Preparation method and application of 3D mixed cell microspheres

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a preparation method and application of 3D mixed cell microspheres. The method comprises the following steps: mixing and inoculating olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells into a low-adsorption culture plate, and carrying out mixed culture to obtain 3D mixed cell microspheres; the two kinds of cells have a certain supporting effect on the states of the olfactory mucosa basal stem cells and the olfactory mucosa mesenchymal stem cells in a 3D mixed culture system, the two kinds of cells support each other, the biological characteristics of the cells are further improved, the cells are more easily colonized at the focus position, and then the anti-apoptosis capacity of the 3D mixed cell microspheres is improved; the 3D mixed cell microsphere prepared by the preparation method provides a good application prospect for treating respiratory system diseases by stem cells, and provides a basis for applying the stem cells from olfactory mucosa to the field of regenerative medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a preparation method of 3D hybrid cell microspheres and applications thereof. Background Art

[0002] The respiratory tract is one of the organs of the human body that is directly open to the outside world. As the gateway to the respiratory tract, nasal airway epithelial cells are an important physiological defense barrier of the human body, playing an important role in resisting the invasion of pathogenic microorganisms and removing toxic substances. The airway and respiratory alveoli originate from different primordia of the primitive endoderm, namely the tracheal primordium and the pulmonary primordium. The two primordia contain different stem cells, which are called proximal and distal stem cells, respectively. Proximal stem cells are airway basal stem cells, and distal stem cells include bronchiolar basal stem cells, stem cells at the junction of respiratory bronchioles and alveolar ducts, and alveolar type II cells. Studies have shown that there are epithelial stem cells in the distal basal layer of the nasal mucosa, which express stemness surface markers such as p63, KRT5 and NGFR, and can be isolated and cultured in vitro. When induced under air-liquid interface conditions, epithelial stem cells in the basal layer of the nasal mucosa can differentiate into airway pseudostratified epithelial tissue composed of ciliated cells, goblet cells and basal layer cells, but their current culture methods are limited, and there are problems such as poor in vitro proliferation and easy apoptosis.

[0003] Mesenchymal stem cells are a type of adult stem cell with paracrine, immunomodulatory and multidirectional differentiation potentials. Compared with embryonic stem cells, they have advantages such as low immune response, low risk of tumorigenesis, and no ethical issues. At present, some studies have combined mesenchymal stem cells with other types of cells such as hematopoietic stem cells, endothelial cells and podocytes for 3D culture, and found that they can enhance their therapeutic efficacy on disease models. However, the current preparation methods for stem cells for the treatment of the respiratory system mostly use cell suspensions, which are easy to flow and have a single cell structure, making it difficult for them to colonize in the lesion site to exert their effects. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of 3D hybrid cell microspheres, which are beneficial to the mutual support of olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells, improve their proliferation and anti-apoptosis ability, are easier to colonize in the lesion site, and improve the anti-apoptosis ability of the 3D hybrid cell microspheres.

[0005] The present invention provides a method for preparing 3D hybrid cell microspheres, comprising the following steps: inoculating olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells 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 and the olfactory mucosa mesenchymal stem cells is 1:1.

[0008] Preferably, the temperature of 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 comprises DMEM / F12 complete culture medium containing 5-20% v / v FBS.

[0010] Preferably, the inoculation amount of the olfactory mucosa basal stem cells and the olfactory mucosa mesenchymal stem cells is (4-5)×10 7 Pieces / hole.

[0011] Preferably, the method for preparing the olfactory mucosa basal stem cells comprises: 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 culture medium containing 10% v / v FBS.

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

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

[0015] Beneficial effects:

[0016] The present invention provides a preparation method of 3D mixed cell microspheres. The present invention mixes olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells and inoculates them in a low-adsorption culture plate for mixed culture to obtain 3D mixed cell microspheres. The two cells have a certain supporting effect on the state of the olfactory mucosa basal stem cells and the olfactory mucosa mesenchymal stem cells in the 3D mixed culture system. The two cells support each other, further improve their biological characteristics, and are easier to colonize in the lesion site, thereby improving the anti-apoptosis ability of the 3D mixed cell microspheres. The 3D mixed cell microspheres prepared by this preparation method provide good application prospects for stem cell treatment of respiratory diseases and provide a basis for using stem cells derived from the olfactory mucosa in the field of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 The optical microscope images of the olfactory mucosa basal stem cells in Example 1 and the olfactory mucosa mesenchymal stem cells in Example 2;

[0019] Figure 2 The identification results of olfactory mucosa 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 mixed microspheres obtained by mixed culture of olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells in Example 4;

[0022] Figure 5 This is a fluorescent staining image of the 3D mixed microspheres obtained by mixed culture of olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells in Example 4;

[0023] Figure 6 The relative expression and quantitative analysis results of apoptosis-related proteins Bax, Bcl-2, cleave-casepase3, and pro-casepase3 in 3D mixed microspheres in Example 4. DETAILED DESCRIPTION

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

[0025] As an embodiment, the present invention prepares olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells.

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

[0027] As an embodiment, the olfactory mucosa basal stem cells of the present invention are obtained from healthy people. As an embodiment, the nasal mucosa tissue block of the present invention is obtained from the superficial tissue inside the root of the turbinate; in a specific embodiment, the nasal mucosa tissue block is obtained from the superficial layer of the root of the middle turbinate on one side. As an embodiment, the volume of the nasal mucosa tissue block of the present invention is 0.5-1.5mm 3 As another embodiment, the volume of the nasal mucosal tissue block is 0.5mm 3 .

[0028] As an 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. As an embodiment, the cell culture of the present invention is carried out in a humidified incubator, and the temperature of the cell culture is 37°C. As an embodiment, the carbon dioxide concentration of the cell culture is 5%. As an embodiment, the humidity of the cell culture is 80-99%; as another embodiment, the humidity of the cell culture is 95%. As an embodiment, during the cell culture process, a new culture medium is replaced every 3 days.

[0029] As an embodiment, the concentration of the pancreatic enzyme of the present invention can be 0.2-0.5% pancreatic enzyme; as another embodiment, the concentration of the pancreatic enzyme is 0.25%.

[0030] As an embodiment, the preparation steps of the olfactory mucosa mesenchymal stem cells of the present invention can be prepared by referring to the preparation method disclosed in the Chinese patent application number 201410228728.X.

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

[0032] As an embodiment, the low adsorption culture plate of the present invention includes a low adsorption six-well plate. The use of the low adsorption culture plate for the mixed culture has the advantages of preventing cell adhesion and promoting cell suspension. As an embodiment, the inoculation density ratio of the olfactory mucosa basal stem cells and the olfactory mucosa mesenchymal stem cells of the present invention is 1:1. The inoculation amounts of the olfactory mucosa basal stem cells and the olfactory mucosa mesenchymal stem cells of the present invention are (4-5)×10 7In one embodiment, the mixed culture of the present invention is carried out in a humidified incubator, and the temperature of the mixed culture is 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] As one embodiment, the culture medium for mixed culture of the present invention comprises DMEM / F12 complete culture medium containing 5-20% (v / v) FBS; as another embodiment, the proportion of the FBS is 10% (v / v).

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

[0035] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1

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

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

[0039] After signing the informed consent form, preoperative preparation was performed 3 days before the operation, including cleaning the nasal hair, washing the nasal cavity, and dripping 3 to 5 drops of chloramphenicol eye drops. Before obtaining, insert a tetracaine cotton strip into the nasal cavity under endoscopy until the lower part of the superior turbinate for intranasal anesthesia. Under nasal endoscopy, use a sieve bite forceps to clamp 1 to 2 tissue blocks on the inner side of the root of the middle turbinate on one side, with a diameter of about 5 to 8 mm. After the operation, use cotton balls to compress and stop bleeding. Use ophthalmic scissors to cut into 0.5 mm 3 After the small tissue pieces are separated, they are transferred to a centrifuge tube and allowed to settle naturally for 5 minutes.

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

[0041] (3) The cells in step (2) were transferred to a six-well plate for KRT5 and P63 (the source and dilution of the antibodies are shown in Table 2) cell immunofluorescence identification, the steps of which were as follows: the cells were soaked in PBS three times, each time for 3 minutes, and then fixed with sucrose-PFA solution (5%) for 15 minutes; the fixative was removed and the cells were soaked in PBS three times, each time for 3 minutes; 5% bovine serum albumin powder (Bsa) powder + 0.1% PBST phosphate buffered saline (0.1% Triton) solution was used for blocking and permeabilization for 15 minutes; PBS was soaked three times, each time for 3 minutes; the primary antibody solution was added dropwise, incubated at 4°C overnight, and the next day, the cells were washed with PBS solution three times, each time for 3 minutes; the cells were incubated with secondary antibody solution at room temperature in the dark for 1 hour, and the cells were washed with PBS solution three times, each time for 3 minutes, and observed under a fluorescence microscope. Figure 2 ,in Figure 2 A and B in the figure represent the results of KRT5 staining and P63 staining, respectively.

[0042] Table 1 Culture medium of olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells

[0043] Culture medium composition Olfactory Mucosa Basal Stem Cell Culture Medium DMEM + 10% FBS Olfactory Mucosa Mesenchymal Stem Cell Culture Medium DMEM / F12+10%FBS

[0044] Table 2 Related information of KRT5 and P63 antibodies

[0045] Antibody Name brand Dilution multiple KRT5 Abcam 1：100 P63 Abcam 1：200 Rabbit secondary antibody Proteintech 1：200

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

[0047] Example 2

[0048] The steps for the isolation, culture and identification of olfactory mucosa mesenchymal stem cells (OM-MSCs) are as follows:

[0049] Olfactory mucosal mesenchymal stem cells (such as Figure 1As shown in B), and the identification method disclosed in the above patent was used for identification, the steps of which are: when the cell density in the culture bottle is about 80%, wash it twice with sterile PBS solution, add 0.25% trypsin solution after washing for digestion, observe the cell digestion under an optical microscope, and then use complete culture medium to terminate the digestion. Collect the suspension into a centrifuge tube and centrifuge it at 1000rpm for 5 minutes. Discard the supernatant, wash it twice with sterile PBS solution, and collect the cells. Divide the cells into 6 tubes, and add CD34, CD45, CD73, CD90, and CD105 antibodies respectively. The sources and dilutions of the antibodies are shown in Table 3, and incubate at room temperature in the dark for 30 minutes. Wash it twice with sterile PBS solution and resuspend the cells, then use the flow cytometer to detect it, and the results are as shown Figure 3 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 MSCs-related markers CD73, CD90 and CD105, do not express CD34 and CD45, and the purity is as high as over 99%.

[0054] Example 3

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

[0056] When the density of olfactory mucosal basal cells and olfactory mucosal mesenchymal stem cells was 80%, the cell suspension was collected by conventional digestion, and the cell number was determined by a cell counter. The olfactory mucosal basal cells and olfactory mucosal mesenchymal stem cells were prepared at (4-5)×10 7 Basal stem cells and mesenchymal stem cells were seeded in a conventional six-well plate at a density of 1:1, and DMEM / F12 solution containing 10% (v / v) FBS was added to resuspend the cells and evenly distribute them. The plates were 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 above two types of cells is about 80%, the cell suspension is collected by conventional digestion, and the cell number is determined by a cell counter. Olfactory mucosal basal cells and olfactory mucosal mesenchymal stem cells (4-5)×10 7 Basal stem cells and mesenchymal stem cells were seeded in a low-adsorption six-well plate at a density of 1:1. 10% (v / v) FBS DMEM / F12 solution was added to resuspend the cells and evenly distribute them. The cells were placed in a humidified incubator (37°C, 5% CO2) for further culture. After 3 days, a 3D mixed cell sphere with a circular shape was visible 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 be mixed to form 3D microspheres.

[0061] At the same time, the inventors also conducted a series of comparative experiments. For example, the 3D hybrid cell spheroids were cultured using the steps of step (1), with the only difference being that the number of culture days 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 spheroids all contained dead cells.

[0062] Alternatively, the inventors simultaneously conducted a series of comparative experiments, for example, culturing 3D mixed cell spheroids using the procedure of step (1), with 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 mixed cell spheroids were relatively small and contained a large number of suspended single cells.

[0063] Alternatively, the 3D mixed cell spheroids were cultured using the same procedure as in step (1), except that the number of basal stem cells was 1×10 8 and mesenchymal stem cells 1×10 8 The cells were seeded at a density of 1.547 W in a low-adsorption six-well plate. The results also showed that there were dead cells in the center of the obtained 3D mixed cell spheroids.

[0064] (2) The 3D hybrid microspheres cultured by the culture method of the present invention in step (1) were subjected to cell immunofluorescence staining with CD90 and KRT5 antibodies. The sources and dilution multiples of the antibodies are shown in Table 4, and the staining steps are the same as in Example 1. Under a fluorescence microscope, CD90 and KRT5 staining marks were on the same cell sphere, confirming that the cultured 3D hybrid microspheres were mixed spheres of mesenchymal stem cells and basal stem cells. Figure 5 shown.

[0065] Table 4 Sources and dilutions of CD90 and KRT5 antibodies

[0066] Antibody Name brand Dilution multiple CD90 Proteintech 1：200 KRT5 Abcam 1：100 Mouse secondary antibody Proteintech 1：200 Rabbit secondary antibody Proteintech 1：200

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

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

[0069] 2) Protein concentration detection: Follow the instructions of 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 with RIPA lysis buffer in a 50% gradient, add 25 μL of the diluted standard and blank sample to the standard well of the 96-well plate, and add 25 μL of the sample to the sample well of the 96-well plate. Add 200 μL of BCA working solution to each well and place at 37°C for 30 minutes. Use an enzyme reader to measure the absorbance value at wavelengths between 5 and 50 nm, and calculate the protein concentration based on the standard curve;

[0070] 3) SDS-PAGE electrophoresis: Select the appropriate gel concentration according to the molecular weight of the target protein, and prepare the separation gel and concentrated gel for SDS-PAGE according to the instructions. Prepare 10% separation gel, add TEMED and shake it immediately to cast the gel. After casting the gel, seal the gel with isopropanol. When the dividing line of the gel caster does not change when it is slightly tilted, it means that the gel has solidified. Wait for another 3 minutes for the gel to fully solidify, then pour off the isopropanol on the top of the gel and absorb it with filter paper. Then prepare 4.8% concentrated gel, add TEMED and shake it immediately to cast the gel. Insert the comb into the glass plate, fill the remaining space with concentrated gel, and wait for the gel to solidify. Take 300μL of protein supernatant, add 75μL 5× loading buffer and mix well, boil in boiling water for 5 minutes, and put it in an ice box for medium-speed cooling. According to the results of protein quantification, add 2μL of marker to the first well, and 10-20μL of denatured protein to each of the other wells. Start electrophoresis at a constant voltage of 75V for 130 minutes. When bromophenol blue reaches the bottom of the gel, stop electrophoresis.

[0071] 4) Transfer: Cut the gel according to the molecular weight of the target protein, prepare 6 filter papers of the same size as the gel and 1 NC membrane according to the size of the gel segment after segmentation, and put the NC membrane and filter paper into the transfer buffer until they are completely soaked. Put 3 filter papers, NC membrane, gel, and another 3 filter papers in order to avoid bubbles. Cover the instrument, turn on the power, and transfer the membrane at a constant current of 300mA. After the transfer is completed, take out the membrane and wash it once in 1×PBST;

[0072] 5) Blocking: Use 1×PBST to prepare 5% skim milk powder, immerse the membrane in it, and leave it at room temperature for 90 minutes;

[0073] 6) Antibody incubation (the source and dilution of the antibody 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, incubate at 4°C overnight, and place at room temperature for 30 minutes the next day. After the incubation, wash 3 times with 1×PBST, 10 minutes 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 minutes. After the incubation, wash 3 times with 1×PBST, 15 minutes 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 film, expose it to X-ray film in a dark box for 1-30 min, and develop and rinse. The results showed that compared with 2D, the expression of anti-apoptotic protein Bcl-2 in the 3D group increased significantly, and the pro-apoptotic protein Bax and apoptosis-related index cleave-casepase 3 showed a downward trend, as shown in Figure 2. Figure 6 ,in Figure 6 “**” and “****” indicate significant differences (p<0.01) and (p<0.0001), respectively.

[0075] Table 5 Sources and dilutions of antibodies

[0076] Antibody Name brand Dilution multiple Bax Abcam 1：2000 BCL-2 Proteintech 1：2000 Caspase3 Proteintech 1：2000 β-actin Proteintech 1：5000 Mouse secondary antibody Abiowell 1：5000 Rabbit secondary antibody Abiowell 1：5000

[0077] From the results in Example 4, it can be seen that the 3D mixed culture system can improve its anti-apoptotic effect against the external harsh microenvironment.

[0078] It can be concluded from the above examples that the 3D hybrid cell microspheres of the present invention are beneficial to the mutual support of olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells, improve their biological characteristics, are easier to colonize in the lesion site, and improve the anti-apoptosis ability of the 3D hybrid cell microspheres.

[0079] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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 method comprises the following steps: mixing and inoculating olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells in a low-adsorption 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 comprises a low-adsorption six-well plate.

3. The preparation method according to claim 1, characterized in that: The inoculation density ratio of the olfactory mucosa basal stem cells and the olfactory mucosa mesenchymal stem cells is 1:

1.

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

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

6. The preparation method according to any one of claims 1 to 3, characterized in that: The inoculation amount of the olfactory mucosa basal stem cells and olfactory mucosa mesenchymal stem cells is (4-5)×10 7 Pieces / hole.

7. The preparation method according to claim 1, characterized in that: The preparation method of the olfactory mucosa basal stem cells comprises: inoculating nasal mucosa tissue blocks into olfactory mucosa basal stem cell culture medium for cell culture, replacing new culture medium every 2 to 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 culture 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 is 37°C, the carbon dioxide concentration is 5%, and the humidity is 80-99%.

10. Use of the 3D mixed cell microspheres prepared by the preparation method according to any one of claims 1 to 9 in improving the anti-apoptosis ability of olfactory mucosa mesenchymal stem cells and / or olfactory mucosa basal stem cells.

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