Preparation method of 3D mixed cell microspheres and application thereof

Abstract

This invention belongs to the field of cell culture technology, specifically relating to a method for preparing 3D hybrid cell microspheres and their applications. The invention involves co-seeding olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells in a low-absorption culture plate to obtain 3D hybrid cell microspheres. In the 3D hybrid culture system, the two cell types provide support for the state of the olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells, mutually supporting each other and further enhancing their biological characteristics, making them easier to colonize at lesion sites, thereby improving the anti-apoptotic ability of the 3D hybrid cell microspheres. The 3D hybrid cell microspheres prepared by this method offer promising applications for stem cell therapy of respiratory diseases and provide a foundation for using olfactory mucosal-derived stem cells in regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for preparing 3D hybrid cell microspheres and its application. Background Technology

[0002] The respiratory tract is one of the organs directly exposed to the external environment. Nasal airway epithelial cells, as the gateway to the respiratory tract, are an important physiological defense barrier, playing a crucial role in resisting the invasion of pathogenic microorganisms and clearing toxins. The airway and respiratory alveoli originate from different primordia of the primitive endoderm: the tracheal primordia and the lung primordia. These two primordia contain different types of stem cells, called proximal and distal stem cells, respectively. Proximal stem cells are airway basal stem cells, while distal stem cells include bronchiolar basal stem cells, stem cells at the junction of respiratory bronchioles and alveolar ducts, and type II alveolar cells. Studies have shown that the basal layer of the distal nasal mucosa contains epithelial stem cells expressing stem cell surface markers such as p63, KRT5, and NGFR, which can be isolated and expanded in vitro. When induced under air-liquid interface conditions, nasal mucosal basal layer epithelial stem cells can differentiate into airway pseudostratified epithelial tissue composed of ciliated cells, goblet cells, and basal cells. However, current culture methods are limited, leading to challenges such as poor in vitro proliferation and easy apoptosis.

[0003] Mesenchymal stem cells (MSCs) are adult stem cells with paracrine, immunomodulatory, and multi-lineage differentiation potential. Compared to embryonic stem cells, they have advantages such as lower immune response, lower tumorigenesis risk, and no ethical issues. Some studies have combined MSCs with other cell types, such as hematopoietic stem cells, endothelial cells, and podocytes, in 3D culture, finding that this enhances their therapeutic efficacy in disease models. However, current methods for preparing stem cells for respiratory system treatments mostly use cell suspensions, which are highly mobile and have a simple, homogeneous cell structure, making it difficult for them to colonize at lesion sites and exert their effects. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing 3D hybrid cell microspheres and their application. The 3D hybrid cell microspheres facilitate mutual support between olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells, enhance their proliferation and anti-apoptotic abilities, and make them easier to colonize at lesion sites, thereby improving the anti-apoptotic ability of the 3D hybrid cell microspheres.

[0005] This invention provides a method for preparing 3D hybrid cell microspheres, comprising the following steps: seeding olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells together in a low-adsorption culture plate for mixed culture to obtain the 3D hybrid cell microspheres.

[0006] Preferably, the low-adsorption culture plate comprises a low-adsorption six-well plate.

[0007] Preferably, the seeding density ratio of the olfactory mucosa basal stem cells to the olfactory mucosa mesenchymal stem cells is 1:1.

[0008] Preferably, the temperature for the mixed culture is 37°C, the concentration of carbon dioxide is 5%, and the humidity is 80-99%.

[0009] Preferably, the culture medium for the mixed culture includes DMEM / F12 complete medium containing 5-20% v / v FBS.

[0010] Preferably, the seeding amounts of the olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells are (4-5) × 10⁻⁶. 7 One hole / hole.

[0011] Preferably, the method for preparing the olfactory mucosa basal stem cells includes: inoculating nasal mucosa tissue blocks into an olfactory mucosa basal stem cell culture medium for cell culture, replacing the culture medium every 2 to 5 days until the cell density reaches more than 90%, thereby obtaining the olfactory mucosa basal stem cells.

[0012] Preferably, the olfactory mucosa basal stem cell culture medium comprises DMEM high-glucose complete medium containing 10% v / v FBS.

[0013] Preferably, the volume of the nasal mucosal tissue block is 0.5–1.5 mm. 3 The cell culture temperature was 37°C, the carbon dioxide concentration was 5%, and the humidity was 80-99%.

[0014] The present invention also provides the application of 3D hybrid cell microspheres prepared by the preparation method described above in improving the anti-apoptotic ability of olfactory mucosal mesenchymal stem cells and / or olfactory mucosal basal stem cells.

[0015] Beneficial effects:

[0016] This invention provides a method for preparing 3D hybrid cell microspheres. The method involves co-seeding olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells in a low-absorption culture plate to obtain 3D hybrid cell microspheres. In the 3D hybrid culture system, the two cell types provide support for the state of the olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells, mutually supporting each other and further enhancing their biological characteristics, making them easier to colonize at lesion sites, thereby improving the anti-apoptotic ability of the 3D hybrid cell microspheres. The 3D hybrid cell microspheres prepared by this method offer promising application prospects for stem cell therapy of respiratory diseases and provide a foundation for using olfactory mucosa-derived stem cells in the field of regenerative medicine. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 Optical microscope images of olfactory mucosal basal stem cells in Example 1 and olfactory mucosal mesenchymal stem cells in Example 2;

[0019] Figure 2 The identification results of olfactory mucosal basal stem cells in Example 1;

[0020] Figure 3 The identification results of olfactory mucosal mesenchymal stem cells in Example 2;

[0021] Figure 4 This is an optical microscope image of the 3D hybrid microspheres obtained by co-culturing olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells in Example 4.

[0022] Figure 5 This is a fluorescence staining image of the 3D hybrid microspheres obtained by co-culturing olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells in Example 4;

[0023] Figure 6 The results show the relative expression and quantitative analysis of apoptosis-related proteins Bax, Bcl-2, cleave-casepase3, and pro-casepase3 in the 3D hybrid microspheres in Example 4. Detailed Implementation

[0024] This invention provides a method for preparing 3D hybrid cell microspheres, comprising the following steps: seeding olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells together in a low-adsorption culture plate for mixed culture to obtain the 3D hybrid cell microspheres.

[0025] In one embodiment, the present invention prepares olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells.

[0026] As one embodiment, the method for preparing olfactory mucosal basal stem cells according to the present invention includes: inoculating nasal mucosal tissue blocks into olfactory mucosal basal stem cell culture medium for cell culture, replacing the culture medium every 2 to 5 days until the cell density reaches more than 90%, and then digesting and centrifuging the obtained cells with trypsin, collecting the precipitate, and obtaining the olfactory mucosal basal stem cells.

[0027] In one embodiment, the olfactory mucosal basal stem cells of the present invention are obtained from healthy individuals. In another embodiment, the nasal mucosal tissue block of the present invention is obtained from the superficial layer of the medial aspect of the nasal turbinate near its root; in a specific embodiment, the nasal mucosal tissue block is obtained from the superficial layer of the middle turbinate near its root on one side. In another embodiment, the volume of the nasal mucosal tissue block of the present invention is 0.5–1.5 mm. 3 In another embodiment, the volume of the nasal mucosal tissue block is 0.5 mm. 3 .

[0028] In one embodiment, the olfactory mucosal basal stem cell culture medium of the present invention is a DMEM high-glucose complete medium containing 10% (v / v) FBS. In another embodiment, the cell culture of the present invention is carried out in a humidified incubator at a temperature of 37°C. In yet another embodiment, the carbon dioxide concentration of the cell culture is 5%. In one embodiment, the humidity of the cell culture is 80-99%; in another embodiment, the humidity is 95%. In yet another embodiment, the culture medium is replaced every 3 days during the cell culture process.

[0029] In one embodiment, the concentration of the pancreatic enzyme in this invention can be 0.2-0.5%; in another embodiment, the concentration of the pancreatic enzyme is 0.25%.

[0030] As one embodiment, the preparation steps of the olfactory mucosal mesenchymal stem cells of the present invention can be carried out with reference to the preparation method disclosed in Chinese Patent Application No. 201410228728.X.

[0031] After obtaining the olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells, the present invention seeded the olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells together in a low-absorption culture plate for mixed culture to obtain the 3D mixed cell microspheres.

[0032] In one embodiment, the low-adsorption culture plate of the present invention comprises a low-adsorption six-well plate. Using the low-adsorption culture plate for the mixed culture has the advantage of preventing cell adhesion and promoting cell suspension. In one embodiment, the seeding density ratio of olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells in the present invention is 1:1. The seeding amounts of olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells in the present invention are (4-5) × 10⁻⁶. 7Cells / well. In one embodiment, the mixed culture of the present invention is carried out in a humidified incubator at a temperature of 37°C. In one embodiment, the carbon dioxide concentration of the mixed culture is 5%. In one embodiment, the humidity of the mixed culture is 80-99%; in another embodiment, the humidity of the mixed culture is 95%.

[0033] In one embodiment, the culture medium for mixed culture according to the present invention comprises DMEM / F12 complete medium containing 5-20% (v / v) FBS; in another embodiment, the proportion of FBS is 10% (v / v).

[0034] The present invention also provides the application of 3D hybrid cell microspheres prepared by the preparation method described above in improving the anti-apoptotic ability of olfactory mucosal mesenchymal stem cells and / or olfactory mucosal basal stem cells.

[0035] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] Isolation, culture and identification of olfactory mucosal basal stem cells

[0038] (1) Samples were taken from the nasal mucosa of healthy volunteers:

[0039] After signing the informed consent form, preoperative preparations are performed 3 days prior to the procedure, including cleaning nasal hairs, rinsing the nasal cavity, and administering 3-5 drops of chloramphenicol eye drops. Before harvesting, a tetracaine tampon is inserted into the nasal cavity under endoscopic guidance, up to the lower part of the superior turbinate, for intranasal anesthesia. Under endoscopic guidance, 1-2 tissue fragments, approximately 5-8 mm in diameter, are harvested from the medial aspect of the root of one side of the middle turbinate using cribriform forceps. Postoperatively, hemostasis is achieved by applying pressure with cotton balls. The tissue fragments are then cut into 0.5 mm pieces using ophthalmic scissors. 3 After separating the tissue into small pieces, transfer them to centrifuge tubes and allow them to settle naturally for 5 minutes.

[0040] (2) The tissue blocks obtained in step (1) were evenly seeded at the bottom of a culture flask, and olfactory mucosal basal stem cell culture medium (Table 1) was added. The flask was then placed in a humidified incubator (37℃, 5% CO2) and cultured, with the culture medium replaced every 3 days. When the cell density reached more than 90%, the morphology under an optical microscope was observed. Figure 1 As shown in Figure A, it can be passaged for future use or subjected to immunofluorescence identification.

[0041] (3) Transfer the cells from step (2) to a six-well plate for KRT5 and P63 cell immunofluorescence identification (antibody source and dilution are shown in Table 2). The steps are as follows: soak the cells in PBS three times for 3 min each time, then fix them with sucrose-PFA solution (5%) for 15 min; remove the fixative, soak the cells in PBS three times for 3 min each time; block and permeate the cells with 5% bovine serum albumin powder (Bsa) powder + 0.1% PBST phosphate buffered saline solution (0.1% Triton) for 15 min; soak the cells in PBS three times for 3 min each time; add the primary antibody solution, incubate overnight at 4°C, wash the cells with PBS solution three times for 3 min each time the next day; incubate the cells with the secondary antibody solution at room temperature in the dark for 1 hour, wash the cells with PBS solution three times for 3 min each time, and observe the cells under a fluorescence microscope. Figure 2 ,in Figure 2 In the diagram, A and B represent the results of KRT5 staining and P63 staining, respectively.

[0042] Table 1. Culture media for olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells.

[0043] culture medium composition Olfactory mucosal basal stem cell culture medium DMEM + 10% FBS Olfactory mucosal mesenchymal stem cell culture medium DMEM / F12+10%FBS

[0044] Table 2. Relevant information on KRT5 and P63 antibodies.

[0045] Antibody name brand Dilution factor KRT5 Abcam 1：100 P63 Abcam 1：200 Rabbit II Anti- Proteintech 1：200

[0046] Depend on Figure 2 It can be concluded that the obtained cells are olfactory mucosal basal stem cells.

[0047] Example 2

[0048] The isolation, culture, and identification of olfactory mucosal mesenchymal stem cells (OM-MSCs) follow these steps:

[0049] Olfactory mucosal mesenchymal stem cells (e.g., a method for isolating and culturing human olfactory mucosal mesenchymal stem cells) were obtained according to the steps in Example 1 of Chinese Patent Application No. 201410228728.X, entitled "A Method for Isolating and Culturing Human Olfactory Mucosal Mesenchymal Stem Cells". Figure 1As shown in Figure B), the cells were identified using the identification method disclosed in the aforementioned patent. The steps were as follows: When the cell density in the culture flask was approximately 80%, the cells were washed twice with sterile PBS solution. After washing, 0.25% trypsin solution was added for digestion. The cell digestion was observed under an optical microscope, and then digestion was terminated using complete culture medium. The suspension was collected into centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with sterile PBS solution and collected. The cells were divided into 6 tubes, and CD34, CD45, CD73, CD90, and CD105 antibodies were added respectively. The source and dilution of the antibodies are shown in Table 3. The cells were incubated at room temperature in the dark for 30 min. The cells were washed twice with sterile PBS solution and resuspended, and then analyzed using a flow cytometer. The results are shown in Table 3. Figure 3 As shown.

[0050] Table 3. Sources and dilutions of CD34, CD45, CD73, CD90, and CD105 antibodies.

[0051]

[0052]

[0053] Depend on Figure 3 It can be concluded that OM-MSCs express the relevant markers of MSCs, CD73, CD90 and CD105, but do not express CD34 and CD45, and the purity is as high as 99% or more.

[0054] Example 3

[0055] The steps for culturing 2D hybrid cells are as follows:

[0056] When the density of both olfactory mucosal basal cells and olfactory mucosal mesenchymal stem cells is 80%, the cell suspension is collected by routine digestion, and the cell count is determined by a cell counter. (4–5) × 10⁶ cells are then prepared for each type of olfactory mucosal basal cell and olfactory mucosal mesenchymal stem cell. 7 Basal stem cells and mesenchymal stem cells were seeded at a density of 1:1 in conventional six-well plates. The cells were resuspended in DMEM / F12 solution containing 10% (v / v) FBS and evenly distributed. The plates were then placed in a humidified incubator (37°C, 5% CO2) and cultured for 3 days to obtain 2D mixed cells.

[0057] Example 4

[0058] The steps for culturing, identifying, and characterizing 3D hybrid microspheres (i.e., 3D hybrid cell microspheres) are as follows:

[0059] (1) When the density of the two types of cells is approximately 80%, the cell suspension is collected by routine digestion, and the cell count is determined by a cell counter. Then, 4–5 × 10⁶ olfactory mucosal basal cells and olfactory mucosal mesenchymal stem cells are prepared separately. 7 Basal stem cells and mesenchymal stem cells were seeded at a 1:1 density in low-absorption six-well plates. The cells were resuspended in DMEM / F12 solution containing 10% (v / v) FBS and evenly distributed. The plates were then incubated in a humidified incubator (37°C, 5% CO2) for 3 days. After 3 days, approximately spherical 3D hybrid cell spheroids were observed under an optical microscope. The results are as follows: Figure 4 .

[0060] Depend on Figure 4 It can be concluded that olfactory mucosal basal cells and olfactory mucosal mesenchymal stem cells can mix to form 3D microspheres.

[0061] Meanwhile, the inventors also conducted a series of comparative experiments. For example, they used the steps in step (1) to culture 3D hybrid cell spheres, with the only difference being that the number of days of culture was adjusted from 3 days to 5 or 7 days. The results showed that the cells in the center of the obtained 3D hybrid cell spheres all had dead cells.

[0062] Alternatively, the inventors conducted a series of comparative experiments, for example, using the steps of step (1) to culture 3D hybrid cell spheres, the only difference being that the number of days of culture was adjusted from 3 days to 1 or 2 days. The results showed that the obtained 3D hybrid cell spheres were smaller and contained a large number of suspended single cells.

[0063] Alternatively, 3D hybrid cell spheroids can be cultured using the steps in (1), the only difference being that the number of basal stem cells is 1×10⁻⁶. 8 The number of mesenchymal stem cells is 1×10 8 When the cells were seeded at a density of 100% in a low-adsorption six-well plate, the same result was observed: dead cells were found in the center of the obtained 3D hybrid cell spheres.

[0064] (2) The 3D hybrid microspheres cultured using the method of the present invention in step (1) were subjected to CD90 and KRT5 antibody immunofluorescence staining. The source and dilution factor of the antibodies are shown in Table 4, and the staining steps are the same as in Example 1. Observation under a fluorescence microscope showed that CD90 and KRT5 staining were applied to the same cell spheres, confirming that the cultured 3D hybrid microspheres were a mixture of mesenchymal stem cells and basal stem cells. Figure 5 As shown.

[0065] Table 4. Sources and dilution factors of CD90 and KRT5 antibodies

[0066] Antibody name brand Dilution factor CD90 Proteintech 1：200 KRT5 Abcam 1：100 Mouse secondary antibody Proteintech 1：200 Rabbit II Anti- Proteintech 1：200

[0067] (3) Antiapoptotic ability test of mesenchymal stem cells and basal stem cells derived from human olfactory mucosa: 2D and 3D mixed cell spheroids prepared in Examples 3 and 4 were treated with the apoptosis inducer 250 nM STS, respectively. Subsequently, apoptosis-related indicators such as Bax, Bcl-2, pro-casepase 3 and cleave-casepase 3 were detected by Western blotting to evaluate their antiapoptotic ability. The steps are as follows:

[0068] 1) Protein sample preparation: Wash cells with pre-cooled PBS solution, then add 500 μL LIPA lysis buffer, scrape cells with a scraper, sonicate for 1.5 min, lyse on ice for 10 min, then centrifuge at 4℃, 12000 rpm for 15 min, and transfer the supernatant after centrifugation to a sterile centrifuge tube.

[0069] 2) Protein Concentration Detection: Follow the instructions for the BCA protein quantification kit to determine the protein concentration. Prepare an appropriate amount of BCA working solution according to the sample volume, mix thoroughly, and stabilize at room temperature for 24 hours. Completely dissolve the protein standard to a concentration of 2 mg / mL. Dilute the standard serially with RIPA lysis buffer at a concentration of 50%. Add 25 μL of the diluted standard and blank sample to the standard wells of a 96-well plate, and add 25 μL of the sample to the sample wells of the 96-well plate. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 minutes. Measure the absorbance at wavelengths between 550 nm using a microplate reader, and calculate the protein concentration based on the standard curve.

[0070] 3) SDS-PAGE electrophoresis: Select an appropriate gel concentration based on the molecular weight of the target protein, and prepare the separating and stacking gels for SDS-PAGE according to the instructions. Prepare a 10% separating gel, add TEMED, and immediately mix before pouring. After pouring, seal the gel with isopropanol. When the dividing line no longer changes when the gel is slightly tilted, the gel has solidified. Wait another 3 minutes to allow the gel to fully solidify, then pour off the top layer of isopropanol and blot dry with filter paper. Next, prepare a 4.8% stacking gel, add TEMED, and immediately mix before pouring. Insert the comb into the glass plate, fill the remaining space with the stacking gel, and wait for the gel to solidify. Take 300 μL of protein supernatant, add 75 μL of 5× loading buffer, mix well, boil in water for 5 minutes, and then rapidly cool in an ice box for later use. Based on the protein quantification results, spot 2 μL of marker in the first well, and 10-20 μL of denatured protein in each of the other wells. Start electrophoresis at a constant voltage of 75V for 130 minutes. Electrophoresis was stopped when the bromophenol blue reached the bottom of the gel.

[0071] 4) Transfer: Cut the gel to the molecular weight of the target protein. Prepare 6 sheets of filter paper the same size as the gel and 1 NC membrane, based on the size of the gel fragments after separation. Place the NC membrane and filter paper together in the transfer buffer until completely saturated. Arrange the gel in the following order: 3 sheets of filter paper, NC membrane, gel, and another 3 sheets of filter paper, avoiding air bubbles. Cover the instrument, turn on the power, and transfer at a constant current of 300mA. After transfer, remove the membrane and wash it once in 1×PBST.

[0072] 5) Sealing: Prepare 5% skim milk powder using 1×PBST, immerse the membrane in the solution, and let it stand at room temperature for 90 minutes;

[0073] 6) Antibody incubation (antibody sources and dilution ratios are shown in Table 5): Dilute the primary antibody solution with 1×PBST according to a certain ratio, incubate the membrane with the primary antibody overnight at 4°C, and place at room temperature for 30 min the next day. After incubation, wash three times with 1×PBST, 10 min each time. Then dilute the HRP-labeled secondary antibody with 1×PBST, and incubate the diluted secondary antibody solution with the membrane at room temperature for 90 min. After incubation, wash three times with 1×PBST, 15 min each time.

[0074] 7) Development: Incubate the membrane with ECL chemiluminescent solution for 1 min, absorb the liquid with filter paper, wrap the hybridization membrane with plastic wrap, and expose it to X-ray film in a dark box for 1-30 min. Develop and rinse. Results showed that compared to 2D, the expression of the anti-apoptotic protein Bcl-2 was significantly increased in the 3D group, while the expression of the pro-apoptotic protein Bax and the apoptosis-related marker cleave-casepase 3 showed a decreasing trend. (See...) Figure 6 ,in Figure 6 In the text, "**" and "****" indicate significant differences, p < 0.01 and p < 0.0001, respectively.

[0075] Table 5. Sources and dilution factors of antibodies

[0076] Antibody name brand Dilution factor Bax Abcam 1：2000 BCL-2 Proteintech 1：2000 Caspase 3 Proteintech 1：2000 β-actin Proteintech 1：5000 Mouse secondary antibody Abiowell 1：5000 Rabbit II Anti- Abiowell 1：5000

[0077] As can be seen from the results in Example 4, the 3D hybrid culture system can enhance its anti-apoptotic effect against harsh external microenvironments.

[0078] From the above embodiments, it can be concluded that the 3D hybrid cell microspheres of the present invention facilitate mutual support between olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells, improve their biological characteristics, make them easier to colonize at the lesion site, and enhance the anti-apoptotic ability of the 3D hybrid cell microspheres.

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing 3D hybrid cell microspheres, characterized in that, The process includes the following steps: olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells are mixed and seeded in a low-absorption culture plate for mixed culture to obtain the 3D mixed cell microspheres.

2. The preparation method according to claim 1, characterized in that, The low-adsorption culture plate includes a low-adsorption six-well plate.

3. The preparation method according to claim 1, characterized in that, The seeding density ratio of olfactory mucosal basal stem cells to olfactory mucosal mesenchymal stem cells is 1:

1.

4. The preparation method according to claim 1, characterized in that, The mixed culture was conducted at a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of 80-99%.

5. The preparation method according to claim 1, characterized in that, The culture medium for the mixed culture includes DMEM / F12 complete medium containing 5-20% v / v FBS.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The seeding amounts of the olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells were (4~5)×10⁻⁶. 7 One hole / hole.

7. The preparation method according to claim 1, characterized in that, The method for preparing the olfactory mucosa basal stem cells includes: inoculating nasal mucosa tissue blocks into an olfactory mucosa basal stem cell culture medium for cell culture, replacing the culture medium every 2-5 days until the cell density reaches more than 90%, thereby obtaining the olfactory mucosa basal stem cells.

8. The preparation method according to claim 7, characterized in that, The olfactory mucosa basal stem cell culture medium includes DMEM high-glucose complete medium containing 10% v / v FBS.

9. The preparation method according to claim 7, characterized in that, The volume of the nasal mucosal tissue block is 0.5~1.5 mm. 3 The cell culture temperature was 37°C, the carbon dioxide concentration was 5%, and the humidity was 80-99%.

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