In vitro culture method for olfactory ensheathing cells

Abstract

The present invention relates to the technical field of cell proliferation, and specifically provides an in vitro culture method for olfactory ensheathing cells. The method comprises: co-culturing primary olfactory ensheathing cells with fibroblasts in a primary olfactory ensheathing cell culture medium; and when the co-cultured cells reach a confluence of 70%-90%, continuing culturing and inhibiting the growth of the fibroblasts, thereby obtaining the proliferated olfactory ensheathing cells, wherein the fibroblasts account for 75%-80% of the total number of cells in the co-culture. The method can maintain the native characteristics of the olfactory ensheathing cells and significantly enhance the proliferation rate of the olfactory ensheathing cells, and the obtained olfactory ensheathing cells further have the advantages of high purity and high viability, thereby providing an efficient cell source for repair of nerve injuries.

Description

An in vitro culture method for olfactory ensheathing cells Technical Field

[0001] This invention relates to the field of cell proliferation technology, and more specifically, to a method for in vitro culture of olfactory ensheathing cells. Background Technology

[0002] Olfactory ensheathing cells (OECs) are a special type of glial cell that functionally falls between Schwann cells and oligodendrocytes. They possess neurotrophic, glial proliferation-inhibiting, scar-forming, and sheath-forming functions. They provide a suitable microenvironment for axonal growth and exhibit strong migration properties, making them one of the ideal candidate cells for promoting central nervous system regeneration.

[0003] Olfactory ensheathing cells (OECCs) play a crucial role in nerve repair, promoting nerve regeneration and improving injury healing outcomes. However, existing OECC culture techniques suffer from low proliferation efficiency and poor cell viability, limiting their potential for clinical application. To overcome these challenges, a technique for efficiently proliferating OECCs in vitro is needed to meet the demands of large-scale cell preparation. Summary of the Invention

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

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

[0006] This invention provides an in vitro culture method for olfactory ensheathing cells, wherein primary olfactory ensheathing cells and fibroblasts are mixed and cultured in a primary olfactory ensheathing cell culture medium. When the confluence of the mixed cultured cells reaches 70% to 90%, the culture is continued and the growth of the fibroblasts is inhibited to obtain proliferated olfactory ensheathing cells.

[0007] The percentage of fibroblasts in the mixed culture is 75% to 80% of the total number of cells.

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

[0009] Furthermore, the method for inhibiting the growth of the fibroblasts involves culturing the mixed-cultured cells in a culture medium containing menaquinone.

[0010] Furthermore, during the continued culture period, the culture medium containing menaquinone is replaced every 2 to 3 days, and the continued culture period is 12 to 15 days. After the continued culture is completed, the purity of the proliferated olfactory ensheathing cells is 97% to 99%.

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

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

[0013] Furthermore, the mixed culture includes the following steps:

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

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

[0016] S3. The single-cell suspension is cultured and incubated on a culture plate.

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

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

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

[0020] Furthermore, in step S1, nasal mucosal tissue fragments are first obtained, and after digestion and filtration, the single-cell suspension is obtained.

[0021] The beneficial effects of this invention are as follows:

[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 cells in mixed culture reaches a certain degree, which can effectively ensure the purity and activity of 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 the repair of nerve damage. Attached Figure Description

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

[0027] Figure 2 shows the in vitro culture method of olfactory ensheathing cells of the present invention. In Example 2, the staining results of cells in single-cell suspension are shown. In Figure 2, a is the staining diagram of cell nuclear staining agent, b is the staining diagram of olfactory ensheathing cells, c is the staining diagram of universal cell staining agent, and d is the overlapping staining diagram.

[0028] Figure 3 shows the in vitro culture method of olfactory ensheathing cells of the present invention. In Example 2, the cell count of olfactory ensheathing cells is statistically analyzed.

[0029] Figure 4 shows the in vitro culture method of olfactory ensheathing cells of the present invention. In Example 2, the cell viability test results are shown. In Figure 4a, live cells are shown and dead cells are shown.

[0030] Figure 5 shows the in vitro culture method of olfactory ensheathing cells of the present invention. In Example 2, the fluorescence electron micrograph of olfactory ensheathing cells treated with menaquinone and the olfactory ensheathing cell-specific proteins is shown. Detailed Implementation

[0031] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0032] The in vitro culture method for olfactory ensheathing cells of the present invention involves co-culturing primary olfactory ensheathing cells and fibroblasts in a primary olfactory ensheathing cell culture medium. When the confluence of the co-cultured cells reaches 70% to 90%, i.e., when the cell count is approximately 1 million in a T25 culture flask, the culture is continued while the growth of fibroblasts is inhibited to obtain proliferated olfactory ensheathing cells. The percentage of fibroblasts in the co-cultured cells is 75% to 80% of the total number of cells.

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

[0034] While olfactory ensheathing cells maintain a stable proportion when cultured under high-purity conditions, their proliferation rate decreases significantly. Under high-purity (e.g., 98% or higher) conditions, the composition of olfactory ensheathing cells does not change significantly when cultured alone. This is because purification removes most non-olfactory ensheathing cells (such as fibroblasts) and impurities, allowing olfactory ensheathing cells to maintain their state as a single type. The presence of fibroblasts typically secretes certain cytokines (such as FGF and EGF) or provides 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 enter a slower proliferation mode.

[0035] The aforementioned method of culturing cells alone, due to the lack of growth-promoting signals from heterogeneous cells (such as fibroblasts), makes olfactory ensheathing cells prone to approaching a state of "contact inhibition" or undergoing a longer cell cycle, thus hindering large-scale expansion. Furthermore, under high-purity conditions, the metabolic needs of the cells may not be fully met, further limiting 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 menaquinone.

[0037] Menquinone (commonly known as vitamin K2) is a fat-soluble vitamin and an important member of the vitamin K family. It primarily functions in bone health and blood clotting. It is a natural compound widely found in fermented foods and certain animal products. Menquinone can inhibit fibroblast growth.

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

[0039] The value of n ranges from 1 to 6.

[0040] Preferably, during the continued culture period, the culture medium containing menaquinone is replaced every 2 to 3 days, and the culture period is 12 to 15 days. After the continued culture is completed, 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 obtains high purity olfactory ensheathing cells, which has good application prospects.

[0041] Preferably, in the specific process of the above-mentioned continued culture, the molar concentration of menaquinone in the culture medium containing menaquinone is 450-550 nM.

[0042] More preferably, the molar concentration of menaquinone is 500 nM.

[0043] Preferably, the culture medium containing menadione also includes fetal bovine serum, penicillin, streptomycin, and DMEM / F-12 basal culture medium.

[0044] The mixed culture of the present invention includes the following steps:

[0045] S1. Obtain a single-cell suspension containing primary olfactory ensheathing cells and fibroblasts.

[0046] S2. Detect the number of primary olfactory ensheathing cells and fibroblasts.

[0047] The specific detection method involves using immunofluorescence labeling technology to label OECs (such as p75^NTR^) and fibroblasts (such as Thy-1) with specific antibodies, combined with fluorescent dyes, and then observing them under a fluorescence microscope and counting them using image analysis software (such as ImageJ).

[0048] S3. Incubate and culture the single-cell suspension on a culture plate.

[0049] Preferably, the culture plate is a 6-well culture plate coated with poly-L-L, which can effectively enhance the cell adhesion properties.

[0050] Preferably, during culture, 2 mL of primary olfactory ensheathing cell culture medium is added to each well of the culture plate.

[0051] Preferably, the primary olfactory ensheathing cell culture medium comprises fetal bovine serum, penicillin, streptomycin, and DMEM / F-12 basal culture medium.

[0052] Further preferred, the primary olfactory ensheathing cell culture medium has the following composition: with the volume of DMEM / F-12 basal culture medium as 100%, the volume percentage of fetal bovine serum is 10%, and the concentration of penicillin in the culture medium is 50 I.U. / mL, and the concentration of streptomycin is 50 μg / mL.

[0053] The in vitro culture method for olfactory ensheathing cells of the present invention is applicable to various types of samples. For example, primary olfactory ensheathing cells and fibroblasts can be obtained and prepared into a cell suspension according to the cell number ratio of the present invention, and then subjected to subsequent culture.

[0054] For example, nasal mucosa tissue can be obtained, chopped, and then subjected to pretreatment steps such as digestion and filtration to obtain a single-cell suspension containing primary olfactory ensheathing cells and fibroblasts. In this case, after pretreatment, the cell ratio of primary olfactory ensheathing cells to fibroblasts in the single-cell suspension needs to be detected. If the detected cell ratio meets the requirements of this invention, the culture can continue; if the detected cell ratio does not meet the requirements of this invention, the cell number can be adjusted.

[0055] Specifically, if the proportion of olfactory ensheathing cells is low, the recovery rate of olfactory ensheathing cells can be improved by obtaining new nasal mucosa 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.

[0056] In addition, the aforementioned nasal mucosa tissue can be human nasal mucosa tissue or animal (mouse) nasal mucosa tissue, and its source can be through purchase, volunteer donation, etc.

[0057] The present invention will be illustrated by specific embodiments below.

[0058] In the following examples, all culture plates and flasks used must be coated with poly-L-lysine (PLL) before cell culture begins. The coating process involves first thawing a 50 μL aliquot of sterile frozen poly-L-lysine and then adding it to 6 mL of sterile water. For a 6-well culture plate, 1 mL of diluted poly-L-lysine is added to each well, and the plate is rotated to evenly coat the surface. For a T25 culture flask, 3 mL of poly-L-lysine is added and the plate is rotated to evenly coat the surface. For a T75 culture flask, 75 μL of poly-L-lysine is added to 5 mL of sterile water and then coated.

[0059] After coating, incubate the culture plate or flask at 37°C for 4 to 6 hours or overnight. Before use, poly-L-lysine must be removed and the plate or flask rinsed twice with 1 mL HBSS to ensure all residues are removed, as poly-L-lysine is toxic to cells. The coated culture plate or flask can be stored at 37°C for up to 4 days, with the addition of 2 mL HBSS during storage.

[0060] One to two hours before biopsy, thaw 300 μL of 10 mg / mL Dispase II aliquots and add them to 700 μL of DMEM / F-12, diluting to 2.4 units / mL. Store Dispase at 2–8°C for a maximum of 4 hours.

[0061] Example 1: Extraction and culture of mouse olfactory ensheathing cells

[0062] (1) Olfactory mucosa collection: Select healthy C57BL / 6 mice aged 6 to 8 days, anesthetize them with isoflurane or other suitable anesthetics, and begin the procedure after ensuring that the mice have completely lost their pain response. Under sterile conditions, fix the mice on the dissection table, perform craniotomy, and collect the olfactory mucosa from the nasal cavity.

[0063] (2) Processing and digestion: The extracted olfactory mucosa tissue should be cut into pieces of about 1 mm in size under aseptic conditions. 3Small pieces. Under aseptic conditions, each biopsy sample was placed in a sterile disposable culture dish containing 2 mL of culture medium to rinse the sample. Each biopsy tissue was processed separately, transferred to a new culture dish, and rinsed again. The samples were observed and cleaned using a dissecting microscope to remove blood, mucus, and cartilage.

[0064] The chopped tissue pieces were transferred to an enzyme solution containing 1 mg / mL collagenase I and 0.25% trypsin, and incubated at 37°C for 30 to 60 minutes, gently pipetting every 10 minutes to promote tissue breakdown. After digestion, an equal volume 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 filter to remove incompletely digested 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.

[0065] (3) Mixed culture: Single-cell suspensions were seeded into culture plates, such as those coated with polylysine, to enhance cell adhesion. Cells were cultured in an incubator at 37°C and 5% CO2, with the culture medium changed periodically. The culture medium consisted of a DMEM / F12 mixed medium supplemented with 10% FBS and 1% penicillin-streptomycin. After several days of culture, olfactory ensheathing cells gradually adhered and grew. Cell purity and characteristics could be further verified by morphological observation, immunofluorescence labeling, or flow cytometry.

[0066] Continue culturing until the cells reach approximately 80% confluence, at which point they can be passaged (the cell count is approximately 1 million).

[0067] (4) Continued culture: Primary olfactory ensheathing cells were passaged into new T25 culture dishes and cultured further using medium containing Menquinone. The final molar concentration of Menquinone in the medium was 500 nM.

[0068] During the continued culture period, the medium containing 500 nM Menquinone was changed every 3 days, and the olfactory ensheathing cell count was continuously monitored. After 12 days of continued culture, the purity of the olfactory ensheathing cells was 97%.

[0069] Example 2: Extraction and culture of human olfactory ensheathing cells

[0070] The experimental procedure of this embodiment is shown in Figure 1.

[0071] (1) Olfactory ensheathing cell extraction: Under general anesthesia, the biopsy donor lies supine on the operating table with their head slightly tilted back and fixed. The nasal cavity is rinsed with sterile isotonic sodium chloride solution, followed by disinfection with hydrogen peroxide solution. The surgery employs a transnasal, transseptal submucosal periosteal microsurgery technique, aiming as close as possible to the anterior end of the cribriform plate and the nasal septum (the location where olfactory ensheathing cells are concentrated). Guided by intranasal surgical instruments and a cranial navigation instrument, unilateral nasal mucosa is removed. Finally, 2 to 3 cells with an average area of ​​0.5 cm² are collected. 2 Mucosal fragments.

[0072] (2) Processing and digestion: The processed sample was transferred to a culture dish containing 1 mL of 2.4 units / mL Dispase II and incubated at 37°C for 20 minutes. Subsequently, the basal layer tissue was scraped and minced to approximately 0.05 mm. 2 Size. Tissue was digested with 1 mg / mL collagenase H and incubated at 37°C until the tissue was completely separated into a single-cell suspension.

[0073] The cell count in the single-cell suspension was detected, and the results are shown in Figures 2 and 3. As can be seen from Figures 2 and 3, the percentage of olfactory ensheathing cells in the single-cell suspension obtained from the treatment and digestion in this embodiment is 25%.

[0074] (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 incubated at 37°C. The culture medium consisted of DMEM / F12 mixed medium with 10% FBS and 1% penicillin-streptomycin added.

[0075] After 24 hours, check the cell attachment. If the cells are attached well, continue culturing and change the culture medium. Continue culturing until the cells reach approximately 80% confluence. Once the cells reach 80% confluence, they can be passaged (the cell count is approximately 1 million).

[0076] (4) Continued culture: Primary olfactory ensheathing cells were passaged into new T25 culture dishes, and Menquinone was added to the culture medium to a final molar concentration of 500 nM. Menquinone can inhibit fibroblast growth. The culture medium containing 500 nM Menquinone was then replaced every 3 days, and the olfactory ensheathing cell count was continuously monitored during continued culture. After 14 days of continued culture, the purity of the olfactory ensheathing cells was 98%.

[0077] The cell viability test obtained in this embodiment includes the detection of the number of viable cells and the detection of the expression of olfactory ensheathing cell-specific protein (P75nrt).

[0078] (1) Viable cell count detection: First, prepare an incubator and set the temperature to 22℃. Thaw the required reagents (Calcein AM and BOBO-3 Iodide). Next, transfer the liquid from the green reagent bottle to the red reagent bottle, with a total volume of 1 mL, and then vortex 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 (containing olfactory ensheathing cells). Incubate the 96-well plate at 22℃ for 15 minutes, then replace with fresh culture medium to complete the procedure.

[0079] As shown in Figure 4, the test results showed that after further culture in a medium containing Menquinone, the ratio of live to dead olfactory ensheathing cells was 99.59%, indicating that the cultured olfactory ensheathing cells had good activity.

[0080] (2) P75^NTR expression: The expression of P75^NTR was detected by fluorescent immunolabeling, and the results are shown in Figure 5. As can be seen from Figure 5, olfactory ensheathing cells treated with Menquinone still expressed P75^NTR.

[0081] Comparative Example 1: Mixed culture of olfactory ensheathing cells in different proportions

[0082] In this comparative example, primary olfactory ensheathing cells and fibroblasts were cultured together. The source of the olfactory ensheathing cells was the same as in Example 2, and the treatment process was also the same as in Example 2.

[0083] This comparative example includes two experimental groups. In the first experimental group, the percentage of fibroblasts accounted for 50% of the total number of cells in the mixed culture, and in the second experimental group, the percentage of fibroblasts accounted for 90% of the total number of cells in the mixed culture.

[0084] The two experimental groups were then mixed and cultured, and the specific process was the same as in Example 2.

[0085] The number of olfactory ensheathing cells was monitored during the culture process. Monitoring showed that after 14 days of continued culture, the purity of olfactory ensheathing cells in the first experimental group was 10%, while in the second experimental group it was 3%.

[0086] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro culture of olfactory ensheathing cells, characterized in that, Primary olfactory ensheathing cells and fibroblasts were co-cultured in primary olfactory ensheathing cell culture medium. When the confluence of the co-cultured cells reached 70%–90%, the cells were cultured further and the growth of the fibroblasts was inhibited to obtain proliferated olfactory ensheathing cells. The percentage of fibroblasts in the mixed culture is 75% to 80% of the total number of cells.

2. The method for in vitro culture 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 menaquinone.

3. The method for in vitro culture of olfactory ensheathing cells according to claim 2, characterized in that, During the continued culture period, the culture medium containing menaquinone is replaced every 2 to 3 days, and the continued culture period 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 method for in vitro culture of olfactory ensheathing cells according to claim 3, characterized in that, The molar concentration of menaquinone in the culture medium containing menaquinone is 450–550 nM.

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

6. A method for in vitro culture of olfactory ensheathing cells according to any one of claims 1 to 5, characterized in that, The mixed culture includes the following steps: S1. Obtain a single-cell suspension containing the primary olfactory ensheathing cells and the fibroblasts; S2. Detect the number of the primary olfactory ensheathing cells and the fibroblasts; S3. The single-cell suspension is cultured and incubated on a culture plate.

7. The method for in vitro culture 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-L-lysine.

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

9. The method for in vitro culture of olfactory ensheathing cells according to claim 8, characterized in that, The primary olfactory ensheathing cell culture medium consists of fetal bovine serum, penicillin, streptomycin, and DMEM / F-12 basal culture medium.

10. A method for in vitro culture of olfactory ensheathing cells according to claim 6, characterized in that, In step S1, nasal mucosal tissue fragments are first obtained, and after digestion and filtration, the single-cell suspension is obtained.

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