. The bony labyrinth of the inner ear is a structure that conducts and senses sound waves (such as Figure 1 (As shown). The English name of the cochlea, Cochlea, means "snail shell" in Latin, which is derived from its shape similar to a snail shell. The cochlea is an important component of the peripheral auditory system. Its core part is the Organ of Corti, which is the auditory transduction organ responsible for converting sound signals from the middle ear into corresponding neural electrical signals, which are then transmitted to the central auditory system in the brain, ultimately realizing sound perception [1].

[0003] The organ of Corti is an auditory receptor that senses sound waves and is composed of supporting cells and hair cells (such as Figure 2 As shown in the figure, the supporting cells play a structural role in supporting the hair cells, while the hair cells are sound wave sensing cells. Each hair cell forms a synaptic connection with the nerve fibers and is the key to the perception function of the organ of Corti [2]. There is a tympanic membrane above the hair cells, which is in contact with the cilia of the hair cells. When sound occurs, the sound waves are transmitted to the tympanic membrane through the external auditory canal, amplifying the vibration of the tympanic membrane and transmitting it to the oval window of the inner ear. The vibration of the oval window causes the perilymph in the inner ear cochlea to vibrate. The vibration of the perilymph causes the relative movement between the tectorial membrane and the static cilia of the hair cells. This movement is called shear movement. The deflection of the cilia causes the mechanically gated ion channels (such as the MET channel) on the hair cells to open, allowing ions to flow, thereby generating electrical signals on the hair cells. The auditory nerve fibers transmit the electrical signals to the auditory center, allowing the brain to perceive the sound.

[0004] Cochlear lesions are closely related to various hearing impairments. Hair cells in the cochlea of ​​animals such as fish, birds, and amphibians can regenerate after damage, but once damaged, the hair cells of mammals cannot regenerate; cell therapy is an emerging treatment method that involves transplanting cells into the patient's body to repair or replace damaged tissues and organs. In the treatment of sensorineural hearing loss (SNHL), cell therapy mainly focuses on regenerating or protecting the hair cells and spiral ganglion neurons in the inner ear. By inducing ESCs or iPSCs to differentiate into hair cell-like cells and then transplanting them into the damaged inner ear, hearing can be restored [3]. Therefore, clarifying the developmental mechanism of hair cells is the key to solving the problem of hearing impairment caused by cochlear lesions. In vitro culture of hair cells is a prerequisite for studying the differentiation and development mechanism of cochlear hair cells in the auditory system. The growth and development of hair cells generally takes 20 days [4]. If the hair cells survive in vitro for less than this time, their complete differentiation and development process cannot be studied. At the same time, in vitro gene therapy research related to hearing requires the collection of 28 days of in vitro animal treatment data, but the currently reported in vitro culture methods of cochlear hair cells can only maintain hair cell growth for 3 to 10 days. For example, the Chinese patent "A method for culturing mouse cochlear hair cells" (publication number: CN 109182250 A) provides a special culture medium that can be used for in vitro culture of mammalian cochlear hair cells and stably maintain the activity of cochlear hair cells, but the culture medium formula is complex and can only culture hair cells for a maximum of 5 days; the Chinese patent "A method for isolating and culturing inner ear hair cells in vitro" (publication number: CN 109280634 A) provides a formula for high-glucose DMEM medium containing 5% horse serum at a final concentration, 5% fetal bovine serum at a final concentration by volume, and 1% N2 cytokine supplement at a final concentration by volume. While this medium can achieve a morphology closer to that of in vivo basilar membrane hair cells, it only prolongs in vitro culture time to 10 days and requires medium replacement every 24 hours. Therefore, current hair cell culture protocols fail to meet these requirements. Furthermore, the required medium components are complex, costly, and laborious, requiring frequent medium changes, increasing the risk of contamination. Therefore, a simple method is urgently needed to significantly prolong the survival of cochlear hair cells in vitro. Summary of the Invention

[0005] In response to the above needs, the present invention provides a method for prolonging the in vitro culture time of cochlear hair cells, which belongs to the field of cell culture. The method comprises the following steps: (1) dissecting the temporal bone: peeling off the mouse forebrain, cerebellum and brainstem to isolate the cochlea; (2) extracting the cochlea: separating the spiral ligament on the organ of Corti from the rest of the cochlea, peeling off the Reissner membrane, and removing the tectorial membrane above the organ of Corti; (3) culturing the cochlea in vitro: placing the cochlear hair cells upward on a cell culture plate containing a cell slide and culture medium, wherein the cochlear hair cell culture medium is a MEM high-glucose culture medium containing 0.615% glucose, 1% N2 supplement and 100U / mL penicillin G sodium, and culturing the hair cells in a cell culture incubator, with the culture medium replaced every 72 hours. The in vitro culture method of cochlear hair cells provided by the present invention is the first reported method in China and abroad, and can culture hair cells in vitro for 30 days. It can meet the needs of exploring the mechanism of hair cell development and differentiation and collecting data on in vitro gene therapy for hearing impairment.

[0006] On the one hand, the present invention provides a method for prolonging the in vitro culture time of cochlear hair cells, wherein the cochlear hair cells are adhered to a cell slide and cultured using a high-glucose culture medium containing glucose.

[0007] Adhering cochlear hair cells to a fixture helps promote the growth of cells in a multicellular or localized tissue state. The physical and chemical factors created provide the cells with an environment that is closest to the in vivo environment of the cells, which in turn helps maintain the cell morphology and structure, and also helps prolong the survival time of cochlear hair cells in vitro; prevents cell autolysis and tissue corruption, while maintaining the cell's antigenicity to facilitate subsequent experiments and analysis. Currently, the conventional method of adhering tissues is to use adhesion proteins and polyornithine. This adhesion method requires pretreatment before it can be used for tissue adhesion, namely incubation at 37°C for 30 minutes, and then rinsing the cochlear hair cell tissue three times with PBS buffer. This is time-consuming, labor-intensive, and cumbersome operation. Therefore, the present invention explores a new method of fixing cells to prolong the survival time of cochlear hair cells cultured in vitro.

[0008] A cell slide is a glass cover slip with a thickness of approximately 0.17 mm that can be immersed in cell culture medium to allow cells to grow on the slide. However, currently, cell slides are mainly used for tests such as cell immunofluorescence and in situ hybridization. The present invention, based on the understanding of the adhesion principle of cell slides, attempts to apply it to tissue adhesion and verifies its feasibility through experiments. For the first time, cell slides were applied to culture tissue containing cochlear hair cells, and it was found that the use of cell slides can significantly prolong the time of in vitro hair cell culture. This is because cell slides can fix the tissue to the bottom of the culture plate, preventing the tissue from floating, which in turn helps cell growth and prolongs cell survival. More specifically, under normal circumstances, biochemical adhesives can be toxic to cells or affect the cell cycle and differentiation, thereby shortening the survival time of cells in vitro; the surface of the glass cover slip has a permanent cationic charge, which can adsorb tissue or cells through electrostatic action, adhering to the tissue or cells in a more gentle and firm manner. In addition, the use of cell slides to adhere tissue cells is also more conducive to the observation of some upright microscope immunofluorescence experiments.

[0009] In addition, carbon source is usually an important limiting factor in cell metabolism. There are many substances that can be used as carbon sources for culture media, such as glucose, fructose, etc., and glucose is the most common carbon source in cell culture. Therefore, the present invention explored the supplementation of high-sugar culture medium and found that adding low concentrations of glucose can significantly prolong the time of hair cells cultured in vitro.

[0010] Furthermore, the high-glucose culture medium is DMEM culture medium or MEM culture medium.

[0011] DMEM (Dulbecco's Modified Eagle Medium) and MEM (Minimum Essential Medium) are both commonly used cell culture media. DMEM was developed based on MEM. The amino acid content of DMEM is twice that of MEM culture medium and contains non-essential amino acids such as glycine. In addition, the vitamin content of DMEM is four times that of MEM culture medium and contains pyruvate, an important substance in the glycolysis pathway. In contrast, MEM is a minimum essential medium with added nutrients, containing only 12 essential amino acids, L-glutamine and 8 vitamins, and has a lower sugar content. Due to the different components of the two culture media, DMEM and MEM also have different applications. High-glucose DMEM is suitable for fast-growing adherent cells, such as tumor cells; low-glucose MEM is suitable for slower-growing adherent cells, especially after the addition of serum, and can be used to culture a variety of monolayer-growing cells, such as fibroblasts.

[0012] In some specific embodiments, the high glucose culture medium is DMEM culture medium.

[0013] Furthermore, the high-glucose medium further comprises any one or more of N1 supplement, N2 supplement, penicillin G sodium, FBS, and BSA. These reagents are all commonly used supplements in cell culture. N1 supplement typically contains animal-derived components such as serum, while N2 supplement is serum-free and has a defined chemical composition, making it more suitable for cell culture requiring serum-free conditions. It is understood that experimenters can use the high-glucose DMEM medium containing glucose provided by the present invention in combination with the cell culture target and requirements.

[0014] In some specific embodiments, the high-glucose medium is DMEM high-glucose medium containing glucose, N2 supplement, and penicillin G sodium. While the two high-glucose media offer comparable culture performance, N2 supplement is preferred because its chemical composition is more defined and the insulin in N2 supplement is recombinant human insulin rather than animal-derived insulin, which helps avoid potential issues associated with animal-derived ingredients.

[0015] Furthermore, the content of glucose is 0.5% to 0.8%; preferably, 0.6% to 0.7%; more preferably, 0.615%; and the concentration of penicillin G sodium is 100 U / mL.

[0016] In some preferred embodiments, the concentrations of glucose, N2 supplement and penicillin G sodium are 0.615%, 1% N2 and 100 U / mL, respectively; penicillin G sodium at this concentration can effectively inhibit the growth of Gram-positive bacteria and some Gram-negative bacteria.

[0017] Furthermore, the method for prolonging the in vitro culture time of cochlear hair cells provided by the present invention comprises the following steps:

[0018] (1) Isolate and extract the mouse cochlea;

[0019] (2) Placing cochlear hair cells on a cell culture plate containing a cell slide and a high-glucose medium containing glucose, wherein the medium containing glucose is the same as described above;

[0020] (3) Every 72 hours, replace half of the culture medium, that is, discard half of the old culture medium and add half of the new culture medium.

[0021] Furthermore, in step (2), the hair cells are placed on the cell slide in the following orientation: the area on which the tectorial membrane of the hair cells is removed faces upward, and the basement membrane faces downward. The orientation of the hair cells is a necessary condition for their normal growth.

[0022] Furthermore, in step (2), before culturing the hair cells with a high-glucose medium containing glucose, the freshly isolated hair cells need to be cultured with a traditional culture medium (80% DMEM, 20% FBS, and 100 U / mL penicillin G) for 24 hours. This is because the FBS in the traditional culture medium can provide attachment factors for mammalian cells, and the attachment factors have the function of promoting cell adhesion and spreading. Therefore, pre-culturing the hair cells with a traditional culture medium is more conducive to the cochlear hair cell tissue adhering to the cell slide and growing.

[0023] On the other hand, the present invention provides a use of a cell slide for prolonging the in vitro culture time of cochlear hair cells.

[0024] On the other hand, the present invention provides a use of glucose for prolonging the in vitro culture time of cochlear hair cells.

[0025] The beneficial effects of the present invention include:

[0026] 1. The present invention significantly extends the in vitro culture time of cochlear hair cells to 30 days, while conventional culture methods can only culture for a maximum of 10 days. This facilitates research on the mechanism of cochlear hair cell differentiation and development, and provides a theoretical basis for the treatment of hearing impairment caused by cochlear hair cell damage.

[0027] 2. It also meets the need for collecting data when using in vitro gene therapy to treat hearing impairment;

[0028] 3. The in vitro culture method for cochlear hair cells provided by the present invention has simple steps, convenient operation, low experimental cost, and great application prospects;

[0029] 4. Cell slides and glucose-containing culture medium have a synergistic effect on the in vitro culture of cochlear hair cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 : Diagram of ear structure

[0032] Figure 2 :Schematic diagram of the structure of the organ of Corti

[0033] Figure 3 :The cell slide used in the present invention

[0034] Figure 4 : Preliminary separation of the cochlear region

[0035] Figure 5 :Snail case of cochlea

[0036] Figure 6 : Cochlear explant

[0037] Figure 7 : The hair cell tissue cultured in vitro for 5 days as described in Example 3

[0038] Figure 8 : The hair cell tissue cultured in vitro for 10 days as described in Example 3

[0039] Figure 9 : The hair cell tissue cultured in vitro for 15 days as described in Example 3

[0040] Figure 10 : The hair cell tissue cultured in vitro for 30 days as described in Example 3

[0041] Figure 11 : The hair cell tissue cultured in vitro for 10 days in Comparative Example 1

[0042] Figure 12 : The hair cell tissue cultured in vitro for 10 days in Comparative Example 2

[0043] Figure 13 : Hair cell tissue cultured in vitro for 10 days under different nitrogen source conditions as described in Example 5

[0044] Figure 14 : Hair cell tissue cultured in vitro for 30 days under different glucose concentrations as described in Example 6 DETAILED DESCRIPTION

[0045] The present invention is further elaborated below in conjunction with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. 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.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0047] Example 1

[0048] The method provided by the present invention for significantly prolonging the in vitro culture time of cochlear hair cells comprises the following steps:

[0049] 1. Preparation of Instruments Before Dissection

[0050] Disinfect the operating table with 75% medical alcohol (Lierkang); prepare an instrument tray, including an instrument tray and a 60 mm diameter culture dish, heat-sterilized surgical scissors (az, CrJ21190), a surgical knife handle (RWD, S32003-12), a micro knife and forceps (two each of No. 4 (RWD, F11020-11) and No. 55 (FST, 11255-20)); prepare a sterile #15 scalpel blade (Hangzhou Huawei Medical Supplies Co., Ltd.), a sealable plastic bag or container (for handling mouse corpses), and ice-cold, 1× sterile Hank's balanced salt solution (HBSS, Solarbio, H1025), and divide 500 mL of HBSS solution into 50 mL experimental plastic tubes (Corning Incorporated, 430829) and store them on ice.

[0051] 2. Preparation of Culture Materials

[0052] Prepare a 24-well cell culture plate (Corning Incorporated, 3524) and place a 10 mm diameter square cell slide ( Figure 3 ; Absin, abs7032).

[0053] Prepare the required culture medium, as shown in Table 1 for details. In this example, culture medium A and culture medium B are mainly prepared, and culture medium C and culture medium D are used for comparative examples 1 and 2, respectively; MEM culture medium is purchased from gibco (10370-021), and DMEM culture medium is purchased from gibco (10313-021); glucose is purchased from Sangon (C6H1206); N1 supplement is purchased from sigma (N6530-5ML); penicillin G sodium is purchased from sigma (P3032-1MU); FBS is purchased from VivaCell (C04001-500); BSA is purchased from McLean (B824162-100g); the liquid culture medium is equilibrated to room temperature before use.

[0054] Table 1 All culture medium formulas and uses involved in the present invention

[0055]

[0056] 3. Obtaining the Rodent Cochlea

[0057] Take a culture dish with a diameter of 60 mm and spray 70% ethanol on the lid until the surface of the lid is moist; pour the cold HBSS solution in the 50 mL centrifuge tube into the above-mentioned bottom culture dish; place the above-mentioned 50 mL centrifuge tube, culture dish and its lid on ice to keep the mouse tissue low in temperature when not being dissected; place newborn mice 1 to 5 days after birth on ice, cut off their fingers with latex gloves, and wait 1-3 minutes until they lose consciousness due to hypothermia; then quickly cut off the mouse's head with surgical scissors, and place the severed mouse head in the above-mentioned pre-cooled culture dish containing HBSS solution, and place the remaining body in a sealed plastic bag.

[0058] Use a No. 15 scalpel to make a superficial incision from the front to the back of the mouse's head (directly above the sagittal suture) to remove the skull skin; use a scalpel to cut the external auditory canals on both sides of the mouse and fold the skin forward to expose the entire skull; use a scalpel to open the skull along the sagittal suture. The skull of a newborn mouse has not yet ossified, so it is easy to cut. Be sure to cut the skull from front to back, gently moving the blade up and down to avoid compressing the cochlea; use a No. 15 scalpel to make a vertical incision directly behind the eye socket, remove and discard the nose. At this time, the back of the skull should still contain the brain and cochlea; use No. 4 forceps to bluntly dissect the forebrain, cerebellum, and brainstem.

[0059] The magnification of the stereomicroscope (Nanjing Jiangnan Yongxin Optical Co., Ltd., SZ6100) was adjusted to 6.5 times, and the light source was adjusted, and forceps were placed under the microscope to optimize illumination and focus. The two halves of the skull were placed in a 60 mm diameter culture dish filled with cold HBSS solution. The cochlea was completely exposed, adjacent to the stapedial artery (a curved artery in the temporal bone), and the cochlea was directly separated from the temporal bone using No. 4 forceps to fully expose the cochlea (as shown in Figure 2). Figure 4 ).

[0060] Transfer the cochlea to a new 60mm culture dish and place the dish under a microscope. Meanwhile, place the 60mm plastic culture dish containing the other half of the skull on ice. Remove the cochlea from the vestibular system and carefully dissect the cochlear volute (the cochlear volute of the mouse at this stage is usually cartilaginous) with No. 4 forceps (e.g. Figure 5 All subsequent experimental steps used No. 55 forceps.

[0061] Continue to process the inner ear tissue, use a micro knife and two 55# fine forceps to carefully separate the spiral ligament adhered to the Corti organ from the rest of the cochlea; cut the darker translucent area in the gap between the spiral ligament and the hair cell area, grasp the spiral ligament on the most basal side, and gently untie the spiral ligament while moving at the top for subsequent removal; use No. 4 forceps to grasp the side opposite to the hair cells, fix the mouse cochlea on the bottom of the culture dish, and position the specimen, that is, using the longitudinal and sagittal views of the cochlea as a reference, use a micro knife to cut the cochlea into three sections: the apex, the middle, and the base; remove the tissue around the hair cells and nerve fibers to obtain a cochlear explant that can be placed horizontally on the culture dish (such as Figure 5 ), it should be noted that the appropriate size for stable adhesion on the culture dish is about half of a cochlear spiral turn; use tweezers to gently peel off the tectorial membrane, which is a very thin, translucent layer located above the Corti organ; use tweezers to touch both sides of the vestibular membrane (Reissner's membrane) and peel it off. The membrane constitutes the upper wall of the membranous cochlear duct, and the inner end starts from the bony spiral plate, and ends upward and outward at the outer wall of the bony cochlear duct, attached to the top of the spiral ligament. At the same time, use a microknife to cut off any damaged hair cell area on the outside to obtain the cochlear hair cell tissue for in vitro culture.

[0062] 4. Fixing and culturing cochlear hair cells

[0063] Wash the 24-well cell culture wells with 50 μL of sterile water to remove the tissue adhesive solution in the wells, repeat 3 to 5 times, and use a pipette (Eppendorf) to aspirate the remaining liquid in the wells; use a 1 mL pipette (Eppendorf) to draw 120 μL of DMEM culture medium to wet the pipette tip to prevent the specimen from adhering to the inside of the tip; use a 1 mL pipette to transfer the isolated cochlear specimens one by one into a 24-well culture dish, with only one specimen in each culture well. Then, draw 70 μL of cold HBSS solution and add it to the culture wells so that the specimens are immersed in HBSS solution; under a microscope at 50x magnification, check whether the specimen is oriented correctly, that is, ensure that the area on which the hair cells have their tectorial membrane removed is facing up and the basement membrane is facing down, and adhere to the cell slide. The placement method of the hair cells greatly affects their in vitro culture time, because floating in the culture medium without the support of the coverslip may cause their degeneration. If the specimen is found to be upside down during inspection, deposit the HBSS solution remaining in the pipette tip into the inlay and reposition the specimen until it is correctly oriented; after the cochlear hair cells are correctly placed, aspirate the excess HBSS solution with a 1 mL pipette and wait 15 seconds.

[0064] Use a pipette to draw 300 μL of prepared culture medium B (80% DMEM, 20% FBS and 100 U / mL penicillin G sodium) equilibrated to room temperature, and add the culture medium to the culture well containing the specimen. It should be noted that when adding the culture medium, the pipette cannot touch the specimen. Incubate overnight at 37°C and 5% CO2. On the second day, replace with high-glucose culture medium A (see Table 1 for the formula), the volume of which is 500 μL. On the third day, add 500 μL of high-glucose culture medium A. Thereafter, replace half of the culture medium with new culture medium A every 72 hours, that is, discard half of the old culture medium A and add half of the new culture medium A, adding 300 μL of culture medium A to each well each time. Finally, return all the stock solutions to the appropriate location, dispose of the corpse and residual waste, disinfect the operating table (such as using ethanol or hypochlorite), clean and autoclave the instruments, and discard sharp objects in appropriate containers.

[0065] Example 2

[0066] The steps for observing the morphology of cochlear hair cells by immunofluorescence are as follows:

[0067] Blocking solution (containing 94% Phosphate-Buffered Saline (Shanghai Sangon Biotechnology Co., Ltd., E607008-0500), 5% horse serum (NHS Gibco, 16050-130), and 1% nonionic surfactant Triton X-100 (Sigma, T8787-50ML) and antibody dilution solution (98.6% PBS, 1% NHS, 0.4% nonionic surfactant Triton X-100) were prepared. The antibody dilution solution was used to dilute rabbit anti-myosin 7a (Myo7a) antibody (Proteus 25-6790) at a dilution ratio of 1:400. This antibody is suitable for the observation of inner hair cells and outer hair cells.

[0068] At the same time, use a pipette to absorb the culture medium in the culture well. Note that the pipette cannot touch the specimen during the absorption process; rinse the cochlear hair cell explants twice with sterile PBS buffer (pH = 7.4), that is, immerse the sample tissue in PBS buffer, place the specimen on a shaker for 5 minutes, aspirate the PBS buffer, and repeat the above steps twice; transfer the washed tissue to 4 inlays in a 4-well culture dish with a diameter of 35 mm, immerse and fix the tissue with 4% paraformaldehyde (PFA-CAUTION) (Sigma, 6148-500G), fix it on a shaker for 20 minutes, and then aspirate the residual PFA solution; rinse the cochlear hair cell explants 3 times with sterile PBS buffer (pH = 7.4), that is, immerse the sample tissue in PBS buffer, place the specimen on a shaker for 5 minutes, aspirate the PBS buffer, and repeat the above steps 3 times, each time using 60 μL of PBS buffer; place the cochlear hair cells in the prepared blocking solution (94% PBS, 5% NHS, 1% nonionic surfactant Triton X-100) and placed on a shaker for blocking for 30 minutes.

[0069] The cochlear hair cells were placed in the primary antibody Myo7a (vortexed before use) at room temperature overnight. The antibody was diluted with the prepared antibody diluent (98.6% PBS, 1% NHS, 0.4% non-ionic surfactant) at a ratio of 1:400. It should be noted that the culture dish needs to be placed in a humidified box to keep the culture dish moist and prevent the antibody solution from evaporating overnight. The cochlear hair cell explants were rinsed 3 times with sterile PBS buffer (pH = 7.4), that is, after the sample tissue was immersed in PBS buffer, the specimen was placed on a shaker for 5 minutes, the PBS buffer was aspirated, and the above steps were repeated 3 times to wash away the residual primary antibody solution. Goat anti-rabbit IgG H&L (Alexa Fluor 500 Immunofluorescence ... 488)(Abcam, AB150077) / goat anti-rabbit IgG (H+L) ( Cochlear hair cells were incubated with Alexa Fluor 594 (Alexa Fluor 594, 115-585-003) secondary antibody for 1.5 hours. During the incubation, the culture dish containing the secondary antibody and cochlear hair cells was placed in a black humidified box, which was placed on a shaker and protected from light. The cochlear hair cell explants were rinsed three times with sterile PBS buffer (pH = 7.4) to remove residual secondary antibody solution. After immersing the sample tissue in PBS buffer, the specimen was placed on a shaker for 5 minutes, the PBS buffer was aspirated, and the above steps were repeated three times, with 60 μL of PBS buffer used each time.

[0070] Use tweezers to remove the coverslip with the specimen from the 4-well plate and immerse it in a solution filled with distilled water; drop a drop of anti-fade mounting medium (VECTASHIED H-1000) on the microscope slide, turn the coverslip over so that the tissue facing down is immersed in the medium; lay the slide flat in a covered place away from light for 15-20 minutes until it is dry, then put it in a box or observe the tissue under a fluorescence microscope (OLYMPUS CKX41). It should be noted that the best way to preserve fluorescent staining is to store the slides at 4 or -20°C.

[0071] When three clearly visible rows of outer hair cells and one row of inner hair cells are observed under a fluorescence microscope, and the cells in each row are neatly arranged and closely connected, it indicates that the cochlear hair cells are in a normal growth state at this time, that is, the culture conditions can still maintain the normal physiological activities of the hair cells; when the cells are observed to be discrete and falling off, it means that the culture conditions are not suitable for their growth, and the cells will gradually lyse (no fluorescence can be detected at this time). Therefore, the time point when the hair cell morphology is closer to the in vivo state, that is, the time point when the inner and outer hair cells are more completely arranged is the survival time of the hair cells in vitro.

[0072] Example 3

[0073] The cochlear hair cells were cultured in vitro according to the method described in Example 1, and the cochlear hair cells cultured for 5 days, 10 days, 15 days and 30 days were detected by the immunofluorescence detection method described in Example 2. The detection results are shown in FIG. Figures 7 to 10 It is important to understand that although Figures 7 to 10 Although the fluorescence colors observed in the experiment were different, the detection results were the same. Observation under a fluorescence microscope revealed that the number, morphology, and structural integrity of hair cells (including three rows of outer hair cells and one row of inner hair cells) cultured for 5 days using the method provided by the present invention were not significantly different from those cultured for 30 days. Both cultures were in a normal growth state, and their morphology was similar to that in vivo. This demonstrates that the method provided in Example 1 can culture cochlear hair cells in vitro for at least 30 days.

[0074] Comparative Example 1

[0075] In this comparative example, a traditional culture medium was used to culture cochlear hair cells in vitro. The steps of isolating, fixing with a cell slide, culturing, and observing cochlear hair cells were the same as those described in Examples 1 and 2, except that after culturing the hair cells with culture medium B for 24 hours, the composition of the culture medium was changed to MEM culture medium C containing 10% BSA (see Table 1 for the formula). The fluorescence imaging results of the cochlear cells after continuous in vitro culture for 10 days are shown in FIG. Figure 11 .Depend on Figure 11It can be seen that compared with the detection results of Example 3, the fluorescence signal of this comparative example is weak and fuzzy, and many hair cells are missing, indicating that the number of cochlear hair cells cultured by this culture method is significantly reduced and the activity is greatly reduced; and the hair cells of this comparative example were only cultured in vitro for 10 days, while those in Example 3 were cultured for 30 days, further demonstrating the superiority of the high-sugar culture medium containing glucose provided by the present invention for culturing hair cells in vitro. On the other hand, under the condition of using only culture medium C without using cell slides to adhere hair cells, the hair cells survived in vitro for 3 to 5 days (this data comes from the patent "A method for isolating and culturing inner ear hair cells in vitro" (patent number: 201811017201.7)), while after using cell slides in this comparative example, the survival time was extended by 7 days, indicating that the use of cell slides is beneficial to prolonging the survival time of hair cells in vitro.

[0076] Comparative Example 2

[0077] In this comparative example, a conventional culture medium was used to culture cochlear hair cells in vitro. The steps of isolating, fixing with a cell slide, culturing, and observing cochlear hair cells were the same as those described in Examples 1 and 2. However, after culturing the hair cells with culture medium B for 24 hours, the composition of the culture medium was changed to a different one. In this example, DMEM culture medium D containing 10% FBS (see Table 1 for the formula) was used instead. The fluorescence imaging results of the cochlear cells after continuous in vitro culture for 10 days are shown in FIG. Figure 12 .Depend on Figure 12 It can be seen that although the test results of this comparative example are slightly better than those of comparative example 1, compared with the test results of Example 3, the fluorescence signal of this comparative example is still weak, and the hair cells are missing more, indicating that the number of cochlear hair cells cultured by this culture method is small. It is very likely that the number of cochlear hair cells will further decrease with the increase of culture time. According to the experience of culturing cells, it is estimated that the hair cells cultured under this condition can survive in vitro for 7 to 8 days; and the hair cells of this comparative example were only cultured in vitro for 10 days, while those in Example 3 were cultured for 30 days, further demonstrating the superiority of the high-sugar medium containing glucose provided by the present invention for culturing hair cells in vitro. On the other hand, under the condition of using only culture medium D without using cell climbing sheets to adhere to hair cells, the hair cells survived in vitro for less than 5 days (the data comes from the patent "A method for isolating and culturing inner ear hair cells in vitro" (patent number: 201811017201.7)), while the use of cell climbing sheets in this comparative example extended the survival time by 12 days, further illustrating that the use of cell climbing sheets is beneficial to prolonging the survival time of hair cells in vitro.

[0078] Example 4

[0079] The adhesion mode of cochlear hair cells largely determines the length of time that hair cells can be cultured in vitro. This example investigates the effect of the type of fixative to which cochlear hair cells adhere on the time that hair cells can be cultured in vitro. The fixative includes a cell slide, tissue adhesive (corning, 354240), laminin (Sigma-Aldrich, 114956-81-9), and polyornithine (Sigma-Aldrich, 27378-49-0). Laminin, polyornithine, and fetal bovine serum are uniformly mixed at a volume ratio of 2:2:1. 50 μL of the mixture is then added to a 24-well plate. The edges of the plate are sealed with sealing film and incubated at 37°C for 30 minutes. The coating solution is then discarded and the plates are washed three times with PBS buffer (pH = 7.4) before use for tissue adhesion. The remaining experimental procedures and methods for detecting hair cell morphology are the same as those described in Examples 1 and 2. The results are shown in Table 2.

[0080] Table 2 Effects of different hair cell adhesion fixatives on the time of hair cells cultured in vitro

[0081] Fixture type Hair cell survival time in vitro / <24h Cell slides >30d tissue adhesives <3d Adhesin and polyornithine ≤10d

[0082] Table 2 shows that compared with other fixative materials, the use of cell slides significantly prolonged the survival of hair cells cultured in vitro. This is likely due to the permanent cationic charge on the slide surface, which creates electrostatic forces between the glass and the tissue, thereby adhering the tissue to the slide. At the same time, under certain conditions, adhesives can adversely affect cell growth and development, thereby shortening the lifespan of hair cells. In summary, compared with other adhesives, cell slides adhere to tissues through physical electrostatic forces, which is more conducive to cell growth and prolongs cell survival.

[0083] Example 5

[0084] This example investigates the effects of supplements in a high-glucose medium on the in vitro culture time of hair cells. The high-glucose medium is DMEM medium containing 1% N2 supplement and 100 U / mL penicillin G sodium. The supplements include carbon sources (glucose) and nitrogen sources (fetal bovine serum, L-glutamine, and bovine serum albumin (BSA)) commonly used in mammalian cell culture. Fetal bovine serum was purchased from BI, L-glutamine was purchased from Shanghai Shenggong Bioengineering Co., Ltd., and bovine serum albumin (BSA) was purchased from WISSEN LifeScience. The remaining experimental procedures and methods for detecting hair cell morphology were the same as those described in Examples 1 and 2. The results are shown in Tables 3 and Figure 13 , Figure 13 The immunofluorescence results of cochlear explants cultured continuously for 10 days under different nitrogen sources.

[0085] Table 3 Effects of culture medium supplements on the in vitro culture time of hair cells As shown in Table 3, adding a small amount of carbon source (glucose) to the original high-glucose culture medium can significantly prolong the time of in vitro culture of hair cells. This may be because the carbon source is a very important factor affecting cell metabolism. It is worth noting that adding both carbon and nitrogen sources did not achieve better results. This may be because DMEM basal medium already contains amino acids, and the amino acid content in DMEM medium is twice that of MEM, including essential and non-essential amino acids, which are important nitrogen sources for cell synthesis of proteins and nucleic acids. At the same time, cochlear hair cells have a low demand for nitrogen sources, and the amino acids contained in the basal culture medium have met the basic needs of cochlear hair cells for growth and development, so there is no need to supplement the relevant nitrogen sources. Excessive nitrogen sources will inhibit their growth.

[0086] Depend on Figure 13 It can be seen that after 10 days of in vitro culture, the Figure 8 ), the number of hair cells cultured with additional nitrogen source decreased to varying degrees; moreover, the hair cells cultured with additional 10% FBS or 4mM glutamine were arranged more neatly and tightly than those cultured with 10% BSA. Cell adhesion is an important condition for maintaining normal cell function, so it can be seen that the addition of BSA has the worst effect on hair cell culture, which is consistent with the statistical results in Table 4.

[0087] Therefore, it is preferred to culture hair cells in DMEM medium containing 0.615% glucose, 1% N2 supplement and 100 U / mL penicillin G sodium.

[0088] Example 6

[0089] Combined with the experimental results of Example 5, this example explored the effects of different concentrations of glucose supplements on the survival time of hair cells in vitro. The survival time of hair cells in vitro was the time point at which the fluorescence emitted by the hair cells could no longer be detected. The experimental procedures and the method for detecting hair cell morphology were the same as those described in Examples 1 and 2. The specific results are shown in Tables 4 and Figure 14 , Figure 14 The results of immunofluorescence analysis of cochlear explants cultured continuously for 30 days in the presence of different glucose concentrations.

[0090] Table 4 Effects of different glucose concentrations on the time of in vitro cultured hair cells

[0091] Glucose concentration Hair cell survival time in vitro / <7d 0.5％ <30d 0.6％ >30d 0.7％ >30d 0.8％ <30d

[0092] As shown in Table 4, the survival time of cochlear cells in vitro can be prolonged by using cell slides without adding glucose, which is consistent with the above results. When glucose is present, the survival time is significantly prolonged. It is worth noting that a glucose concentration of 0.6% to 0.7% can achieve the desired effect. On the contrary, as the glucose concentration increases, the survival time of hair cells in vitro will be relatively shortened. Figure 14 It can be clearly observed that the structural integrity and number of hair cells cultured under 0.8% glucose conditions are significantly worse than those cultured under 0.6% to 0.7% glucose concentration. This may be because excessive glucose in the culture medium causes the culture medium to be too viscous, making it difficult for the culture medium to move relative to the cells, and between cells, thereby hindering the material exchange process. In other words, the cells cannot absorb nutrients well, and the "waste" produced by metabolism accumulates around the cells and cannot be discharged, which is not conducive to cell growth or reproduction. Therefore, the optimal concentration of glucose supplement is 0.6% to 0.7%. Furthermore, in order to make the in vitro environment for hair cell growth more favorable (because both excessively high and low glucose concentrations will affect the in vitro growth state of cochlear hair cells), the preferred concentration is 0.615%.

[0093] Combining the experimental results of Examples 3 to 6 and Comparative Examples 1 to 2, it can be seen that extending the survival time of hair cells in vitro to 30 days requires the combination of cell slides and a high-glucose culture medium containing an appropriate concentration of glucose.

[0094] The basic principles, main features and advantages of the present invention are shown and described above. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the appended claims rather than the above description. Therefore, it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any figure signs in the claims should not be regarded as limiting the claims involved.

[0095] References

[0096] [1] Kazmierczak and Muller, Sensing sound: molecules that orchestrate mechanotransduction by hair cells. Trends Neurosci. 2012, 35, 220–229.

[0097] [2]Recent Advancements in the Regeneration of Auditory Hair Cells andHearing Restoration.Frontiers in Molecular Neuroscience 10(2017):236.

[0098] [3]Liu SS,Yang R.Inner Ear Drug Delivery for Sensorineural HearingLoss:Current Challengesand Opportunities.Front Neurosci.2022 May 24;16:867453.doi:10.3389 / fnins.2022.867453.PMID:35685768;PMCID:PMC9170894.

[0099] [4]Single-cell transcriptomic profiling of the mouse cochlea:An atlasfor targeted therapies.JeanP,Proc Natl Acad Sci U S A.2023 Jun 27;120(26):e2221744120.

Claims

1. A method for prolonging the in vitro culture time of cochlear hair cells, characterized in that: The method is to adhere the cochlear hair cells to a cell slide and culture the cochlear hair cells in a high-glucose culture medium containing glucose.

2. The method according to claim 1, characterized in that The high-glucose culture medium is DMEM culture medium or MEM culture medium.

3. The method according to claim 1, characterized in that The high-glucose culture medium further comprises any one or more of N1 supplement solution, N2 supplement solution, penicillin G sodium, FBS, and BSA.

4. The method according to claim 3, characterized in that The high-glucose culture medium is a MEM high-glucose culture medium containing glucose, N2 supplement and penicillin G sodium.

5. The method according to claim 4, characterized in that The content of glucose is 0.45% to 0.8%; the concentration of penicillin G sodium is 100 U / mL.

6. The method according to claim 1, characterized in that The steps of the method are as follows: (1) Isolate and extract the cochlea of ​​mice; (2) placing the cochlear hair cells on a cell culture plate containing a cell slide and a high-glucose medium containing glucose, wherein the medium containing glucose is the same as that described in any one of claims 1 to 5; (3) Every 72 hours, replace half of the culture medium by discarding half of the old culture medium and adding half of the new culture medium.

7. The method according to claim 6, characterized in that In step (2), the hair cells are placed on the cell slide in the following orientation: the area on the side of the hair cells where the tectorial membrane is removed faces upward, and the basement membrane faces downward.

8. The method according to claim 7, characterized in that In step (2), before culturing the hair cells with a high-glucose medium containing glucose, the hair cells need to be cultured with a traditional culture medium for 24 hours, wherein the traditional culture medium contains 80% DMEM, 20% FBS and 100 U / mL penicillin G sodium.

9. Use of a cell slide for prolonging the in vitro culture time of cochlear hair cells.

10. Use of glucose for prolonging the in vitro culture time of cochlear hair cells.

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