Method for promoting cochlea organ maturation in GELMA-MXene matrix based on electroacoustic stimulation

Abstract

The invention discloses a method for promoting maturation of cochlea organs in a GELMA-MXene matrix based on electroacoustic stimulation, and belongs to the field of cochlea organ culture for hair cell regeneration. The preparation method of the cochlea organ culture component comprises the following steps: preparing GelMA; sequentially adding LiF, MAX, pure water and acid into the reaction container; stirring the mixture until the reaction is completed, centrifuging the mixture, and removing supernate and MXene to obtain Ti3C2Tx; dissolving GelMA by using a light curing agent, and adding Ti < 3 > C < 2 > T < x >; and irradiating with blue light to obtain the cochlea organ culture component. Using the cochlea organ culture component to culture inner ear stem cells, and adding a differential medium; and after the differentiation is completed, applying low-frequency electroacoustic stimulation to the differentiated organoid. The advantages of a culture support and electroacoustic stimulation are combined, and differentiation and maturation of cochlea organs are more effectively promoted.

Description

Technical Field

[0001] The present invention relates to the field of cochlear organoid culture for hair cell regeneration, and particularly to a method for promoting the maturation of cochlear organoids in a GELMA-MXene matrix based on electroacoustic stimulation. Background Art

[0002] Sensorineural hearing loss is caused by the dysfunction of the cochlea or spiral ganglion, and the inducing factors include aging, noise exposure, ototoxic drugs, etc. The cochlea is responsible for converting sound vibrations into nerve signals. Hair cells are sensory epithelial cells in the cochlea, divided into outer hair cells and inner hair cells. The former amplifies sound signals, and the latter transmits sound signals to the auditory nerve to form hearing. Mammalian cochlear hair cells cannot regenerate autonomously, so sensorineural hearing loss caused by hair cell defects is irreversible, making the treatment of sensorineural hearing loss quite difficult.

[0003] Cochlear organoids are important in vitro inner ear simulation platforms, promoting inner ear development and pathology research, and also providing a powerful tool for the formulation of treatment strategies for sensorineural deafness and drug screening. The structural function of the cochlea is precise and complex. Although cochlear organoids have made remarkable progress in simulating inner ear development and function, there are still some limitations.

[0004] For example, in the current research on cochlear organoids, the commonly used three-dimensional culture matrix is Matrigel. However, Matrigel has limitations such as unclear composition, large batch differences, and high immunogenicity. Therefore, it is necessary to find a more perfect culture scaffold. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention proposes a method for promoting the maturation of cochlear organoids in a GELMA-MXene matrix based on electroacoustic stimulation.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect of the present invention, it relates to a preparation method of a cochlear organoid culture component, including the following steps:

[0008] Prepare GelMA;

[0009] Add LiF, MAX, pure water and acid to the reaction vessel in sequence; stir the mixture. After the reaction is completed, centrifuge the mixture, remove the supernatant, and remove MXene to obtain Ti 3 C 2 Tx;

[0010] Dissolve GelMA with a photoinitiator, and add Ti 3 C 2Tx; Obtain cochlear organoid culture components by blue light irradiation.

[0011] Optionally, the acid includes hydrochloric acid.

[0012] Optionally, the method for preparing GelMA includes: dissolving gelatin powder in PBS solution, adding methacrylic anhydride and reacting, dialyzing the mixed solution in deionized water, then adjusting the pH value to neutral, and freeze-drying to obtain GelMA.

[0013] Optionally, the MAX phase is Ti 2 AlC.

[0014] A second aspect of the present invention relates to a method for stimulating the maturation of cochlear organoids, including the following steps:

[0015] Using the above-mentioned cochlear organoid culture components, culturing inner ear stem cells and adding a differentiation medium;

[0016] After differentiation is completed, applying low-frequency electroacoustic stimulation to the differentiated organoids.

[0017] Optionally, in the electroacoustic stimulation, the sound frequency is 600 - 650 Hz and the current magnitude is 1900 μA.

[0018] Optionally, the proliferation medium includes: DMEM / F12, N2 Supplement, B-27 Serum-Free Supplement, Epidermal Growth Factor (EGF), Insulin-like Growth Factor (IGF), Fibroblast Growth Factor (FGF), Valproic Acid Sodium Salt (VPA), CHIR99021 (GSK inhibitor), and Ampicillin.

[0019] Optionally, the differentiation medium includes: DMEM / F12, N2 Supplement, B-27 Serum-Free Supplement, CHIR99021 (GSK inhibitor), γ-secretase inhibitor (LY41575), and Ampicillin.

[0020] A third aspect of the present invention relates to an ESA device for culturing cochlear organoids, including:

[0021] The above-mentioned culture components;

[0022] A circuit board, with a number of circuit wires extending from the electrodes on both sides thereof;

[0023] An artificial cochlear implant, which is fixed on both sides of the circuit board respectively through the magnetic attraction between an internal magnet and a microphone;

[0024] A receiving stimulator, which contains electronic components, has one end connected to a coil and the other end transmitting current to the electrode.

[0025] And a metal wire, which is fixed at the bottom and the top cover of the culture vessel and is connected to the wires on both sides of the circuit board respectively to form a complete stimulation circuit.

[0026] The fourth aspect of the present invention relates to the application of the culture components prepared by the above preparation method, the above method for stimulating the maturation of cochlear organoids or the above equipment for culturing cochlear organoids in the preparation or culture of cochlear organoids and the regeneration of inner ear hair cells.

[0027] Advantages of the present invention:

[0028] The present invention constructs a more perfect three-dimensional culture system for cochlear organoids and explores the effects of this system on the proliferation of newborn cells in the organoids and the differentiation of hair cells. The current cochlear organoid culture system is single and has certain limitations. The present invention uses Ti 3 C 2 Tx MXene and GelMA as important components for constructing the culture platform. Secondly, EAS is introduced into the conductive hydrogel to couple Ti 3 C 2 Tx MXene, GelMA and EAS, and the positive effects of the three are integrated to achieve the goal of culturing more mature cochlear organoids under in vitro conditions, and more effectively promote the differentiation and maturation of cochlear organoids. The electrophysiological properties of the hair cells of the cochlear organoids cultured by the present invention are at least equivalent to those of the natural IHCs of wild-type mice at seven days after birth (P7), which lays a foundation for the better development of the in vitro cochlear simulation platform. Description of the Drawings

[0029] The present invention will be further described below with reference to the drawings.

[0030] Figure 1 It is a schematic structural diagram of the EAS device in Embodiment 3 of the present application;

[0031] Figure 2 It is a flow chart of the experimental method of the present application;

[0032] Figure 3 It is for Ti in Embodiment 1 of the present application 3 C 2Characterization of Tx MXene—GelMA composite hydrogel;

[0033] Figure 4 Ti in Example 2 of this application 3 C 2 Tx MXene promotes the proliferation of neonatal cells in cochlear organoids;

[0034] Figure 5 EAS in Example 3 of this application promotes the differentiation and functional maturation of hair cells in cochlear organoids;

[0035] Figure 6 The expression levels of genes related to hair cell differentiation and membrane potential regulation in the organoid cells of the EAS group reflected in Example 4 of this application are increased;

[0036] Figure 7 EAS promotes the functional maturation of hair cells in cochlear organoids as reflected in Example 5 of this application. Detailed implementation manners

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0038] To better study the effect of the Ti 3 C 2 Tx MXene-GelMA-EAS organoid culture system on the proliferation and differentiation of cochlear organoids, the present invention uses the following steps for verification (see the attached Figure 2 )

[0039] Example 1 Successful preparation of Ti 3 C 2 Tx MXene-GelMA composite hydrogel

[0040] 1.1 Preparation of Ti 3 C 2 Tx MXene-GelMA composite hydrogel, and its structure and physical properties are analyzed by SEM images, Raman spectra, and XPS spectra.

[0041] 1.1.1 Preparation of Ti 3 C 2 Tx MXene-GelMA hydrogel

[0042] (1) Preparation of GelMA: Dissolve 10 grams of gelatin powder in 100 milliliters of PBS solution, and then add 10 milliliters of methacrylic anhydride (MA) dropwise to the mixture. After reacting for 2 hours, dialyze the mixed solution in deionized water for one week, changing the water every day. After dialysis is completed, adjust the pH value to neutral, and obtain GelMA after complete freeze-drying.

[0043] (2) Ti 3 C 2 (2) Preparation of Ti (n+1) AX n , in this example, Ti 2 AlC is used. Wrap the reaction vessel with aluminum foil and plastic film, and stir the mixture at 40 °C for 30 hours. After the reaction is completed, centrifuge the mixture. Then increase the rotation speed from 3000 revolutions per minute, centrifuge for 10 minutes each time, and increase by 500 revolutions per minute each time until it reaches 7500 revolutions per minute. Then pour out the supernatant after each operation. During the last centrifugation, after pouring out the supernatant, a black and gray layer separation can be seen. The black layer, that is, MXene, can be washed away.

[0044] (3) Curing of the composite hydrogel: Dissolve GelMA with a photoinitiator, completely dissolve it at 37 °C, add an appropriate amount of Ti 3 C 2 TxMXene to a final concentration of 100 μg / ml, and irradiate with blue light for 45 - 60 s.

[0045] 1.1.2Ti 3 C 2 Characterization of Ti

[0046] (1) Scanning electron microscope (SEM): Freeze-dry the hydrogel, sputter gold after drying, place the clamped sample into the scanning electron microscope, and set appropriate acceleration voltage and working distance. Observe the microstructure of the hydrogel under SEM and collect images.

[0047] (2) Transmission electron microscope (TEM): Dilute Ti 3 C 2 Tx MXene and observe its surface structure under the transmission electron microscope.

[0048] The results show that Ti 3 C 2The Tx MXene-GelMA composite hydrogel was successfully prepared (Appended Figure 3 ).

[0049] Example 2 Ti 3 C 2 Tx MXene promotes the proliferation of newborn cells in cochlear organoids

[0050] 2.1 By using Ti 3 C 2 Wild-type newborn mouse inner ear stem cells were cultured in vitro with the Tx MXene-GelMA composite hydrogel. Proliferation medium was added, and samples were collected on the tenth day of proliferation and observed under bright field. The proliferation was characterized by counting the number and diameter of organoids, and data and image processing and analysis were performed using Prism 8 software.

[0051] 2.1.1 Three-dimensional proliferation culture of organoids

[0052] The cochleae of P1-P2 FVB mice were dissected to obtain the basilar membranes, which were digested with 200 μL of 0.25% trypsin at 37 °C for 6 minutes. An equal volume of trypsin inhibitor was added to terminate the digestion. After thorough pipetting, undigested tissues were filtered through a single-cell filter sieve to obtain the inner ear stem cell filtrate. The filtrate was centrifuged at 1000 rcf for 3 minutes, the supernatant was discarded, 5% GelMA was added, and after mixing, it was divided into two groups. One group was seeded into a 24-well plate with a round glass slide added, and the other group was seeded into a 24-well plate after adding MXene to a final concentration of 100 μg / ml. Both groups were subjected to proliferation culture.

[0053] The conditions of the proliferation culture medium were as follows:

[0054]

[0055]

[0056] 2.1.2 Bright field observation and result statistics

[0057] The organoids proliferated for 10 days were placed under a microscope in bright field for observation and recording. The number and area of the organoids in the two groups were counted and the results were compared.

[0058] The results showed that the number and area of organoids in the Ti 3 C 2 Tx MXene-GelMA group were significantly higher than those in the GelMA group, indicating that Ti 3 C 2 Tx MXene promoted the expansion of organoids (Appended Figure 4 ).

[0059] Example 3 EAS promotes the differentiation of cochlear organoid hair cells

[0060] 3.1 Through Ti 3 C 2 The inner ear stem cells of wild-type neonatal mice were cultured in vitro with Tx MXene-GelMA composite hydrogel. After adding the differentiation medium and differentiating for three days, low-frequency electroacoustic stimulation was applied to the organoids for ten minutes every day for three consecutive days. Two months after the differentiation of the organoids, they were fixed and harvested. Myosin7A was used to label hair cells, and Otoferlin and Prestin were used to label inner and outer hair cells respectively to characterize the differentiation of the organoids. Data and image processing and analysis were performed using software such as Fiji, prism8, and ZEN.

[0061] 3.1.1 Three-dimensional differentiation culture of organoids

[0062] The steps of dissection and stem cell collection were the same as in 2.1.1. Using Ti 3 C 2 Tx MXene-GelMA composite hydrogel was used to resuspend the cells, which were seeded into the electroacoustic stimulation culture dish with circular glass slides added. After adding the proliferation medium and proliferating for one week, the differentiation medium was replaced.

[0063] The conditions of the differentiation culture medium were as follows:

[0064]

[0065] 3.1.2 Applying EAS to the organoids

[0066] As Figure 1 shown, the nerve receiver in the titanium shell of the cochlear implant system was connected to a double-sided printed circuit board through a chip, and the stimulating contacts injection-molded with liquid silicone were placed on the circuit board groove. The entire process from the receiver to the stimulating point was covered with PBS for impedance value adjustment. Circuit lines were extended on both sides of the printed board and connected to the cochlear organoids in the dish through metal wires to form a current loop and apply electroacoustic stimulation. The relevant parameters of the EAS pathway were designed as shown in the following table:

[0067] Stimulation mode Bipolar stimulation Sound frequency 300 - 650 Hz Current magnitude Approximately 1900 μA Impedance range 5 - 10 kΩ Pulse width 30 μs

[0068] 3.1.3 Immunofluorescence staining

[0069] (1) Primary antibody incubation

[0070] Collect the organoids that have been differentiated for 30 days, place the organoid samples in 4% PFA solution, and fix them at room temperature for 1 hour. Wash the fixed samples 3 times with PBS solution, each time for 5 minutes. Place the samples in Blocking medium (prepared by mixing 1 part donkey serum and 9 parts PBS solution containing 0.1% Triton X-100), and block them at room temperature for 1 hour. Subsequently, incubate the samples overnight at 4°C with the hair cell marker Myosin7A diluted 1:1000 and the inner hair cell (IHC) marker Otoferlin antibody (diluted in PBST, prepared by mixing 0.1 part donkey serum and 99.9 parts PBS solution containing 0.1% Triton X-100).

[0071] (2) Incubation with secondary antibody

[0072] Wash 3 times with PBS solution, each time for 5 minutes. Mix the appropriately diluted secondary antibody (1:400) and DAPI (1:1000) in PBST, and incubate at room temperature for 1 hour. Wash the samples 3 times again with PBS solution, each time for 5 minutes. After treating with DAKO fluorescence quencher, place the samples upside down on the glass slides and seal them with nail polish. Collect images of the samples using a confocal microscope.

[0073] The results showed that the number of hair cells in the organoids treated with EAS was significantly higher than that in the control group, and the same result was observed for inner hair cells (attached Figure 5 ).

[0074] Example 4 Expression of genes related to hair cell differentiation in cochlear organoids

[0075] 4.1 By extracting Ti 3 C 2 Tx MXene-GelMA group, Ti 3 C 2 Tx MXene-GelMA-EAS group organoid RNA and total protein, and using experimental methods of mRNA sequencing and real-time quantitative PCR (qPCR), detect the RNA expression levels of genes related to hair cell differentiation in the two groups of organoids.

[0076] 4.1.1 RNA sequencing

[0077] (1) RNA extraction:

[0078] a) Collect the organoids in the culture dish into an EP tube, remove the culture medium after centrifugation, and add Trizol reagent.

[0079] b) Add 200 μL of chloroform to every 1 mL of Trizol reagent, mix well, and let it stand at room temperature for 15 - 20 minutes.

[0080] c) Centrifuge at 12,000 rpm for 15 minutes at 4°C. The sample will be stratified into an organic layer, an intermediate layer, and an aqueous phase. RNA is mainly in the upper aqueous phase.

[0081] d) Carefully aspirate the upper aqueous phase into a new tube, avoiding contamination from the intermediate layer and the organic layer. Add an equal volume of isopropanol to the aqueous phase and incubate at -20°C for 1 hour to precipitate the RNA.

[0082] e) Centrifuge at 12,000 rpm for 10 minutes at 4°C and discard the supernatant. Wash the RNA pellet with 1 ml of 75% ethanol, centrifuge for 5 minutes, and remove the ethanol as much as possible.

[0083] f) Air-dry at room temperature for 5 - 10 minutes, dissolve the RNA pellet with DEPC water or TE buffer, and measure the concentration and purity of the RNA using a spectrophotometer. The initial amount of RNA is 1 - 4 μg.

[0084] (2) PCR enrichment library:

[0085] After fragmenting the RNA, synthesize double-stranded cDNA and amplify the library by PCR for high-throughput sequencing.

[0086] (3) Perform high-throughput sequencing on the library using platforms such as Illumina to obtain sequence data.

[0087] (4) Data analysis:

[0088] Perform quality control, alignment, quantification, filtering, and inter-sample normalization on the sequencing data, as well as differential expression analysis, etc., to obtain differentially expressed genes related to hair cell differentiation and membrane potential regulation in the two groups of organoids.

[0089] The results showed that the expression levels of genes related to hair cell differentiation and membrane potential regulation in the organoid cells of the EAS group were higher than those in the group without EAS application (attached Figure 6 ).

[0090] Example 5 EAS promotes the functional maturation of cochlear organoid hair cells

[0091] 5.1 Perform electrophysiological recordings on the cochlear organoids in the Ti 3 C 2 Tx MXene-GelMA composite hydrogel with EAS applied and the hair cells on the cochlear basilar membrane of P7 wild-type mice, and use Clampfit and GraphPad software for data analysis to compare their functional maturity.

[0092] 5.1.1 Electrophysiological recording

[0093] (1) Hair cell preparation

[0094] Organoid hair cells: Take out the round glass slide on which the cochlear organoids are cultured, place it in the center of a petri dish with an appropriate diameter, and pour in the prepared artificial cerebrospinal fluid to ensure that the organoids are not dispersed.

[0095] Natural hair cells of wild-type mice: Dissect the cochlea of P7 wild-type mice, isolate the basilar membrane under a microscope, place it in the center of a petri dish, and pour in the prepared artificial cerebrospinal fluid.

[0096] (2) Electrode preparation

[0097] Use a microelectrode puller to pull a borosilicate glass capillary into a microelectrode with a tip diameter of 1-2 μm, and fill it with the prepared intracellular solution to ensure that there are no air bubbles at the tip.

[0098] (3) Electrode contact with cells

[0099] Under a microscope, bring the glass electrode into contact with the cell membrane, and form a high-resistance seal through negative pressure aspiration. Adjust the negative pressure to form a stable seal between the electrode tip and the cell membrane.

[0100] (4) Membrane rupture

[0101] By gently aspirating, rupture the patch to connect the electrode with the inside of the cell and enter the whole-cell mode.

[0102] (5) Current measurement

[0103] Keep the hair cells at -50 mV, and in the current-clamp mode, trigger firing through the duration of a series of current steps (10 steps, increment of 10 pA).

[0104] (6) Experimental recording

[0105] The recording was carried out in the voltage-clamp mode, starting from -100 mV, with a voltage step of 500 ms and an increment of 10 mV. Within 150 milliseconds, the calcium current (Ica) under the voltage ramp from -87 mV to +63 mV under voltage clamp was recorded.

[0106] The results showed that there was no significant difference in the resting membrane potential (RMP) between organoid hair cells and cochlear hair cells of P7 mice. The voltage response ability of organoid hair cells was better than that of P7 native IHCs, and organoid hair cells showed an outward rectifying K+ current significantly higher than that of P7 natural IHCs. Applying EAS could improve the functional maturity of cochlear organoid hair cells (Appendix Figure 7 ).

[0107] In the description of this specification, the descriptions referring to the terms "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0108] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A method for preparing cochlear organoid culture components, characterized in that: The following steps are involved: Preparation of GelMA; LiF, MAX, pure water and acid are added to the reaction container in sequence; the mixture is stirred until the reaction is completed, and the mixture is centrifuged to remove the supernatant and MXene to obtain Ti3C2Tx; Use photocuring agent to dissolve GelMA, add Ti3C2Tx, and irradiate with blue light to obtain cochlear organoid culture components.

2. The method for preparing cochlear organoid culture components according to claim 1, characterized in that: The acid includes hydrochloric acid.

3. The method for preparing cochlear organoid culture components according to claim 1, characterized in that: The preparation method of GelMA comprises: dissolving gelatin powder in PBS solution, adding methacrylic anhydride dropwise for reaction, dialyzing the mixed solution in deionized water, adjusting the pH value to neutral, and freeze-drying to obtain GelMA.

4. The method for preparing cochlear organoid culture components according to claim 1, characterized in that: The MAX phase is Ti2AlC.

5. A method for stimulating maturation of cochlear organoids, characterized in that: The following steps are involved: Using the cochlear organoid culture component according to any one of claims 1 to 4 to culture inner ear stem cells, and adding differentiation medium; After differentiation is complete, electroacoustic stimulation is applied to the differentiated organoids.

6. The method for stimulating cochlear organoid maturation according to claim 5, characterized in that: In the electroacoustic stimulation, the sound frequency is 600-650 Hz and the current is 1900 μA.

7. The method of stimulating cochlear organoid maturation according to claim 5, characterized in that: Proliferation medium is added during the culturing of inner ear stem cells, and the proliferation medium includes: DMEM / F12, N2 additive, B-27 serum-free additive, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, sodium valproate, GSK inhibitor and ampicillin.

8. The method of stimulating cochlear organoid maturation according to claim 5, characterized in that: The differentiation medium comprises: DMEM / F12, N2 additive, B-27 serum-free additive, GSK inhibitor, γ-secretase inhibitor and ampicillin.

9. An EAS device for culturing cochlear organoids, characterized in that: include The culture component according to any one of claims 1 to 4; A circuit board, with a plurality of circuit conductors extending from electrodes on both sides thereof; The cochlear implant is fixed on both sides of the circuit board through the magnetic attraction between the internal magnet and the microphone; The receiving stimulation connector contains electronic components, one end of which is connected to the coil and the other end transmits current to the electrode. And, the metal wires are fixed to the bottom and the top cover of the culture vessel and are respectively connected to the wires on both sides of the circuit board to form a complete stimulation circuit.

10. Use of the culture components obtained by the preparation method of any one of claims 1 to 4, the method for stimulating the maturation of cochlear organoids according to claims 5 to 8, or the device for culturing cochlear organoids according to claim 9 in the preparation or cultivation of cochlear organoids and the regeneration of inner ear hair cells.

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