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

Abstract

The present application discloses a culture medium for inducing olfactory mucosa mesenchymal stem cells into olfactory ensheathing cells, and a method for using the culture medium to induce olfactory mucosa mesenchymal stem cells into olfactory ensheathing cells.

Description

A culture medium and culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. Technical Field

[0001] This application belongs to the field of cell culture technology, and relates to a culture medium and culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory sheath cells. Background Technology

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

[0003] Because olfactory ensheathing cells (OECs) secrete neurotrophic factors and axonal growth stimulants, promoting axonal regeneration and myelin formation, they are considered ideal candidate cells for promoting central nervous system regeneration and are currently widely used in neurological diseases such as spinal cord injury, brain injury, and neurodegenerative diseases. Due to the high demand for olfactory ensheathing cells, current scientific research mainly utilizes animal olfactory bulb tissue to extract them, a method that is inefficient and not clinically applicable. On the other hand, methods for directly obtaining and culturing olfactory ensheathing cells from human nasal mucosa tissue are complex, and the tissue source is not readily available for long-term supply. Therefore, there is an urgent need for a convenient, stable, and clinically applicable method for obtaining these cells.

[0004] The method for inducing olfactory mucosal mesenchymal stem cells to differentiate into dopaminergic neurons, known to the inventors of this application, suggests that olfactory mucosal mesenchymal stem cells can be transformed into nerve cells. Olfactory mucosal mesenchymal stem cells possess multipotent differentiation potential, belong to the same ectoderm cell type as olfactory ensheathing cells, and differentiation of olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells does not require crossing germ layers.

[0005] Olfactory mucosal mesenchymal stem cells (OLMSCs) do not require repeated harvesting; a single nasal mucosal tissue sample can yield a large number of OLMSCs. Therefore, OLMSCs can be considered a stable source of olfactory ensheathing cells.

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

[0007] In view of this, the purpose of this application is to provide a culture medium and culture method for inducing olfactory mucosal mesenchymal stem cells into olfactory sheath cells.

[0008] This application provides a cell culture medium for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. The cell culture medium is based on DMEM / F12 and also includes transferrin, insulin, hydrocortisone, sodium selenate, N-2 supplement, and NT3.

[0009] This application also provides a method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells, comprising the following steps: culturing olfactory mucosal mesenchymal stem cells using the aforementioned cell culture medium.

[0010] This application relates to an olfactory ensheathing cell fluid prepared by the above method, wherein the olfactory ensheathing cells express olfactory ensheathing cell markers GFAP and S100β, and their purity reaches more than 65%. Attached Figure Description

[0011] Figure 1 shows the morphology of induced olfactory sheath cells under a light microscope.

[0012] Figure 2 shows the immunofluorescence detection of olfactory ensheathing cell expression markers.

[0013] Figure 3 shows the flow cytometry detection of olfactory ensheathing cell expression markers. Detailed Implementation

[0014] The technical solution of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0015] This application provides a cell culture medium for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. The cell culture medium is based on DMEM / F12 and also includes transferrin, insulin, hydrocortisone, sodium selenate, N-2 supplement, and NT3.

[0016] In an exemplary embodiment, the cell culture medium is based on DMEM / F12 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.

[0017] In another exemplary embodiment, the cell culture medium is based on DMEM / F12 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.

[0018] DMEM / F12 basal medium 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. Olfactory mesenchymal stem cells (MMSCs) require sufficient energy for morphological and functional transformation during induced differentiation. The iron ions transported by transferrin can maintain iron homeostasis inside and outside the cell, promote cell growth and proliferation, and prevent the formation of oxygen free radicals in the extracellular matrix. Simultaneously, transferrin may also participate in cellular signal transduction processes, indirectly regulating cell differentiation. Insulin plays an important role in cellular metabolism, promoting the uptake of glucose and amino acids, lipid production, transport of monovalent cations and phosphates, synthesis of proteins and nucleic acids, and cell proliferation. For the induction of olfactory mesenchymal stem cells into olfactory ensheathing cells, insulin can promote the uptake of nutrients such as glucose, providing sufficient energy and material basis for cell differentiation. Furthermore, insulin can regulate cellular protein synthesis, helping cells construct new protein structures, which is essential for morphological and functional transformation. Hydrocortisone is an adrenocortical hormone with anti-inflammatory and immunomodulatory effects, helping to maintain cellular homeostasis and prolong cell lifespan. In the induction of olfactory mesenchymal stem cells into olfactory ensheathing cells, hydrocortisone may regulate gene expression. It can also regulate cellular inflammatory responses, reducing inflammatory interference during cell culture and promoting stable cell differentiation. Sodium selenate contains selenium, a cofactor for glutathione peroxidase and other important enzymes, possessing antioxidant properties that protect cells from oxidative stress damage. N-2 supplement is a cell culture additive primarily used for nerve cell culture. It contains various components such as saturated transferrin, recombinant insulin, and progesterone, which provide the nutrients and signaling molecules necessary for cell growth, promoting cell survival, proliferation, and differentiation, and facilitating the transformation of olfactory mesenchymal stem cells into olfactory ensheathing cells. NT3, or neurotrophin-3, is a member of the neurotrophic factor family and an important component in promoting nerve cell growth and development. During the induction of olfactory mucosal mesenchymal stem cells (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 intracellular signaling pathways that participate in regulating various cellular processes such as cell proliferation, survival, and differentiation, thus helping 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 their differentiation into olfactory ensheathing cells.

[0019] This application also provides a method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells, comprising the following steps: culturing olfactory mucosal mesenchymal stem cells using the aforementioned cell culture medium.

[0020] In one exemplary embodiment, the method includes the following steps:

[0021] (1) Culture olfactory mucosal mesenchymal stem cells to generation P2;

[0022] (2) Digest the cells with pancreatic enzymes and centrifuge;

[0023] (3) Remove the supernatant from the centrifuge tube and collect the precipitate;

[0024] (4) Mix the cell precipitate with the cell culture medium to obtain a mixed solution;

[0025] (5) Inoculate the mixed solution evenly into the cell plate and place it in an incubator for culture;

[0026] (6) After culturing for 1 day, remove the supernatant and floating cells, add the cell culture medium as described above, and change the medium regularly thereafter; after culturing for 14 consecutive days, olfactory sheath cells are obtained.

[0027] In one embodiment, the cell plate is a six-well plate, and the inoculation volume in the cell plate is 2 mL / well.

[0028] In one embodiment, in step (2), the concentration of the pancreatic enzyme is 0.25% (w / v), the digestion time is 30 seconds, the centrifugation speed is 1000 rpm, and the centrifugation time is 5 minutes.

[0029] In one embodiment, in step (4), the seeding density of cells in the cell culture medium is 10. 5 per ml.

[0030] In one embodiment, in step (1), olfactory mucosal mesenchymal stem cells are cultured in olfactory mucosal mesenchymal stem cell culture medium to a cell confluence of 70-80% to obtain P2 generation. The olfactory mucosal mesenchymal stem cell culture medium consists of DMEM / F12 and 10% (v / v) fetal bovine serum.

[0031] Compared with the prior art, this application has the following beneficial effects:

[0032] This application provides a culture medium and method for inducing olfactory mucosal mesenchymal stem cells into olfactory ensheathing cells. The culture medium used in this application has widely available raw materials and clearly defined components. Following the culture method provided in this application, mesenchymal stem cells can be induced into olfactory ensheathing cells in stages. This method is simple, safe, and efficient. The induced cells express the olfactory ensheathing cell markers GFAP and S100β, and their purity reaches over 65%, effectively solving the problem of olfactory ensheathing cell source and showing promising application prospects in the field of nerve repair.

[0033] To further illustrate this application, the following detailed description is provided through the embodiments and accompanying drawings. All raw materials used in the following embodiments of this application are commercially available products.

[0034] Example 1: Preparation of olfactory ensheathing cell induction culture medium

[0035] The formulation is based on DMEM / F12 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-2 supplement (100X) 0.3 mL / L, and NT3 0.03 μg / L.

[0036] Example 2: Preparation of olfactory mucosal mesenchymal stem cells

[0037] (1) Nasal mucosa donors sign informed consent forms, exclude infectious diseases such as syphilis, AIDS and respiratory viruses, and use chloramphenicol eye drops in the nose for 3 days, 3 times a day, 3 drops each time;

[0038] (2) After nasal disinfection and anesthesia, the superior turbinate and the upper outer part of the middle turbinate mucosa tissue are grasped with ethmoid sinus forceps. The collected human olfactory mucosa samples are cleaned. The cleaning preferably includes washing the collected olfactory mucosa samples with physiological saline solution 3 times until the bloodstains are washed away.

[0039] (3) Cut the cleaned human olfactory mucosa sample into pieces smaller than 1 mm using sterile ophthalmic scissors. 3 Size of tissue blocks;

[0040] (4) The tissue block and mesenchymal stem cell culture medium were thoroughly mixed and planted in a cell culture flask and cultured in an incubator at 37°C and 5% CO2. After changing the medium and passaged regularly, olfactory mucosal mesenchymal stem cells were obtained.

[0041] The mesenchymal stem cell culture medium consisted of DMEM / F12 and 10% (v / v) fetal bovine serum.

[0042] Example 3: Preparation and Detection of Olfactory Ensheathing Cells

[0043] The specific steps are as follows:

[0044] (1) The obtained olfactory mucosal mesenchymal stem cells were cultured to the P2 generation with 70-80% cell confluence.

[0045] (2) Digest the above cells with 0.25% (w / v) trypsin for 30 seconds and centrifuge at 1000 rpm for 5 minutes;

[0046] (3) Remove the supernatant from the centrifuge tube and collect the precipitate;

[0047] (4) Mix the cell pellet with olfactory ensheathing cell culture medium at 10 5 Mix thoroughly at a density of / ml;

[0048] (5) Inoculate the above mixed solution evenly into a six-well plate, 2 mL / well, and incubate in a CO2 humidification incubator at 37℃;

[0049] (6) After culturing for 1 day, remove the supernatant and floating cells, and add olfactory ensheathing cell culture medium again.

[0050] (7) Change the culture medium every two days thereafter, and change half of the medium each time. After culturing for 14 days, olfactory ensheathing cells can be obtained, as shown in Figure 1.

[0051] (8) Collect cells and detect olfactory ensheathing cell markers using immunofluorescence, see Figure 2;

[0052] (9) Collect cells and use flow cytometry to detect olfactory ensheath cell markers, as shown in Figure 3. The purity is above 65%.

[0053] Further explanation: Through extensive and long-term experimental research, the optimal formulation of the olfactory ensheathing cell induction culture 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-2 supplement 0.1–0.5 mL / L, and NT3 0.01–0.05 μg / L. Within this range, the effects described in Example 3 can be obtained.

[0054] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. Various modifications to these embodiments will be readily 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 this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded 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 and also includes transferrin, insulin, hydrocortisone, sodium selenate, N-2 supplement, and NT3.

2. 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 and also 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.

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 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-2 supplement 0.3 mL / L, and NT3 0.03 μg / L.

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

5. The method according to claim 4, characterized in that, Includes the following steps: (1) Culture olfactory mucosal mesenchymal stem cells to generation P2; (2) Digest the cells with pancreatic enzymes and then centrifuge; (3) Remove the supernatant from the centrifuge tube and collect the precipitate; (4) Mix the cell precipitate with the cell culture medium according to any one of claims 1 to 3 to obtain a mixed solution; (5) Inoculate the mixed solution evenly into the cell plate and place it in an incubator for culture; (6) After culturing for 1 day, remove the supernatant and floating cells, and add the cell culture medium as described in any one of claims 1 to 3. Change the medium regularly thereafter; after culturing for 14 consecutive days, olfactory sheath cells are obtained.

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 the trypsin is 0.25 w / v%, the digestion time is 30 seconds, the centrifugation speed is 1000 rpm, and the centrifugation time is 5 minutes.

8. The method according to claim 5, characterized in that, In step (4), the seeding density of cells in the cell culture medium is 10. 5 per mL.

9. The method according to claim 5, characterized in that, In step (1), olfactory mucosal mesenchymal stem cells are cultured in olfactory mucosal mesenchymal stem cell culture medium to a cell confluence of 70-80% to obtain P2 generation. The olfactory mucosal mesenchymal stem cell culture medium consists of DMEM / F12 and 10v / 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, and the olfactory ensheathing cells express the olfactory ensheathing cell markers GFAP and S100β.

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