In-vitro layered culture model construction method for observing dentin cell polarity

Abstract

The invention relates to the technical field of cell culture, and particularly discloses an in-vitro layered culture model construction method for observing dentin cell polarity, and the method comprises the following steps: extracting mandibular incisor neck ring tissue, and separating odontogenic epithelial tissue from dental papilla tissue; respectively digesting the odontogenic epithelial tissue and the dental papilla tissue into single-cell suspensions; the method comprises the following steps: inoculating epithelial cells on the surface of a movable polycaprolactone membrane for culturing to obtain an epithelial cell-polycaprolactone membrane complex; performing adherent culture on the dental papilla cells for proliferation, and performing experimental condition pretreatment on the dental papilla cells in the culture process or after culture; inoculating the pretreated dental papilla cells into the upper chamber; the epithelial cell-polycaprolactone membrane complex is attached to a lower chamber; and the epithelial cell-polycaprolactone membrane complex is removed. Co-culture is realized by attaching an epithelial cell-polycaprolactone membrane to the lower part of the small chamber, and the epithelial cells can be utilized to induce dental papilla cells to establish top-bottom polarity by virtue of gaps of the small chamber.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a method for constructing an in vitro stratified culture model for observing the polarity of odontoblasts. BACKGROUND

[0002] Dentin, as the main structure of teeth, plays a dual role in protecting the dental pulp and supporting the enamel. The dentin tubule structure not only effectively transmits masticatory pressure, but also transmits external stimuli, which has a crucial physiological function. In the case of caries or trauma, the dentin structure is damaged, and external stimuli are transmitted to the dental pulp through the dentin tubules, which can form reparative dentin to block external stimuli. However, when the stimulus is too strong or the defense mechanism is limited, the formed dentin often shows irregular shape, lack of tubular structure, or disordered arrangement, which weakens its physiological function, such as Figure 1 Therefore, it is a key scientific problem to explore the molecular mechanism of tubular dentin formation and promote its regeneration.

[0003] During tooth development, the dental papilla cells of the tooth germ are induced by the dental epithelium, and differentiate into pre-odontoblasts. The cells gradually elongate and polarize to form odontoblasts with top-bottom polarity, and then move to the pulp cavity side after secreting dentin matrix, and the secretion end forms odontoblast processes, which remain in the mineralized matrix to form tubular dentin, as shown in Figure 2 Therefore, the top-bottom polarity of odontoblasts mediates the formation of tubular dentin. In odontoblasts, the top-bottom polarity is characterized by cell elongation to form a high column, the nucleus is far from the dentin side, and the Golgi apparatus and matrix vesicles are aggregated near the dentin side to promote the directional secretion and mineralization of dentin organic matrix, as shown in Figure 3 However, the regulatory mechanism of the top-bottom polarity of odontoblasts is not clear at present. In order to further understand the regulatory factors of tubular dentin formation and provide more theoretical basis for tooth development research and tubular dentin regeneration, it is necessary to explore the related research model of tooth germ development.

[0004] At present, there are many methods for studying the top-bottom polarity of odontoblasts. In vivo culture methods include gene knockout mice, subrenal capsule culture of tooth germs, and subcutaneous tooth fragment culture in nude mice. In vitro culture methods include tooth germ organ culture, tooth germ section culture, and organoid culture. However, the above research methods have their own limitations in meeting the research conditions we expect.

[0005] Gene knockout mice are currently common research models. Using gene editing technology, we can knock out the target gene in the dental papilla cells at a certain stage to study its effect on dental papilla polarization / differentiation, and have a clear developmental phenotype and can track the impact at different developmental stages. However, conditional knockout mice have a long construction period, high cost, and large workload, and require a large number of animals for breeding and genotyping, making it difficult to meet the needs of high-throughput or mechanistic research.

[0006] The tooth germ kidney capsule culture technique is to extract the developing mouse molar tooth germ, condition it, and then implant it under the mouse kidney capsule for 2-3 weeks. After removal, the tooth germ development is observed by histological experiments. This technique can provide a good in vivo environment, which is conducive to the differentiation and polarity formation of dental papilla cells. However, it has the following two shortcomings: when specific treatment (gene intervention or drug stimulation) is needed for the dental papilla, the epithelium and dental papilla cells often need to be separated under a microscope for separate treatment of the dental papilla cells, which significantly increases the technical difficulty and workload of the operator; due to the longer culture time, it is often necessary to use efficient, stable, and long-term effective treatment methods: such as silencing the target gene, often requiring high-efficiency, stable expression, and long-term effective lentivirus transfection to intervene in the dental papilla tissue, and then recombining the epithelium-dental papilla tissue before implantation. This greatly increases the difficulty and failure rate of the entire model construction.

[0007] Subcutaneous tooth fragment culture in nude mice is to separate the developing mouse molar tooth germ into small tissue blocks of dental papilla and epithelium under a microscope, and directly implant them subcutaneously in the back of nude mice to support the development of heterologous tooth tissue in the immunodeficient environment for 2-4 weeks. Compared with kidney capsule transplantation, the operation is simpler, the host is more tolerant, and early polarity and dentinogenic differentiation can be obtained. However, the subcutaneous site lacks the rich blood supply and signal microenvironment of the kidney capsule, and the development efficiency is low and the variation is large; and like the aforementioned in vivo models, once gene intervention or drug treatment is needed for the dental papilla, it must be isolated, virus transfected, and then recombined and implanted, which is a tedious and low success rate process, making it difficult to achieve high-throughput or fine mechanism research.

[0008] Tooth germ section culture is to cut the developing tooth germ tissue into 200 μm thin sections and culture them in vitro. This culture method can partially retain the interaction between epithelium and dental papilla, and observe the dentinogenic cell apical-basal polarity of dentinogenic cells under in vivo conditions. However, since the section tissue is a complete complex, it is difficult to independently intervene in the dental papilla cells, limiting its application in mechanism research.

[0009] In recent years, although a three-dimensional co-culture system based on dental papilla stem cells and epithelial cells has been gradually established, and a culture method for forming a dental organoid model with a tooth-derived structure has been gradually explored, the exploration of dental organoids by researchers is still limited, which is manifested in that, whether for a three-dimensional culture model of dental papilla cells or for a mixed culture of epithelial-dental papilla stem cells, the obtained organoids can only observe the epithelial / dental pulp stem cell structure in vitro, and cannot observe the odontoblast apical-polar characteristics, which is difficult to be used for the study of the establishment mechanism of polarity. Therefore, it is necessary to develop a model for observing the odontoblast apical-polar characteristics under in vitro conditions by treating dental papilla cells under experimental conditions. SUMMARY

[0010] The purpose of the present application is to provide an in vitro layered culture model construction method for observing the polarity of odontoblast cells, so as to solve the problems presented in the background art.

[0011] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The in vitro layered culture model construction method for observing the polarity of odontoblast cells comprises the following steps: Extracting mandibular incisor neck ring tissue, separating dental epithelial tissue and dental papilla tissue; Digesting the dental epithelial tissue and the dental papilla tissue into single-cell suspensions, respectively; Seeding epithelial cells on the surface of a movable polycaprolactone film for culture to obtain an epithelial cell-polycaprolactone film complex; Subsequently, adherent culture is performed on the dental papilla cells to proliferate, and experimental conditions are pretreated on the dental papilla cells during or after the culture; Digesting, counting and seeding the pretreated dental papilla cells into the upper chamber of a Transwell chamber, and adhering the epithelial cell-polycaprolactone film complex to the lower chamber of the Transwell chamber to perform interface co-culture of the epithelial cells and the dental papilla cells in the Transwell chamber; After co-culturing for a predetermined time, removing the epithelial cell-polycaprolactone film complex, fixing and staining the dental papilla cells on the Transwell chamber membrane, and observing the apical-polar morphology of the odontoblast cells.

[0012] As a further scheme of the present application, the separation operation specifically comprises: after the incisor neck ring tissue is digested with 0.75 mg / mL Dispase II, the epithelial and dental papilla tissues are mechanically separated using a micro-instrument.

[0013] As a further scheme of the present application, 0.25% trypsin-EDTA is used to digest the epithelial tissue, and 3 mg / mL collagenase type I is used to digest the dental papilla tissue.

[0014] As a further scheme of the present application, the experimental condition pre-treatment comprises at least one of gene silencing, siRNA transfection, and drug stimulation.

[0015] As a further scheme of the present application, the seeding density of the dental papilla cells is 160,000 cells per 200 μL of culture medium; and the co-culture condition is 37°C, 5% CO2, and the culture medium is F12 / DMEM culture medium containing 10% serum and 1% penicillin-streptomycin-amphotericin B.

[0016] As a further scheme of the present application, the polycaprolactone film is tightly attached to the bottom of the lower chamber of the transwell chamber by the buoyancy generated by adding the culture medium to the lower chamber.

[0017] As a further scheme of the present application, the polycaprolactone film is suspended on the surface of the culture medium before use, and the epithelial cells are seeded on the film at a density of 80,000 cells per 150 μL.

[0018] Compared with the prior art, the present application has the beneficial effect that the co-culture is achieved by attaching the epithelial cell-polycaprolactone film to the lower chamber of the transwell chamber, and the epithelial cells can induce the dental papilla cells to establish apical-basal polarity by relying on the gap of the transwell chamber.

[0019] The polycaprolactone film for culturing epithelial cells can be removed at any time, ensuring that the observation of the dental papilla cells is not affected by the epithelial cells. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application.

[0021] Figure 1 The tooth body tissue structure and function mode diagram provided for the embodiments of the present application.

[0022] Figure 2 The odontoblast polarization-differentiation process diagram provided for the embodiments of the present application.

[0023] Figure 3 The odontoblast apical-basal polarity characteristic diagram provided for the embodiments of the present application.

[0024] Figure 4 The layered culture mode diagram provided for the embodiments of the present application.

[0025] Figure 5 The mouse mandibular incisor cervical loop extraction and tooth epithelial-dental papilla tissue separation process diagram provided for the embodiments of the present application.

[0026] Figure 6 A hierarchical culture operation process diagram provided for the embodiment of the present application.

[0027] Figure 7 An immunofluorescence image of mouse dental papilla cells on a hierarchical culture model Transwell upper chamber provided for the embodiment of the present application.

[0028] Figure 8 A hierarchical culture model three-dimensional fluorescence image observation diagram of each orthogonal plane provided for the embodiment of the present application.

[0029] Figure 9 A cell contour segmentation diagram provided for the embodiment of the present application.

[0030] Figure 10 A morphological difference diagram of polarized and non-polarized cells provided for the embodiment of the present application.

[0031] Figure 11 A dental papilla cell polarization characteristic performance diagram after hierarchical culture provided for the embodiment of the present application. DETAILED DESCRIPTION

[0032] In order to make the technical problems to be solved by the present application, the technical solutions and beneficial effects more clear and explicit, the present application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0033] The method for constructing an in vitro hierarchical culture model for observing odontoblast polarity comprises: Extracting mandibular incisor neck ring tissue, separating dental epithelial tissue and dental papilla tissue; Digesting the dental epithelial tissue and the dental papilla tissue into single cell suspensions, respectively; Seeding epithelial cells on the surface of a movable polycaprolactone film for culture to obtain an epithelial cell-polycaprolactone film complex; Proliferating dental papilla cells by adherent culture, and pre-treating the dental papilla cells under experimental conditions during or after culture; Digesting, counting and seeding the pre-treated dental papilla cells in the upper chamber of a Transwell chamber; and adhering the epithelial cell-polycaprolactone film complex to the lower chamber of the Transwell chamber to co-culture the epithelial cells and the dental papilla cells in the Transwell chamber; After co-culturing for a predetermined time, removing the epithelial cell-polycaprolactone film complex, fixing and staining the dental papilla cells on the Transwell chamber film, and observing the apical-basal polarity morphology of odontoblasts.

[0034] In the present embodiment, as shown in Figure 1 dentin is formed by regularly polarized odontoblasts, which can transmit external stimuli and make the pulp respond; under pathological conditions such as caries, bacteria invade along the dentin tubules, and the tubular structure collapses, which can transmit the stimuli to the pulp tissue and form reparative dentin, which often exhibits irregular morphology, lack of tubular structure, or disordered arrangement.

[0035] As shown in Figure 2 , after the dental papilla cells are induced by the dental germ epithelium, they differentiate into pre-odontoblasts, gradually elongate and polarize to form odontoblasts with top-bottom polarity, move to the pulp cavity side after secreting dentin matrix, and form odontoblast cell processes at the secretion end, which remain in the mineralized matrix to form tubular dentin.

[0036] As shown in Figure 3 , the top-bottom polarity of odontoblasts is characterized by cell elongation to form a high column, with the nucleus far from the dentin side and the Golgi apparatus accumulating near the dentin side.

[0037] The extraction and assembly process is shown in Figure 4 , the cervical loop tissue is extracted from the mouse mandible, the epithelial-dental papilla tissue is separated and digested into single cells, the epithelial cells are cultured on a movable polycaprolactone film, and the dental papilla cells are cultured adherently for cell proliferation. After the proliferation is completed, the dental papilla cells can be pretreated with drugs or siRNA.

[0038] The proliferated or pretreated dental papilla cells are digested and inoculated into the upper chamber of the transwell, then the epithelial cell-polycaprolactone film is taken out and attached to the lower chamber of the transwell, and the film is always attached to the lower chamber by the buoyancy of the lower layer of culture medium, so as to play the role of epithelial induction. After 7 days of culture, the epithelial cell-polycaprolactone film is removed, the transwell film is cut, and the top-bottom polarity of the dental papilla cells after odontogenic differentiation is observed by immunofluorescence staining.

[0039] Preparation before extraction of dental germ: before use, the culture dishes, gun heads, gun head boxes and body microscopes are wiped with alcohol and placed in a sterile operation table, and are irradiated with ultraviolet light for 30 minutes; all consumables entering the sterile operation table need to be disinfected with alcohol; Prepare 1 60mm cell culture dish, add about 5mL of DPBS containing 1% triton X-100 in the culture dish, and prepare 5-8 60mm cell culture dishes, each containing 5mL of D-PBS. Then pre-cool the culture dishes at 4°C. Prepare 0.75mg / mL Dispase II and 30μg / mL DNase in advance to neutralize the enzyme.

[0040] As a preferred embodiment of the present application, the separation operation specifically comprises: after the incisor cervical loop tissue is enzymatically digested with 0.75 mg / mL Dispase II, the epithelium and dental papilla tissue are mechanically separated using a micro-instrument.

[0041] In this embodiment, the mouse molar embryo is extracted: 5-7-day-old C57 mice are euthanized, the head and body parts of the embryo are separated, the mandible is cut and separated from the line connecting the mouth corner to the ear, and the separated mandible is transferred to a pre-cooled 60-mm D-PBS cell culture dish. Under a stereomicroscope, the mandible is observed, and the lingual side of the mandibular incisor cervical loop can be seen.

[0042] Next, the tooth germ is washed twice with D-PBS, the incisor is carefully separated from the mandible using a micro-ophthalmic clamp, the mineralized part of the incisor is removed, the incisor cervical loop is retained, and it is placed in a culture dish and stored on ice.

[0043] Each tooth cervical loop embryo is incubated in 20 μL of Dispase II (concentration of 0.75 mg / mL) for 10 minutes, and then 40 μL of DNase-containing medium is used to neutralize the enzyme and prevent cell aggregation; the epithelium and dental papilla tissue are then separated using a micro-clamp, and the separated tissue is placed in DPBS and stored on ice (see Figure 5 ).

[0044] As a preferred embodiment of the present application, 0.25% trypsin-EDTA is used to digest the epithelial tissue, and 3 mg / mL type I collagenase is used to digest the dental papilla tissue.

[0045] In this embodiment, the preparation before culture: the cell culture box is carefully wiped with alcohol, sterile water is added to the bottom tray, and the box is adjusted to 37°C, 5% CO2, and 80-90% humidity; before use, the culture dish, gun head, gun head box, and body microscope are wiped with alcohol and placed in the sterile operating table, and are irradiated with ultraviolet light for 30 minutes; all consumables entering the sterile operating table need to be disinfected with alcohol; The epithelium and dental papilla tissue are digested with 0.25% trypsin-EDTA and 3 mg / mL type I collagenase for 15 minutes and 40 minutes, respectively, at 37°C, 5% CO2, in a culture box, and the digestion is stopped using medium after 300g centrifugation for 5 minutes. The number of cells is counted using a cell counter.

[0046] Single-cell culture before layered culture: Pre-configuration of cell culture medium before digestion is stopped: add 10% serum, 1% penicillin-streptomycin-amphotericin B to F12 / DMEM medium, use 1 mL of medium to stop the digestion of the digested cells, stop the digestion and resuspend by blowing, pipette the cell suspension into a 1.5 mL centrifuge tube, then centrifuge at 300g for 5 minutes, remove the centrifuge tube, discard the supernatant, add culture medium, mix gently by blowing and transfer to the culture plate, then supplement the culture medium to 2 mL; seed the dental papilla cells in a 6-well plate, mix evenly on the sterile operation table 8, observe under an inverted microscope, and culture at 37°C, 5% CO2. At the same time, add 0.5 mL of culture medium to the dish, and then suspend the polycaprolactone film on the surface of the culture medium. Resuspend the epithelial cells to 80,000 / 150uL of culture medium and seed on the polycaprolactone film for layered culture. Observe the cell morphology and contamination once every 24 hours; culture the cells for 3 days, and replace the culture medium every other day.

[0047] Layered culture construction method: After 3 days of primary cell culture, remove the culture dish for epithelial culture and the 6-well plate for dental papilla cell culture from the incubator, and observe the cell state, cell growth, and contamination degree under an inverted microscope; Wipe with alcohol and place in a sterile operation table, carefully pipette the culture medium, add 3 mL of PBS for washing 1-2 times to remove floating dead cells; add 200uL of 0.25% trypsin-EDTA to the 6-well plate to digest the dental papilla cells, and place in the incubator for 1 min of digestion; when microscopy shows that the cells are spherical and floating, it indicates that the digestion is complete; add 4 mL of culture medium to stop the digestion and resuspend by blowing, pipette the cell suspension into a 15 mL centrifuge tube, and add culture medium to the 6-well plate for washing twice, and transfer the liquid to the centrifuge tube. Balance the centrifuge tube and centrifuge at 1500 rpm for 5 minutes. Remove the centrifuge tube, discard the supernatant, resuspend with 1 mL of culture medium, and then perform cell counting, seed 160,000 / 200uL of dental papilla cells on the upper chamber of the transwell insert, and at the same time, adsorb the polycaprolactone film for culturing primary epithelial cells to the lower layer of the transwell insert by adsorption force, add culture medium to the lower chamber of the transwell, and culture in a 37°C, 5% CO2 incubator for 7 days. Replace the organ culture medium every other day, and ensure the adsorption of the polycaprolactone film for primary epithelial cells during the replacement process (see Figure 6 ).

[0048] After 7 days of culture, the transwell membrane was cut using microscissors and placed in a 6-well plate containing 4% paraformaldehyde to fix the cells. After 30 minutes of fixation, the fixative was discarded, and the cells were washed with 2 mL of PBS. This process was repeated three times. After discarding the PBS, 2 mL of 2% BSA was added for blocking for 1 hour. Vimentin / GM130 antibody (1:200 ratio) was prepared using 2% BSA and incubated overnight at 4°C. The next day, the primary antibody was discarded, and the cells were washed with 2 mL of PBS. This process was repeated three times. Then, secondary antibody was prepared using PBS and incubated for 2 hours. 40 μL of DAPI staining solution was added to stain the cell nuclei, and after 15 minutes of incubation, the DAPI staining solution was discarded, and the cells were washed with 2 mL of PBS. This process was repeated three times. The transwell membrane was placed in a confocal microscope, and 200 μL of anti-quenching mounting medium was added. A coverslip was placed on top, and the apical-basal polarity of odontoblasts was observed under a confocal microscope. The results were then analyzed using Imaris 10.2 software.

[0049] As a preferred embodiment of the present invention, the experimental pretreatment includes at least one of gene silencing, siRNA transfection, and drug stimulation.

[0050] Figure 7 Immunofluorescence images of dental papilla cells from the upper chamber of a Transwell mouse stratified culture model: green fluorescent GM130 labels the Golgi apparatus, red phalloidin delineates the cytoskeleton, and blue DAPI locates the cell nucleus, with clear cell images visible.

[0051] like Figure 8 As shown, the Ortho Slicer tool in the 3D View module and the Section module in Imaris 10.1.0 software are used to observe the three-dimensional fluorescence images of the layered culture model in various orthogonal planes.

[0052] like Figure 9 As shown, the Cell tool in the 3D View module of Imaris 10.1.0 software was used to segment the cell outline by distinguishing the background and signal based on the fluorescence signal intensity in different color channels. The results showed that dental papilla cells cultured without epithelial induction exhibited a flat cell body morphology and did not have polarization characteristics; while the epithelial induction conditions provided in this culture model could promote the polarization of mouse dental papilla cells, which was manifested as the cells extending slender protrusions.

[0053] like Figure 10As shown, the observation range is trimmed using the Clipping Plane tool in the 3D View module in Imaris 10.1.0 software, and the morphological differences between polarized and non-polarized cells are observed at the single-cell level. It can be seen that the polarized cells extend long cell processes, and the nuclei and Golgi apparatus are columnar, showing top-bottom polarity in three-dimensional planes (as shown in the box in figure D). The non-polarized cells are just flat on the culture membrane, and the nuclei and Golgi apparatus are distributed in the plane.

[0054] As shown in the figure, Figure 11 As shown, after layered culture, the dental papilla cells polarize, and the polarization characteristics are that the cells extend long cell processes, and the nuclei and Golgi apparatus are columnar, showing top-bottom polarity in three-dimensional planes.

[0055] Before the experiment, the gene knockout, siRNA transfection or drug stimulation of the dental papilla cells in the ordinary culture plate is completed, the experimental condition intervention of the dental papilla cells alone can be realized without affecting the epithelial cells, so as to further clarify the related factors for regulating the polarization and differentiation of the dental papilla cells.

[0056] The epithelial cells are pasted on the polycaprolactone membrane below the transwell chamber to realize co-culture, and the dental papilla cells can establish top-bottom polarity by relying on the gap of the transwell chamber by using the epithelial cells to induce the dental papilla cells.

[0057] The polycaprolactone membrane for culturing epithelial cells can be taken out at any time, so as to ensure that the observation of the dental papilla cells is not affected by the epithelial cells.

[0058] In summary, with the advantages of free pretreatment and short culture time, the system provides an instant and visualized way for screening the polarization-related genes, signal pathways and small molecule interventions of the odontoblast cells.

[0059] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing an in vitro layered culture model for observing odontoblast polarity, characterized in that, The method includes: Extract the cervical ring tissue of the mandibular incisors and separate the dental epithelial tissue from the dental papilla tissue; The dental epithelial tissue and dental papilla tissue were digested into single-cell suspensions, respectively. Epithelial cells were seeded onto the surface of a movable polycaprolactone membrane and cultured to obtain an epithelial cell-polycaprolactone membrane complex. Dental papilla cells were cultured in an adherent environment to promote proliferation, and experimental conditions were pretreated on the dental papilla cells during or after culture. The pretreated dental papilla cells were digested, counted, and seeded into the upper chamber of the Transwell chamber; the epithelial cell-polycaprolactone membrane complex was attached to the lower chamber of the Transwell chamber, so that the epithelial cells and dental papilla cells were co-cultured at the interface in the Transwell chamber. After co-culturing for a predetermined time, the epithelial cell-polycaprolactone membrane complex was removed, and the dental papilla cells on the Transwell chamber membrane were fixed and stained to observe the apical-basal polarity morphology of odontoblasts.

2. The method for constructing an in vitro layered culture model for observing odontoblast polarity according to claim 1, characterized in that, The separation operation specifically includes: digesting the incisor cervical ring tissue with 0.75 mg / mL Dispase II, and then mechanically separating the epithelium and dental papilla tissue using microsurgical instruments.

3. The method for constructing an in vitro layered culture model for observing odontoblast polarity according to claim 1, characterized in that, During the digestion process, 0.25% trypsin-EDTA was used to digest the epithelial tissue, and 3 mg / mL type I collagenase was used to digest the dental papilla tissue.

4. The method for constructing an in vitro layered culture model for observing odontoblast polarity according to claim 1, characterized in that, The experimental pretreatment conditions include at least one of gene silencing, siRNA transfection, and drug stimulation.

5. The method for constructing an in vitro layered culture model for observing odontoblast polarity according to claim 1, characterized in that, The seeding density of the dental papilla cells was 160,000 cells / 200 μL of culture medium; the co-culture conditions were 37°C, 5% CO2, and the culture medium was F12 / DMEM medium containing 10% serum and 1% penicillin-streptomycin-amphoteric B.

6. The method for constructing an in vitro layered culture model for observing odontoblast polarity according to claim 1, characterized in that, The buoyancy generated by adding culture medium to the lower chamber allows the polycaprolactone membrane to adhere tightly to the bottom of the lower chamber of the Transwell chamber.

7. The method for constructing an in vitro layered culture model for observing odontoblast polarity according to claim 1, characterized in that, The polycaprolactone membrane is suspended on the surface of the culture medium before use, and epithelial cells are seeded onto the membrane at a density of 80,000 cells / 150 μL.

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