Mouse middle ear epithelium 3D organoid and construction method thereof

Abstract

The invention provides a mouse middle ear epithelium 3D organoid and a construction method thereof, and is characterized in that the method comprises the following steps: a, taking a bubble structure of a mouse middle ear tissue, cleaning, digesting, filtering, centrifuging filtrate, collecting precipitate, and separating primary cells of the mouse middle ear; and b, inoculating the digested single cells and cell agglomerates into a 24-pore plate, wrapping with matrigel, and carrying out 3D culture, wherein a medium is a middle ear epithelium amplification medium. And culturing for about 3-5 days to obtain the mouse 3D middle ear proliferation type organoid. And performing differentiation culture by using a middle ear epithelium differentiation medium to obtain the mouse middle ear epithelium organ. According to the method, the 3D mouse middle ear epithelium organ is successfully constructed, the physiological and pathological processes of the middle ear are conveniently analyzed and researched, the interaction between the middle ear microenvironment and cells can be more accurately simulated, and the application range is wide as a model.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to a 3D organoid of mouse middle ear epithelium and a construction method thereof. Background Art

[0002] Organoids are three-dimensional cell cultures that closely resemble their corresponding human or animal organs, reproducing key cell types and structural features of the organ they represent. These "mini-organs" offer highly relevant tissue modeling and complex spatial morphology, as well as the ability to demonstrate cell-cell and cell-matrix interactions. This allows scientists to investigate complex scientific questions in vitro. Compared to 2D cell cultures, 3D cell models such as organoids and spheroids offer researchers the opportunity to better understand complex biology in a physiologically relevant context. By combining high physiological relevance with the ease of in vitro manipulation, organoids have the potential to complement or replace in vitro experiments using primary cells, immortalized cell lines, and animals in many cases. Furthermore, organoids are highly genetically stable in culture, maintaining the genotype and phenotype of the tissue of origin. Therefore, research using organoids, spheroids, and 3D cell models holds great potential for numerous applications, including disease modeling and regenerative medicine.

[0003] The middle ear is a functional organ that requires adequate ventilation to effectively transmit sound from the external auditory canal to the cochlea and auditory nerve. It consists of the tympanic cavity, the superior antrum, and the mastoid cavity. Clinically, otitis media is defined as an infection of the middle ear space that causes inflammation. It is a series of diseases that include acute otitis media (AOM), otitis media with effusion (OME), and otitis media. In most animals, the middle ear is a relatively inaccessible organ lined by a thin mucociliary epithelium. The lack of a complete in vitro research model has been a long-standing problem that has plagued scientific research, making the study of early disease processes in the middle ear very challenging. Therefore, the development of an animal-derived middle ear organoid for in vitro drug screening and scientific research is extremely urgent. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art, such as the limited sample sources during the construction of middle ear organoids and the many ethical constraints during use, which limit the development of research work. A method for constructing mouse 3D middle ear organoids is provided, which can achieve efficient construction, passage and expansion of mouse 3D middle ear organoids.

[0005] The method is characterized in that:

[0006] a) Isolation of primary middle ear cells: The bulla structure of the mouse middle ear tissue was obtained, washed, digested, filtered, and the filtrate was centrifuged to collect the precipitate to isolate the primary mouse middle ear cells, with single cells as the main population;

[0007] b) Collecting middle ear epithelial cells by differential adhesion method: The primary middle ear cells of mice obtained in step a were inoculated into a six-well plate.

[0008] Culture at 37°C for 3-6 hours in DMEM / F12 medium; fibroblasts will quickly adhere to the wall, while non-adherent cells are mainly epithelial cells; collect non-adherent cells and inoculate them into six-well plates, then culture them in suspension using middle ear expansion medium.

[0009] c) 3D middle ear organoid differentiation: After 3-5 days of culture, cells are harvested for 3D culture. The specific steps are as follows: After digestion, cells are inoculated into Matrigel at a Matrigel concentration of 50-95% (v / v). 3D expansion is performed first, and after 1-5 days, 3D cell spheres containing solid vesicles and a mixture are grown. The culture medium is then replaced with a middle ear epithelial differentiation medium and cultured for 10-14 days to obtain mature middle ear organoids.

[0010] The digestion solution in step a comprises: Dispase II 0.05% (0.05 g Dispase II / 100 ml DMEM / F12).

[0011] The particle size of the bulla structure of the mouse middle ear tissue in step a is 3-4 mm; the amount and time of the digestion solution in step a are: 5 ml of digestion solution, digestion at 37° C. for 5 hours, then transfer to 4° C. for overnight digestion; the amount of mouse middle ear primary cells in step a is 10,000-100,000; and the filtration in step a uses a 50-100 μm cell filter.

[0012] The inoculation concentration in step b is: 8,000 to 30,000 cells / well in a 24-well plate.

[0013] The middle ear epithelial expansion medium in step b comprises: GlutaMax 1% to 5% vol / vol, HEPES 1% to 7% vol / vol, P / S 1% to 5% vol / vol, insulin 5 to 50 ug / ml, EGF 10 to 100 ng / ml, transferrin 5 to 10 ug / ml, retinoic acid 0.01 to 0.09 uM, and Y27632 5 to 20 uM, supplemented with DMEM / F12 to the final volume of the required medium.

[0014] The composition of the middle ear epithelial differentiation medium in step c includes: GlutaMax 1% to 7% vol / vol, HEPES 1 to 3% vol / vol, P / S 1% to 8% vol / vol, BSA 1 to 20 mg / ml, EGF 5 to 20 ng / ml, transferrin 5 to 10 ug / ml, retinoic acid 0.01 to 0.1 uM, and DMEM / F12 is added to the final volume of the required culture medium.

[0015] The above method was used to construct the mouse middle ear epithelium 3D middle ear organoid.

[0016] The application of the mouse middle ear epithelial 3D middle ear organoids constructed by the above method in screening and developing drugs for preventing and / or treating middle ear diseases.

[0017] Through the above technical solution, the present invention can achieve at least the following beneficial technical effects:

[0018] (1) The method provided by the present invention uses mouse middle ears as raw materials, which are widely available and avoid the ethical restrictions brought about by human experimental materials. Moreover, specific strains of mouse middle ears can be selected for organoid preparation according to different research needs, which has the advantage of wide product applicability.

[0019] (2) The method provided by the present invention is simple, rapid, and efficient for preparing 3D mouse middle ear organoids, effectively improving experimental efficiency. Furthermore, in the method provided by the present invention, the same culture medium is used for the construction, expansion, and differentiation of mouse middle ear organoids, reducing the possibility of experimental errors caused by the use of different culture media at different stages in most current organoid culture methods.

[0020] (3) The method provided by the present invention has a high success rate when used to prepare mouse middle ear organoids. By optimizing the addition of growth factors and nutrients, the success rate and quality of constructing mouse middle ear epithelial 3D organoids are improved. At the same time, during the cell culture process, technical details such as cell density, culture time, and the timing of replacing the culture medium are mastered to reduce the incidence of cell death problems. Finally, mouse middle ear epithelial 3D organoids are constructed, which facilitates the analysis and study of the physiological and pathological processes of the middle ear. The model of the present invention has higher biological performance and can more accurately simulate the middle ear microenvironment and cell-cell interactions, providing a promising approach for studying the middle ear. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are bright field images of primary mouse middle ear cells (the scale bar in the figure is 200 μm).

[0022] Figure 2Shown is a schematic diagram of the primary cell expansion and growth process (the scale bar in the figure is 200 μm).

[0023] Figure 3 Shown are the final morphological images of organoids, where A represents a schematic diagram of the organoid culture and passage process (the scale bar in the figure is 200 μm); B represents HE staining and Ki67 immunohistochemical staining of organoids during the proliferation period (the scale bar in the figure is 10 μm); C represents HE staining of organoids during the differentiation period (the scale bar in the figure is 10 μm); D represents immunofluorescence staining during the differentiation period (the scale bar in the figure is 20 μm). DETAILED DESCRIPTION

[0024] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0025] The specific embodiments of the present invention are further described below in conjunction with the examples. The following examples are only used to more clearly illustrate the technical embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0026] Example 1 Extraction of the bulla structure of mouse middle ear tissue and isolation of mouse middle ear primary cells

[0027] The tympanic bulla of mouse middle ear tissue was washed with PBS and digested with 20 ml of digestion buffer per gram of tissue at 37°C for 5 hours, followed by overnight digestion at 4°C. The digestion buffer recipe is as follows (manufacturer: solarbio, catalog number: D6430). The tissue was filtered through a 100 μm cell strainer (manufacturer: bkmam). The filtrate was centrifuged at 300 g for 3 minutes to obtain the pellet, which is the primary cell pellet.

[0028] Example 2 Construction of Mouse Middle Ear Epithelial 3D Organoids

[0029] The cells were seeded into a 24-well plate at a concentration of 8,000-30,000 cells per well, corresponding to 25-30 μL of matrix gel. (Manufacturer: corning, item number: 3516) and the situation was observed. The inoculated well plate was placed upside down in the incubator to solidify for 15 to 30 minutes. After the matrix gel solidified, 500 μL of middle ear epithelial expansion medium was added to each well and cultured in a constant temperature incubator at 37°C and 5% CO2. The medium was changed every other day. After growing into a 3D cell ball, it indicates that the mouse middle ear epithelial organoid was successfully constructed. The final morphology of the successfully constructed organoid is shown in the figure below. Figure 3 The present invention adds matrigel to establish a 3D cell structure to maintain cell stemness, which has higher survival efficiency than 2D, and successfully constructs mouse middle ear organoids.

[0030] In the method provided by the present invention, the source of the murine primary middle ear cells used is non-immunodeficient mice.

[0031] According to a preferred embodiment of the present invention, the method further comprises the step of preparing primary mouse middle ear cells. Any method known in the art for preparing primary cells from mouse middle ear tissue can be applied to this step of the present invention. This step preferably comprises digesting the mouse middle ear tissue otic vesicle using a digestive solution.

[0032] Preferably, this step further comprises sieving the tissue suspension obtained after digestion using a sieve with a pore size of 100 μm.

[0033] The digestive fluid used in the present invention is used to digest and dissociate the otic vesicles of the mouse middle ear tissue into single cells. Any substance or composition that can achieve this purpose can be used as a digestive fluid and is suitable for use in the method provided by the present invention.

[0034] After a large number of experiments, the inventors of the present invention cleverly discovered that by using a combination of specific digestive enzymes, the digestion efficiency of the otic vesicle of the mouse middle ear tissue can be further improved, while ensuring the activity of the obtained mouse middle ear primary cells, thereby correspondingly improving the success rate and efficiency of the subsequent construction of mouse middle ear organoids.

[0035] According to a preferred embodiment of the present invention, the digestive fluid comprises: Disase II, DMEM / F12

[0036] Preferably, the digestive fluid has a concentration of 0.05%.

[0037] To further improve the digestion efficiency of mouse skin tissue while ensuring the viability of mouse middle ear primary cells, according to a preferred embodiment of the present invention, the digestion conditions include a 5:1 volume ratio of digestion fluid to mouse otic bulla, a digestion temperature of 37°C, a digestion time of 5 hours, and then a transfer to 4°C for overnight digestion. To ensure sufficient contact between the mouse otic bulla and the digestion fluid, improve digestion efficiency, and avoid overdigestion of localized tissue, digestion is preferably performed under a shaking speed of 100-200 rpm. To prevent overdigestion of mouse middle ear tissue, which could lead to decreased viability or mass cell death, tissue digestion is preferably observed under a microscope every 5 minutes during the digestion process. When more than 80% of the tissue is digested, PBS or culture medium can be added to terminate the digestion. To achieve better digestion results and increase the yield of mouse middle ear primary cells, preferably, 6-10 pieces of mouse tympanic bulla tissue correspond to 5 ml of digestion fluid, with 8 pieces of tympanic bulla tissue being the preferred amount.

[0038] According to a preferred embodiment of the present invention, the organoid culture method includes: expanding primary mouse middle ear cells, coating them with matrigel, and then culturing them in mouse middle ear organoid culture medium. The matrigel used in the present invention can be any matrigel that can be used for organoid or cell 3D culture in the art. It can be a related product prepared according to existing technology or a related product directly purchased from a commercial source (for example, Matrigel matrix produced by Corning).

[0039] Preferably, the culture conditions include: replacing the culture medium every 2 to 3 days.

[0040] Preferably, the amount of murine middle ear organoid culture medium used does not exceed 30% of the culture vessel volume, preferably 15-25%. The culture vessel volume refers to the maximum volume of the culture vessel used to construct the murine middle ear organoid, such as the volume of a culture bottle or dish, or the volume of a single well in a culture plate. For example, when using a 24-well cell culture plate, the amount of murine middle ear organoid culture medium used can be 600-800 μL / well.

[0041] According to a preferred embodiment of the present invention, the method of using Matrigel to coat the expanded mouse primary middle ear cells includes: mixing a suspension of mouse primary middle ear cells cooled on ice with Matrigel, adding the obtained suspension to a culture container (e.g., a low-adhesion cell culture plate), and solidifying the suspension at 36-38°C.

[0042] Preferably, the content of primary murine middle ear cells in the suspension is 8,000 to 30,000 cells / mL, preferably 20,000 cells / mL.

[0043] The second aspect of the present invention provides a culture medium for murine middle ear organoids used in the aforementioned method. The characteristics of the culture medium are as described above and will not be repeated here.

[0044] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for constructing a 3D organoid of mouse middle ear epithelium, characterized by: The method comprises the following steps: a) Isolation of primary middle ear cells: The bulla structure of the mouse middle ear tissue was obtained, washed, digested, filtered, and the filtrate was centrifuged to collect the precipitate to isolate the primary mouse middle ear cells, with single cells as the main population; b) Collect middle ear epithelial cells using the differential adhesion method: The primary mouse middle ear cells obtained in step a were seeded into a six-well plate and cultured at 37°C for 3-6 hours in DMEM / F12 medium. Fibroblasts will rapidly adhere to the wall, while non-adherent cells are primarily epithelial cells. Collect the non-adherent cells, seed them into a six-well plate, and then culture them in suspension in middle ear expansion medium. c) 3D middle ear organoid differentiation: After 3-5 days of culture, cells are harvested for 3D culture. The specific steps are as follows: After digestion, cells are inoculated into Matrigel at a Matrigel concentration of 50-95% (v / v). 3D expansion is performed first, and after 1-5 days, 3D cell spheres containing solid vesicles and a mixture are grown. The culture medium is then replaced with a middle ear epithelial differentiation medium and cultured for 10-14 days to obtain mature middle ear organoids.

2. The construction method according to claim 1, characterized in that The digestion solution in step a comprises: Dispase II 0.05% (0.05 g Dispase II / 100 ml DMEM / F12).

3. The construction method according to claim 1, characterized in that The particle size of the bulla structure of the mouse middle ear tissue in step a is 3-4 mm; the amount and time of the digestion solution in step a are: 5 ml of digestion solution, digestion at 37° C. for 5 hours, then transfer to 4° C. for overnight digestion; the amount of mouse middle ear primary cells in step a is 10,000-100,000; and the filtration in step a uses a 50-100 μm cell filter.

4. The construction method according to claim 1, characterized in that The inoculation concentration in step b is: 8,000 to 30,000 cells / well in a 24-well plate.

5. The construction method according to claim 1, characterized in that The middle ear epithelium expansion culture medium in step b comprises: GlutaMax 1%~5% vol / vol, HEPES 1%~7% vol / vol, P / S 1%~5% vol / vol, insulin 5~50ug / ml, EGF 10~100ng / ml, transferrin 5~10ug / ml, retinoic acid 0.01~0.09uM, Y27632 5~20uM, add DMEM / F12 to the final volume of the required culture medium.

6. The construction method according to claim 1, characterized in that The composition of the middle ear epithelial differentiation culture medium in step c includes: GlutaMax 1%~7%vol / vol, HEPES1~3%vol / vol, P / S1%~8%vol / vol, BSA 1~20mg / ml, EGF 5-20 ng / ml, transferrin 5-10 ug / ml, retinoic acid 0.01-0.1 uM, add DMEM / F12 to the final volume of the required culture medium.

7. A 3D middle ear organoid of mouse middle ear epithelium constructed by the method of any one of claims 1 to 7.

8. Use of the middle ear organoid according to any one of claims 1 to 6 or the ear organoid according to claim 7 in screening and developing drugs for preventing and / or treating middle ear diseases.

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