Serum-free culture medium, 3D stem cell microsphere induction method and application

Abstract

The application belongs to the technical field of cell culture, and particularly relates to a serum-free culture medium, a 3D stem cell microsphere induction method and application. The serum-free culture medium provided in the application can induce the 3D stem cell microsphere to have the abilities of bone formation, cartilage formation and dental pulp regeneration in vivo and in vitro, and solves the technical problem that the existing 3D stem cell microsphere induction method lacks a serum-free culture medium.

Description

Technical Field

[0001] This application belongs to the field of cell culture technology, and in particular relates to a serum-free culture medium, a method for inducing 3D stem cell microspheres, and their application. Background Technology

[0002] Repair and reconstruction of soft and hard tissues such as bone, cartilage, and dental pulp are challenging problems in clinical medicine today. Traditional treatment strategies suffer from various problems, such as limited availability of stem cells and insufficient regenerative induction capacity. In recent years, stem cell therapy has become the most promising regenerative treatment strategy.

[0003] Compared with traditional 2D cultured stem cells, 3D stem cell microspheres can better simulate intercellular interactions, thus attracting much attention from researchers. However, 3D stem cell microspheres are generally used in tumor treatment research and are rarely seen in regenerative medicine research.

[0004] Meanwhile, existing methods for inducing 3D stem cell microspheres involve induction in a culture medium containing serum and factors, which is complex and costly, thus limiting its promotion and application. Summary of the Invention

[0005] In view of this, this application provides a serum-free culture medium, a method for inducing 3D stem cell microspheres, and its application, to solve the technical problem that the existing methods for inducing 3D stem cell microspheres lack serum-free culture medium.

[0006] The first aspect of this application provides a serum-free culture medium composed of antibiotics, dexamethasone, sodium β-glycerophosphate, ascorbic acid, neodymium-doped silica nanomaterials, and metformin.

[0007] Preferably, the serum-free culture medium contains 1% (by mass) of the double antibiotics.

[0008] The concentration of dexamethasone is 10 nmol / L;

[0009] The concentration of the sodium β-glycerophosphate is 10 mmol / L;

[0010] The concentration of ascorbic acid is 50 mg / L;

[0011] The mass fraction of the neodymium-doped silicon oxide nanomaterial is 0.01–1%.

[0012] The concentration of metformin is 10 nM to 50 μM / L.

[0013] In this patent application, "double antibiotics" refers to penicillin and streptomycin.

[0014] The second aspect of this application provides a method for inducing 3D stem cell microspheres using the above-mentioned serum-free culture medium, the induction method comprising the steps of:

[0015] Step S1: Seed the suspension of stem cells to be induced into an agarose culture plate to obtain self-aggregating 3D stem cell microspheres seeded in the microwells of the agarose culture plate;

[0016] Step S2: Add the serum-free culture medium to the self-aggregating 3D stem cell microspheres for induction.

[0017] Preferably, in step S1, the agarose culture plate has a micropore diameter of 200 μm, a height of 150 μm, and a spacing of 100 μm.

[0018] Preferably, in step S1, the agarose culture plate has 2000 microwells;

[0019] Each self-aggregating 3D stem cell microsphere contains 250 cells.

[0020] Preferably, in step S1, the stem cell suspension to be induced is a dental pulp stem cell suspension to be induced.

[0021] Preferably, in step S2, during the induction process, half of the serum-free culture medium is replaced every 3 to 4 days.

[0022] It should be noted that agarose culture dishes are non-cytotoxic and do not carry cell adhesion sites, thus isolating cells from the substrate and promoting cell aggregation to form 3D cell microspheres. In addition, the induction process of the 3D stem cell microspheres provided in this patent application does not require an external scaffold, simplifying the preparation process.

[0023] Preferably, in step S1, the method for preparing neodymium-doped silicon oxide nanomaterials in the serum-free culture medium includes the following steps:

[0024] Step S11: Tetraethyl orthosilicate and neodymium nitrate are stirred and reacted under the catalysis of ammonia water to obtain neodymium-doped silicon oxide precipitate;

[0025] Step S12: The neodymium-doped silicon oxide precipitate is dried and calcined sequentially to obtain neodymium-doped silicon oxide nanomaterials.

[0026] The third aspect of this application provides the application of the aforementioned self-aggregating 3D stem cell microspheres in regenerative medicine.

[0027] Preferably, the application in regenerative medicine specifically refers to its application in dental pulp regeneration medicine.

[0028] Preferably, the application in regenerative medicine specifically refers to its application in cartilage regeneration medicine.

[0029] Preferably, the application in regenerative medicine specifically refers to its application in bone regeneration medicine.

[0030] It should be noted that the self-aggregating 3D stem cell microspheres provided in this patent application have the potential for osteogenic, chondrogenic, and dental pulp regeneration, and can therefore be widely used in the field of regenerative medicine for the repair and reconstruction of soft and hard tissue diseases such as bone, cartilage, and dental pulp.

[0031] In summary, this application provides a serum-free culture medium, a method for inducing 3D stem cell microspheres, and its application. The serum-free culture medium is composed of antibiotics, dexamethasone, sodium β-glycerophosphate, ascorbic acid, neodymium-doped silica nanomaterials, and metformin. It can induce the formation of self-aggregating 3D stem cell microspheres for osteogenic, chondrogenic, and dental pulp regeneration without the addition of serum or its factors. This simplifies the induction method of 3D stem cell microspheres and reduces the induction cost, thereby solving the technical problem of the lack of serum-free culture medium in the induction methods of 3D stem cell microspheres in the prior art. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram of the agarose culture plate provided in Example 2 of this patent application;

[0034] Figure 2 This is a schematic diagram of the induction of self-aggregating 3D stem cell microspheres provided in Embodiment 2 of this patent application;

[0035] Figure 3 This is a schematic diagram of the neodymium-doped silicon oxide nanoparticle material provided in Embodiment 2 of this patent application;

[0036] Figure 4 This is a schematic diagram of osteogenic expression induced by self-aggregating 3D stem cell microspheres provided in Example 2 of this patent application;

[0037] Figure 5 This is a schematic diagram of the chondrogenic differentiation of self-aggregating 3D stem cell microspheres provided in Embodiment 2 of this patent application;

[0038] Figure 6 This is a schematic diagram of pulp regeneration induced by self-aggregating 3D stem cell microspheres provided in Embodiment 2 of this patent application;

[0039] Figure 3In the image, 3-a shows the structural characterization of neodymium-doped silicon dioxide nanoparticles, 3-b shows a fluorescence image of the nanoparticles being phagocytosed by cells, and 3-c shows a TEM image of the nanoparticles being phagocytosed by cells.

[0040] Figure 4 In the diagram, 4-a shows that self-aggregating 3D stem cell microspheres promote osteogenic gene expression, 4-b shows that they promote osteogenic protein expression, 4-c shows that they promote new bone formation in vivo, and 4-d shows that H&E staining demonstrates increased new bone formation in vivo and Masson's staining indicates increased cartilage formation.

[0041] Figure 5 In the table, 5-a represents safranin O staining, 5-b represents alisin blue staining, 5-c represents chondrogenic gene expression, and 5-d represents chondrogenic protein expression.

[0042] Figure 6 In the images, 6-a is a SEM image of dentin, 6-b is the treatment of the implanted tooth, 6-c is the subcutaneous implantation in nude mice, 6-d is the post-implantation image, and 6-e is the H&E staining of the segmental surface. Detailed Implementation

[0043] This application provides a serum-free culture medium, a method for inducing 3D stem cell microspheres, and its application, which solves the technical problem that the existing methods for inducing 3D stem cell microspheres lack serum-free culture medium.

[0044] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0045] Example 1

[0046] Given that the existing methods for inducing 3D stem cell microspheres involve induction in a culture medium containing serum and factors, the induction conditions are complex and the induction cost is high, thus limiting its promotion and application, Example 1 of this application provides a serum-free culture medium. The serum-free culture medium is composed of antibiotics, dexamethasone, sodium β-glycerophosphate, ascorbic acid, neodymium-doped silica nanomaterials, and metformin. The use of neodymium-doped silica nanomaterials and metformin in the serum-free culture medium is the core of this invention, realizing serum-free induction. The serum-free culture medium provided in this patent application does not contain added serum and its factors, which simplifies the induction method of 3D stem cell microspheres and reduces the induction cost, thereby solving the technical problem of the lack of serum-free culture medium in the existing methods for inducing 3D stem cell microspheres.

[0047] Regarding the concentrations of each component in the serum-free culture medium provided in this patent application, the mass fraction of the double antibiotics in the serum-free culture medium is 1%, the concentration of dexamethasone is 10 nmol / L, the concentration of sodium β-glycerophosphate is 10 mmol / L, the concentration of ascorbic acid is 50 mg / L, the mass fraction of neodymium-doped silicon oxide nanomaterials is 0.01–1%, and the concentration of metformin is 10 nM–50 μM / L.

[0048] Example 2

[0049] Example 2 of this application provides an example of inducing 3D stem cell microspheres using the serum-free culture medium provided in Example 1, including raw material preparation, preparation of neodymium-doped silica nanomaterials, cell culture, preparation of 3D stem cell microspheres, and induction of 3D stem cell microspheres.

[0050] The preparation of raw materials includes: preparing the required basic culture medium, trypsin, centrifuge tubes (50mL and 15mL) and centrifuge, microscope, cell counter, T75 cell culture flask, tetraethyl orthosilicate, neodymium nitrate, metformin drug and other required chemical reagents.

[0051] The preparation of neodymium-doped silicon oxide nanomaterials includes: adding 250 mL of deionized water to a beaker at room temperature and starting stirring; adding 4.5 mL of tetraethyl orthosilicate and a certain amount of 40 mg of neodymium nitrate to the central opening; adding 10 mL of ammonia water for catalysis; stirring at room temperature for 12 h; collecting the precipitate by centrifugation; then centrifuging the precipitate again, removing the supernatant, washing the precipitate three times with ethanol solution, drying it, and calcining it in a muffle furnace at 600 degrees Celsius for 2 h to obtain neodymium-doped silicon oxide nanomaterials. The amount of neodymium nitrate added can also be selected from 10 mg, 20 mg, or 80 mg.

[0052] Cell culture includes: taking osteoblast stem cells as an example, dental pulp stem cells are collected and washed, and then expanded and cultured. After expanding to 5-7 generations, they are digested with trypsin, centrifuged, the supernatant is removed, and then 5 mL of preheated 1×PBS (37℃) is added to wash the cells. The cells are repeatedly mixed to remove residual collagenase, and then centrifuged again to collect the cells and disperse them into centrifuge tubes for later use.

[0053] The preparation of 3D stem cell microspheres includes: resuspending the above cell suspension in cell culture medium and adjusting the cell density to 1×10⁻⁶. 6 Cells / mL were added to an agarose culture plate with 200-micron pores; the agarose culture plate structure is as follows. Figure 1 As shown, each agarose culture plate contains approximately 2,000 microwells, with 500,000 cells seeded per plate. After fusion, each 3D stem cell microsphere contains approximately 250 cells.

[0054] The induced 3D stem cell microspheres consist of: differentiating cell aggregates into microtissues in a serum-free culture medium containing 1% penicillin and streptomycin, 10 nmol / L dexamethasone, 10 mmol / L sodium β-glycerophosphate and 50 mg / L ascorbic acid, a certain concentration (0.01%–1%) of neodymium-doped silica nanomaterials, and a certain concentration (10 nM–50 μM) of metformin, with half the volume of the culture medium replaced every 3–4 days.

[0055] Experimental Example 1

[0056] Example 1 of this experiment was used to test the effect of serum-free culture medium on the induction of self-aggregating 3D stem cell microspheres, including the promotion of in vivo and in vitro bone regeneration by self-aggregating 3D stem cells, the induction of chondrogenic differentiation of self-aggregating 3D stem cells into dental pulp regeneration by self-aggregating 3D stem cells.

[0057] Among them, the self-aggregating 3D stem cell microspheres have a significant effect on promoting in vivo and in vitro bone regeneration. Figure 4 As shown, the effect of inducing chondrogenic differentiation of self-aggregated 3D stem cells is as follows: Figure 5 As shown, the effect of self-aggregating 3D stem cells inducing dental pulp regeneration is as follows: Figure 6 As shown, from Figure 4-6 It can be seen that the self-aggregating 3D stem cell microspheres provided in this application have the potential for osteogenic, chondrogenic and dental pulp regeneration under serum-free culture medium induction.

[0058] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A serum-free culture medium, characterized in that, It is composed of bispecific antibodies, dexamethasone, sodium β-glycerophosphate, ascorbic acid, neodymium-doped silica nanomaterials, and metformin, wherein the bispecific antibodies are penicillin and streptomycin; The mass fraction of the bispecific antibody is 1%; The concentration of dexamethasone was 10 nmol / L; The concentration of the sodium β-glycerophosphate is 10 mmol / L; The concentration of ascorbic acid is 50 mg / L; The mass fraction of the neodymium-doped silicon oxide nanomaterial is 0.01% to 1%. The concentration of metformin is 10 nM~50 μM / L.

2. A method for inducing 3D stem cell microspheres in a serum-free culture medium, characterized in that, Including the following steps: Step S1: Seed the suspension of stem cells to be induced into an agarose culture plate to obtain self-aggregating 3D stem cell microspheres seeded in the microwells of the agarose culture plate; Step S2: Add the self-aggregating 3D stem cell microspheres to the serum-free culture medium of claim 1 for induction.

3. The method for inducing 3D stem cell microspheres in a serum-free culture medium according to claim 2, characterized in that, In step S1, the agarose culture plate has a micropore diameter of 200 μm, a height of 150 μm, and a spacing of 100 μm.

4. The method for inducing 3D stem cell microspheres in a serum-free culture medium according to claim 2, characterized in that, In step S1, the stem cell suspension to be induced is a dental pulp stem cell suspension to be induced.

5. The method for inducing 3D stem cell microspheres in a serum-free culture medium according to claim 2, characterized in that, In step S1, the agarose culture plate has 2000 microwells; Each self-aggregating 3D stem cell microsphere contains 250 cells.

6. The method for inducing 3D stem cell microspheres in a serum-free culture medium according to claim 2, characterized in that, In step S2, during the induction process, half of the serum-free culture medium is replaced every 3-4 days.

7. The method for inducing 3D stem cell microspheres in a serum-free culture medium according to claim 2, characterized in that, In step S1, the method for preparing neodymium-doped silicon oxide nanomaterials in the serum-free culture medium includes the following steps: Step S11: Tetraethyl orthosilicate and neodymium nitrate are stirred and reacted under the catalysis of ammonia water to obtain neodymium-doped silicon oxide precipitate; Step S12: The neodymium-doped silicon oxide precipitate is dried and calcined sequentially to obtain neodymium-doped silicon oxide nanomaterials.

8. The application of the self-aggregating 3D stem cell microspheres according to any one of claims 2-7 in the preparation of regenerative medicine products, characterized in that, The regenerative medicine mentioned here specifically refers to pulp regeneration medicine, cartilage regeneration medicine, or bone regeneration medicine.

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