Method for constructing in vitro stratification culture model for observing odontoblast polarity

Abstract

The application relates to the technical field of cell culture, and particularly discloses a method for constructing an in-vitro layered culture model for observing the polarity of odontoblasts, which comprises the following steps: extracting mandibular incisor neck ring tissues, separating dental epithelial tissues and dental papilla tissues; separately digesting the dental epithelial tissues and the dental papilla tissues into single-cell suspensions; inoculating epithelial cells on the surface of movable polycaprolactone films for culture to obtain an epithelial cell-polycaprolactone film complex; carrying out adherent culture on dental papilla cells to proliferate, and pre-treating the dental papilla cells under experimental conditions during or after the culture; inoculating the pre-treated dental papilla cells in an upper chamber; adhering the epithelial cell-polycaprolactone film complex to a lower chamber; and removing the epithelial cell-polycaprolactone film complex. By adhering the epithelial cell-polycaprolactone film to the lower chamber for co-culture, the epithelial cells can induce the dental papilla cells to establish top-bottom polarity by relying on the gap of the chamber.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for constructing an in vitro stratified culture model for observing the polarity of odontoblasts. Background Technology

[0002] Dentin, as the main structure of teeth, plays a dual role in protecting the dental pulp and supporting the enamel. The dentinal tubules not only effectively transmit chewing pressure but also conduct external stimuli, possessing crucial physiological functions. In cases of caries or trauma, the dentin structure is damaged, and external stimuli are transmitted to the dental pulp through the dentinal tubules, allowing for the formation of reparative dentin to block these stimuli. However, when the stimulus is too strong or the defense mechanism is limited, the formed dentin often exhibits irregular morphology, a lack of tubular structures, or disordered arrangement, weakening its physiological functions. Figure 1 As shown; therefore, exploring the molecular mechanisms of tubular dentin formation and promoting its regeneration is a key scientific problem that urgently needs to be solved.

[0003] During tooth development, the tooth germ papilla cells, after being induced by the tooth germ epithelium, differentiate into pre-odontoblasts. These cells gradually elongate and polarize, forming odontoblasts with apical-basal polarity. After secreting dentin matrix, they migrate towards the pulp cavity, with the secretory end forming odontoblast processes. These processes remain in the mineralized matrix, forming tubular dentin. Figure 2 As shown; therefore, the apical-basal polarity of odontoblasts mediates the formation of tubular dentin. In odontoblasts, the apical-basal polarity is characterized by cell elongation forming tall columnar structures, with the nucleus located away from the dentin side, and the Golgi apparatus and matrix vesicles accumulating on the proximal dentin side to promote the directed secretion and mineralization of the dentin organic matrix, such as... Figure 3 As shown; however, the regulatory mechanism of apical-basal polarity of odontoblasts is still unclear. In order to gain a deeper understanding of the regulatory factors of tubular dentin formation and to provide more theoretical basis for tooth development research and tubular dentin regeneration, it is necessary to explore research models related to tooth germ development.

[0004] Currently, there are various methods for studying the apical-basal polarity of odontoblasts. In vivo culture methods include gene knockout mice, subcapsular culture of tooth germ kidneys, and subcutaneous tooth fragment culture of nude mice. In vitro culture methods include tooth germ organ culture and tooth germ section culture for organoids. However, all of these research methods have their own limitations for the research conditions we hope to meet.

[0005] Gene knockout mice are a common research paradigm. Using gene editing technology, we can knock out target genes in dental papilla cells at a specific time to study their effects on dental papilla polarization / differentiation. These mice exhibit a clear developmental phenotype and allow for tracking of the effects at different developmental stages. However, constructing conditional knockout mice is time-consuming, costly, and labor-intensive, requiring large-scale breeding and genotyping of animals, making it difficult to meet the needs of high-throughput or mechanistic studies.

[0006] Tooth germ renal capsule culture involves extracting developing mouse molar germs, conditioning them, and then implanting them under the renal capsule for 2-3 weeks. After removal, histological experiments are performed to observe tooth germ development. This technique provides a favorable in vivo environment, promoting the differentiation and polarity formation of dental papilla cells. However, it has two main drawbacks: When specific treatments (genetic intervention or drug stimulation) are needed for the dental papilla, it is often necessary to separate epithelial and dental papilla cells under direct microscopic observation for individual treatment of the dental papilla cells, significantly increasing the technical difficulty and workload for the operator; given the long culture time, highly efficient, stable, and long-term effective treatment methods are often required. For example, when silencing a target gene, it is often necessary to transfect the dental papilla tissue with a highly efficient, stable, and long-term effective lentivirus, followed by epithelial-dental papilla tissue recombination and implantation. This greatly increases the difficulty and failure rate of the entire model construction.

[0007] Subcutaneous tooth fragment culture in nude mice involves microscopically separating developing mouse molar germs into small tissue blocks of dental papillae and epithelium, which are then directly implanted subcutaneously into the back of nude mice. The immunodeficient environment of the mice supports the continued development of the heterologous tooth tissue for 2–4 weeks. Compared to renal capsule transplantation, this method is simpler, has better host tolerance, and can yield results in odontoblast differentiation and early polarity indicators. However, the subcutaneous site lacks the rich blood supply and signal microenvironment of the renal capsule, resulting in low developmental efficiency and high variability. Furthermore, similar to the aforementioned in vivo models, any gene intervention or drug treatment of the dental papillae requires prior in vitro isolation, viral transfection, and recombinant implantation—a cumbersome process with low success rates, hindering high-throughput or detailed mechanistic studies.

[0008] Tooth germ section culture involves cutting developing tooth germ tissue into 200 μm thin slices and culturing them in vitro. This method can partially preserve the interaction between the epithelium and the dental papilla, and allows observation of the apical-basal polarity of odontoblasts under in vivo conditions. However, because the sectioned tissue is a complete complex, it is difficult to independently intervene in dental papilla cells, limiting its application in mechanistic studies.

[0009] In recent years, although three-dimensional co-culture systems based on dental papilla stem cells and epithelial cells have been gradually established, and culture methods for forming organoid models with tooth-derived structures have been explored, researchers' exploration of dental organoids remains relatively limited. This is evident in the fact that, regardless of whether it's a three-dimensional culture model of dental papilla cells or a co-culture of epithelial-dental papilla stem cells, the resulting organoids only allow for the observation of epithelial / dental pulp stem cell structures in vitro, failing to reveal the apical-basal polarity characteristics of odontoblasts, making them unsuitable for studying polarity establishment mechanisms. Therefore, it is essential to develop a model that can experimentally treat dental papilla cells under in vitro conditions and observe the apical-basal polarity of odontoblasts. Summary of the Invention

[0010] The purpose of this invention is to provide a method for constructing an in vitro layered culture model for observing the polarity of odontoblasts, so as to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] A method for constructing an in vitro layered culture model for observing odontoblast polarity, the method comprising:

[0013] Extract the cervical ring tissue of the mandibular incisors and separate the dental epithelial tissue from the dental papilla tissue;

[0014] The dental epithelial tissue and dental papilla tissue were digested into single-cell suspensions, respectively.

[0015] Epithelial cells were seeded onto the surface of a movable polycaprolactone membrane and cultured to obtain an epithelial cell-polycaprolactone membrane complex.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] As a further embodiment of the present invention, the separation operation specifically includes: enzymatically 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.

[0020] As a further embodiment of the present invention, the epithelial tissue is digested with 0.25% trypsin-EDTA and the dental papilla tissue is digested with 3 mg / mL type I collagenase.

[0021] As a further embodiment of the present invention, the experimental pretreatment includes at least one of gene silencing, siRNA transfection, and drug stimulation.

[0022] As a further embodiment of the present invention, the seeding density of the dental papilla cells is 160,000 cells / 200 μL of culture medium; the co-culture conditions are 37°C, 5% CO2, and the culture medium is F12 / DMEM medium containing 10% serum and 1% penicillin-streptomycin-amphoteric B.

[0023] As a further embodiment of the present invention, 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.

[0024] As a further embodiment of the present invention, the polycaprolactone membrane is suspended on the surface of the culture medium before use, and epithelial cells are seeded on the membrane at a density of 80,000 cells / 150 μL.

[0025] Compared with the prior art, the beneficial effects of the present invention are: by co-culturing epithelial cells-polycaprolactone membrane attached below the transwell chamber, it is possible to use epithelial cells to induce dental papilla cells to establish top-bottom polarity by relying on the transwell chamber space.

[0026] The polycaprolactone membrane used for culturing epithelial cells can be removed at any time, ensuring that the dental papilla cells are not affected by the epithelial cells during observation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention.

[0028] Figure 1 This is a schematic diagram of the dental tissue structure and function provided in an embodiment of the present invention.

[0029] Figure 2 This diagram illustrates the polarization-differentiation process of odontoblasts, as provided in an embodiment of the present invention.

[0030] Figure 3 This is a diagram illustrating the apical-basal polarity characteristics of odontoblasts provided in an embodiment of the present invention.

[0031] Figure 4A diagram illustrating a stratified culture model provided in an embodiment of the present invention.

[0032] Figure 5 This diagram illustrates the process of extracting the cervical ring of the mouse mandibular incisor and separating the dental epithelium-dental papilla tissue, as provided in an embodiment of the present invention.

[0033] Figure 6 A diagram illustrating the stratified culture process provided in an embodiment of the present invention.

[0034] Figure 7 Immunofluorescence images of dental papilla cells from the upper chamber of a Transwell mouse, a stratified culture model provided in this embodiment of the invention.

[0035] Figure 8 These are observation diagrams of the three-dimensional fluorescence images of the layered culture model provided in this embodiment of the invention, showing the views of each orthogonal plane.

[0036] Figure 9 The cell outline segmentation diagram provided in the embodiments of the present invention.

[0037] Figure 10 This is a diagram illustrating the morphological differences between polarized and non-polarized cells, provided as an embodiment of the present invention.

[0038] Figure 11 This is a diagram illustrating the polarization characteristics of posterior tooth papilla cells cultured in a layered manner, as provided in an embodiment of the present invention. Detailed Implementation

[0039] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0040] A method for constructing an in vitro layered culture model for observing odontoblast polarity, the method comprising:

[0041] Extract the cervical ring tissue of the mandibular incisors and separate the dental epithelial tissue from the dental papilla tissue;

[0042] The dental epithelial tissue and dental papilla tissue were digested into single-cell suspensions, respectively.

[0043] Epithelial cells were seeded onto the surface of a movable polycaprolactone membrane and cultured to obtain an epithelial cell-polycaprolactone membrane complex.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In this embodiment, as Figure 1 As shown, under physiological conditions, dentin is differentiated from regularly polarized odontoblasts to form tubular structures that can transmit external stimuli and make the dental pulp respond. Under pathological conditions such as caries, bacteria invade along the dentinal tubules, the tubular tissue collapses, and the tubular tissue transmits the stimuli to the dental pulp tissue, which can form reparative dentin, often showing characteristics such as irregular shape, lack of tubular structures or disordered arrangement.

[0048] like Figure 2 As shown, after being induced by the tooth germ epithelium, dental papilla cells differentiate into pre-odontoblasts. The cells gradually elongate and polarize to form odontoblasts with apical-basal polarity. After secreting dentin matrix, they move toward the pulp cavity, and the secretory end forms an odontoblast process, which remains in the mineralized matrix to form tubular dentin.

[0049] like Figure 3 As shown, the apical-basal polarity of odontoblasts is characterized by cell elongation to form a tall columnar shape, with the nucleus located away from the dentin side and the Golgi apparatus clustered near the dentin side.

[0050] Cell extraction and assembly process, such as Figure 4 As shown, cervical ring tissue was extracted from the mouse mandible, the epithelial-dental papilla tissue was isolated and digested into single cells, the epithelial cells were cultured on a mobile polycaprolactone membrane, and the dental papilla cells were cultured in an adherent culture to allow for cell proliferation. After proliferation, the dental papilla cells could be pretreated with drugs or siRNA.

[0051] Dental papilla cells that have undergone proliferation or pretreatment are digested and seeded into the upper chamber of a transwell. The epithelial cell-polycaprolactone membrane is then removed and attached tightly to the lower chamber of the transwell. The membrane is kept in contact with the lower chamber by the buoyancy of the lower culture medium to induce epithelialization. After 7 days of culture, the epithelial cell-polycaprolactone membrane is removed, the transwell membrane is cut off, and the apical-basal polarity of the dental papilla cells after odontogenic differentiation is observed by immunofluorescence staining.

[0052] Preparation before tooth germ extraction: Before using the sterile operating table, wipe the culture dish, pipette tip, pipette tip box and stereo microscope with alcohol and place them in the sterile operating table for 30 minutes of ultraviolet irradiation; all consumables entering the sterile operating table must be wiped and disinfected with alcohol.

[0053] Prepare one 60mm cell culture dish and add approximately 5mL of DPBS containing 1% triple antibody. Prepare another 5-8 60mm cell culture dishes, adding 5mL of D-PBS to each dish. Pre-cool the culture dishes to 4°C. Prepare 0.75mg / mL Dispase II and 30μg / mL DNase in centrifuge tubes beforehand to neutralize the enzyme.

[0054] In a preferred embodiment of the present invention, 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.

[0055] In this embodiment, the extraction of mouse molar embryos was performed by euthanizing C57 mice aged 5-7 days. The head and body of the embryos were separated, and the mandible was cut along a line from the corner of the mouth to the ear. The separated mandible was transferred to a pre-cooled 60 mm D-PBS cell culture dish. The mandible was observed under a stereomicroscope, and the cervical ring of the mandibular incisors was visible on the lingual side.

[0056] Next, the tooth germ was washed twice with D-PBS, and the incisor was carefully separated from the mandible with micro-ophthalmic forceps. The mineralized part of the incisor was removed, the incisor neck ring was retained, and it was placed in a petri dish and stored on ice.

[0057] Each tooth cervical ring embryo was incubated in 20 μL of Dispase II (0.75 mg / mL) for 10 minutes, followed by neutralization of the enzyme and prevention of cell aggregation with 40 μL of DNase-containing medium; the epithelium and dental papilla tissue were then separated using microforceps, and the separated tissues were placed in DPBS and temporarily stored on ice (see details). Figure 5 ).

[0058] In a preferred embodiment of the present invention, the epithelial tissue is digested with 0.25% trypsin-EDTA and the dental papilla tissue is digested with 3 mg / mL type I collagenase.

[0059] In this embodiment, the preparation before culture is as follows: the cell culture incubator is carefully wiped and disinfected with alcohol, sterile water is added to the tray, and the temperature inside the incubator is adjusted to 37°C, 5% CO2, and 80-90% humidity; before using the sterile operating table, the culture dishes, pipette tips, pipette tip boxes, and stereomicroscope are wiped with alcohol and placed in the sterile operating table and irradiated with ultraviolet light for 30 minutes; all consumables entering the sterile operating table must be wiped and disinfected with alcohol.

[0060] Epithelial and dental papilla tissues were digested in a 37°C, 5% CO2 incubator with 0.25% trypsin-EDTA and 3 mg / mL type I collagenase for 15 minutes and 40 minutes, respectively. Digestion was stopped with culture medium, and the cells were centrifuged at 300g for 5 minutes. The number of cells was counted using a cell counter.

[0061] Single-cell culture before stratified culture:

[0062] Before stopping digestion, prepare the cell culture medium in advance: Add 10% serum and 1% penicillin-streptomycin-amphotericidal B to F12 / DMEM medium. Stop digestion of the digested cells with 1 mL of medium, resuspend the cells by pipetting, and transfer the cell suspension to a 1.5 mL centrifuge tube. Centrifuge at 300g for 5 minutes, remove the centrifuge tube, discard the supernatant, add culture medium, gently mix by pipetting, and transfer to a culture plate. Add culture medium to 2 mL. Seed the dental papilla cells into 6-well plates, mix by figure-eight maneuvering on a sterile operating table, observe under an inverted microscope, and culture at 37°C with 5% CO2. At the same time, add 0.5 mL of culture medium to a small dish, and then suspend a polycaprolactone membrane on the surface of the medium. Resuspend epithelial cells at a ratio of 80,000 cells / 150 μL of medium and seed them onto the polycaprolactone membrane for stratified culture. Observe cell morphology and contamination after 24 hours. Culture the cells for 3 days, changing the medium every other day.

[0063] Hierarchical culture construction method:

[0064] Three days after primary cell culture, the culture dishes containing epithelial cells and the 6-well plates containing dental papilla cells were removed from the incubator and observed under an inverted microscope to assess cell status, cell growth, and degree of contamination.

[0065] Wipe the cells with alcohol and place them in a sterile operating table. Carefully aspirate the culture medium with a pipette, add 3 mL of PBS and wash 1-2 times to remove floating dead cells. Add 200 μL of 0.25% trypsin-EDTA to a 6-well plate to digest the dental papilla cells, and incubate for 1 min. Digestion is complete when microscopic examination shows that the cells are spherical and floating. Add 4 mL of culture medium to stop digestion and resuspend the cells by pipetting. Transfer the cell suspension to a 15 mL centrifuge tube, and wash twice more with culture medium in the 6-well plate. Transfer all liquids to centrifuge tubes. Balance the centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Remove the centrifuge tubes, discard the supernatant, add 1 mL of culture medium to resuspend the cells, and then count the cells. Seed 160,000 dental papilla cells / 200 μL in the upper chamber of the transwell insert. Simultaneously, adsorb the polycaprolactone membrane of cultured primary epithelium to the lower layer of the transwell insert using adsorption. Add culture medium to the lower chamber of the transwell and incubate for 7 days at 37°C and 5% CO2. The organ culture medium should be changed every other day, ensuring the adsorption of polycaprolactone membrane from the primary epithelium during the change (see details). Figure 6 ).

[0066] 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.

[0067] As a preferred embodiment of the present invention, the experimental pretreatment includes at least one of gene silencing, siRNA transfection, and drug stimulation.

[0068] 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.

[0069] like Figure 8As 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.

[0070] 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.

[0071] like Figure 10 As shown, the Clipping Plane tool in the 3D View module of Imaris 10.1.0 software was used to trim the observation range, enabling the observation of morphological differences between polarized and non-polarized cells at the single-cell level. It can be seen that polarized cells extend slender cell processes, and the cell nucleus and Golgi body are columnar, showing top-bottom polarity in a three-dimensional plane (as shown in the box in Figure D), while non-polarized cells simply lie flat on the culture membrane, with the cell nucleus and Golgi body distributed in a plane.

[0072] like Figure 11 As shown, after stratified culture, dental papilla cells undergo polarization. The polarization is characterized by the extension of slender cellular processes, and the nucleus and Golgi apparatus appearing columnar, exhibiting top-bottom polarity in a three-dimensional plane.

[0073] Before the experiment, the present invention performs gene knockout, siRNA transfection or drug stimulation on dental papilla cells in ordinary culture plates. This allows for experimental intervention on dental papilla cells without affecting epithelial cells, thereby further clarifying the relevant factors regulating the polarization and differentiation of dental papilla cells.

[0074] Co-culture of epithelial cells with a polycaprolactone membrane attached below the transwell chamber allows for the induction of dental papilla cells to establish apical-basal polarity via the transwell chamber space.

[0075] In practice, the polycaprolactone membrane used for culturing epithelial cells can be removed at any time, ensuring that the observation of dental papilla cells is not affected by the epithelial cells.

[0076] In summary, thanks to its advantages of free pretreatment and short culture time, this system provides an immediate and visualized approach for screening odontoblast polarization-related genes, signaling pathways, and small molecule interventions.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

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, allowing the epithelial cells and dental papilla cells to be co-cultured at the interface in the Transwell chamber. The buoyancy generated by the culture medium added to the lower chamber was used to ensure that the polycaprolactone membrane was tightly attached to the bottom of the lower chamber of 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 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 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.

Images