Culture medium and culture method for inducing olfactory mucosa mesenchymal stem cells into olfactory ensheathing cells

Abstract

The invention discloses a culture medium and a culture method for inducing olfactory mucosa mesenchymal stem cells into olfactory ensheathing cells, and belongs to the technical field of cell induction and culture. The main components of the culture medium provided by the invention are cell nutritional factors and do not contain animal proteins, and all the components act together, so that a suitable growth and differentiation environment is provided for the olfactory mucosa mesenchymal stem cells. The invention also provides a method for inducing the olfactory mucosa mesenchymal stem cells into the olfactory ensheathing cells by using the culture medium. According to the culture method provided by the invention, the mesenchymal stem cells can be induced into the olfactory ensheathing cells in stages. The method is simple, convenient, safe and efficient, the induced cell expresses the olfactory ensheathing cell marker, the problem of the source of the olfactory ensheathing cell is solved, and the method has a good application prospect in the field of neural restoration.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and relates to a culture medium and a culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. Background Art

[0002] Olfactory ensheathing cells (OECs) are a special type of glial cell that is functionally intermediate between Schwann cells and oligodendrocytes. OECs can not only secrete BDNF, NGF neurotrophic factor-3 (NT3) and NGF neurotrophic factor-4 (NT-4), but also express fibronectin, laminin, L1, S100, glial-derived connexins and neural cell adhesion molecules, all of which can support the extension of axons.

[0003] Since OECs secrete neurotrophic factors and axon growth stimulating substances, they can promote axon regeneration and myelin formation, making them one of the ideal candidate cells for promoting central nervous system regeneration. They have been widely used in the treatment of neurological diseases such as spinal cord injury, brain injury, and neurodegenerative diseases. Due to the large demand for olfactory ensheathing cells, current scientific research mainly uses animal olfactory bulb tissue to extract olfactory ensheathing cells, which is inefficient and cannot be used clinically. On the other hand, the method of directly obtaining and culturing olfactory ensheathing cells based on human nasal mucosal tissue is complicated and the tissue source is not easy to provide for a long time. Therefore, there is an urgent need for a method of obtaining olfactory ensheathing cells that is convenient and stable in source and can be used clinically in the future.

[0004] The inventor of this application previously authorized a method for inducing olfactory mucosal mesenchymal stem cells to differentiate into dopaminergic neurons in the patent CN109735493B, which suggests that olfactory mucosal mesenchymal stem cells can be transformed into nerve cells. Olfactory mucosal mesenchymal stem cells have multidirectional differentiation potential and belong to the same ectoderm as olfactory ensheathing cells. Olfactory mucosal mesenchymal stem cells do not need to cross germ layers to differentiate into olfactory ensheathing cells.

[0005] The source of olfactory mucosa mesenchymal stem cells does not need to be repeatedly obtained. A large number of olfactory mucosa mesenchymal stem cells can be obtained by taking nasal mucosa tissue once. Therefore, olfactory mucosa mesenchymal stem cells can be called a stable source of olfactory ensheathing cells.

[0006] Therefore, there is an urgent need to provide a culture medium and a culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells to provide strong support for their future clinical application. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a culture medium and a culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In one aspect, the present invention provides a cell culture medium for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. The cell culture medium uses DMEM / F12 as a basic culture medium and further includes transferrin, insulin, hydrocortisone, sodium selenate, N-2Supplement, and NT3.

[0010] The basal medium DMEM / F12 is a widely used basal medium that provides rich nutrients and trace elements to support cell growth and differentiation. It can be used to support the growth of many types of mammalian cells. Transferrin is a protein that can bind and transport iron ions. During the induction of differentiation, olfactory mucosal mesenchymal stem cells require sufficient energy to transform cell morphology and function. The iron ions transported by transferrin can maintain the balance of iron inside and outside the cell, promote cell growth and proliferation, and avoid the formation of oxygen free radicals in the extracellular matrix. At the same time, transferrin may also participate in the signal transduction process of cells and play an indirect regulatory role in cell differentiation. Insulin plays an important role in cell metabolism. It can promote the absorption of glucose and amino acids, lipogenesis, the transport of monovalent cations and phosphates, the synthesis of proteins and nucleic acids, and promote cell proliferation. For the induction of olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells, insulin can promote the cell uptake of nutrients such as glucose, providing sufficient energy and material basis for cell differentiation. In addition, insulin can also regulate cell protein synthesis and help cells build new protein structures, which is essential for the transformation of cell morphology and function. Hydrocortisone is an adrenal cortex hormone with anti-inflammatory and immunomodulatory effects, which can help maintain the homeostasis of cells and prolong cell life. In the process of inducing olfactory mucosal mesenchymal stem cells to olfactory ensheathing cells, hydrocortisone may regulate gene expression. It can also regulate the inflammatory response of cells, reduce inflammatory interference during cell culture, and facilitate stable differentiation of cells. Selenium in sodium selenate is a cofactor of glutathione peroxidase and some other important enzymes, has antioxidant effects, and can protect cells from oxidative stress damage. N-2Supplement is a cell culture additive, mainly used for the culture of nerve cells. It contains a variety of ingredients such as saturated transferrin, recombinant insulin, progesterone, etc. These ingredients can provide nutrients and signal molecules required for cell growth, promote cell survival, proliferation and differentiation, and facilitate the transformation of olfactory mucosal mesenchymal stem cells to olfactory ensheathing cells. NT3, or neurotrophin-3, is a member of the neurotrophin family and one of the important ingredients that promote the growth and development of nerve cells. In the process of inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells, NT3 can stimulate cell differentiation and promote the transformation of cells into olfactory ensheathing cells. It can activate a series of signal pathways in cells, which are involved in regulating various cell processes such as cell proliferation, survival, and differentiation, and help olfactory mucosal mesenchymal stem cells to better differentiate into olfactory ensheathing cells. These components work together to provide a suitable growth and differentiation environment for olfactory mucosal mesenchymal stem cells, thereby inducing them to differentiate into olfactory ensheathing cells.

[0011] Preferably, the cell culture medium is based on DMEM / F12 as the basic culture medium, and further includes transferrin 0.1-0.5 mg / L, insulin 0.5-1.0 μL / L, hydrocortisone 0.5-1.0 μL / L, sodium selenate 0.1-0.5 μg / ml, N-2 Supplement 0.1-0.5 mL / L, and NT3 0.01-0.05 μg / L.

[0012] Preferably, the cell culture medium is based on DMEM / F12 as the basal medium, and further includes transferrin 0.2 mg / L, insulin 0.5 μL / L, hydrocortisone 0.8 μL / L, sodium selenate 0.4 μg / ml, N-2Supplement 0.3 mL / L, and NT3 0.03 μg / L.

[0013] Furthermore, the present invention also provides a method for inducing olfactory mucosa mesenchymal stem cells into olfactory ensheathing cells, comprising the following steps: culturing the olfactory mucosa mesenchymal stem cells using the cell culture medium.

[0014] Preferably, the method comprises the following steps:

[0015] ① Culture olfactory mucosal mesenchymal stem cells to P2;

[0016] ②Use trypsin to digest the cells and centrifuge;

[0017] ③Remove the supernatant from the centrifuge tube and collect the precipitate;

[0018] ④ Evenly mixing the cell precipitate with the cell culture medium to obtain a mixed solution;

[0019] ⑤Evenly inoculate the mixed solution into the cell plate and place it in an incubator for culture;

[0020] ⑥ After culturing for 1 day, remove the supernatant and floating cells, add the cell culture medium again, and change the medium regularly; after continuous culture for 14 days, obtain olfactory ensheathing cells.

[0021] Preferably, the cell plate is a six-well plate, and the inoculation volume in the cell plate is 2 mL / well.

[0022] Preferably, in step ②, the concentration of pancreatic enzyme is 0.25 w / v%, the digestion time is 30 seconds, the centrifugal speed is 1000 rpm, and the centrifugal time is 5 minutes.

[0023] Preferably, in step ④, the inoculation density of cells in the cell culture medium is 10 5 Pieces / ml.

[0024] Preferably, in step ①, the olfactory mucosa mesenchymal stem cells are cultured in an olfactory mucosa mesenchymal stem cell culture medium to a cell confluence of 70-80% to obtain the P2 generation, wherein the olfactory mucosa mesenchymal stem cell culture medium consists of DMEM / F12 and 10 v / v% fetal bovine serum.

[0025] The invention relates to an olfactory ensheathing cell fluid, which is prepared by the method. The olfactory ensheathing cells express olfactory ensheathing cell markers GFAP and S100β, and the purity thereof reaches more than 65%.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a culture medium and a culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. Compared with the existing technology, the culture medium has a wide source of raw materials and clear culture medium components. The cells obtained by the method express olfactory ensheathing cell markers GFAP and S100β, and the purity reaches more than 65%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The morphology of olfactory ensheathing cells after induction under light microscope.

[0029] Figure 2 Immunofluorescence was used to detect the markers expressed by olfactory ensheathing cells.

[0030] Figure 3 Flow cytometry was used to detect the markers expressed by olfactory ensheathing cells. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In order to further illustrate the present invention, the following examples and drawings are provided for detailed description. The raw materials used in the following examples of the present invention are all commercially available products.

[0033] Example 1 Preparation of Olfactory Ensheathing Cell Induction Culture Medium

[0034] The formula is based on DMEM / F12 as the basic culture medium, and also includes transferrin 0.2 mg / L, insulin 0.5 μL / L, hydrocortisone 0.8 μL / L, sodium selenate 0.4 μg / mL, N-2Supplement (100X) 0.3 mL / L, and NT3 0.03 μg / L.

[0035] Example 2 Preparation of olfactory mucosal mesenchymal stem cells

[0036] ① The nasal mucosa donor signed an informed consent form to exclude infectious diseases such as syphilis, AIDS and respiratory viruses, and used chloramphenicol eye drops in the nose for 3 days, 3 times a day, 3 drops each time;

[0037] ② After disinfection and anesthesia of the nasal cavity, the superior turbinate and the outer mucosal tissue of the middle turbinate are clamped with ethmoid sinus forceps, and the collected human olfactory mucosa sample is cleaned. The cleaning preferably includes cleaning the collected olfactory mucosa sample with physiological saline solution for 3 times until the blood is washed away;

[0038] ③ Use sterile ophthalmic scissors to cut the cleaned human olfactory mucosa sample into pieces less than 1 mm. 3 Size of tissue blocks;

[0039] ④ The tissue blocks were fully mixed with the mesenchymal stem cell culture medium and planted in a cell culture bottle and cultured in an incubator at 37°C and 5% CO2. The olfactory mucosa mesenchymal stem cells were obtained after regular medium replacement and subculturing.

[0040] The mesenchymal stem cell culture medium consists of DMEM / F12 and 10% (v / v) fetal bovine serum.

[0041] Example 3 Preparation and detection of olfactory ensheathing cells

[0042] The specific steps are as follows:

[0043] ① Culture the obtained olfactory mucosal mesenchymal stem cells to P2 generation, with a cell confluence of 70-80%;

[0044] ② Digest the cells with 0.25 w / v% trypsin for 30 seconds and centrifuge at 1000 rpm for 5 minutes;

[0045] ③Remove the supernatant from the centrifuge tube and collect the precipitate;

[0046] ④ Mix the cell pellet and olfactory ensheathing cell culture medium at 10 5 / ml density and mix evenly;

[0047] ⑦ The mixed solution was evenly inoculated into a six-well plate, 2 mL / well, and cultured in a CO2 humidified incubator at 37°C;

[0048] ⑧After culturing for 1 day, remove the supernatant and floating cells, and add the olfactory ensheathing cell culture medium again.

[0049] ⑨ After that, the culture medium was changed every two days, and half of the medium was changed each time. After 14 days of continuous culture, olfactory ensheathing cells were obtained. Figure 1 ;

[0050] ⑩ Collect cells and detect olfactory ensheathing cell markers by immunofluorescence. Figure 2 ;

[0051] Cells were collected and olfactory ensheathing cell markers were detected by flow cytometry. Figure 3 , its purity is above 65%.

[0052] Further description: After a long period of extensive experimental exploration, the optimal formula of the olfactory ensheathing cell induction medium was determined to be: transferrin 0.1-0.5 mg / L, insulin 0.5-1.0 μL / L, hydrocortisone 0.5-1.0 μL / L, sodium selenate 0.1-0.5 μg / ml, N-2Supplement 0.1-0.5 mL / L, NT3 0.01-0.05 μg / L. Within this range, the effects described in Example 3 can be obtained.

[0053] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A cell culture medium for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells, characterized in that: The cell culture medium is based on DMEM / F12 as the basic culture medium and further comprises transferrin, insulin, hydrocortisone, sodium selenate, N-2Supplement and NT3.

2. The culture medium for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells according to claim 1, characterized in that: The cell culture medium is based on DMEM / F12 as a basic culture medium and further comprises 0.1-0.5 mg / L transferrin, 0.5-1.0 μL / L insulin, 0.5-1.0 μL / L hydrocortisone, 0.1-0.5 μg / ml sodium selenate, 0.1-0.5 μg / ml N-2Supplement, and 0.01-0.05 μg / L NT3.

3. The cell culture medium for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells according to claim 1, characterized in that: The cell culture medium is based on DMEM / F12 as a basic culture medium and further includes 0.2 mg / L transferrin, 0.5 μL / L insulin, 0.8 μL / L hydrocortisone, 0.4 μg / mL sodium selenate, 0.3 mL / L N-2 Supplement, and 0.03 μg / L NT3.

4. A method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells, characterized in that: The method comprises the following steps: using the cell culture medium according to any one of claims 1 to 3 to culture olfactory mucosal mesenchymal stem cells.

5. The method according to claim 4, characterized in that The following steps are involved: ① Culture olfactory mucosal mesenchymal stem cells to P2; ②Use trypsin to digest the cells and centrifuge; ③Remove the supernatant from the centrifuge tube and collect the precipitate; ④ Evenly mixing the cell precipitate with the cell culture medium according to any one of claims 1 to 3 to obtain a mixed solution; ⑤Evenly inoculate the mixed solution into the cell plate and place it in an incubator for culture; ⑥ After culturing for 1 day, remove the supernatant and floating cells, add the cell culture medium according to any one of claims 1 to 3 again, and change the medium regularly thereafter; after continuous culturing for 14 days, obtain olfactory ensheathing cells.

6. The method according to claim 5, characterized in that The cell plate is a six-well plate, and the inoculation volume in the cell plate is 2 mL / well.

7. The method according to claim 5, characterized in that In step ②, the concentration of pancreatic enzyme is 0.25 w / v%, the digestion time is 30 seconds, the centrifugal speed is 1000 rpm, and the centrifugal time is 5 minutes.

8. The method according to claim 5, characterized in that In step ④, the inoculation density of cells in the cell culture medium is 10 5 Pieces / mL.

9. The method according to claim 5, characterized in that In step ①, the olfactory mucosa mesenchymal stem cells are cultured in an olfactory mucosa mesenchymal stem cell culture medium to a cell confluence of 70-80% to obtain the P2 generation, wherein the olfactory mucosa mesenchymal stem cell culture medium consists of DMEM / F12 and 10 v / v% fetal bovine serum.

10. An olfactory ensheathing cell fluid, characterized in that: The olfactory ensheathing cells are prepared by the method according to any one of claims 4 to 9, wherein the olfactory ensheathing cells express olfactory ensheathing cell markers GFAP and S100β.

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