In-vitro culture method of olfactory ensheathing cells

Abstract

The invention relates to the technical field of cell proliferation, in particular to an in-vitro culture method of olfactory ensheathing cells. The method comprises the following steps: performing mixed culture on primary olfactory ensheathing cells and fibroblasts in a primary olfactory ensheathing cell culture solution, and when the growth confluence degree of the cells subjected to mixed culture is 70-90%, continuing culture and inhibiting the growth of the fibroblasts to obtain proliferated olfactory ensheathing cells; the number percentage of the fibroblasts accounts for 75-80% of the total number of the cells subjected to mixed culture. According to the method, the natural characteristics of the olfactory ensheathing cells can be maintained, the proliferation speed of the olfactory ensheathing cells is remarkably increased, the obtained olfactory ensheathing cells also have the advantages of high purity and strong activity, and an efficient cell source is provided for repairing nerve injury.

Description

Technical Field

[0001] The present invention relates to the technical field of cell proliferation, and in particular to an in vitro culture method of olfactory ensheathing cells. Background Art

[0002] Olfactory ensheathing cells (OECs) are a special type of glial cells that are functionally intermediate between Schwann cells and oligodendrocytes, and have neurotrophic, glial hyperplasia inhibition, scar formation, and sheathing effects. They provide a suitable microenvironment for axon growth and have strong migration characteristics, making them one of the ideal candidate cells for promoting central nervous system regeneration.

[0003] Olfactory ensheathing cells play an important role in nerve repair, promoting nerve regeneration and improving injury repair. However, existing olfactory ensheathing cell culture technologies have problems such as low proliferation efficiency and poor cell activity, which limits their potential in clinical applications. In order to overcome these challenges, it is necessary to develop a technology that can efficiently proliferate olfactory ensheathing cells in vitro to meet the needs of large-scale cell preparation. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an in vitro culture method for olfactory ensheathing cells.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides an in vitro culture method of olfactory ensheathing cells, wherein primary olfactory ensheathing cells and fibroblasts are mixed and cultured in a primary olfactory ensheathing cell culture solution, and when the growth confluence of the mixed cultured cells is 70% to 90%, the culture is continued and the growth of the fibroblasts is inhibited to obtain proliferated olfactory ensheathing cells;

[0007] Wherein, the number percentage of the fibroblasts accounts for 75% to 80% of the total number of cells in the mixed culture.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the method for inhibiting the growth of the fibroblasts is to continue culturing the mixed cultured cells in a culture medium containing menadione.

[0010] Furthermore, during the continued culture, the culture medium containing menadione is replaced every 2 to 3 days, the continued culture time is 12 to 15 days, and after the completion of the continued culture, the purity of the proliferated olfactory ensheathing cells is 97% to 99%.

[0011] Furthermore, in the culture medium containing menadione, the molar concentration of menadione is 450-550 nM.

[0012] Furthermore, the culture medium containing menadione also includes fetal bovine serum, penicillin, streptomycin and DMEM / F-12 basic culture fluid.

[0013] Further, the mixed culture comprises the following steps:

[0014] S1. obtaining a single cell suspension containing the primary olfactory ensheathing cells and the fibroblasts;

[0015] S2. detecting the number of the primary olfactory ensheathing cells and the fibroblasts;

[0016] S3. Culture and incubate the single cell suspension on a culture plate.

[0017] Furthermore, in step S3, the culture plate is a 6-well culture plate coated with poly-lysine.

[0018] Furthermore, during the culture, 2 mL of the primary olfactory ensheathing cell culture medium was added to each well of the culture plate.

[0019] Furthermore, the components of the primary olfactory ensheathing cell culture medium include fetal bovine serum, penicillin, streptomycin and DMEM / F-12 basic culture medium.

[0020] Furthermore, in step S1, nasal mucosal tissue fragments are first obtained, and then digested and filtered to obtain the single cell suspension.

[0021] The beneficial effects of the present invention are:

[0022] (1) The in vitro culture method of olfactory ensheathing cells of the present invention maintains the mixed culture ratio of olfactory ensheathing cells and fibroblasts, which can not only maintain the natural characteristics of olfactory ensheathing cells, but also significantly enhance the proliferation rate of olfactory ensheathing cells;

[0023] (2) The in vitro culture method of olfactory ensheathing cells of the present invention inhibits the growth of fibroblasts when the confluence of the mixed cultured cells reaches a certain level, thereby effectively ensuring the purity and activity of the olfactory ensheathing cells;

[0024] (3) The in vitro culture method of olfactory ensheathing cells of the present invention uses menadione to inhibit the growth of fibroblasts, which not only has a good inhibitory effect, but also does not affect the continued proliferation and activity of olfactory ensheathing cells;

[0025] (4) The in vitro culture method of olfactory ensheathing cells of the present invention provides an efficient cell source for repairing nerve damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The in vitro culture method of olfactory ensheathing cells of the present invention is a schematic diagram of the experimental process of Example 2;

[0027] Figure 2 In the in vitro culture method of olfactory ensheathing cells of the present invention, in Example 2, the cell staining results in the single cell suspension are: Figure 2 Figure a is the staining picture of cell nucleus dye. Figure 2 Middle b is the staining picture of olfactory ensheathing cells. Figure 2 The middle c is the staining picture of universal cell stain. Figure 2 The middle d is the coincident coloring picture;

[0028] Figure 3 The in vitro culture method of olfactory ensheathing cells of the present invention, in Example 2, is a statistical result diagram of the number of olfactory ensheathing cells;

[0029] Figure 4 In the in vitro culture method of olfactory ensheathing cells of the present invention, in Example 2, the cell activity test results are: Figure 4 The figure a in the middle is a living cell picture. Figure 4 Middle b is a picture of dead cells;

[0030] Figure 5 The in vitro culture method of olfactory ensheathing cells of the present invention is a fluorescence electron microscopic image of olfactory ensheathing cell-specific proteins of olfactory ensheathing cells treated with menadione in Example 2. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] The in vitro culture method of olfactory ensheathing cells of the present invention comprises: mixing primary olfactory ensheathing cells and fibroblasts in a primary olfactory ensheathing cell culture medium; when the growth confluence of the mixed cultured cells is 70% to 90%, that is, when the cell amount in a T25 culture flask is about 1 million, continuing to culture and inhibiting the growth of fibroblasts to obtain proliferated olfactory ensheathing cells; wherein the number percentage of fibroblasts accounts for 75% to 80% of the total number of cells in the mixed culture.

[0033] The in vitro culture method of the olfactory ensheathing cells of the present invention maintains the mixed culture ratio of the olfactory ensheathing cells and fibroblasts, ensuring that they occupy 75% to 80% of the total number of cells in the subsequent culture process. This mixed culture not only maintains the natural characteristics of the olfactory ensheathing cells, but also significantly enhances the proliferation rate of the olfactory ensheathing cells, providing an efficient cell source for the repair of nerve damage.

[0034] When olfactory ensheathing cells are cultured under high-purity conditions, although they can maintain a stable ratio, the proliferation rate will drop significantly. Under high-purity culture conditions (such as 98% or above), when olfactory ensheathing cells are cultured alone, the proportion of their components will not change significantly. This is because most non-olfactory ensheathing cells (such as fibroblasts) and impurities have been removed after purification, and the olfactory ensheathing cells maintain their state as a single type. The presence of fibroblasts can usually secrete certain cytokines (such as FGF, EGF) or provide physical support, which helps promote the proliferation and expansion of olfactory ensheathing cells. Under high-purity conditions, due to the lack of these exogenous supports, olfactory ensheathing cells will enter a slower proliferation mode.

[0035] In the above-mentioned single culture method, since olfactory ensheathing cells lack growth-promoting signals from heterogeneous cells (such as fibroblasts), the cells are prone to approaching a "contact inhibition" state or undergoing a longer cell cycle, making it difficult to achieve large-scale expansion. In addition, under high-purity conditions, the metabolic needs of cells may be difficult to fully meet, which will further limit the expansion capacity.

[0036] In the in vitro culture method of the present invention, the method for inhibiting the growth of fibroblasts is to continue culturing the mixed cultured cells in a culture medium containing menadione.

[0037] Menquinone (commonly known as vitamin K2) is a fat-soluble vitamin and an important member of the vitamin K family. It plays a major role in human bone health and blood coagulation. It is a natural compound that is widely found in fermented foods and certain animal products. Menquinone can inhibit the growth of fibroblasts.

[0038] The chemical formula of menadione is shown in formula (1):

[0039]

[0040] Here, the value of n is 1 to 6.

[0041] Preferably, during the continued culture, the culture medium containing menadione is replaced every 2 to 3 days, and the continued culture time is 12 to 15 days. After the completion of the continued culture, the purity of the proliferated olfactory ensheathing cells is 97% to 99%. It can be seen that the in vitro culture method of olfactory ensheathing cells of the present invention has a fast proliferation rate of olfactory ensheathing cells, and the obtained olfactory ensheathing cells have high purity, and have good application prospects.

[0042] Preferably, in the specific process of continued cultivation, the molar concentration of menadione in the culture medium containing menadione is 450-550 nM.

[0043] Further preferably, the molar concentration of menadione is 500 nM.

[0044] Preferably, the culture medium containing menadione also includes fetal bovine serum, penicillin, streptomycin and DMEM / F-12 basic culture fluid.

[0045] The mixed culture of the present invention comprises the following steps:

[0046] S1. Obtain a single cell suspension containing primary olfactory ensheathing cells and fibroblasts.

[0047] S2. Detect the number of primary olfactory ensheathing cells and fibroblasts.

[0048] The specific detection method is to use immunofluorescence labeling technology, use specific antibodies to label OECs (such as p75^NTR^) and fibroblasts (such as Thy-1) respectively, combine with fluorescent dyes for labeling, observe under a fluorescence microscope and count using image analysis software (such as ImageJ).

[0049] S3. Culture and incubate the single cell suspension on a culture plate.

[0050] Preferably, the culture plate is a 6-well culture plate coated with poly-lysine. Coating with poly-lysine (PLL) can effectively enhance the cell's wall adhesion properties.

[0051] Preferably, during the culture, 2 mL of primary olfactory ensheathing cell culture medium is added to each well of the culture plate.

[0052] Preferably, the components of the primary olfactory ensheathing cell culture medium include fetal bovine serum, penicillin, streptomycin and DMEM / F-12 basic culture medium.

[0053] Further preferably, the specific composition of the primary olfactory ensheathing cell culture medium is as follows: the volume of DMEM / F-12 basic culture medium is 100%, the volume percentage of fetal bovine serum is 10%, the concentration of penicillin in the culture medium is 50 I.U. / mL, and the concentration of streptomycin is 50 μg / mL.

[0054] The in vitro culture method of olfactory ensheathing cells of the present invention can be applied to samples in various forms. For example, primary olfactory ensheathing cells and fibroblasts can be obtained, and the two can be prepared into a cell suspension according to the cell number ratio of the present invention, and then subsequently cultured.

[0055] For another example, nasal mucosal tissue can be obtained, minced, and then subjected to pre-treatment steps such as digestion and filtration to obtain a single cell suspension containing primary olfactory ensheathing cells and fibroblasts. In this case, after the pre-treatment, it is necessary to first detect the cell number ratio of primary olfactory ensheathing cells and fibroblasts in the single cell suspension. When the cell number ratio obtained by the detection meets the requirements of the present invention, the culture is continued. When the cell number ratio obtained by the detection does not meet the requirements of the present invention, the cell number can be adjusted.

[0056] Specifically, if the proportion of OECs is low, the recovery rate of OECs can be improved by re-obtaining nasal mucosal samples or optimizing the separation and processing steps. If the proportion still does not meet the requirements, the original tissue sample should be reprocessed and the digestion, separation or purification steps should be optimized.

[0057] In addition, the above-mentioned nasal mucosa tissue can be human nasal mucosa tissue or animal (mouse) nasal mucosa tissue, and its source can be purchased, donated by volunteers, etc.

[0058] The present invention is illustrated below by means of specific examples.

[0059] In the following examples, all culture plates and flasks used must be coated with poly-lysine (PLL) before starting cell culture. The coating method is as follows: first, thaw a 50 μL aliquot of sterile frozen poly-lysine and then add it to 6 mL of sterile water. For a 6-well culture plate, add 1 mL of diluted poly-lysine to each well and rotate to evenly coat the surface. For a T25 culture flask, add 3 mL of poly-lysine and rotate to evenly coat. For a T75 culture flask, add 75 μL of poly-lysine to 5 mL of sterile water and coat.

[0060] After coating, incubate the plates or flasks at 37°C for 4 to 6 hours or overnight. Before use, the poly-lysine must be removed and rinsed twice with 1 mL of HBSS to ensure that all residues are removed, as poly-lysine is toxic to cells. Coated plates or flasks can be stored at 37°C for up to 4 days, adding 2 mL of HBSS when storing.

[0061] 1-2 hours before biopsy, thaw a 300 μL aliquot of 10 mg / mL Dispase II and add to 700 μL of DMEM / F-12 to dilute to 2.4 units / mL. Store Dispase at 2-8°C for up to 4 hours.

[0062] Example 1 Extraction and culture of mouse olfactory ensheathing cells

[0063] (1) Olfactory mucosa collection: Select healthy C57BL / 6 mice that are 6 to 8 days old and anesthetize them with isoflurane or other suitable anesthetics. Ensure that the mice have completely lost their pain response before starting the operation. Under sterile conditions, fix the mice on the dissection table, perform a craniotomy, and collect the olfactory mucosa from the nasal cavity.

[0064] (2) Processing and digestion: The removed olfactory mucosal tissue should be cut into pieces of about 1 mm using scissors under sterile conditions. 3 Under sterile conditions, place each biopsy specimen in a sterile disposable dish with 2 mL of culture medium to rinse the specimen. Process each biopsy separately, move to a new dish, and rinse again. View and clean the specimen under a dissecting microscope to remove blood, mucus, and cartilage.

[0065] The chopped tissue pieces were transferred to an enzyme solution containing 1 mg / mL collagenase I and 0.25% trypsin, incubated at 37°C for 30 to 60 minutes, and gently pipetted and mixed every 10 minutes to promote tissue decomposition. After digestion, an equal amount of culture medium containing 10% fetal bovine serum (FBS) was added to terminate the enzyme reaction. Subsequently, the digestion product was filtered through a 40 μm cell strainer to remove incompletely decomposed tissue pieces. The filtered cell suspension was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in fresh culture medium to obtain a single cell suspension.

[0066] (3) Mixed culture: Inoculate a single cell suspension into a culture plate, such as a culture dish coated with polylysine, to enhance the cell's wall adhesion properties. Culture cells in an incubator at 37°C and 5% CO2, and change the culture medium regularly. The culture medium consists of a DMEM / F12 mixed culture medium supplemented with 10% FBS and 1% penicillin-streptomycin. After several days of culture, the olfactory ensheathing cells will gradually adhere to the wall and the cell purity and characteristics can be further verified by morphological observation, immunofluorescence labeling, or flow cytometry.

[0067] The culture was continued until the cells reached about 80% confluence, at which point the cells could be passaged (the cell number was about 1 million).

[0068] (4) Continued culture: The primary olfactory ensheathing cells were subcultured to a new T25 culture dish and continued to be cultured using a culture medium containing Menquinone. The final molar concentration of Menquinone in the culture medium was 500 nM.

[0069] During the continued culture period, the culture medium containing 500 nM Menquinone was replaced every 3 days, and the number of olfactory ensheathing cells was continuously monitored during the continued culture period. After 12 days of continued culture, the purity of the olfactory ensheathing cells was 97%.

[0070] Example 2 Extraction and culture of human olfactory ensheathing cells

[0071] The experimental process of this embodiment is as follows Figure 1 shown.

[0072] (1) Extraction of olfactory ensheathing cells: Under general anesthesia, the biopsy tissue donor lies supine on the operating table with the head slightly tilted back and fixed. The nasal cavity is rinsed with sterile isotonic sodium chloride solution and then disinfected with hydrogen peroxide solution. The surgery uses a submucosal periosteal microsurgery method through the nose and septum, as close as possible to the front end of the cribriform plate and the position of the nasal septum (where the human olfactory ensheathing cells are concentrated). Under the guidance of intranasal surgical tools and skull navigation instruments, the unilateral nasal mucosa is removed. Finally, 2 to 3 samples with an average area of ​​0.5 cm are collected. 2 of mucosal fragments.

[0073] (2) Treatment and digestion: The treated samples were transferred to a culture dish containing 1 mL of 2.4 units / mL Dispase II and incubated at 37°C for 20 min. The basal tissue was then scraped and minced into approximately 0.05 mm 2 Digest the tissue with 1 mg / mL collagenase H and incubate at 37°C until the tissue is completely dissociated into a single cell suspension.

[0074] The number of cells in the single cell suspension was detected, and the results were as follows Figure 2 and 3 As shown. Figure 2 and Figure 3 It can be seen that in the single cell suspension obtained by the treatment and digestion in this example, the percentage of olfactory ensheathing cells is 25%.

[0075] (3) Mixed culture: The obtained single cell suspension was transferred to a 6-well culture plate coated with PLL, 2 mL of culture medium was added to each well, and the cells were incubated at 37° C. The culture medium consisted of a DMEM / F12 mixed culture medium supplemented with 10% FBS and 1% penicillin-streptomycin.

[0076] After 24 hours, check the attachment of cells. If the cells are well attached, continue culturing and replace the medium. Cultivate until the cells reach about 80% confluence, at which point the cells can be passaged (cell quantity is about 1 million).

[0077] (4) Continued culture: The primary olfactory ensheathing cells were subcultured into a new t25 culture dish, and Menquinone was added to the culture medium. The final molar concentration of Menquinone was 500 nM. Menquinone can inhibit the growth of fibroblasts. Thereafter, the culture medium containing 500 nM Menquinone was replaced every 3 days, and the number of olfactory ensheathing cells was continuously monitored during the continued culture. After monitoring, the purity of the olfactory ensheathing cells was 98% after 14 days of continued culture.

[0078] The cell activity test obtained in this example includes the detection of the number of living cells and the detection of the expression of the olfactory ensheathing cell-specific protein (P75nrt).

[0079] (1) Detection of viable cell count: First, prepare an incubator and set the temperature to 22°C. Thaw the reagents required for the experiment (Calcein AM and BOBO-3 Iodide). Next, transfer the liquid in the green reagent bottle to the red reagent bottle with a total volume of 1 mL, and then shake to mix for 45 seconds. Subsequently, mix 1 mL of the prepared reagent with 1 mL of cell culture medium. Aliquot 100-150 μL of the diluted reagent into the wells of a 96-well plate (the wells contain olfactory ensheathing cells). Incubate the 96-well plate at 22°C for 15 minutes, then replace with fresh culture medium to complete the operation.

[0080] like Figure 4 As shown, the test results show that after continued cultivation in the culture medium containing Menquinone, the ratio of the number of live cells to the number of dead cells of the olfactory ensheathing cells is 99.59%, indicating that the cultured olfactory ensheathing cells have good activity.

[0081] (2) P75^NTR expression: The expression of P75nrt was detected by fluorescent immunolabeling. The test results are as follows Figure 5 As shown. Figure 5 It can be seen that the olfactory ensheathing cells still express P75^NTR after Menquinone treatment.

[0082] Comparative Example 1 Mixed culture of olfactory ensheathing cells in different proportions

[0083] In this comparative example, primary olfactory ensheathing cells and fibroblasts were mixed and cultured. The source of the olfactory ensheathing cells was the same as that in Example 2, and the treatment process was also the same as that in Example 2.

[0084] This comparative example includes two experimental groups. In the first experimental group, the percentage of fibroblasts accounts for 50% of the total number of cells in the mixed culture. In the second experimental group, the percentage of fibroblasts accounts for 90% of the total number of cells in the mixed culture.

[0085] The two experimental groups were subjected to mixed culture and continued culture, respectively, and the specific process was the same as that in Example 2.

[0086] The number and content of olfactory ensheathing cells were monitored during the culture process. After 14 days of culture, the purity of the olfactory ensheathing cells in the first experimental group was 10%, and the purity of the olfactory ensheathing cells in the second experimental group was 3% after 14 days of culture.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for culturing olfactory ensheathing cells in vitro, characterized in that: Mixing primary olfactory ensheathing cells and fibroblasts in a primary olfactory ensheathing cell culture medium, and when the growth confluence of the mixed cultured cells reaches 70% to 90%, continuing the culture and inhibiting the growth of the fibroblasts to obtain proliferated olfactory ensheathing cells; Wherein, the number percentage of the fibroblasts accounts for 75% to 80% of the total number of cells in the mixed culture.

2. The in vitro culture method of olfactory ensheathing cells according to claim 1, characterized in that: The method for inhibiting the growth of the fibroblasts is to continue culturing the mixed cultured cells in a culture medium containing menadione.

3. The in vitro culture method of olfactory ensheathing cells according to claim 2, characterized in that: During the continued culture, the culture medium containing menadione is replaced every 2 to 3 days. The continued culture time is 12 to 15 days. After the continued culture is completed, the purity of the proliferated olfactory ensheathing cells is 97% to 99%.

4. The in vitro culture method of olfactory ensheathing cells according to claim 3, characterized in that: In the culture medium containing menadione, the molar concentration of menadione is 450-550 nM.

5. The in vitro culture method of olfactory ensheathing cells according to claim 4, characterized in that: The culture medium containing menadione also includes fetal bovine serum, penicillin, streptomycin and DMEM / F-12 basic culture fluid.

6. The in vitro culture method of olfactory ensheathing cells according to any one of claims 1 to 5, characterized in that: The mixed culture comprises the following steps: S1. obtaining a single cell suspension containing the primary olfactory ensheathing cells and the fibroblasts; S2. detecting the number of the primary olfactory ensheathing cells and the fibroblasts; S3. Culture and incubate the single cell suspension on a culture plate.

7. The in vitro culture method of olfactory ensheathing cells according to claim 6, characterized in that: In step S3, the culture plate is a 6-well culture plate coated with poly-lysine.

8. The in vitro culture method of olfactory ensheathing cells according to claim 7, characterized in that: During the culture, 2 mL of the primary olfactory ensheathing cell culture medium was added to each well of the culture plate.

9. The in vitro culture method of olfactory ensheathing cells according to claim 8, characterized in that: The components of the primary olfactory ensheathing cell culture fluid include fetal bovine serum, penicillin, streptomycin and DMEM / F-12 basic culture fluid.

10. The in vitro culture method of olfactory ensheathing cells according to claim 6, characterized in that: In step S1, nasal mucosal tissue fragments are first obtained, and then digested and filtered to obtain the single cell suspension.

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