Smell mucosa mesenchymal stem cells with efficient differentiation potential and preparation method of smell mucosa mesenchymal stem cells

Abstract

The invention discloses olfactory mucosa mesenchymal stem cells with efficient differentiation potential and a preparation method of the olfactory mucosa mesenchymal stem cells, and relates to the technical field of cell culture. According to the method, firstly, a complete culture medium special for the olfactory mucosa mesenchymal stem cells is prepared, after chondroitin sulfate, valproic acid, a fibroblast growth factor (FGF) and a nerve growth factor (NGF) are added into the culture medium, the in-vitro proliferation speed of the human olfactory mucosa mesenchymal stem cells is greatly increased by promoting expression of syndecan 1, and the survival rate of the human olfactory mucosa mesenchymal stem cells is increased; meanwhile, it is guaranteed that the stem cells still maintain an undifferentiated state after multi-generation proliferation, and the stem cells have high stem cell property. The olfactory mucosa mesenchymal stem cells pretreated by using the culture medium have stronger capability of differentiating to neurons, and can be better applied to diseases related to neural restoration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell preparation, in particular to an olfactory mucosa mesenchymal stem cell with high differentiation potential and a preparation method thereof. BACKGROUND

[0002] Human olfactory mucosa mesenchymal stem cells are promising "seed" cells for treating nervous system diseases, originating from the ectoderm, having the characteristics of easy cultivation in vitro, permanent proliferation, easy differentiation into the nervous system, low immune rejection, etc., and can be obtained by culturing autologous olfactory mucosa tissue in vitro, which is lower in difficulty and safer than obtaining tissue from the spinal cord or embryo, and can avoid the ethical issues involved in using embryos or spinal cords. Human olfactory mucosa mesenchymal stem cells have been proven to have neuroprotective effects and the ability to promote nerve regeneration, and repair nerve damage in the spinal cord or brain through different mechanisms, and rebuild neural networks. Therefore, how to quickly obtain a large number of stable human olfactory mucosa mesenchymal stem cells in vitro is a prerequisite for affecting the treatment effect.

[0003] At present, DMEM / F12 medium is usually used to culture human olfactory mucosa mesenchymal stem cells, but this culture method will cause the problems of small amount of cell differentiation, easy cell aging, and difficulty in long-term maintenance of biological characteristics, and long-term culture is still needed to obtain pure cells, thereby prolonging the culture time and increasing the risk of cell differentiation. When such cells are applied to treatment, it is difficult to ensure the survival of olfactory mucosa mesenchymal stem cells in vivo, and the surviving cells mainly improve the inflammatory microenvironment through paracrine matrix, so that they cannot fully exert the advantages of olfactory mucosa mesenchymal stem cells. SUMMARY

[0004] In view of the above problems, the present application aims to provide an olfactory mucosa mesenchymal stem cell with high differentiation potential and a preparation method thereof. The olfactory mucosa mesenchymal stem cells are cultured by pretreating the culture medium, which can make the olfactory mucosa mesenchymal stem cells adapt to the microenvironment shaped by the neuron secretion factors in the brain in advance on the basis of promoting the proliferation of the olfactory mucosa mesenchymal stem cells, so that the olfactory mucosa mesenchymal stem cells can be differentiated into neurons more quickly and efficiently after entering the damaged area in the brain.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions: A complete culture medium for culturing olfactory mucosa mesenchymal stem cells, the complete culture medium contains DMEM / F12, 1-2 % (v / v) L-glutamine, 2-5 % (v / v) serum substitute, 10-400 μg / ml chondroitin sulfate, 2-5 mM valproic acid, 10-20 ng / mL FGF and 50-100 ng / mL NGF.

[0006] Preferably, the complete medium contains DMEM / F12, 1% (v / v) L-glutamine, 2% (v / v) serum replacement, 400 μg / ml chondroitin sulfate, 5 mM valproic acid, 20 ng / mL FGF and 100 ng / mL NGF.

[0007] Preferably, the complete medium contains DMEM / F12, 1% (v / v) L-glutamine, 2% (v / v) serum replacement, 400 μg / ml chondroitin sulfate, 5 mM valproic acid, 20 ng / mL FGF and 100 ng / mL NGF.

[0008] The present application further provides the use of the complete medium in at least one of the following ①-④: ① for culturing olfactory mucosa mesenchymal stem cells with high differentiation potential in vitro; ② for inhibiting the aging of olfactory mucosa mesenchymal stem cells in vitro; ③ for promoting the proliferation of olfactory mucosa mesenchymal stem cells; ④ for preparing an olfactory mucosa mesenchymal stem cell culture solution.

[0009] Preferably, the differentiation potential is neuronal differentiation potential.

[0010] Further, the present application relates to an olfactory mucosa mesenchymal stem cell with high differentiation potential, which is obtained by culturing the complete medium.

[0011] The preparation method of the olfactory mucosa mesenchymal stem cell comprises the following steps: inoculating olfactory mucosa mesenchymal stem cells into the complete medium according to any one of claims 1-2 for culture.

[0012] Preferably, the culture time is at least 24 hours.

[0013] Preferably, the culture time is 48 hours.

[0014] Preferably, the cell culture conditions are temperature 37℃, humidity RH 97%, CO2 volume concentration 5%, and oxygen volume concentration 21%.

[0015] Compared with the prior art, the present application has the following beneficial effects: The present application determines that after adding chondroitin sulfate, valproic acid, FGF and NGF in the original culture system, the cell proliferation can be promoted, the cell aging can be inhibited, and the cell has high differentiation potential, and is more easily differentiated into neurons. The olfactory mucosa mesenchymal stem cells cultured by using the culture medium have advantages in the transplantation treatment of nerve repair diseases.

[0016] The method employed significantly improves the efficiency of obtaining mucosal mesenchymal stem cells, successfully cultivating a large population of cells with high differentiation potential within a short period (48 hours). This method not only shortens the traditional culture cycle, but also yields cells that exhibit excellent proliferative capacity and multi-lineage differentiation potential, providing a stable and reliable cell source for subsequent regenerative medicine research and clinical applications.

[0017] While olfactory mucosal mesenchymal stem cells can be successfully induced into neurons in vitro, the induction process is lengthy and transplantation is difficult. However, olfactory mucosal mesenchymal stem cells cultured using this method can be induced into neurons in vitro in a short period of time. Attached Figure Description

[0018] Figure 1 Flow cytometry diagram of olfactory mucosal mesenchymal stem cells.

[0019] Figure 2 Cell proliferation in the control group and the experimental group.

[0020] Figure 3 Proliferation of olfactory mucosal mesenchymal stem cells cultured with different concentrations of chondroitin sulfate.

[0021] Figure 4 Syndecan 1 protein and gene expression levels in the control and experimental groups.

[0022] Figure 5 TERT gene expression levels in cells of different generations in the control group and experimental group.

[0023] Figure 6 The expression levels of BDNF and MAP2 genes in the blank group and the experimental group after neuronal induction and differentiation. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0025] The DMEM / F12 described in the following examples is a 1:1 DMEM / F12 liquid culture medium.

[0026] Example 1: Effect of culture medium on the proliferation of olfactory mucosal mesenchymal stem cells Olfactory mucosa tissue was extracted from the nasal cavity of healthy volunteers, washed, and cut into small pieces. These pieces were then transferred to culture flasks. Complete culture medium A (DMEM / F12 medium supplemented with 1% L-glutamine + 2% serum substitute) was slowly added to the culture flasks. The flasks were incubated, with the medium changed every 2–3 days, until cell confluence reached 70%–80%, yielding P0 cells. Cells were cultured at a rate of 1.4 × 10⁻⁶ cells / day.5 2.1 x 10 5 4 cells / mL were resuspended with complete culture medium A and transferred into culture flasks, and then cultured in an incubator until the confluence reached 70-80% to obtain P1 generation cells. The above steps were repeated to obtain P4 generation olfactory mucosa mesenchymal stem cells, which were identified to meet the characteristics of mesenchymal stem cells. Figure 1

[0027] The P4 generation olfactory mucosa mesenchymal stem cells were divided into an experimental group and a blank group, and were plated in a 12-well plate at a concentration of 1 x 10 4 cells per well, and then culture medium (blank group: complete culture medium A, experimental group: complete culture medium A added with 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF) was added, and the cells were cultured at a temperature of 37°C, humidity RH 97%, CO2volume concentration 5%, and oxygen volume concentration 21% for 24 h and 48 h, and then cell counting was performed to determine the cell proliferation.

[0028] The results showed that the cells obtained in the blank group and the experimental group met the characteristics of mesenchymal stem cells identified by flow cytometry. Compared with the blank group, the number of cells in the experimental group significantly increased after 48 h of culture. Figure 2

[0029] Example 2: Effect of chondroitin sulfate on the proliferation of olfactory mucosa mesenchymal stem cells Based on the culture steps of Example 1, the cells were cultured to P4 generation, and then the olfactory mucosa mesenchymal stem cells were plated in a 12-well plate at a concentration of 1 x 10 4 cells per well, and culture medium containing different concentrations of chondroitin sulfate (complete culture medium A added with 0-400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF) was added, and the cells were cultured at a temperature of 37°C, humidity RH 97%, CO2volume concentration 5%, and oxygen volume concentration 21% for 48 h, and then cell counting was performed to determine the cell proliferation.

[0030] The results showed that chondroitin sulfate had a significant promoting effect on the proliferation of olfactory mucosa mesenchymal stem cells, and 150 μg / ml and 400 μg / ml chondroitin sulfate had the most significant effect on the proliferation of olfactory mucosa mesenchymal stem cells.

[0031] Example 3: Effect of culture medium on syndecan-1 protein and gene of olfactory mucosa mesenchymal stem cells Based on the culture steps of Example 1, the cells were cultured to P4 generation, and then the olfactory mucosa mesenchymal stem cells were plated in a 12-well plate at a concentration of 1 x 10 10 cells per well, and culture medium containing different concentrations of chondroitin sulfate (complete culture medium A added with 0-400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF) was added, and the cells were cultured at a temperature of 37°C, humidity RH 97%, CO2volume concentration 5%, and oxygen volume concentration 21% for 48 h, and then cell counting was performed to determine the cell proliferation.​​Cells were seeded at a concentration of [specific concentration not specified] per well in a 12-well plate and randomly divided into a control group (cultured in complete medium A) and an experimental group (cultured in complete medium A supplemented with 400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF). After adding the medium, the cells were cultured at 37℃, RH 97%, CO2 5%, and Oxygen 21% for 48 h. The expression of the transmembrane receptor protein syndecan-1 was then detected using Western blotting and qPCR. In short, olfactory mesenchymal stem cell proteins were extracted using a mixture of cell lysis buffer and protease inhibitors. After incubation with syndecan-1 primary antibody and specific secondary antibody, and with β-actin as an internal control, protein expression was observed after imaging. Cellular RNA was extracted using RIPA, and cDNA was synthesized via reverse transcription. Syndecan-1 primers were constructed, and qPCR was performed using the cDNA as a template to determine the expression level.

[0032] Table 1 Primer sequences for gene detection

[0033] The results showed that ( Figure 4 The experimental group was able to promote the expression of syndecan-1 protein and gene.

[0034] Example 4: Effects of culture medium on senescence of olfactory mucosal mesenchymal stem cells Based on the culture steps in Example 1, cells were cultured to passages P4 and P2, respectively. 10 Generation, according to 1×10 4 Cells were seeded at a concentration of 100 cells / well into 12-well plates, and complete culture medium was added. The plates were cultured at 37°C, RH 97%, CO2 5%, and oxygen 21% for 48 hours. RNA was then extracted for qPCR to detect the relative expression level of the TERT gene.

[0035] Experimental Groups: MRC-5 group: MRC-5 cells 293-T group: 293-T cells P4 generation OMMSC group (P4): P4 generation OMMSC cells were supplemented with complete culture medium A (DMEM / F12 medium supplemented with 1% L-glutamine + 2% serum substitute). P4 generation OMMSCs + complete medium (P4+): P4 generation OMMSCs are supplemented with complete medium B (complete medium A is supplemented with 400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF). P 10Passage 4 (P4) OMMSCs were added with complete medium A (1% L-glutamine + 2% serum replacement in DMEM / F12 medium) 10 ): P 10 Passage 4 (P4) OMMSCs were added with complete medium B (complete medium A added with 400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF) P 10 Passage 4 (P4) OMMSCs were added with complete medium B (complete medium A added with 400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF) 10 ): P 10 Passage 4 (P4) OMMSCs were added with complete medium B (complete medium A added with 400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF) Table 2 primer sequences of detection genes

[0036] TERT gene overexpression can promote the repair of cell telomeres and inhibit cell aging, but excessive expression can appear tumor-like performance. The results show that (Table 6) Figure 5 ), all groups of OMMSCs show similar TERT gene expression as MRC-5 cells, and there is a significant difference with 293-T cell expression. This shows that the proliferation ability of OMMSCs, whether P4 or P 10 With the increase of the number of passages, the expression of TERT gene will be inhibited, indicating that the cell begins to age, but after adding chondroitin sulfate, valproic acid, FGF and NGF, the expression of TERT can be restored, and the aging of the cell can be inhibited.

[0037] Example 5 According to the method of Example 4, P4 cells were plated into a 12-well plate at a concentration of 1 × 10 4 cells / well, and complete medium A (blank group) or complete medium B (experimental group) was added, and then incubated at a temperature of 37°C, humidity RH 97%, CO2volume concentration 5%, oxygen volume concentration 21% for 48h, then the complete medium was replaced with neural induction differentiation medium (a commercial product, STEMCELL Technologies, item number: 05835), and the induction differentiation medium was replaced every 2-3 days according to the steps of the induction differentiation medium. After 28 days of induction, the expression of BDNF gene and MAP2 gene was detected by qPCR technology. BDNF is a brain-derived growth factor used to promote the growth of neurons, and MAP2 is a key component of the cytoskeleton of neuronal cells.

[0038] Table 3 primer sequences of detection genes

[0039] The results show that the olfactory mucosa mesenchymal stem cells cultured in the complete medium B have high differentiation potential. The experimental group can significantly promote the differentiation of OMMSCs into neurons and promote the high expression of BDNF and MAP2.

[0040] Further, the skilled person can also obtain the effect of the experimental groups of the preceding embodiments 3-5 through the following embodiments: Embodiment 6 Complete medium B: DMEM / F12 medium added with 2 % L-glutamine + 5 % serum substitute + 400 μg / ml chondroitin sulfate + 5 mM valproic acid + 20 ng / mL FGF + 100 ng / mL NGF.

[0041] The olfactory mucosa mesenchymal stem cells are plated into the 12-well plate at a concentration of 1 x 10 4 The olfactory mucosa mesenchymal stem cells are plated into the 12-well plate at a concentration of 1 x 10

[0042] Embodiment 7 Complete medium B: DMEM / F12 medium added with 1 % L-glutamine + 2 % serum substitute + 400 μg / ml chondroitin sulfate + 2 mM valproic acid + 10 ng / mL FGF + 100 ng / mL NGF.

[0043] The olfactory mucosa mesenchymal stem cells are plated into the 12-well plate at a concentration of 1 x 10 4 The olfactory mucosa mesenchymal stem cells are plated into the 12-well plate at a concentration of 1 x 10

[0044] Embodiment 8 Complete medium B: DMEM / F12 medium added with 1 % L-glutamine + 2 % serum substitute + 400 μg / ml chondroitin sulfate + 2 mM valproic acid + 10 ng / mL FGF + 50 ng / mL NGF.

[0045] The olfactory mucosa mesenchymal stem cells are plated into the 12-well plate at a concentration of 1 x 10 4 The olfactory mucosa mesenchymal stem cells are plated into the 12-well plate at a concentration of 1 x 10

[0046] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A complete culture medium for culturing olfactory mucosal mesenchymal stem cells, characterized in that, The complete culture medium contains DMEM / F12, 1-2% (v / v) L-glutamine, 2-5% (v / v) serum substitute, 10-400 μg / ml chondroitin sulfate, 2-5 mM valproic acid, 10-20 ng / mL FGF and 50-100 ng / mL NGF.

2. The complete culture medium according to claim 1, characterized in that, The complete culture medium contains DMEM / F12, 1% (v / v) L-glutamine, 2% (v / v) serum substitute, 150–400 μg / mL chondroitin sulfate, 5 mM valproic acid, 20 ng / mL LFGF and 100 ng / mL NGF.

3. The complete culture medium according to claim 1, characterized in that, The complete culture medium contained DMEM / F12, 1% (v / v) L-glutamine, 2% (v / v) serum substitute, 400 μg / ml chondroitin sulfate, 5 mM valproic acid, 20 ng / mL FGF and 100 ng / mL NGF.

4. The application of the complete culture medium according to any one of claims 1 to 3, characterized in that, Includes at least one of the following ① to ④: ① Used for in vitro culture of olfactory mucosal mesenchymal stem cells with high differentiation potential; ② Used to inhibit the senescence of olfactory mucosal mesenchymal stem cells in vitro; ③ Used to promote the proliferation of olfactory mucosal mesenchymal stem cells; ④ Used to prepare olfactory mucosal mesenchymal stem cell culture medium.

5. The application according to claim 4, characterized in that, The differentiation potential refers to the differentiation potential of neurons.

6. An olfactory mucosal mesenchymal stem cell with highly efficient differentiation potential, characterized in that, The olfactory mucosal mesenchymal stem cells are obtained by culturing in the complete culture medium as described in any one of claims 1 to 3.

7. The method for preparing olfactory mucosal mesenchymal stem cells according to claim 6, characterized in that, Includes the following steps: Olfactory mucosal mesenchymal stem cells are seeded into the complete culture medium described in any one of claims 1 to 3 and cultured.

8. The preparation method according to claim 7, characterized in that, The incubation period is at least 24 hours.

9. The preparation method according to claim 7, characterized in that, The incubation period is 48 hours.

10. The preparation method according to claim 7, characterized in that, The cell culture conditions were 37°C, RH 97%, CO2 volume concentration 5%, and oxygen volume concentration 21%.

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