Deciduous tooth pulp stem cell culture system without fetal bovine serum and application thereof

Abstract

The application discloses a fetal bovine serum-free milk tooth dental pulp stem cell culture system and application thereof. The application realizes that fetal bovine serum is replaced by low-dose human serum, the milk tooth dental pulp stem cell is cultured by being added into a basic culture medium, and the proliferation of the milk tooth dental pulp stem cell is not inhibited in the culture process. Further, CGF is added and combined with HS to replace FBS in a conventional stem cell culture medium, and low-concentration HS and CGF can promote the in-vitro proliferation and differentiation of SHED, and a sufficient amount of CGF can be extracted to expand SHED without extracting a large amount of blood. Therefore, the application provides a FBS substitute which is derived from human body, has high safety and has few ethical disputes, foreign factors in the SHED culture system are completely eliminated, the influence of external factors contained in FBS on the in-vitro expansion of SHED is eliminated, and SHED can be expanded to a clinical scale without affecting the stemness of SHED.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, more specifically, relates to a fetal bovine serum-free deciduous tooth pulp stem cell culture system and its application. BACKGROUND

[0002] Regenerative medicine using bone tissue engineering aims to restore the structure and function of damaged bone tissue. Bone tissue regeneration requires three basic components, namely stem cells, bioactive molecules and scaffolds. Stem cells, such as mesenchymal stem cells (MSCs), can differentiate into various cell types, such as osteoblasts, chondrocytes and myoblasts. By utilizing specific growth factors and culture conditions, MSCs have been widely studied as seed cells in cell therapy, which includes replacing dysfunctional or dead cells to restore specific body functions. In addition, under the stimulation of inflammation or chemotactic factors (such as Vascular Endothelial Growth Factor (VEGF) or Hepatocyte Growth Factor (HGF)), MSCs show the "homing" feature, i.e. migrating to the injury site, making them a potential tool for stem cell therapy in spinal cord injury. Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells isolated from exfoliated deciduous teeth, which have the characteristics of less ethical controversy and low immunogenicity. SHED has the potential of self-renewal and multi-directional differentiation, and compared with mature dental pulp stem cells (DPSC), SHED has a higher level of stemness. Therefore, SHED is very potential in stem cell therapy for various diseases (such as bone tissue engineering). However, due to the relatively small amount of SHED in adult tissues, it takes weeks of in vitro expansion period to produce a sufficient number of SHED for clinical application. In addition, due to the inherent characteristics of stem cells, such as proliferation and differentiation, changes may occur in vitro, therefore special care is needed to ensure the safety and regenerative efficacy of pre-cultured SHED when applied in vivo.

[0003] Fetal bovine serum (FBS) is a common standard ingredient in stem cell culture medium. FBS helps cell growth in vitro because it contains hormones (such as cortisol, insulin, growth hormone), proteins (such as albumin, globulin), growth factors (such as epidermal growth factor, fibroblast growth factor), lipids (such as endotoxin), inorganic substances and some undefined small molecules directly involved in cell adhesion and growth. However, FBS as a supplement to the culture medium has obvious disadvantages. First, the composition and quality of FBS vary with serum batch, which may hinder the reproducibility of experimental data. In addition, the chemical properties of FBS have not been clearly defined. Second, FBS is a xenogenic material in human medicine. Third, the risk of FBS being contaminated with endotoxin, virus and prion after heat inactivation is still high. Therefore, the safety of FBS in clinical application is controversial, and it is not suitable for in vitro cell expansion before clinical application. Fourth, the method of collecting FBS is controversial in ethics. Based on these major negative factors, the present application aims to find a FBS substitute derived from human body, with high safety and less ethical controversy, to eliminate the influence of external factors (such as endotoxin and heterologous proteins) in FBS on SHED in vitro expansion.

[0004] Human serum (HS) is suggested as a substitute for FBS. However, the inhibitory effect of HS on MSC proliferation has been confirmed in some studies. When 10% allogeneic HS is supplemented in the cell expansion system, the proliferation rate of bone marrow-derived MSCs is reduced compared with the culture system supplemented with 10% FBS, and the time required for cells to reach confluence is prolonged. In addition, the same metabolic inhibitory effect is also observed in MSCs cultured with 5% or 7.5% HS (allogeneic / autologous). While there are currently 15% HS used in the culture system to replace fetal bovine serum for the culture of dental pulp stem cells, to culture stem cells to a clinically useful scale, the high dose of HS generates great cost and pressure in practical application. Therefore, it is urgent to provide a low-dose FBS substitute derived from human body, with high safety and less ethical controversy, to eliminate the influence of external factors (such as endotoxin and heterologous proteins) in FBS on SHED in vitro expansion, so that SHED can be expanded to a clinical scale without affecting its stemness. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides a deciduous tooth dental pulp stem cell culture system without fetal bovine serum.

[0006] The second object of the present application is to provide the use of the deciduous tooth dental pulp stem cell culture system.

[0007] The above-mentioned object of the present application is realized by the following technical scheme:

[0008] The present application provides a fetal bovine serum-free milk tooth pulp stem cell (stem cells from human exfoliated deciduous teeth, SHED) culture system, comprising a basic culture medium and an additive component; the additive component is 4.5-5.5% human serum (HS) and 0.002-0.008 g / mL concentrated growth factor (CGF) based on the volume fraction of the basic culture medium.

[0009] Among them, the concentrated growth factor (CGF) is a third-generation platelet concentrate (PC), which is a fibrin biomaterial rich in growth factors. It is obtained by centrifuging autologous blood at an alternating speed without adding other substances (such as anticoagulants). CGF serves as a cytokine library containing high concentrations of growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor beta-1 (TGF-β1), insulin-like growth factor-1 (IGF-1), VEGF and fibroblast growth factor (FGF). These growth factors are closely involved in the regulation of cell proliferation, differentiation and angiogenesis, which is crucial for hard and soft tissue regeneration.

[0010] Deciduous tooth pulp stem cells (SHED) are mesenchymal stem cells with the ability to self-renew and multidirectionally differentiate, but their proliferation and differentiation abilities are insufficient for large-scale application in bone tissue engineering. In order to expand SHED to a clinical application scale, they are usually pre-cultured with fetal bovine serum (FBS). However, when SHED are transplanted into the human body, this pre-culture method may bring potential contamination or increase the risk of altering stem cell viability and growth. The present invention achieves the replacement of fetal bovine serum with low-dose (4.5-5.5%) human serum, which is added to the basal culture medium to culture deciduous tooth pulp stem cells. During the culture process, there is no inhibitory effect on the proliferation of deciduous tooth pulp stem cells. Low-dose human serum also has the effect of significantly enhancing the osteogenic differentiation potential of SHED. Further adding CGF and combining it with HS as a substitute for FBS in conventional stem cell culture medium, low concentrations of HS and CGF replacing FBS can promote the proliferation and differentiation of SHED in vitro, and sufficient CGF can be extracted to expand SHED without extracting a large amount of blood. The present invention provides an FBS substitute that is derived from the human body, is highly safe, and has less ethical controversy. It completely eliminates exogenous factors in the SHED culture system and eliminates the effects of external factors (such as endotoxins and heterologous proteins) contained in FBS on the in vitro expansion of SHED.

[0011] Furthermore, the added components are 5% human serum and 0.002-0.008 g / mL concentrated growth factor in the volume fraction of the basic culture medium.

[0012] Preferably, the added components are 5% human serum and 0.002 g / mL concentrated growth factor in the volume fraction of the basal culture medium.

[0013] Preferably, the concentrated growth factor CGF is prepared by centrifuging venous blood to obtain a serum layer, a gel-like CGF layer and a red blood cell layer from top to bottom; removing excess liquid from the CGF layer to obtain a CGF membrane, which is then freeze-dried.

[0014] Furthermore, the basal culture medium is DMEM-high glucose culture medium.

[0015] Furthermore, the deciduous tooth dental pulp stem cell culture system also contains antibiotics.

[0016] Preferably, the antibiotic is penicillin and / or streptomycin.

[0017] The present invention provides use of any of the above-mentioned deciduous tooth dental pulp stem cell culture systems in the in vitro proliferation of deciduous tooth dental pulp stem cells.

[0018] The present invention provides the use of any of the above-mentioned deciduous tooth dental pulp stem cell culture systems in inducing differentiation of deciduous tooth dental pulp stem cells in vitro.

[0019] Further, the differentiation is one or more of osteogenic differentiation, adipogenic differentiation, or chondrogenic differentiation.

[0020] The present application also provides a method for culturing stem cells from human exfoliated deciduous teeth, which is cultured by using the stem cell culture system from human exfoliated deciduous teeth as described above.

[0021] Further, the culture is proliferation culture or induced differentiation culture.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application provides a stem cell culture system from human exfoliated deciduous teeth without fetal bovine serum, which comprises a basic culture medium and an additive; the additive is 4.5-5.5% human serum in volume fraction of the basic culture medium and 0.002-0.008 g / mL concentrated growth factor. The present application realizes the replacement of fetal bovine serum with low-dose human serum, and the stem cells from human exfoliated deciduous teeth are cultured in the basic culture medium. The proliferation of the stem cells from human exfoliated deciduous teeth is not inhibited in the culture process. Further, the CGF is added and combined with the HS, which is used as a substitute for the fetal bovine serum in the conventional stem cell culture medium. The low-concentration HS and CGF can promote the in-vitro proliferation and differentiation of the stem cells from human exfoliated deciduous teeth, and a sufficient amount of CGF can be extracted to expand the stem cells from human exfoliated deciduous teeth without extracting a large amount of blood. Therefore, the present application provides a substitute for the fetal bovine serum, which is derived from human body, has high safety, and has less ethical controversy. The exogenous factors in the stem cell culture system from human exfoliated deciduous teeth are completely removed, the influence of the external factors (such as endotoxin and heterologous protein) in the fetal bovine serum on the in-vitro expansion of the stem cells from human exfoliated deciduous teeth is eliminated, and the stem cells from human exfoliated deciduous teeth are expanded to the clinical scale without affecting the stemness. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 4 is a diagram of the proliferation effect of the stem cells from human exfoliated deciduous teeth stimulated by the conditioned medium containing CGF (2x, 4x, 8x) on the 1st, 3rd, 5th and 7th days; wherein "*" indicates a significant difference compared with 10% FBS, p<0.05.

[0025] Figure 2 Figure 5 is a diagram of the colony morphology (A) and the number of colony cells (B) of the stem cells from human exfoliated deciduous teeth cultured in the basic culture medium containing 5% HS or 5% HS+2x CGF for 10 days.

[0026] Figure 3Figure 3 shows the effect of 2×CGF on SHED cell migration; A is crystal violet staining of migrating cells from different groups after culturing in 10% FBS, 5% HS medium with or without 2×CGF for 24 h; B is statistical analysis of the average number of migrating cells per visual field from different groups at 24 h, n=9, "*" indicates p<0.05; C is an image of scratch healing in 10% FBS, 5% HS medium with or without 2×CGF; D is quantitative analysis of scratch healing area using ImageJ software, scale bar = 200 μm, "*" indicates significant difference compared with the 10% FBS group, p<0.05.

[0027] Figure 4 Figure 3 ALP staining (A) and quantification (B) of SHED on days 4 and 7 after stimulation with osteogenic medium containing CGF (2×, 4×, 8×); “*” indicates p<0.05, and “**” indicates p<0.01, which are significantly different from those in the 10% FBS group.

[0028] Figure 5 The osteogenic medium containing 2×CGF induced the mRNA expression of osteogenic markers RUNX2, COL1α1 and OCN in SHED on days 7 and 14; "*" indicates p < 0.05, and "**" indicates p < 0.01, which are significantly different compared with 10% FBS.

[0029] Figure 6 Figures 2A and 2B show the results of cell surface markers of SHED after culture in 5% HS in the presence or absence of 2×CGF (A) and the results of karyotype analysis of SHED at passage 3 cultured with 10% FBS, 5% HS and 2×CGF for 3 days (B).

[0030] Figure 7 SHED exhibited multilineage differentiation ability under different culture conditions; A was the Alizarin Red staining result of SHED after osteogenic differentiation, B was the Calcium Blue staining result of SHED after chondrogenic differentiation, and C was the Oil Red O staining result of SHED after adipogenic differentiation. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0033] Example 1

[0034] Preparation of a xenogenic serum / exogenous growth factor-free culture system containing CGF and HS:

[0035] The inclusion criteria for the donation of concentrated growth factors (CGF) and human serum (HS) were: 1) age: 20-30 years old; 2) non-smoker; 3) physically healthy, no infectious disease; 4) no blood disease, normal platelet count; 5) no drugs affecting platelet function within 3 months before blood collection. All donors signed the informed consent form.

[0036] Venous blood was collected and immediately centrifuged in Medifuge MF200 to obtain three layers from top to bottom, serum layer, CGF layer in gel form and red blood cell layer. The serum in the upper layer was collected and stored in a refrigerator at minus 80 degrees Celsius for later use. The CGF in the middle layer was pressed with sterile gauze to absorb excess liquid, so that the gel-like CGF became a film-like structure, called "CGF film". This film was freeze-dried in a vacuum freeze-drier overnight and ground into CGF powder using a planetary ball mill. The obtained CGF powder was suspended in 50 mL of culture medium, and after incubation at 4°C for 24 hours, the suspension was centrifuged at 453 x g for 5 minutes to separate the red blood cells from the culture medium. Finally, the CGF-containing culture medium was collected, and 5% HS and 1% antibiotics were added.

[0037] The concentration of CGF is defined as follows: 1 x CGF represents CGF extracted from 10 mL of venous blood and placed in 50 mL of culture medium; or 1 x CGF is about 0.05 g of lyophilized CGF dissolved in 50 mL of culture medium.

[0038] A xenogenic serum / exogenous growth factor-free culture system containing CGF and HS was constructed by adding the corresponding HS and CGF and antibiotics to the DMEM-high glucose-based medium.

[0039] Example 2

[0040] Extraction and identification of SHED:

[0041] The primary teeth were all collected from children in the Department of Pediatric Dentistry, Stomatology Hospital of Guangzhou Medical University. The inclusion criteria for the collection of primary teeth were: 1) children aged 6-8 years; 2) the root resorption rate of the primary teeth was less than 1 / 3; 3) no caries and no periapical disease; 4) the child donors had no history of systemic and genetic diseases. The ethical approval for the use of SHEDs was provided by the Ethics Committee of Stomatology Hospital of Guangzhou Medical University (No. LCYJ2021025). The informed consent was obtained from the parents / guardians of the children who donated the primary teeth. The SHEDs isolated from the dental pulp of the children's primary teeth were grown and maintained in DMEM-high glucose medium supplemented with 10% FBS, 1% penicillin-streptomycin at 37°C. After detecting the SHED surface markers by flow cytometry and identifying the SHEDs with multi-differentiation ability of osteogenic and adipogenic differentiation, the SHEDs were determined as dental pulp stem cells. The passage 3-5 SHEDs were used for all experiments of the present application. First, the effects of HS and CGF on the proliferation, clonogenicity and migration ability of SHEDs were explored:

[0042] 1. Effects of HS and CGF on the proliferation ability of SHEDs:

[0043] Control group: DMEM + 10% FBS (v / v) + 1% penicillin-streptomycin; DMEM + 5% HS (v / v) + 1% penicillin-streptomycin;

[0044] Experimental group: DMEM + 5% HS (v / v) + 1% penicillin-streptomycin + CGF (2x, 4x or 8x).

[0045] SHEDs were seeded at 1x10 3 cells per well of a 96-well plate and cultured for 1, 3, 5 and 7 days. The total number of cells was determined using a cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The optical density was measured at 450 nm using a microplate reader. Three independent experiments were repeated.

[0046] 2. Effects of CGF on the clonogenicity of SHEDs:

[0047] Positive control group: DMEM + 10% FBS (v / v) + 1% penicillin-streptomycin;

[0048] Negative control group: DMEM + 5% HS (v / v) + 1% penicillin-streptomycin;

[0049] Experimental group: DMEM + 5% HS (v / v) + 1% penicillin-streptomycin + CGF.

[0050] SHED cells were seeded at 100 cells per well of a 6-well plate and cultured for 10 days without changing the medium. SHED colonies were stained with crystal violet and counted visually. SHED colonies with a diameter greater than 1 mm were considered a single colony unit. Three independent experiments were performed.

[0051] 3. Detection of the effect of CGF on SHED migration ability:

[0052] 1) Transwell cell migration assay

[0053] Transwell migration assay was used to evaluate the effect of CGF on the three-dimensional (3D) migration ability of SHED.

[0054] Positive control group: DMEM+10% FBS+1% penicillin-streptomycin;

[0055] Negative control group: DMEM+5% HS+1% penicillin-streptomycin;

[0056] Experimental group: DMEM+5% HS+1% penicillin-streptomycin+CGF.

[0057] The experiment was performed using a 24-well transwell chamber. 4 SHEDs / 200 μL of serum-free medium were added to the upper chamber, and 600 μL of complete medium containing 10% FBS and 5% HS complete medium with or without CGF were added to the lower chamber. The cells were incubated at 37°C for 24 h. Migrated SHEDs were fixed with 4% paraformaldehyde for 30 min, washed with deionized water, stained with crystal violet, and washed again with deionized water. The cells inside the upper chamber were gently wiped clean with a cotton swab to prevent counting errors. Six randomly selected microscopic fields were moved at 10× magnification, and the number of migrated cells was counted. The same field selection strategy was used for each replicate experiment. The number of cells per field was counted using ImageJ software, and the average number of cells per field was determined using GraphPad Prism 9.0.2 software.

[0058] 2) Scratch test

[0059] The effect of CGF on the planar migration of SHED was evaluated using a scratch assay.

[0060] Positive control group: DMEM+10% FBS+1% penicillin-streptomycin;

[0061] Negative control group: DMEM+5% HS+1% penicillin-streptomycin;

[0062] Experimental group: DMEM+5% HS+1% penicillin-streptomycin+CGF.

[0063] SHED was cultured in 6-well plates until cell confluence reached 90%. Scratches were made using a sterile plastic 200 μL micropipette tip. After washing with PBS, the culture medium containing 2% FBS, 1% HS, and 1% HS+CGF was replaced, respectively. Photos of the scratch area were taken at 0, 6, 12, and 24 h using a 10x optical upright microscope. Ensure that photos were taken and measured from the same scratch position at each time point. The scratch area was assessed using ImageJ software. Scratch recovery rate (%) = (original scratch area - scratch area at the measurement point) / original scratch area × 100%.

[0064] Here are the results:

[0065] Effect of 5% HS+CGF on the proliferation ability of SHED ( Figure 1 ): No cytotoxic effect was observed at different concentrations of CGF (2×, 4× and 8×), and all concentrations of CGF had a positive effect on the proliferation of SHED. In the control group, 5% HS was slightly better than 10% FBS in promoting SHED proliferation on the 3rd day, and slightly lower than 10% FBS on the 5th and 7th days, but there was no significant difference. CGF (2×, 4× and 8×) combined with 5% HS can show the same effect of promoting SHED proliferation as 10% FBS. CGF (2×, 4× and 8×) combined with 5% HS showed a stronger proliferation-promoting effect than 5% HS alone on the 5th and 7th days (p<0.05). It is worth noting that compared with 2×CGF, 4× and 8× concentrations of CGF did not show a stronger proliferation effect.

[0066] Effect of 5% HS+CGF on SHED clone formation ability: The results are as follows Figure 2 Each bar represents the mean ± SD of CFU from three independent experiments. Two SHED batches were analyzed by independent t-test. The colony morphology (A) and colony number (B) of SHED cultured under the conditions of 10% FBS, 5% HS, or 5% HS + 2× CGF are shown in Figure 2. Figure 3 As shown, the SHED colonies formed in the presence of 5% HS + 2×CGF were denser and larger on day 10, and the number of colonies had no statistical difference from the positive control group, while the negative control group also had no statistical difference from the positive control group.

[0067] Effect of 5% HS+CGF on SHED migration ability ( Figure 3 ): Transwell experiment results show ( Figure 3A, B) After 24h of culture, the number of migrated cells in the 2xCGF group was significantly higher than in the negative control group (2.3-fold; p<0.0001) and in the positive control group (1.6-fold; p<0.0001). The results of the scratch test showed that Figure 3 C, D) After 24h of culture, the 2xCGF group showed a significant reduction in the scratch area compared to the negative control group (1.5-fold; p<0.0001). Thus, the results showed that 5% HS + 2xCGF can effectively promote the horizontal and vertical migration of SHED.

[0068] In summary, in the culture of SHED, the substitution of 10% FBS with 5% HS or 5% HS + CGF did not affect the proliferation and clonogenic capacity of SHED, while 5% HS + 2xCGF significantly enhanced the migration capacity of SHED.

[0069] Example 3

[0070] 1. Detection of the effect of CGF on the osteogenic differentiation potential of SHED

[0071] Positive control group: DMEM + 10% FBS by volume + 1% penicillin-streptomycin + 10 nM dexamethasone + 10 mM beta-glycerophosphate + 50 pg / mL ascorbic acid;

[0072] Negative control group: DMEM + 5% HS by volume + 1% penicillin-streptomycin + 10 nM dexamethasone + 10 mM beta-glycerophosphate + 50 pg / mL ascorbic acid;

[0073] Experimental group: DMEM + 5% HS by volume + 1% penicillin-streptomycin + 10 nM dexamethasone + 10 mM beta-glycerophosphate + 50 pg / mL ascorbic acid + CGF.

[0074] 1) Alkaline phosphatase staining (ALP)

[0075] The osteogenic phenotype of SHED cultured for 4 and 7 days was evaluated using alkaline phosphatase (ALP) protein staining.

[0076] SHED were seeded in 48-well culture plates at 2.0 x 10 4 cells per well and cultured for 4 and 7 days in osteogenic induction medium with or without CGF in 5% HS, changing the medium every 3 days. For ALP staining, cells were fixed in 4% paraformaldehyde for 30 min, washed three times in deionized water and stained for ALP protein using the 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT) ALP chromogenic kit. The entire well was observed and photographed using a stereomicroscope at 0.82x magnification. The optical density of the stained ALP protein was measured using ImageJ software to semi-quantify the ALP protein.

[0077] 2) RNA isolation and real-time quantitative PCR (RT-qPCR) analysis

[0078] SHEDs were cultured in osteogenic induction medium supplemented with 10% FBS, 5% HS with or without CGF for 7 or 14 days. Total RNA of SHEDs was extracted using RNA extraction kit and reverse transcribed. TB Green Premix Ex Taq was used for RT-qPCR analysis. Premix Ex Taq TM II Detection of relative mRNA expression of target genes (ALP, RUNX2, COL1a1 and OCN). Phosphoglyceraldehyde dehydrogenase (GAPDH) was used to normalize the expression level of mRNA.

[0079] 2. Detection of the effect of CGF on SHED surface markers and karyotype analysis

[0080] Control group: DMEM + 5% HS (v / v) + 1% penicillin-streptomycin;

[0081] Experimental group: DMEM + 5% HS (v / v) + 1% penicillin-streptomycin + CGF.

[0082] Flow cytometry was used to determine whether CGF affects SHED phenotype (CD90, CD73, CD105, CD14, CD34 and HLA-DR). Karyotype analysis was performed to determine the possible effect of CGF on stem cell karyotype.

[0083] Cell samples (5.0 x 10 5 Cells were incubated with CD90-PE-CY5, CD73-FITC, CD105-PE, CD14-FITC, CD34-PE and HLA-DR-FITC for 30 minutes at 4°C. After incubation, cells were washed and suspended in PBS. Flow cytometry was used to analyze the cell surface expression of CD90, CD73, CD105, CD14, CD34 and HLA-DR markers.

[0084] SHEDs were cultured in medium supplemented with 10% FBS, 5% HS with 2x CGF for 72h. SHED samples were then sent to a biological company for karyotype analysis.

[0085] 3. Detection of the effect of CGF on SHED multipotential differentiation potential

[0086] Positive control group: DMEM + 10% FBS (v / v) + 1% penicillin-streptomycin;

[0087] Negative control group: DMEM + 5% HS (v / v) + 1% penicillin-streptomycin;

[0088] Experimental group: DMEM + 5% v / v HS + 1% penicillin-streptomycin + CGF.

[0089] 1) Osteogenic differentiation (mineralized nodule detection, using Alizarin Red staining)

[0090] SHED were seeded at 2.5 x 10 4 cells per well of a 48-well plate. When the cell confluence reached 80-90%, the medium was changed to osteogenic differentiation medium. The medium was changed every 3 days for 21 days. To detect mineralization, cells were fixed in 4% formaldehyde for 30 min. Then, cells were washed with deionized water and stained with 0.5% Alizarin Red S staining solution for 5 min at room temperature. Images were acquired using a stereomicroscope.

[0091] 2) Chondrogenic differentiation (cartilage sulfated glycosaminoglycan detection, using Alcian Blue staining)

[0092] 1 x 10 5 SHED suspension was dropped into a 48-well plate at 10 μΐ per well, and after 2 h for SHED to form cell micromasses, they were cultured in DMEM-high glucose medium containing 10% FBS, 1% penicillin and streptomycin for 1 day. Then, the medium was changed to chondrogenic differentiation using the Human Mesenchymal Stem Cell Chondrogenic Differentiation Kit for 14 days, changing the medium every 3 days. Subsequently, micromasses were fixed in 4% glutaraldehyde for 30 min and stained with Alcian Blue. Staining depth was evaluated by microscopic photographs (8x) to determine the concentration of glycosaminoglycans.

[0093] 2) Adipogenic differentiation (intracellular lipid droplet detection, using Red Oil O staining)

[0094] SHED were seeded into a 6-well plate and cultured using the Human Related Stem Cell Adipogenic Differentiation Kit. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, they were stained with Oil Red O for 30 min. Photographs were taken with a 20x magnifying lens to evaluate intracellular lipid droplets.

[0095] Results are as follows:

[0096] Effect of CGF on SHED osteogenic differentiation potential was tested Figure 4 , 5): ALP staining and quantification of SHED at day 4 and day 7, results are expressed as the mean ± SD of three measurements from three independent experiments, ALP staining Figure 4 A) and semi-quantitative experiment Figure 4B) The results showed that the 5% HS group significantly outperformed the 10% FBS group in stimulating SHED to produce ALP at day 7, indicating that 5% HS significantly increased the osteogenic differentiation potential of SHED compared to 10% FBS; compared to the 10% FBS group and the 5% HS group, the osteogenic medium containing CGF (2x, 4x, 8x) stimulated SHED to produce ALP at day 4 and day 7, indicating that the addition of CGF can significantly increase the osteogenic differentiation potential of SHED. The osteogenic-related mRNA expression results showed that Figure 5 ), the relative gene expression levels were expressed as the mean ± SD of three measurements from three independent experiments. Compared to the 10% FBS group, the 5% HS group increased the mRNA expression of the early osteogenic marker COL1a1 in SHED at day 7; compared to the 10% FBS group and the 5% HS group, the osteogenic medium containing 2x CGF induced a significant increase in the mRNA expression of the early osteogenic markers RUNX2 and COL1a1 in SHED at day 7 and day 14, and a significant increase in the mRNA expression of the late osteogenic marker OCN at day 14.

[0097] Detection of the effect of CGF on SHED surface markers and karyotype analysis: Figure 6 A is the flow cytometry detection of cell surface markers after culturing SHED with 5% HS with or without 2x CGF; Figure 6 B is the karyotype analysis of the third generation of SHED cultured with 10% FBS and 5% HS containing 2x CGF for 3 days. The results showed that the expression of SHED surface markers was stable and the chromosome karyotype was normal under the culture conditions in the presence of CGF.

[0098] Detection of the effect of CGF on the multi-directional differentiation potential of SHED: SHED showed multi-lineage differentiation ability under different culture conditions. Figure 7 The results of alizarin red (A), cartilage blue staining (B) and oil red O (C) staining after SHED were induced to differentiate into osteoblasts, chondrocytes and adipocytes. The results showed that SHED had multi-directional differentiation potential to differentiate into osteoblasts, chondrocytes and adipocytes under the culture conditions in the presence of CGF. In summary, CGF does not affect the stem cell characteristics of SHED.

Claims

1. A fetal bovine serum-free culture system for deciduous dental pulp stem cells, characterized in that: The invention comprises a basic culture medium and additional components; the additional components are 4.5-5.5% human serum and 0.002-0.008 g / mL concentrated growth factor, accounting for 4.5-5.5% of the volume fraction of the basic culture medium.

2. The deciduous tooth dental pulp stem cell culture system according to claim 1, characterized in that: The added components are 5% human serum and 0.002-0.008 g / mL concentrated growth factor, accounting for 5% of the volume fraction of the basic culture medium.

3. The deciduous tooth dental pulp stem cell culture system according to claim 1, characterized in that: The basic culture medium is DMEM-high glucose culture medium.

4. The deciduous tooth dental pulp stem cell culture system according to claim 1, characterized in that: Also contains antibiotics.

5. The deciduous tooth dental pulp stem cell culture system according to claim 4, characterized in that: The antibiotics are penicillin and / or streptomycin.

6. Use of the deciduous tooth dental pulp stem cell culture system according to any one of claims 1 to 5 in the in vitro proliferation of deciduous tooth dental pulp stem cells.

7. Use of the deciduous tooth dental pulp stem cell culture system according to any one of claims 1 to 5 in inducing differentiation of deciduous tooth dental pulp stem cells in vitro.

8. The application according to claim 7, characterized in that: The differentiation is one or more of osteogenic differentiation, adipogenic differentiation or chondrogenic differentiation.

9. A method for culturing deciduous tooth dental pulp stem cells, characterized in that: The deciduous tooth dental pulp stem cells are cultured using the culture system according to any one of claims 1 to 5.

10. The method according to claim 9, characterized in that: The culture is proliferation culture or differentiation-induced culture.

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