Tooth root organoid, construction method and application thereof, and induction medium

Abstract

The invention discloses a tooth root organ, a construction method and application thereof and an induction medium, and belongs to the technical field of biomedicine. The construction method disclosed by the invention comprises the following steps: reviving and resuspending engineered dental epithelial cells to prepare a dental epithelial cell suspension; dental sac stem cells are taken, re-suspended and then inoculated into a cell culture plate, the culture plate is centrifuged, the dental sac stem cells are gathered at the bottoms of culture holes to form dental sac stem cell clusters, a culture medium is added, culture is conducted, and dental sac stem cell microspheres are formed; and centrifuging the culture plate, absorbing the supernatant, adding a dental epithelial cell suspension, standing, and adding a culture medium for continuous culture, so that the dental epithelial cells are compounded on the surfaces of the dental capsule stem cell microspheres to form tooth root organs. The invention discloses the tooth root organoid obtained by adopting the construction method and application of the tooth root organoid. The invention further discloses an induction culture medium for inducing and differentiating the tooth root organoid.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the field of oral biomedical engineering, and specifically, to tooth root organoids, their construction methods, applications and induction culture media. Background Art

[0002] The root is an important part of the tooth tissue. It is connected to the alveolar socket through the periodontal membrane and has the functions of retention, support, transmission and perception of occlusal stress. Increased root brittleness and even root fracture caused by loss of pulp function after root canal treatment, root resorption caused by orthodontic tooth movement, root fracture caused by various traumas, and root dysplasia caused by various congenital and acquired factors can all lead to loss of root function and even tooth loss, which in turn affects chewing, pronunciation function and facial changes. At present, the clinical treatment of root injury is generally based on local bonding, fiber post reinforcement, etc. For severely fractured or broken roots, they can only be surgically extracted and repaired with implants. All of them are prosthetic repairs, and physiological root function regeneration cannot be achieved. Tissue regeneration based on the principle of organ development has been a hot topic in tissue engineering regeneration in recent years, that is, analyzing the cell and molecular signals in the process of organ development and applying them to tissue regeneration. The development of the tooth root is that after the development of the crown is completed, the inner enamel epithelial cells and outer enamel epithelial cells at the neck ring of the tooth germ grow toward the future apical foramen to form Hertwig's epithelial root sheath (HERS). HERS surrounds the dental papilla cells and, through epithelial-mesenchymal interactions, induces the dental papilla cells to differentiate into the pulp-dentin complex, forming the dentin and pulp cavity and pulp tissue at the root. In this process, HERS, as the germinal center of the tooth root, plays a vital role in the development of the tooth root. HERS induces the differentiation of dental papilla cells through the HH signaling pathway, the BMP signaling pathway, etc., and regulates the formation of the tooth root.

[0003] Organoids are an emerging biotechnology that has developed rapidly in recent years. They mainly self-assemble stem cells from various sources and induce differentiation under artificial three-dimensional culture conditions in vitro to form tissue complexes with structural and functional characteristics. Organoids have all or part of the key structural and functional characteristics of the source tissue or organ. At present, researchers have successfully established a variety of tissue organoid models such as brain, liver, kidney, intestine, etc. Organoid technology shows broad application prospects in the fields of organ development, disease simulation, drug screening and regenerative medicine. As mentioned above, the tooth root is mainly formed by the mutual induction of HERS and dental mesenchymal cells during development. HERS cells are epithelial cells that exist in stages during tooth development. After the development of the tooth root is completed, they remain in the periodontal ligament in the form of a small amount of Malassez epithelial remnants, which are not easy to obtain and purify. At present, there is no research on simulating the principle of tooth root development (i.e., using the principle of mutual induction between HERS cells and dental mesenchyme) to construct tooth root organoids. Through literature search, the only research on tooth root organoids is an article published in Tissue Engineering Part A in 2024, which studied the construction of tooth root organoids (Calabrese TC, et al. Tissue Eng Part A. 2024May; 30(9-10): 404-414. doi: 10.1089 / ten.TEA.2023.0219.) This study was based on the self-assembly of periodontal ligament (PDL) stem / progenitor cells (PDLSCs) and dental pulp stem / progenitor cells (DPSCs), which cannot fully simulate the epithelial-mesenchymal interaction mediated by HERS cells during tooth root development.

[0004] Therefore, providing a method for constructing root organoids that can simulate the epithelial-mesenchymal interaction mediated by HERS cells during root development and provide an in vitro model or bioactive transplant material for exploring the mechanism of root development and physiological root regeneration has become an urgent problem to be solved by technicians in this field. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for constructing tooth root organoids, which can form tooth root organoids within 3-4 weeks, and has important application value in the research of tooth root development mechanism, pathogenesis of root dysplasia, biological tooth root regeneration, etc.

[0006] The second objective of the present invention is to provide tooth root organoids constructed by the construction method.

[0007] The third objective of the present invention is to provide applications of the tooth root organoid.

[0008] A fourth object of the present invention is to provide an induction culture medium for inducing differentiation of tooth root organoids.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The present invention discloses a method for constructing tooth root organoids, comprising the following steps:

[0011] Step 1. Cell thawing: thawing the engineered dental epithelial cells stored in liquid nitrogen, resuspending them to prepare a dental epithelial cell suspension for later use;

[0012] Step 2. Cultivating dental follicle stem cell microspheres: dental follicle stem cells are obtained, resuspended and then seeded into a cell culture plate, the culture plate is centrifuged to allow the dental follicle stem cells to aggregate at the bottom of the culture well to form dental follicle stem cell clusters, culture medium is added, and culture is performed to form dental follicle stem cell microspheres;

[0013] Step 3. Take the culture plate after the dental follicle stem cell microspheres are formed in step 2, centrifuge, remove the supernatant, add the dental epithelial cell suspension, let it stand, and then add the culture medium to continue culturing, so that the dental epithelial cells are complexed on the surface of the dental follicle stem cell microspheres to form tooth root organoids.

[0014] In some embodiments of the present invention, the method for constructing tooth root organoids further comprises a three-dimensional culture step, specifically:

[0015] Step 4. Collect the dental epithelial / mesenchymal stem cell spheres formed by the culture in step 3, wash them, and transfer them to a three-dimensional rotating culture system for further culture to form three-dimensional cultured tooth root organoids;

[0016] Preferably, the three-dimensional rotating culture system rotates at a speed of 12r-14r / min, and is placed in an incubator at 37°C and 5% CO2 for 5-7 days to form three-dimensional cultured tooth root organoids.

[0017] In some embodiments of the present invention, the engineered dental epithelial cells include an engineered dental epithelial cell line HERS-H1;

[0018] The cell concentration of the dental epithelial cell suspension in step 1 is 10 3 ~10 4 pcs / 100μL.

[0019] The engineered dental epithelial cell line HERS-H1 described in the present invention is deposited in the China Center for Type Culture Collection, and its name is: SD rat tooth germ Hertwig epithelial root sheath cell line HERS-H1 (Hertwig's epithelial root sheath, HERS-H1), the deposit number is CCTCC NO: C2017249, the deposit time is November 21, 2017, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0020] In some embodiments of the present invention, the dental follicle stem cells in step 2 are 3rd or 4th generation dental follicle stem cells;

[0021] Preferably, dental follicle stem cells are obtained, resuspended in culture medium, and the cell concentration is adjusted to 10 3 ~10 4 pcs / 100μL, inoculated in culture plates;

[0022] Preferably, the culture plate is centrifuged at 800-1500 rpm for 2-10 min to allow the cells to aggregate at the bottom of the culture well to form a dental follicle stem cell cluster;

[0023] Preferably, the culture plate after centrifugation is placed in an incubator at 37° C. and 5% CO 2 for culture, and dental follicle stem cell microspheres are formed after continuous culture for 3-5 days.

[0024] In some embodiments of the present invention, in step 3, the culture plate after culturing to form dental follicle stem cell microspheres in step 2 is taken, the culture plate is centrifuged at 800-1500 rpm for 2-10 min, the supernatant is removed, and a dental epithelial cell suspension is added; preferably, after adding the dental epithelial cell suspension, the culture plate is transferred to an incubator at 37° C. and 5% CO2 and allowed to stand for 1-2 hours, and then the culture medium is added to continue culturing;

[0025] More preferably, the culture plate with added culture medium is placed in an incubator at 37° C. and 5% CO 2 for 3-5 days to allow the dental epithelial cells to be complexed on the surface of the dental follicle stem cell microspheres to form dental epithelial / mesenchymal stem cell microspheres.

[0026] The present invention discloses tooth root organoids obtained by the above-mentioned construction method.

[0027] The applications of the tooth root organoids disclosed in the present invention include applications in preparing in vitro models or bioactive transplant materials.

[0028] The present invention discloses an induction culture medium for inducing differentiation of the tooth root organoids, comprising a basal culture medium and components added to the basal culture medium; the basal culture medium is an epithelial cell medium, and the components and contents added to the basal culture medium are as follows:

[0029] 50-200μg / L bFGF, 200-400μg / L PDGF, 100-300μg / L BMP4, 50-200μg / L WNT10a, 0.5-2mg / L recombinant human insulin, 50-200mg / L vitamin C, 50-200nM γ-secretase inhibitor LY411575, 5-20nM DNA methylase inhibitor 5-Aza-dC, 5-20nM dexamethasone, 0.5-2mM sodium β-glycerophosphate.

[0030] In some embodiments of the present invention, the components and contents added to the basal medium are as follows:

[0031] 100μg / L bFGF, 300μg / L PDGF, 200μg / L BMP4, 100μg / L WNT10a, 1mg / L recombinant human insulin, 100mg / L vitamin C, 100nM γ-secretase inhibitor, 10nM DNA methylase inhibitor, 10nM dexamethasone, and 1mM sodium β-glycerophosphate.

[0032] Preferably, the γ-secretase inhibitor is LY411575;

[0033] Preferably, the DNA methylase inhibitor is 5-Aza-dC.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention utilizes the engineered dental epithelial cell Hertwig epithelial cell line (HERS-H1) constructed by the inventors in the early stage as the source of HERS cells, and dental follicle stem cells as the source of dental mesenchymal cells to construct tooth root organoids, providing an in vitro model or bioactive transplant material for the exploration of tooth root development mechanisms and physiological tooth root regeneration.

[0036] The tooth root organoids constructed by the present invention have similar tissue morphology and cell components to those of normally developing tooth root tissue.

[0037] The Chinese names corresponding to the English abbreviations in this invention are:

[0038] bFGF: basic fibroblast growth factor

[0039] PDGF: Platelet-derived growth factor

[0040] BMP4: human bone morphogenetic protein-4

[0041] WNT10a: wingless-type MMTV integration site protein family member 10a

[0042] 5-Aza-dC: Decitabine, also known as 5-aza-2'-deoxycytidine. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the process of constructing tooth root organoids of the present invention;

[0044] Figure 2 This is the morphology of HERS-H1 cells;

[0045] Figure 3 This is the immunofluorescence staining image of HERS-H1 cells;

[0046] Figure 4 This is the CK14 positive staining image of HERS-H1 cells;

[0047] Figure 5 This is a morphological diagram of dental follicle stem cells;

[0048] Figure 6 This is the result of flow cytometry detection of dental follicle stem cells;

[0049] Figure 7 The morphology of dental follicle stem cell microspheres after 3 days of culture;

[0050] Figure 8 This is the immunofluorescence staining of dental follicle stem cell microspheres;

[0051] Fig. 9 This is a micrograph of tooth root organoids formed by dental follicle stem cell microspheres combined with HERS-H1 cells;

[0052] Fig.10 This is a hematoxylin staining image of a frozen section of a tooth root organoid;

[0053] Fig.11 Immunofluorescence staining of frozen sections of tooth root organoids;

[0054] Fig.12 This is a section of rat tooth germ tissue;

[0055] Fig.13 This is a micrograph of tooth root organoids after differentiation induction culture;

[0056] Fig.14 This is a scanning electron micrograph of tooth root organoids after differentiation induction culture;

[0057] Fig.15 This is a microCT scan of tooth root organoids after differentiation induction culture;

[0058] Fig.16 HE staining of tooth root organoids after differentiation induction culture;

[0059] Fig.17 This is a comparison of immunohistochemical staining of tooth root organoids after differentiation induced culture and tooth root tissue of normally developing rats. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0061] Example 1

[0062] This embodiment discloses a method for constructing tooth root organoids of the present invention, and its flow chart is as follows: Figure 1 As shown, the specific steps are as follows:

[0063] S1. Dental epithelial cell resuscitation

[0064] The engineered dental epithelial cell line HERS-H1 cells stored in liquid nitrogen were taken out and quickly revived with warm water at 37°C-40°C. The cell suspension after cell resuscitation in each tube was transferred to a 15 mL centrifuge tube, and 5 mL of Epithelial cell medium was added to resuspend. After centrifugation at 1000 rpm-1500 rpm for 5 min, the supernatant was discarded, and 2 mL of Epithelial cell medium containing 2% fetal bovine serum was added to resuspend for later use.

[0065] like Figure 2 As shown, HERS-H1 cells exhibit typical cobblestone-like epithelial cell morphology when cultured on adherent surfaces; Figure 3 and Figure 4 As shown, immunofluorescence staining showed negative staining for Vimentin and positive staining for CK14, indicating that HERS-H1 belonged to odontogenic epithelial cells.

[0066] S2. Preparation of dental follicle stem cells

[0067] S21. The tooth germs of third molars (aged 15-20 years old) that were extracted for orthodontic purposes, whose roots were not fully developed and had no obvious inflammation were removed. The tooth germs were washed twice with normal saline containing 1% double antibiotics (penicillin and streptomycin). The dental follicle tissue was carefully peeled off with micro forceps under a stereomicroscope and transferred to a sterile penicillin bottle for later use.

[0068] S22. Use ophthalmic scissors to quickly cut the dental follicle tissue into pieces of 1-2 mm in size, and wash twice with normal saline; transfer the tissue fragments to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0069] S23. Add 1 mL collagenase and 1 mL Dispase to each centrifuge tube, digest for 30 min at 37°C, add 5 mL DMEM medium containing 10% serum to terminate digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 5 mL DMEM medium containing 10% serum to resuspend;

[0070] S24. Transfer the resuspension in step 1.3 to a 6-well plate and evenly spread it on the bottom of the plate. Add 1 mL of DMEM medium containing 10% serum to each well. Transfer the culture plate to a cell culture incubator at 37°C and 5% CO2 and culture overnight. Replace the medium every 2 days until the cells grow and fuse to about 90% and the dental follicle stem cells are long fusiform or spindle-shaped ( Figure 5 );

[0071] S25. Discard the culture medium, wash twice with PBS, add appropriate amount of trypsin digestion solution and digest for 1-3 minutes until the cells become round, then add DMEM medium containing 10% serum to terminate digestion, transfer to a 15 mL centrifuge tube after pipetting, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant;

[0072] S26. Add DMEM medium containing 10% serum to the cells, pipette to prepare cell suspension, and inoculate into T75 culture flasks for further expansion and passaging.

[0073] Flow cytometry showed that dental follicle stem cells were positive for CD29 and CD44, and negative for CD34 and CD45 ( Figure 6 ), indicating that dental follicle stem cells meet the surface marker protein characteristics of mesenchymal stem cells.

[0074] S3. Cultivation of dental follicle stem cell microspheres

[0075] Dental follicle stem cells are obtained, resuspended and then seeded into a cell culture plate, the culture plate is centrifuged to allow the dental follicle stem cells to aggregate at the bottom of the culture well to form a dental follicle stem cell cluster, culture medium is added, and culture is performed to form dental follicle stem cell microspheres;

[0076] The 3rd or 4th generation dental follicle stem cells were taken and resuspended in DMEM medium containing 10% serum to adjust the cell concentration to 1000 cells / 100 μL. 100 μL of the cell suspension was inoculated into an M-type 96-well ultra-low adhesion cell culture plate (1000 cells / well). The culture plate was centrifuged at 800-1500 rpm for 5 min to allow the cells to aggregate at the bottom of the culture well to form dental follicle stem cell clusters. 150 μL of DMEM medium containing 10% serum was added to each well (the total culture medium volume was 250 μL). The culture plate was placed in an incubator at 37°C and 5% CO2 for culture. After 3 days of continuous culture, dental follicle stem cell microspheres ( Figure 7), microsphere immunofluorescence staining showed Vimentin positive staining ( Figure 8 ), indicating that the constructed cell microspheres maintained the characteristics of mesenchymal cells.

[0077] S4. Cultivation of tooth root organoids

[0078] S41. Take the cells from step S1, digest them into single cells and resuspend them. Count the resuspended HERS-H1 cells, transfer an appropriate amount of cells to another sterile centrifuge tube, add an appropriate amount of culture medium, and adjust the cell concentration to 1000 / 100 μL of epithelial cell suspension for later use.

[0079] S42. Take the culture plate after culturing to form dental follicle stem cell microspheres in step S3, centrifuge the culture plate at 800-1500rpm for 5min, carefully remove the supernatant, add 100μL of the dental epithelial cell suspension prepared in step S41 to each well, transfer the culture plate to a 37°C, 5% CO2 incubator and let it stand for 1 hour, then add 150μL of Epithelial cell medium containing 5% fetal bovine serum to each well (total culture medium volume is 250μL), place the culture plate in a 37°C, 5% CO2 incubator and culture for 3 days to allow the HERS-H1 cells to gradually compound on the surface of the dental follicle stem cell microspheres to form tooth root organoids with dental epithelial cells on the outer layer and dental follicle mesenchymal stem cells on the inner layer.

[0080] S5. 3D culture of tooth root organoids

[0081] The dental epithelial / mesenchymal stem cell spheres were collected, washed twice with 0.01M sterile PBS, and then transferred to a three-dimensional rotating culture system (RCCS rotating culture system) for further culture. In the three-dimensional rotating culture system, the cell spheres were cultured in Epithelial cell medium containing 5% fetal bovine serum. The three-dimensional rotating culture system rotated at a speed of 12r-14r / min and was placed in an incubator at 37°C and 5% CO2. After 5 days of culture, the tooth root organoids were collected ( Fig. 9 ).

[0082] Test Example 1

[0083] This test example discloses an immunofluorescence staining test on HERS-H1 cells.

[0084] The specific steps of immunofluorescence staining are as follows:

[0085] (1) HERS-H1 cells were seeded in a confocal microplate and cultured overnight in a cell culture incubator at 37°C and 5% CO2. The culture medium was removed, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde at room temperature for 10 min.

[0086] (2) Aspirate the fixative and wash with PBS 2-3 times, 5 min each time.

[0087] (3) Treat with 0.1% Triton X-100 (in PBS) at room temperature for 5 min, and wash with PBS three times, each time for 5 min.

[0088] (4) Primary antibodies were added to each group of cells: Anti-Vimentin (1:200) and Anti-CK14 (1:200), and incubated at 37°C for 1 h.

[0089] (5) Remove the primary antibody and wash with PBS three times, 5 min each time. Add fluorescently labeled secondary antibody (1:500) to each well in a dark room and incubate at room temperature in the dark for 1 h.

[0090] (6) Remove the secondary antibody and wash with PBS three times, 5 min each time. Add anti-fluorescence quencher containing DAPI to the well plate, store in dark, and observe and collect images under an inverted fluorescence microscope.

[0091] The results are as follows Figure 3 and Figure 4 As shown, HERS-H1 cells negatively expressed the mesenchymal cell marker Vimentin and positively expressed the epithelial cell marker CK14, indicating that HERS-H1 cells belong to odontogenic epithelial cells.

[0092] Test Example 2

[0093] This test example discloses a test for detecting dental follicle stem cells according to Example 1 using flow cytometry, and the steps are as follows:

[0094] (1) Select dental follicle stem cells with good growth status, aspirate the culture medium, wash with PBS, and then add 1 mL of trypsin for digestion for 1 min. When most cells are observed to be obviously wrinkled and rounded, add an equal amount of complete culture medium to terminate digestion, rinse the bottom of the culture flask, fully collect the cells into the suspension, and centrifuge at 1000 rpm for 5 min.

[0095] (2) Discard the supernatant and resuspend the cells in PBS solution (buffer) supplemented with 2% BSA. Count the cells using a cell counting plate and adjust the cell concentration to 1×10 6 / mL, mix thoroughly, dispense 300μL of cell suspension into each EP tube, centrifuge and discard the supernatant. Wash the cells once with buffer solution, centrifuge to obtain cell pellet for staining.

[0096] (3) Each tube was resuspended with 300 μL buffer, and fluorescent dye-labeled flow cytometry antibodies (CD29, CD44, CD34, CD45) were added to the dental follicle stem cell suspension and incubated at 4°C in the dark for 30 min.

[0097] (4) After the incubation, the cell suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with flow cytometry buffer to remove unbound antibodies. After washing, 300 μL of flow cytometry buffer was added to each tube for resuspending, and the cells were mixed by pipetting and then detected by flow cytometry.

[0098] The results of flow cytometry analysis of cells were as follows Figure 6 As shown, dental follicle stem cells positively expressed mesenchymal stem cell surface markers CD29 and CD44, and negatively expressed hematopoietic cell surface markers CD34 and CD45, indicating that dental follicle stem cells meet the surface marker protein characteristics of mesenchymal stem cells.

[0099] Test Example 3

[0100] This test example discloses the immunofluorescence staining of dental follicle stem cell microspheres. The specific test steps are as follows:

[0101] (1) The dental follicle stem cell microspheres prepared according to the method of Example 1 were collected and centrifuged at 800 rpm for 5 min. The culture medium was removed by aspiration, and the microspheres were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min.

[0102] (2) Aspirate the fixative and wash with PBS 2-3 times, 5 min each time.

[0103] (3) Treat with 10% sucrose solution (prepared with PBS) at room temperature for 1 h. Discard the sucrose solution by centrifugation. Add an appropriate amount of 4% agarose solution cooled to 37°C, shake gently to mix, and place in a 4°C refrigerator until the agarose solidifies.

[0104] (4) Remove the solidified agarose containing the cell microspheres, embed them in OCT, and make 5 μm frozen sections.

[0105] (5) After the sections were hydrated, the primary antibody was added: Anti-Vimentin (1:200) and incubated at 37°C for 1 h.

[0106] (6) Remove the primary antibody and wash twice with PBS, 5 min each time. Add fluorescently labeled secondary antibody (1:500) to each well in a dark room and incubate at room temperature in the dark for 1 h.

[0107] (7) Remove the secondary antibody and wash with PBS three times, 5 min each time. Add anti-fluorescence quencher containing DAPI to the well plate, store in dark, and observe and collect images under an inverted fluorescence microscope.

[0108] The results are as follows Figure 8 The results showed that the cell microspheres positively expressed Vimentin, indicating that the constructed cell microspheres maintained the characteristics of mesenchymal cells.

[0109] Test Example 4

[0110] In this test example, the tooth root organoids constructed in Example 1 were identified.

[0111] 1. Staining

[0112] The tooth root organoids after three-dimensional culture in Example 1 were collected, frozen sections were prepared, and hematoxylin staining and immunofluorescence staining were performed.

[0113] 1.1 The steps for hematoxylin staining of tooth root organoids are as follows:

[0114] (1) Root organoids were collected and centrifuged at 800 rpm for 5 min. The culture medium was removed and the organoids were washed twice with PBS. They were then fixed with 4% paraformaldehyde at room temperature for 30 min.

[0115] (2) Aspirate the fixative and wash with PBS 2-3 times, 5 min each time.

[0116] (3) Treat with 10% sucrose solution (prepared with PBS) at room temperature for 1 h. Discard the sucrose solution by centrifugation. Add an appropriate amount of 4% agarose solution cooled to 37°C, shake gently to mix, and place in a 4°C refrigerator until the agarose solidifies.

[0117] (4) Remove the solidified agarose containing the organoids, embed them in OCT, and make 5 μm frozen sections.

[0118] (5) After the sections are hydrated, add hematoxylin staining solution for 1 min, absorb the hematoxylin, and rinse with PBS twice, each time for 2 min;

[0119] (6) The sections were dehydrated with graded alcohol, transparentized with xylene, and then sealed with neutral resin. The sections were observed under a microscope and photographed.

[0120] 1.2 Immunofluorescence staining steps of tooth root organoids:

[0121] (1) Collect tooth root organoids and prepare frozen sections, following steps 1-4 above;

[0122] (2) After the slices were hydrated, Anti-Vimentin antibody (1:100, mouse source) was added, incubated at 37°C for 1 h, and rinsed twice with PBS, each time for 10 min; Anti-CK14 (1:100, rabbit source) was added to the slices, incubated at 37°C for 1 h, and rinsed twice with PBS, each time for 10 min;

[0123] (3) In a dark room, add red fluorescently labeled goat anti-mouse secondary antibody (1:500) to the sections, wash twice with PBS, 10 min each time, then add green fluorescently labeled goat anti-rabbit secondary antibody (1:500), incubate at room temperature in the dark for 1 h, and then wash twice with PBS, 10 min each time;

[0124] (4) DAPI-containing anti-fluorescence quencher was added to the slices, stored in the dark, and observed and imaged under an inverted fluorescence microscope.

[0125] The results of hematoxylin staining showed that the outer layer of the tooth root organoids was composed of dense epithelial cells, and the inner layer was composed of spindle-shaped dental mesenchymal cells. Fig.10 shown.

[0126] The results of immunofluorescence staining showed that the outer layer of tooth root organoids were CK14-positive dental epithelial cells, and the inner layer was Vimentin-positive dental mesenchymal cells. Fig.11 shown.

[0127] 2. Comparison of rat tooth germ tissue sections

[0128] The tooth germ tissue of the rats with normal development at 7 days after birth was taken and tissue sections were prepared ( Fig.12 ), the developing tooth root tissue was observed under a light microscope. The results showed that the developing tooth root area was composed of a broken HERS epithelial cell layer on the outer layer, and a densely arranged dental mesenchymal cell area on the inner side. Fig.12 shown.

[0129] The tooth root organoids constructed by the present invention have similar tissue morphology and cell components to those of normally developing tooth root tissue.

[0130] Example 2

[0131] This embodiment discloses a method for inducing and identifying differentiation of tooth root organoids.

[0132] The differentiation induction medium used in this embodiment is based on Epithelial cell medium, and is supplemented with a growth factor and small molecule inhibitor composition including bFGF, PDGF, BMP4, WNT10a, recombinant human insulin, Vitamin C, γ-secretase inhibitor LY411575, DNA methylase inhibitor 5-Aza-dC, dexamethasone and β-glycerophosphate; wherein the content of bFGF is 100 μg / L; the content of PDGF is 300 μg / L; the content of BMP4 is 200 μg / L; the content of WNT10a is 100 μg / L; the content of recombinant human insulin is 1 mg / L; the content of Vitamin C is 100 mg / L; the content of γ-secretase inhibitor LY411575 is 100 nM; the content of DNA methylase inhibitor 5-Aza-dC is 10 nM; the content of dexamethasone is 10 nM; and the content of β-glycerophosphate is 1 mM.

[0133] The tooth root organoid differentiation induction medium of the present invention is used to induce differentiation into the dental pulp dentin stage after the tooth root organoid is constructed, specifically as follows:

[0134] The tooth root organoids constructed by three-dimensional culture according to the method of Example 1 were collected, washed twice with 0.01M sterile PBS, and transferred to a three-dimensional rotary culture system (RCCS rotary culture system) for differentiation induction culture with differentiation induction medium. The three-dimensional rotary culture system rotates at a speed of 12r-14r / min and is placed in an incubator at 37°C and 5% CO2. The tooth root organoids were collected after 15 days of culture.

[0135] Test Example 5

[0136] In this experimental example, tooth root organoids induced by differentiation were identified in Example 2.

[0137] 1. Microscopic observation

[0138] The micrograph of tooth root organoids after differentiation induction culture is shown in the attached figure. Fig.13 As shown, its diameter is about 400 μm, and scanning electron microscopy shows an irregular spherical structure, such as Fig.14 shown.

[0139] 2. microCT Scanning

[0140] After the differentiation induction was completed, the tooth root organoids were collected, fixed with 4% paraformaldehyde, and then scanned with microCT. Fig.15 Display: The outer layer of the tooth root organoid forms a high-density mineralized structure, and the inner layer shows a low-density soft tissue shadow.

[0141] 3. Section Staining

[0142] After decalcification, tooth root organoids were sliced ​​and subjected to HE staining and immunohistochemical staining.

[0143] 3.1 HE staining

[0144] The steps for HE staining of tooth root organoids are as follows:

[0145] (1) The tooth root organoids after differentiation induction were collected and centrifuged at 800 rpm for 5 min. The culture medium was removed and the organs were washed twice with PBS. 4% paraformaldehyde was added for fixation at room temperature overnight. After centrifugation, the fixative was removed and 10% EDTA (prepared with 4% paraformaldehyde) was added for decalcification for 2 weeks.

[0146] (2) Remove the decalcification solution by centrifugation and wash with PBS 2-3 times, each time for 10 min.

[0147] (3) Treat with 10% sucrose solution (prepared with PBS) at room temperature for 1 h. Discard the sucrose solution by centrifugation. Add an appropriate amount of 4% agarose solution cooled to 37°C, shake gently to mix, and place in a 4°C refrigerator until the agarose solidifies.

[0148] (4) The solidified agarose containing the tooth root organoids was removed, embedded in OCT, and 5 μm frozen sections were prepared.

[0149] (5) After the sections were hydrated, hematoxylin staining solution was added for 1 min, hematoxylin was aspirated, and the sections were rinsed with PBS twice, each time for 2 min, and eosin staining solution was added for 5 min.

[0150] (6) The sections were dehydrated with graded alcohol, transparentized with xylene, and then sealed with neutral resin. The sections were observed under a microscope and photographed.

[0151] The results of HE staining are shown in the attached Fig.16 Shown: Its outer layer forms a mineralized tissue similar to dentin, and the inner side is a structure similar to pulp tissue.

[0152] 3.2 Immunohistochemical staining of DMP1 in tooth root organoids

[0153] 3.2.1 The steps of DMP1 immunohistochemical staining of tooth root organoids are as follows:

[0154] (1) Collecting induced tooth root organoids and preparing frozen sections, following the same steps as 1-4 above;

[0155] (2) After the slices were hydrated, Anti-DMP1 antibody (1:300) was added, incubated at 37°C for 1 h, and then transferred to 4°C for overnight incubation. The slices were rinsed with PBS twice, each time for 10 min. After rinsing, goat serum was added to the slices and incubated at 37°C for 30 min.

[0156] (3) Excess goat serum was removed, and horseradish peroxidase-labeled secondary antibody (1:300) was added to the slices and incubated at 37°C for 1 h. The slices were washed twice with PBS, each time for 10 min. DAB color developing solution was added to the slices for 1 min, and the color development was terminated by rinsing with PBS.

[0157] (4) The sections were counterstained with hematoxylin, dehydrated with graded alcohol, transparentized with xylene, and sealed with neutral resin. The sections were observed under a microscope and photographed. The results of immunohistochemical staining are shown in the attached figure. Fig.17 As shown in the left picture.

[0158] 3.2.2. Comparison of DMP1 immunohistochemical staining in rat tooth root tissue

[0159] The tooth germ tissue of the rats with normal development at 7 days after birth was obtained and DMP1 immunohistochemical staining was performed according to the procedure in 3.2.1. The results are shown in the attached figure. Fig.17 As shown in the right figure.

[0160] By the attached Fig.17 It can be seen that the mineralized tissue formed by the induced tooth root organoids and the pulp-like tissue adjacent to it both positively expressed DMP1 ( Fig.17 , left) (DMP1 is one of the marker proteins of dentin and odontoblasts). Its morphological structure and DMP1 expression pattern are very similar to those of normal developing rat tooth root tissue ( Fig.17 , right).

[0161] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A method for constructing tooth root organoids, characterized in that: The steps include: Step 1. Cell thawing: thawing the engineered dental epithelial cells stored in liquid nitrogen, resuspending them to prepare a dental epithelial cell suspension for later use; Step 2. Cultivating dental follicle stem cell microspheres: dental follicle stem cells are obtained, resuspended and then seeded into a cell culture plate, the culture plate is centrifuged to allow the dental follicle stem cells to aggregate at the bottom of the culture well to form dental follicle stem cell clusters, culture medium is added, and culture is performed to form dental follicle stem cell microspheres; Step 3. Take the culture plate after the dental follicle stem cell microspheres are formed in step 2, centrifuge, remove the supernatant, add the dental epithelial cell suspension, let it stand, and then add the culture medium to continue culturing, so that the dental epithelial cells are complexed on the surface of the dental follicle stem cell microspheres to form tooth root organoids.

2. The method for constructing tooth root organoids according to claim 1, characterized in that: It also includes three-dimensional culture steps, specifically: Step 4. Collect the dental epithelial / mesenchymal stem cell spheres formed by the culture in step 3, wash them, and transfer them to a three-dimensional rotating culture system for further culture to form three-dimensional cultured tooth root organoids; Preferably, the three-dimensional rotating culture system rotates at a speed of 12r-14r / min, and is placed in an incubator at 37°C and 5% CO2 for 5-7 days to form three-dimensional cultured tooth root organoids.

3. A method for constructing tooth root organoids according to claim 1 or 2, characterized in that: The engineered dental epithelial cells include an engineered dental epithelial cell line HERS-H1; The cell concentration of the dental epithelial cell suspension in step 1 is 10 3 ~10 4 pcs / 100μL.

4. A method for constructing tooth root organoids according to claim 1 or 2, characterized in that: The dental follicle stem cells in step 2 are the third or fourth generation dental follicle stem cells; Preferably, dental follicle stem cells are obtained, resuspended in culture medium, and the cell concentration is adjusted to 10 3 ~10 4 pcs / 100μL, inoculated in culture plates; Preferably, the culture plate is centrifuged at 800-1500 rpm for 2-10 min to allow the cells to aggregate at the bottom of the culture well to form a dental follicle stem cell cluster; Preferably, the culture plate after centrifugation is placed in an incubator at 37° C. and 5% CO 2 for culture, and dental follicle stem cell microspheres are formed after continuous culture for 3-5 days.

5. The method for constructing tooth root organoids according to claim 1 or 2, characterized in that: In step 3, the culture plate after culturing the dental follicle stem cell microspheres in step 2 is taken, the culture plate is centrifuged at 800-1500 rpm for 2-10 minutes, the supernatant is removed, and the dental epithelial cell suspension is added; Preferably, after adding the dental epithelial cell suspension, the culture plate is transferred to an incubator at 37° C. and 5% CO 2 and allowed to stand for 1-2 hours, and then the culture medium is added to continue the culture; More preferably, the culture plate with added culture medium is placed in an incubator at 37° C. and 5% CO 2 for 3-5 days to allow the dental epithelial cells to be complexed on the surface of the dental follicle stem cell microspheres to form dental epithelial / mesenchymal stem cell microspheres.

6. Tooth root organoids obtained by the construction method described in any one of claims 1-5.

7. The use of tooth root organoids according to claim 6, characterized in that: The application includes application in preparing in vitro models or bioactive transplant materials.

8. An induction culture medium for inducing differentiation of the tooth root organoid according to claim 6, characterized in that: It includes a basic culture medium and components added to the basic culture medium; the basic culture medium is an epithelial cell medium, and the components and contents added to the basic culture medium are as follows: 50-200μg / L bFGF, 200-400μg / L PDGF, 100-300μg / L BMP4, 50-200μg / L WNT10a, 0.5-2mg / L recombinant human insulin, 50-200mg / L vitamin C, 50-200nM γ-secretase inhibitor LY411575, 5-20nM DNA methylase inhibitor 5-Aza-dC, 5-20nM dexamethasone, 0.5-2mM sodium β-glycerophosphate.

9. The induction culture medium for inducing differentiation of the tooth root organoid according to claim 6 according to claim 8, characterized in that: The components and contents added to the basal medium are as follows: 100 μg / L bFGF, 300 μg / L PDGF, 200 μg / L BMP4, 100 μg / L WNT10a, 1 mg / L recombinant human insulin, 100 mg / L vitamin C, 100 nM γ-secretase inhibitor, 10 nM DNA methylase inhibitor, 10 nM dexamethasone, 1 mM sodium β-glycerophosphate; Preferably, the γ-secretase inhibitor is LY411575; Preferably, the DNA methylase inhibitor is 5-Aza-dC.

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