Method for 3D culture purification of olfactory ensheathing cells and olfactory ensheathing cell spheroids

Abstract

Provided are a method for 3D culture purification of olfactory ensheathing cells and olfactory ensheathing cell spheroids. The method comprises subjecting a mixed solution containing primary olfactory ensheathing cells to separation culture to obtain mixed cell spheroids; incubating the mixed cell spheroids on a low-adhesion culture plate to obtain purified olfactory ensheathing cell spheroids; the surface of the low-adhesion culture plate being a hydrophobic surface. The method can avoid the use of unstable cell markers, and effectively achieve the separation and purification of olfactory ensheathing cells. The purified olfactory ensheathing cells have good biological activity and can more effectively promote research and medical applications related to olfactory ensheathing cells.

Description

A method for 3D culture and purification of olfactory ensheathing cells and olfactory ensheathing cell spheroids Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically, to a method for 3D culture and purification of olfactory ensheathing cells and olfactory ensheathing cell spheroids. Background Technology

[0002] Olfactory ensheathing cells (OECs) have become a promising candidate for neural regeneration cell transplantation therapy in recent years. OECs belong to a subtype of glial cells distributed in the olfactory mucosa and olfactory bulb, and can continuously aid in the regeneration of olfactory neurons in mammals throughout adulthood. Studies have shown that when OECs are transplanted into animals with central and peripheral nerve injuries, they possess therapeutic properties, such as phagocytizing cell debris and secreting neurotrophic factors. Furthermore, OECs have been applied in cutting-edge three-dimensional (3D) cell transplantation therapy. OECs have demonstrated significant potential in 3D cell culture, forming large numbers of 3D grafts within 24 hours with significantly higher cell viability. Combining OECs with 3D cell culture technology is expected to provide a rapid and effective treatment option for patients with traumatic nerve injuries in the near future.

[0003] However, current olfactory ensheathing cell culture methods hinder the effectiveness of olfactory ensheathing cell-based therapies. To ensure a good proliferation rate for olfactory ensheathing cells, primary olfactory ensheathing cells are generally cultured using a mixed culture method. Furthermore, in some methods of obtaining primary olfactory ensheathing cells from the olfactory nervous system, other cells, particularly olfactory neurofibroblasts, are mixed in, all of which can affect the efficacy of olfactory ensheathing cell-based therapies.

[0004] Improving the purity of olfactory ensheathing cells (OECCs) is crucial for cell therapy that promotes the repair of the central nervous system. Typically, OECC purification involves selective culture media or immunoubiquitination, but this process is highly challenging and necessitates novel approaches. For example, in one selective culture media method, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) are added to the OECC culture medium to increase the proportion of OECCs in primary cell cultures. However, the yield of OECCs obtained in selective media is insufficient; the final OECC density in media containing NT3 and BDNF is 85.00 ± 3.512 cells / mm³, respectively. 2 and 169.0 ± 1.528 cells / mm 2 .

[0005] Furthermore, immunoubiquitination of cells typically leads to decreased cell viability and unstable cell numbers at harvest. Without robust purification methods, culture results of dissociated olfactory mucosa preparations show an olfactory ensheathing cell content of less than 20%, which severely impacts the subsequent application of olfactory ensheathing cells. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for 3D culture and purification of olfactory ensheathing cells and olfactory ensheathing cell spheroids.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides a 3D culture and purification method for olfactory ensheathing cells, which involves separating and culturing a mixture containing primary olfactory ensheathing cells to obtain mixed cell spheroids; incubating the mixed cell spheroids on a low-adsorption culture plate to obtain purified olfactory ensheathing cell spheroids; wherein the surface of the low-adsorption culture plate is hydrophobic.

[0009] Based on the above technical solution, the present invention can be further improved as follows.

[0010] Furthermore, the mixed cell suspension includes the primary olfactory ensheathing cells and fibroblasts, with a cell ratio of 20:80 between the primary olfactory ensheathing cells and the fibroblasts.

[0011] Furthermore, the hydrophobic surface is hydrophobic polystyrene.

[0012] Furthermore, the separation and culture involves inoculating the mixture onto a low-adsorption 3D cell culture dish for culture.

[0013] Furthermore, both the isolation culture medium and the incubation medium are complete culture media, and the components of the complete culture medium include bovine fetal serum, penicillin, streptomycin and DMEM / F-12.

[0014] Furthermore, the isolation and culture conditions are as follows: cultured at 37°C in an incubator containing 5% carbon dioxide by volume for 24–48 hours.

[0015] Furthermore, the incubation conditions are as follows: the mixed cell spheres are transferred to the low-adsorption culture plate for culture, and cultured at 37°C in an incubator containing 5% carbon dioxide by volume for 36–72 hours.

[0016] Furthermore, in the purified olfactory ensheathing cell spheroids, the number of olfactory ensheathing cells is 70% to 80% of the total number of cells.

[0017] The present invention also provides an olfactory ensheathing cell spheroid, which is prepared by the 3D culture and purification method described above.

[0018] The present invention also provides a nerve injury repair agent, comprising olfactory ensheathing cell spheroids as described above.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) The 3D culture and purification method for olfactory ensheathing cells of the present invention uses a low-adsorption culture plate for incubation. Under this condition, the mixed cell spheres formed will have olfactory ensheathing cells growing in the interior and fibroblasts growing on the exterior, thereby avoiding the use of unstable cell markers and effectively achieving the separation and purification of olfactory ensheathing cells.

[0021] (2) The 3D culture and purification method of olfactory ensheathing cells of the present invention utilizes the high cell proliferation characteristics of unpurified primary olfactory ensheathing cell cultures for culture, separation and purification, which can obtain more olfactory ensheathing cells and improve the culture efficiency of olfactory ensheathing cells.

[0022] (3) The 3D culture and purification method for olfactory ensheathing cells of the present invention can effectively improve the survival rate and migration ability of olfactory ensheathing cells;

[0023] (4) The 3D culture and purification method of olfactory ensheathing cells of the present invention has good biological activity, which can more effectively promote the research and medical application of olfactory ensheathing cells. Attached Figure Description

[0024] Figure 1 shows the experimental results of Example 2 of the 3D culture and purification method for olfactory ensheathing cells of the present invention. In Figure 1, a is the fluorescence signal diagram of the mixed cell spheroids formed by olfactory ensheathing cells after 48 hours of incubation in 3D culture; b is the fluorescence signal diagram of the distribution of cell nuclei in 3D culture; c is the distribution diagram of olfactory ensheathing cells expressing DsRed in 3D culture; d is the staining diagram of the cell nuclei of olfactory ensheathing cells expressing DsRed in 2D cell culture; e is the staining diagram of olfactory ensheathing cells expressing DsRed in 2D cell culture; f is the staining diagram of the universal cell stain in 2D cell culture; g is the overlapping staining diagram in 2D cell culture; and h is the ratio diagram of olfactory ensheathing cells expressing DsRed in 2D and 3D cell cultures.

[0025] Figure 2 shows the 3D culture and purification method for olfactory ensheathing cells of the present invention. In Example 3, the live cell imaging image of cells migrating from the mixed cell spheres is shown. In Figure 2, a is the DsRed channel imaging image, and in Figure 2, b is the bright field and DsRed channel imaging image.

[0026] Figure 3 shows the 3D culture and purification method of olfactory ensheathing cells of the present invention. In Example 3, the cell migration results are shown in Figure 3a, which is the overall fluorescence image of each cell, Figure 3b is the staining image of the cell nucleus, Figure 3c is the staining image of olfactory ensheathing cells, Figure 3d is a schematic diagram of cell migration from the cell sphere, and Figure 3e is a comparison of the migration distance of olfactory ensheathing cells expressing DsRed.

[0027] Figure 4 shows the 3D culture and purification method of olfactory ensheathing cells of the present invention. In Example 4, the live cell imaging diagram of cells migrating from the mixed cell spheres is shown. In Figure 4a, it is a DsRed channel imaging diagram, in Figure 4b, it is a bright field and DsRed channel imaging diagram, and in Figure 4c, it is a schematic diagram of cell migration.

[0028] Figure 5 shows the 3D culture and purification method for olfactory ensheathing cells of the present invention. In Example 4, the experimental results of cell type composition attached to the surface of the culture plate are shown. Figure 5a is a fluorescence image of cell distribution on the culture plate, and Figure 5b is a comparison of the percentage of cells that do not express DsRed in the remaining cells of the culture plate.

[0029] Figure 6 illustrates the 3D culture and purification method for olfactory ensheathing cells of the present invention. In Example 4, the distribution of DsRed-expressing olfactory ensheathing cells in the mixed cell spheres is shown. Figure 6a shows the fluorescence distribution of the mixed cell spheres, Figure 6b shows the staining of the cell nuclei, Figure 6c shows the staining of DsRed-expressing olfactory ensheathing cells, Figure 6d shows the distribution of DsRed-expressing olfactory ensheathing cells before and after hydrophobic low-attachment incubation, and Figure 6e shows a comparison of the proportion of DsRed-expressing olfactory ensheathing cells before and after hydrophobic low-attachment incubation.

[0030] Figure 7 illustrates the 3D culture and purification method for olfactory ensheathing cells of the present invention. In Example 5, a comparison of the characteristics of the mixed cell spheres is shown in Figure 7a, which is a 3D reconstructed image of the mixed cell spheres before low-attachment incubation; b, which is a 3D reconstructed image of the mixed cell spheres after low-attachment incubation; c, which is the horizontal diameter of the cell spheres before and after low-attachment incubation; d, which is the vertical diameter of the cell spheres before and after low-attachment incubation; e, which is the hemispherical thickness of the cell spheres before and after low-attachment incubation; and f, which is the cell density of the cell spheres before and after low-attachment incubation.

[0031] Figure 8 illustrates the 3D culture and purification method for olfactory ensheathing cells of the present invention. In Example 6, the expression of olfactory ensheathing cells expressing DsRed after purification is compared with that of olfactory ensheathing cell marker proteins. Figure 8a shows the cell fluorescence distribution in the primary olfactory ensheathing cell culture, and Figure 8b shows the cell fluorescence distribution after purification using the 3D culture method of the present invention. Detailed Implementation

[0032] 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.

[0033] The 3D culture and purification method for olfactory ensheathing cells of the present invention involves separating and culturing a mixed cell slurry containing primary olfactory ensheathing cells to obtain mixed cell spheres; incubating the mixed cell spheres on a low-adsorption culture plate to obtain purified olfactory ensheathing cell spheres; the surface of the low-adsorption culture plate is hydrophobic.

[0034] The 3D culture and purification method for olfactory ensheathing cells of this invention involves directly culturing mixed cell suspensions into mixed cell spheroids. Under these conditions, olfactory ensheathing cells grow concentrated inside the mixed cell spheroids, while fibroblasts grow on the outside. Fibroblasts are the main contaminating cells in olfactory ensheathing cell culture. The purification process involves removing excess fibroblasts. The cell spheroids are then incubated on a low-adhesion culture plate. Due to their different cell adhesion abilities, other cells, such as fibroblasts, will migrate from the mixed cell spheroids and attach to the surface of the low-adhesion culture plate first. This method avoids the use of unstable cell markers for purification and utilizes the high cell proliferation characteristics of unpurified primary olfactory ensheathing cell cultures to harvest more olfactory ensheathing cells.

[0035] The 3D culture and purification method for olfactory ensheathing cells of the present invention can effectively improve the cell survival rate and migration ability, thereby more effectively promoting research and medical applications related to olfactory ensheathing cells.

[0036] Preferably, after the culture and purification are completed, the floating purified olfactory ensheathing cell spheroids can be collected from the culture medium.

[0037] Preferably, the mixed cell suspension includes primary olfactory ensheathing cells and fibroblasts, with a cell ratio of 20:80.

[0038] Preferably, the hydrophobic surface is hydrophobic polystyrene (PS).

[0039] Furthermore, since the method of the present invention does not require cell culture with specific adhesion characteristics, the surface of the culture dish is usually kept in its original state without the addition of coatings or surface modifications.

[0040] Further preferred, the low-adsorption culture plate is 24-well Polystyrene Clear Flat Bottom Not Treated Cell Culture Plate, uncoated, unpatterned surface.

[0041] Preferably, the culture dish used for isolation and culture is a low-adsorption 3D cell culture dish.

[0042] Further preferred, a low-adsorption 3D cell culture disc is... Spheroid Microplates.

[0043] Preferably, both the isolation culture medium and the incubation medium are complete culture media, and the components of the complete culture medium include bovine fetal serum, penicillin, streptomycin and DMEM / F-12.

[0044] Further preferred, the complete culture medium uses a DMEM / F-12 mixture (1:1) as a base and is supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0045] Preferably, the isolation and culture conditions are as follows: cultured at 37°C in an incubator containing 5% carbon dioxide by volume for 24 to 60 hours.

[0046] Preferably, the incubation conditions are as follows: culture on a low-adsorption culture plate and cultured at 37°C in an incubator containing 5% carbon dioxide by volume for 36 to 72 hours.

[0047] Preferably, the number of olfactory ensheathing cells in the purified olfactory ensheathing cell spheroids is 70% to 80% of the total number of cells.

[0048] The purified olfactory ensheathing cell spheroids of the present invention are prepared using the 3D culture purification method described above.

[0049] The nerve damage repair preparation of the present invention includes purified olfactory ensheathing cell spheroids as described above.

[0050] Further preferred formulations for repairing nerve damage also include pharmaceutically acceptable excipients.

[0051] The present invention will be illustrated by specific embodiments below.

[0052] Example 1: Purification of Olfactory Ensheathing Cells through 3D Culture

[0053] This embodiment uses the method of the present invention to perform 3D culture and purification of olfactory ensheathing cells.

[0054] The primary olfactory ensheathing cells in this embodiment were derived from nasal mucosa tissue, and the pretreatment method after acquisition was as follows:

[0055] After rinsing the nasal mucosa tissue with sterile phosphate-buffered saline (PBS) to remove blood and impurities, it was then minced to approximately 1 mm. 3Size. Subsequently, cells were digested with 0.25% trypsin for 15 minutes, forming a single-cell suspension by gentle pipetting, and filtered through a 100μm cell strainer to remove undigested tissue debris. The cell suspension was centrifuged (1000 rpm, 5 minutes), the supernatant was discarded, and the cells were resuspended in complete culture medium. Cells were seeded into 6-well plates at a density of 50,000–100,000 cells per well, with 2 mL of complete culture medium added. The plates were then incubated at 37°C in a 5% CO2 incubator.

[0056] In this example, the culture medium used was a 1:1 mixture of DMEM / F-12 as a base, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The medium was changed for the first time after 48 hours of culturing the primary olfactory ensheathing cells, and thereafter every 2-3 days to maintain cell viability.

[0057] A mixed cell suspension of primary olfactory ensheathing cells and fibroblasts was seeded into a low-absorption 3D cell culture dish. The cells were cultured at 37°C and 5% CO2 for 48 hours. During culture, the primary olfactory ensheathing cells formed mixed cell spheroids. Under these conditions, the olfactory ensheathing cells concentrated and grew inside the spheroids, while the fibroblasts grew on the outside.

[0058] The mixed cell spheroids were placed in a low-absorption 24-well plate for further incubation. This example uses a hydrophobic polystyrene (PS) culture plate. 24-well Polystyrene Clear Flat Bottom Not Treated Cell Culture Plate, uncoated, unpatterned surface.

[0059] During incubation, fibroblasts growing on the outside of the cell sphere will migrate out of the cell sphere before the olfactory ensheathing cells inside. Separating the olfactory ensheathing cells that are still clustered together at this time can achieve the purpose of purifying the olfactory ensheathing cells.

[0060] Example 2: Comparison of the proportion of olfactory ensheathing cells in 2D and 3D cell cultures

[0061] In this embodiment, primary olfactory ensheathing cells were cultured and purified in 2D and 3D. The sample source and pretreatment were the same as in Example 1. The 2D culture process and the 3D culture and purification process were the same as in Example 1. The difference was that a conventional 96-well cell culture plate was used in the 2D culture process, while a low-adhesion culture plate was used in the 3D culture and purification process.

[0062] The olfactory ensheathing cells used in this embodiment have been modified with transgenes that express fluorescence via the S100β-DsRed promoter. In the olfactory system, olfactory ensheathing cells are cells that highly express S100β. Using S100β-DsRed can accurately identify olfactory ensheathing cells.

[0063] After 5 days of incubation, the mixed cells were seeded into low-adhesion culture plates and conventional 96-well cell culture plates, respectively. After 48 hours of separation culture, the proportion of olfactory ensheathing cells in the mixed cell spheroids obtained from 2D and 3D cell cultures was analyzed. The 3D positions of fluorescence signals (cyan and DsRed) were detected using Imaris software, with cyan representing nuclear staining (Hoechst) and red representing olfactory ensheathing cells expressing DsRed. The scale bar was 100 μm.

[0064] As shown in Figure 1a, the mixed cells formed mixed cell spheroids. Figure 1b shows the distribution of nuclei of olfactory ensheathing cells expressing DsRed and other cells not expressing DsRed within the cell spheroids during 3D culture purification. Figure 1c shows the distribution of olfactory ensheathing cells expressing DsRed within the mixed cell spheroids during 3D culture purification. Figures 1d-g show the distribution of olfactory ensheathing cells expressing DsRed and other cells not expressing DsRed after 48 hours of incubation in a standard 96-well cell culture plate during 2D culture. Figure 1e shows that the proportion of olfactory ensheathing cells expressing DsRed was not significantly different between the 2D and 3D environments. The purity of the olfactory ensheathing cells ranged from 20% to 30%.

[0065] The above analysis involved three batches of primary olfactory ensheathing cell cultures and nine cell spheres.

[0066] Example 3: Cell diffusion from cell spheres on a negatively charged polystyrene surface

[0067] This embodiment investigated how fibroblasts diffuse from mixed cell spheroids in 3D cell culture purification to the surface of a conventional cell culture plate, which is a negatively charged polystyrene surface. It also investigated the migration distance and distribution of olfactory ensheathing cells expressing DsRed.

[0068] In this embodiment, the same sample and culture process as in Example 1 were used to culture mixed cell spheres, each containing 100,000 cells.

[0069] The resulting mixed cell spheroids were transferred to 24-well plates on negatively charged polystyrene surfaces. After 12 hours, fibroblasts migrated from the mixed cell spheroids to the surrounding area. Figure 2 shows the migration of fibroblasts from the mixed cell spheroids. During the initial 24-hour incubation, cells not expressing DsRed migrated out of the cell spheroids first. After 24 hours, olfactory ensheathing cells expressing DsRed began to separate from the cell spheroids. Therefore, compared to cells not expressing DsRed, olfactory ensheathing cells expressed DsRed showed a 12-hour delay in migrating from the mixed cell spheroids to the negatively charged polystyrene surface.

[0070] After being transferred to a negatively charged polystyrene surface, the mixed cell spheres were incubated for 3 days. After 3 days of incubation, fibroblasts migrated from the mixed cell spheres and formed a 2D cell culture surface.

[0071] The image scale bar in Figure 3 is 500 μm. Figure 3a shows the fluorescence distribution of cells migrating from the cell spheres after 72 hours of incubation. The left panel shows the distribution of olfactory ensheathing cells expressing DsRed and those not expressing DsRed. Hoechst staining results are shown. Figures 3b and 3c show the distribution of olfactory ensheathing cells expressing DsRed and those not expressing DsRed. Figure 3d is a schematic diagram of measuring the distance between DsRed-expressing olfactory ensheathing cells and the center of the 2D cell culture surface. Figure 3e shows the migration distance of DsRed-expressing olfactory ensheathing cells in nine replicates.

[0072] Measurements showed that the median migration distance of DsRed-expressing olfactory ensheathing cells ranged from 450 μm to 1000 μm. DsRed-expressing cells occupied the entire inner region of the cell culture. Therefore, after all cells migrated from the 3D cell spheres to the 2D surface, DsRed-expressing olfactory ensheathing cells and non-DsRed-expressing cells (fibroblasts) formed an interleaved structure on the negatively charged polystyrene surface.

[0073] Example 4: Effect of hydrophobic polystyrene surface on cell migration

[0074] This embodiment further evaluated how fibroblasts migrate from hybrid cell spheres on a low-adhesion hydrophobic polystyrene surface, and the impact on the migration of cells that do not express DsRed and cells that express DsRed.

[0075] The samples and specific 3D culture and purification process used in this embodiment are the same as in Example 1, and the primary olfactory ensheathing cells are also modified by transgenic modification that expresses fluorescence through the S100β-DsRed promoter.

[0076] Mixed cell spheroids were transferred into 24-well plates with a low-adhesion hydrophobic polystyrene surface. Figure 4a shows an image of live cells after seeding with the cell spheroids. During the initial 0–12 hours, no cells migrated from the cell spheroids. By 24 hours, cells not expressing DsRed began to migrate from the cell spheroids. By 36 hours, olfactory ensheathing cells expressing DsRed remained in the center of the cell spheroids. Figure 4b shows the process of cell migration from the cell spheroids, where cells not expressing DsRed and olfactory ensheathing cells expressing DsRed were separated by the low-adhesion surface.

[0077] After incubating mixed cell spheres in hydrophobic 24-well plates for 3 days, the cell type composition attached to the polystyrene surface was measured.

[0078] The scale bar in Figure 5a is 200 μm. Figure 5a shows DsRed-expressing cells and non-DsRed-expressing cells remaining on the hydrophobic polystyrene surface. The right-hand area is labeled as DsRed-expressing cells. The right-hand area is labeled as the cell distribution on the hydrophobic surface. More than 90% of the remaining cells are non-DsRed-expressing cells. Therefore, the hydrophobic polystyrene surface is more attractive to non-DsRed-expressing cells than to DsRed-expressing olfactory ensheathing cells. Furthermore, based on the above statistical results, the purity (cell percentage) of olfactory ensheathing cells in the mixed cell spheroids is greater than 70%.

[0079] Figures 6a, b, and c show the distribution of the overall cell population and olfactory ensheathing cells expressing DsRed. Incubation with hydrophobic, low-attachment polystyrene significantly increased the proportion of DsRed-expressing olfactory ensheathing cells from 30% to 70% (Figures 6d and e). The scale bar for the images is 200 μm. Data are expressed as mean ± standard error, n = 9, p < 0.0001, student-T-test.

[0080] This result is consistent with the fact that most cells that do not express DsRed migrate to hydrophobic surfaces during incubation on low-attachment polystyrene.

[0081] Therefore, hydrophobic polystyrene surfaces should have a stronger affinity for cells that do not express DsRed. When mixed cell spheres were incubated on hydrophobic polystyrene surfaces, cells that did not express DsRed attached to the surface faster than olfactory ensheathing cells that expressed DsRed. Consequently, cells that did not express DsRed migrated out of the cell spheres, while olfactory ensheathing cells that expressed DsRed remained in the mixed cell spheres during the 3-day incubation period.

[0082] It is evident that hydrophobic polystyrene surfaces exhibit a lower affinity for DsRed-expressing olfactory ensheathing cells than negatively charged polystyrene surfaces. After transferring mixed cell spheres to hydrophobic polystyrene plates, DsRed-expressing olfactory ensheathing cells remained within the spheres (Figure 6). In contrast, cells not expressing DsRed adhered to the hydrophobic surface (Figure 5). This demonstrates that hydrophobic polystyrene surfaces amplify the adhesion differences between the two cell types.

[0083] Example 5: Changes in the size and shape of mixed cell spheres

[0084] To further evaluate the morphological changes of the mixed cell spheres, this embodiment uses Imaris software to construct 3D reconstructions of the mixed cell spheres before and after incubation on a low-attachment polystyrene surface.

[0085] The samples and specific 3D culture and purification process used in this embodiment are the same as in Example 1, and the primary olfactory ensheathing cells are also modified by transgenic modification that expresses fluorescence through the S100β-DsRed promoter.

[0086] Figure 7 shows characteristic images of the mixed cell spheroids before and after low-attachment incubation. The image scale bar is [value missing], and the side length α of the hexahedron is 50 μm. Data are expressed as mean ± standard error. n = 9, ****p < 0.0001, student T-test.

[0087] Figures a and b show representative images of the cell spheroids, showing variations in size and shape. The average and vertical diameters of the cell spheroids were approximately 483 μm, shorter than the 715 μm before incubation with the low-attachment hydrophobic polystyrene (Figures c and d). The thickness and cell density of the cell spheroids did not change during the low-attachment incubation (Figures 7e and 7f).

[0088] Therefore, cells that do not express DsRed may migrate in both the horizontal (x-axis) and vertical (y-axis) directions. Furthermore, the separation of DsRed-non-expressing cells from DsRed-expressing olfactory ensheathing cells did not affect the cell density of the cell spheroids.

[0089] Example 6: Expression of p75 neurotrophic factor receptor in 3D cultured and purified olfactory ensheathing cells

[0090] p75 neurotrophic factor receptor (p75NTR) is the most commonly used marker for olfactory ensheathing cells. This example aims to determine whether DsRed-expressing olfactory ensheathing cells normally express p75NTR. Furthermore, this example also evaluates p75 expression in olfactory ensheathing cells that migrated from cell spheres after 7 days of culture. The samples used and the specific 3D culture and purification process in this example are the same as in Example 1, and the primary olfactory ensheathing cells were also modified with transgenes expressing fluorescence via the S100β-DsRed promoter.

[0091] Cells were fixed and p75 immunolabeled after 7 days of isolation and culture of primary olfactory ensheathing cells.

[0092] Figure 8 shows the colocalization of p75NTR and DsRed fluorescence. Figure 8a is a representative image of cells in primary olfactory ensheathing cell culture. The culture process uses conventional culture methods, especially the culture plate used is a conventional non-hydrophobic surface. Figure 8b is a representative image of olfactory ensheathing cells purified by 3D culture using the present invention.

[0093] As can be seen, after 3D purification and culture, the olfactory ensheathing cells did not dedifferentiate during culture, indicating that the olfactory ensheathing cells isolated and purified by the method of the present invention maintained good biological activity and can be used for subsequent applications.

[0094] 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 3D culture and purification of olfactory ensheathing cells, characterized in that, The mixture containing primary olfactory ensheathing cells was separated and cultured to obtain mixed cell spheroids; the mixed cell spheroids were incubated on a low-adsorption culture plate to obtain purified olfactory ensheathing cell spheroids; the surface of the low-adsorption culture plate was hydrophobic.

2. The method for 3D culture and purification of olfactory ensheathing cells according to claim 1, characterized in that, The mixture includes primary olfactory ensheathing cells and fibroblasts, with a cell ratio of 20:80 between the primary olfactory ensheathing cells and the fibroblasts.

3. The method for 3D culture and purification of olfactory ensheathing cells according to claim 2, characterized in that, The hydrophobic surface is hydrophobic polystyrene.

4. The method for 3D culture and purification of olfactory ensheathing cells according to claim 2, characterized in that, The separation and culture process involves inoculating the mixture onto a low-adsorption 3D cell culture dish for culture.

5. A method for 3D culture and purification of olfactory ensheathing cells according to any one of claims 2 to 4, characterized in that, Both the isolation culture medium and the incubation medium are complete culture media, and the components of the complete culture medium include bovine fetal serum, penicillin, streptomycin and DMEM / F-12.

6. The method for 3D culture and purification of olfactory ensheathing cells according to claim 5, characterized in that, The isolation and culture conditions were as follows: cultured at 37°C in an incubator containing 5% carbon dioxide by volume for 24–48 hours.

7. The method for 3D culture and purification of olfactory ensheathing cells according to claim 5, characterized in that, The incubation conditions are as follows: the mixed cell spheres are transferred to the low-adsorption culture plate and cultured at 37°C in an incubator containing 5% carbon dioxide by volume for 36–72 hours.

8. The method for 3D culture and purification of olfactory ensheathing cells according to claim 5, characterized in that, In the purified olfactory ensheathing cell spheroids, the number of olfactory ensheathing cells is 70% to 80% of the total number of cells.

9. An olfactory ensheathing cell spheroid, characterized in that, It was prepared using the 3D culture and purification method as described in any one of claims 1 to 8.

10. A nerve injury repair agent, characterized in that, Including the olfactory sheath cell spheroids as described in claim 9.

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