3D culture and purification method for olfactory ensheathing cells and olfactory ensheathing cell spheres
The olfactory sheath cells were incubated on low-adsorption culture plates by 3D culture purification method, and the olfactory sheath cells were isolated and purified by hydrophobic surfaces, which solved the problem of insufficient olfactory sheath cells in the prior art, and achieved efficient olfactory sheath cell purification and proliferation effects.
Patent Information
- Application Number
- CN202510061229.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-18
AI Technical Summary
In the existing olfactory sheath cell culture method, the first generation of olfactory sheath cells are easily doped by other cells such as fibroblasts in mixed culture, resulting in insufficient purity of olfactory sheath cells, affecting their efficacy in central nervous system repair.
Using the 3D culture purification method, the mixture containing the first generation of olfactory sheath cells was isolated and cultured to form mixed cell spheres, and incubated on low-absorption culture plates. The hydrophobic surface was used to allow the olfactory sheath cells to grow concentratedly and fibroblasts were grown extra-grown, thereby achieving the isolation and purification of olfactory sheath cells.
It effectively improves the purity and survival rate of olfactory sheath cells, enhances their migration ability, and promotes research and medical applications related to olfactory sheath cells.
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Figure CN119842615A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell culture, and in particular to a 3D culture and purification method of olfactory ensheathing cells and an olfactory ensheathing cell spheroid. Background Art
[0002] Olfactory ensheathing cells are cell candidates that have attracted much attention in recent years in neural regeneration cell transplantation therapy. Olfactory ensheathing cells are a subtype of glial cells distributed in the olfactory mucosa and olfactory bulb, and can continue to help mammals regenerate olfactory neurons in adulthood. Studies have shown that when OECs are transplanted into animals with central and peripheral nerve injuries, they have therapeutic properties such as phagocytosis of cell debris and secretion of neurotrophic factors. In addition, OECs have been used in cutting-edge three-dimensional (3D) cell transplantation therapies. OECs show significant potential in 3D cell culture, where they can form large numbers of 3D grafts within 24 hours and have significantly higher cell viability. Combining OECs and 3D cell culture technology is expected to provide a rapid and effective treatment for patients with traumatic nerve injury in the near future.
[0003] However, the current culture method of OECs hinders the effectiveness of OEC-based therapy. In order to make OECs have a good proliferation rate, primary OECs are generally cultured in a mixed culture. At the same time, in some methods of obtaining primary OECs from the olfactory nervous system, other cells, especially olfactory nerve fibroblasts, are also mixed in. These cells will affect the effectiveness of OEC-based therapy.
[0004] Improving the purity of OECs is critical for cell therapy to promote central nervous system repair. Typically, the purification of OECs involves selective culture media or immunoubiquitination treatment, but this process is very challenging, so new methods are needed. For example, in a selective culture media method, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) are added to the culture media of OECs to increase the proportion of OECs in primary cell cultures. However, the yield of OECs obtained in selective culture media was insufficient, and the final OEC densities in NT3 and BDNF-containing media were 85.00±3.512 cells / mm, respectively. 2 and 169.0±1.528 cells / mm 2 .
[0005] In addition, immunoubiquitination cell isolation often results in decreased cell viability and unstable cell numbers at harvest. Without a robust purification method, culture results of dissociated olfactory mucosal preparations show that the content of OECs is less than 20%, which seriously affects the subsequent application of OECs. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a 3D culture and purification method for olfactory ensheathing cells and an olfactory ensheathing cell spheroid.
[0007] The technical solution of the present invention to solve the above technical problems is as follows:
[0008] The present invention provides a 3D culture and purification method for olfactory ensheathing cells, which comprises separating and culturing a mixed solution containing primary olfactory ensheathing cells to obtain a mixed cell sphere; incubating the mixed cell sphere on a low-adsorption culture plate to obtain a purified olfactory ensheathing cell sphere; and the surface of the low-adsorption culture plate is a hydrophobic surface.
[0009] Based on the above technical solution, the present invention can also be improved as follows.
[0010] Furthermore, the mixed cell suspension includes the primary olfactory ensheathing cells and fibroblasts, and the cell number ratio of the primary olfactory ensheathing cells to the fibroblasts is 20:80.
[0011] Furthermore, the hydrophobic surface is hydrophobic polystyrene.
[0012] Furthermore, the separation culture is to inoculate the mixed solution on a low-adsorption 3D cell culture dish for culture.
[0013] Furthermore, the culture medium for separation and culture and the culture medium for incubation are both complete culture medium, and the components of the complete culture medium include fetal bovine serum, penicillin, streptomycin and DMEM / F-12.
[0014] Furthermore, the separation and culture conditions are: culturing at 37° C. in an incubator containing 5% by volume of carbon dioxide for 24 to 48 hours.
[0015] Furthermore, the incubation condition is to move the mixed cell spheroids to the low-adsorption culture plate for culturing in an incubator at 37° C. and containing 5% by volume of carbon dioxide for 36 to 72 hours.
[0016] Furthermore, in the purified olfactory ensheathing cell spheroids, the number of olfactory ensheathing cells accounts for 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 purification method as described above.
[0018] The present invention also provides a nerve damage repair preparation, comprising the olfactory ensheathing cell spheroid as described above.
[0019] The beneficial effects of the present invention are:
[0020] (1) The 3D culture and purification method of olfactory ensheathing cells of the present invention uses a low-adsorption culture plate for incubation. Under this condition, the mixed cell spheroids formed have olfactory ensheathing cells concentrated in the inner part and fibroblasts growing outside, 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 the olfactory ensheathing cells of the present invention utilizes the high cell proliferation characteristics of the unpurified primary olfactory ensheathing cell culture to culture, separate and purify the cells, thereby obtaining more olfactory ensheathing cells and improving the culture efficiency of the olfactory ensheathing cells;
[0022] (3) The 3D culture and purification method of 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 can more effectively promote the research and medical applications related to olfactory ensheathing cells by providing purified olfactory ensheathing cells with good biological activity. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The experimental results of Example 2 of the 3D culture and purification method of olfactory ensheathing cells of the present invention are as follows: Figure 1 Middle a is the fluorescence signal diagram of the mixed cell spheroid formed by olfactory ensheathing cells in 3D culture after incubation for 48 hours. Figure 1 Figure b is a fluorescence signal diagram of the distribution of cell nuclei in 3D culture. Figure 1 Middle c is the distribution diagram of olfactory ensheathing cells expressing DsRed in 3D culture. Figure 1 Middle d is the staining image of the nucleus of olfactory ensheathing cells expressing DsRed in 2D cell culture. Figure 1 Middle e is the staining image of olfactory ensheathing cells expressing DsRed in 2D cell culture. Figure 1 Middle f is the staining picture of universal cell stain in 2D cell culture. Figure 1 Middle g is a superimposed staining image in 2D cell culture. Figure 1 Middle h is the ratio of olfactory ensheathing cells expressing DsRed in 2D and 3D cell culture;
[0025] Figure 2 In the 3D culture and purification method of olfactory ensheathing cells of the present invention, in Example 3, the live cell imaging of cells migrating from the mixed cell spheroid is shown. Figure 2 The image a in the middle is the DsRed channel image. Figure 2 Middle b is the bright field and DsRed channel imaging;
[0026] Figure 3 The 3D culture and purification method of olfactory ensheathing cells of the present invention, in Example 3, the cell migration result diagram, Figure 3 a in the middle is the overall fluorescence image of each cell. Figure 3 Figure b is the staining picture of cell nucleus dye. Figure 3 Middle c is the staining picture of olfactory ensheathing cells. Figure 3 Figure d is a schematic diagram of cell migration from a cell spheroid. Figure 3 Middle e is a comparison of the migration distances of olfactory ensheathing cells expressing DsRed;
[0027] Figure 4 In the 3D culture and purification method of olfactory ensheathing cells of the present invention, in Example 4, the live cell imaging diagram of the migration of cells from the mixed cell spheroid is shown. Figure 4 The image a in the middle is the DsRed channel image. Figure 4 Middle b is the bright field and DsRed channel imaging. Figure 4 Middle c is a schematic diagram of cell migration;
[0028] Figure 5 In the 3D culture and purification method of olfactory ensheathing cells of the present invention, the experimental results of the cell type composition attached to the surface of the culture plate in Example 4 are as follows: Figure 5 Figure a is a fluorescent image of cell distribution on the culture plate. Figure 5 Middle b is a comparison chart of the percentage of cells that do not express DsRed in the remaining cells in the culture plate;
[0029] Figure 6 In the 3D culture and purification method of olfactory ensheathing cells of the present invention, in Example 4, the distribution of olfactory ensheathing cells expressing DsRed in the mixed cell spheroids is shown in FIG. Figure 6 Middle a is the fluorescence distribution diagram of mixed cell spheroids. Figure 6 Figure b is the staining picture of cell nucleus dye. Figure 6 Middle c is the staining image of olfactory ensheathing cells expressing DsRed. Figure 6 Middle d is the distribution diagram of DsRed-expressing olfactory ensheathing cells before and after hydrophobic low-attachment incubation. Figure 6 Middle e is a comparison of the proportion of DsRed-expressing olfactory ensheathing cells before and after hydrophobic low-attachment incubation;
[0030] Figure 7 In the 3D culture and purification method of olfactory ensheathing cells of the present invention, the characteristics of the mixed cell spheroids in Example 5 are compared. Figure 7 Middle a is a 3D reconstructed image of mixed cell spheroids before low attachment incubation. Figure 7 Middle b is a 3D reconstructed image of mixed cell spheroids after low attachment incubation. Figure 7 Middle c is the horizontal diameter of the cell sphere before and after low attachment incubation. Figure 7 The middle d is the vertical diameter of the cell sphere before and after low attachment incubation. Figure 7 Middle e is the hemispherical thickness of the cell sphere before and after low attachment incubation. Figure 7 Middle f is the cell density of cell spheres before and after low attachment incubation;
[0031] Figure 8 The present invention is a method for 3D culture and purification of olfactory ensheathing cells. In Example 6, the expression of olfactory ensheathing cells expressing DsRed after purification is compared with the expression of olfactory ensheathing cell marker proteins. Figure 8 Middle a is the cell fluorescence distribution diagram in primary olfactory ensheathing cell culture. Figure 8 Middle b is a fluorescence distribution diagram of cells after 3D culture purification using the present invention. DETAILED DESCRIPTION
[0032] 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.
[0033] The 3D culture and purification method of olfactory ensheathing cells of the present invention comprises separating and culturing a mixed cell fluid containing primary olfactory ensheathing cells to obtain a mixed cell sphere; incubating the mixed cell sphere on a low-adsorption culture plate to obtain a purified olfactory ensheathing cell sphere; and the surface of the low-adsorption culture plate is a hydrophobic surface.
[0034] The 3D culture and purification method of olfactory ensheathing cells of the present invention directly uses the mixed cell suspension for the culture of mixed cell spheroids. In the mixed cell spheroids formed under this condition, the olfactory ensheathing cells will grow concentratedly inside, and the fibroblasts will grow outside. Fibroblasts are the main contaminating cells in the culture of olfactory ensheathing cells. The process of purifying olfactory ensheathing cells is to remove excess fibroblasts. Then, the cell spheroids are incubated on a low-adsorption culture plate. Due to different cell adhesion abilities, other cells such as fibroblasts will first migrate from the mixed cell spheroids and adhere to the surface of the low-adsorption culture plate. This method can avoid the use of unstable cell markers for purification, and utilize 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 of olfactory ensheathing cells of the present invention can effectively improve the survival rate and migration ability of cells, thereby more effectively promoting research and medical applications related to olfactory ensheathing cells.
[0036] Preferably, after the culture purification is completed, the floating purified olfactory ensheathing cell spheres can be collected from the culture medium.
[0037] Preferably, the mixed cell suspension includes primary olfactory ensheathing cells and fibroblasts, and the cell number ratio of primary olfactory ensheathing cells to fibroblasts is 20:80.
[0038] Preferably, the hydrophobic surface is hydrophobic polystyrene (PS).
[0039] Further preferably, since the method of the present invention does not require cell culture with specific adherence properties, the surface of the culture dish is usually kept in its original state without adding a coating or performing surface modification.
[0040] Further preferably, the low adsorption culture plate is 24-well Polystyrene Clear FlatBottom Not Treated Cell Culture Plate, uncoated, non-micropatterned surface.
[0041] Preferably, the culture dish used for separation and culture is a low-adsorption 3D cell culture dish.
[0042] Further preferably, the low adsorption 3D cell culture dish is Spheroid Microplates.
[0043] Preferably, the culture medium for separation culture and the culture medium for incubation are both complete culture medium, and the components of the complete culture medium include fetal bovine serum, penicillin, streptomycin and DMEM / F-12.
[0044] More preferably, 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 culturing at 37° C. in an incubator containing 5% by volume carbon dioxide for 24 to 60 hours.
[0046] Preferably, the incubation condition is to culture on a low-adsorption culture plate in an incubator at 37° C. and containing 5% by volume carbon dioxide for 36 to 72 hours.
[0047] Preferably, in the purified olfactory ensheathing cell spheroids, the number of olfactory ensheathing cells accounts for 70% to 80% of the total number of cells.
[0048] The purified olfactory ensheathing cell spheroids of the present invention are prepared by the above-mentioned 3D culture purification method.
[0049] The nerve damage repair preparation of the present invention comprises the purified olfactory ensheathing cell spheroids as described above.
[0050] Further preferably, the nerve damage repair preparation also includes a pharmaceutically acceptable excipient.
[0051] The present invention is illustrated below by means of specific examples.
[0052] Example 1 3D Culture and Purification of Olfactory Ensheathing Cells
[0053] In this example, the method of the present invention is used to perform 3D culture and purification of olfactory ensheathing cells.
[0054] The primary olfactory ensheathing cells of this embodiment are derived from nasal mucosal tissue, and the pre-treatment method after acquisition is as follows:
[0055] The nasal mucosal tissue was rinsed with sterile phosphate buffered saline (PBS) to remove blood and impurities, and then cut into pieces of about 1 mm 3 Size. Subsequently, 0.25% trypsin was used for digestion for 15 minutes, a single cell suspension was formed by gentle pipetting, and filtered with a 100 μm cell strainer to remove undigested tissue debris. After the cell suspension was centrifuged (1000 rpm, 5 minutes), the supernatant was discarded, the cells were resuspended in complete medium, and inoculated into a 6-well plate at a density of 50,000-100,000 cells per well, and 2 mL of complete medium was added. The culture dish was placed in an incubator at 37°C and 5% CO2 for culture.
[0056] The culture medium of this example uses a DMEM / F-12 mixture (1:1) as the base, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After 48 hours of primary olfactory ensheathing cells culture, the culture medium is replaced for the first time, and then the medium is replaced every 2-3 days to maintain cell activity.
[0057] The mixed cell suspension of primary olfactory ensheathing cells and fibroblasts was inoculated into a low-adsorption 3D cell culture dish. Cultured in an incubator at 37°C and 5% CO2 for 48 hours. During the culture process, the primary olfactory ensheathing cells will form mixed cell spheres. In the mixed cell spheres formed under this condition, the olfactory ensheathing cells will grow concentrated inside the sphere, and the fibroblasts will grow outside.
[0058] The mixed cell spheroids are placed in a low-adsorption 24-well plate for further incubation. In this example, a hydrophobic polystyrene (PS) culture plate is used. 24-well Polystyrene Clear FlatBottom Not Treated Cell Culture Plate, uncoated, non-micropatterned surface.
[0059] During the incubation process, the fibroblasts growing outside the cell spheroid will migrate out of the cell spheroid before the olfactory ensheathing cells inside. At this time, separating the olfactory ensheathing cells that are still aggregated together 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 culture
[0061] In this example, 2D and 3D cell culture purification were performed on primary olfactory ensheathing cells, respectively. The sample source and pretreatment were the same as in Example 1, and the 2D culture process and 3D culture purification process were the same as in Example 1, except that a conventional 96-well cell culture plate was used in the 2D culture process, and a low-adhesion culture plate was used in the 3D culture purification.
[0062] The olfactory ensheathing cells used in this example are genetically modified by the S100β-DsRed promoter to express fluorescence. In the olfactory system, the olfactory ensheathing cells are cells that highly express S100β in the olfactory system. The use of S100β-DsRed can accurately identify the olfactory ensheathing cells.
[0063] After 5 days of incubation, the mixed cells were seeded into low-adhesion culture plates and regular 96-well cell culture plates, respectively. After 48 hours of separation and culture, the proportion of olfactory ensheathing cells in the mixed cell spheroids obtained by 2D and 3D cell culture was analyzed. The 3D position of the fluorescent signal (cyan and DsRed) was detected by Imaris software, cyan for nuclear staining (Hoechst), and red for olfactory ensheathing cells expressing DsRed. The scale bar is 100 μm.
[0064] like Figure 1 As can be seen in a, the mixed cells formed a mixed cell spheroid. Figure 1 Middle b shows the distribution of nuclei of olfactory ensheathing cells expressing DsRed and other cells that do not express DsRed in cell spheres during 3D culture purification. Figure 1 Middle (c) shows the distribution of olfactory ensheathing cells expressing DsRed in mixed cell spheroids during 3D culture purification. Figure 1 Figures d to g show the distribution of OECs expressing DsRed and other cells not expressing DsRed after incubation in a conventional 96-well cell culture plate for 48 hours in 2D culture. Figure 1 There was no significant difference in the proportion of OECs expressing DsRed in 2D and 3D environments. The purity of OECs ranged from 20% to 30%.
[0065] The above analysis process involved three batches of primary OECs culture and nine cell spheroids.
[0066] Example 3 Cell Spreading from a Spheroid on a Negatively Charged Polystyrene Surface
[0067] This example investigates how fibroblasts diffuse from mixed cell spheroids in 3D cell culture purification to the surface of a common cell culture plate, which is a negatively charged polystyrene surface, and also investigates the migration distance and distribution of olfactory ensheathing cells expressing DsRed.
[0068] In this example, the same sample and culture process as in Example 1 were used to culture mixed cell spheroids, each of which contained 100,000 cells.
[0069] The resulting mixed cell spheroids were transferred to a 24-well plate on a negatively charged polystyrene surface. After 12 hours, fibroblasts migrated from the mixed cell spheroids to the surrounding area. Figure 2 Figure 3 shows the migration of fibroblasts from a mixed cell spheroid. In the first 24 hours of incubation, cells that do not express DsRed migrate out of the spheroid first. After 24 hours, OECs that express DsRed begin to detach from the spheroid. Thus, there is a 12-hour delay in the migration of OECs expressing DsRed from a mixed cell spheroid onto a negatively charged polystyrene surface compared to cells that do not express DsRed.
[0070] After the mixed cell spheroids were transferred to the negatively charged polystyrene surface and incubated for 3 days, fibroblasts migrated out of the mixed cell spheroids and formed a 2D cell culture surface.
[0071] Figure 3 The image scale bar is 500 μm. Figure 3 Middle a shows the fluorescence distribution of cells migrating from the cell sphere after 72 hours of incubation. The left panel shows the distribution of DsRed-expressing OECs and cells that do not express DsRed. Hoechst is the nuclear staining result; OECs expressing DsRed are red; and cell membranes are magenta. Figure 3 Panels b and c show the distribution of OECs expressing DsRed and cells not expressing DsRed. Figure 3 (d) is a schematic diagram of measuring the distance between DsRed-expressing OECs and the center of the 2D cell culture surface. Figure 3 Middle (e) shows the migration distance of DsRed-expressing OECs in nine replicate groups.
[0072] The median migration distance of DsRed-expressing OECs was measured to be between 450 μm and 1000 μm. DsRed cells occupied the entire inner area of the cell culture. Therefore, after all cells migrated from the 3D cell spheroids to the 2D surface, OECs expressing DsRed and cells that did not express DsRed (fibroblasts) formed a staggered structure on the negatively charged polystyrene surface.
[0073] Example 4 Effect of hydrophobic polystyrene surface on cell migration
[0074] This example further evaluated how fibroblasts migrate out of mixed cell spheroids on low-adhesion hydrophobic polystyrene surfaces and the effect on the migration of cells that do not express DsRed and cells that express DsRed.
[0075] The samples and specific 3D culture purification process used in this example are the same as those in Example 1, and the primary olfactory ensheathing cells are also genetically modified to express fluorescence through the S100β-DsRed promoter.
[0076] Mixed cell spheroids were transferred to a 24-well plate on a low-adhesion hydrophobic polystyrene surface. Figure 4 Figure a shows live cell images after spheroid seeding. In the first 0-12 hours, no cells migrated out of the spheroid. By 24 hours, cells that did not express DsRed began to migrate out of the spheroid. By 36 hours, OECs that expressed DsRed remained in the center of the spheroid. Figure 4 Middle b shows the process of cell migration from a cell spheroid, in which cells not expressing DsRed and OECs expressing DsRed are separated by a low adhesion surface.
[0077] The composition of cell types attached to the polystyrene surface was measured after incubation of mixed cell spheroids in hydrophobic 24-well plates for 3 days.
[0078] Figure 5 The scale bar in a is 200 μm. Figure 5 Figure a shows cells expressing DsRed and cells not expressing DsRed remaining on the hydrophobic polystyrene surface. The right area is marked with cells expressing DsRed. The right area is marked with the distribution of cells on the hydrophobic surface. More than 90% of the remaining cells are cells that do not express DsRed. Therefore, the hydrophobic polystyrene surface is more attractive to cells that do not express DsRed than to olfactory ensheathing cells that express DsRed. At the same time, according to the above statistical results, it can be concluded that the purity (percentage of cell number) of olfactory ensheathing cells in the mixed cell spheroids is greater than 70%.
[0079] Figure 6 Figures a, b, and c show the distribution of the overall cell population and DsRed-expressing OECs. After incubation with hydrophobic low-attachment polystyrene, the proportion of DsRed-expressing OECs increased significantly from 30% to 70% ( Figure 6 d and e), the scale bar of the images is 200 μm, the data are presented as mean ± standard error, n = 9, ****p < 0.0001, student-T-test.
[0080] This result is consistent with the fact that most cells not expressing DsRed migrated to the hydrophobic surface during incubation on low-attachment polystyrene.
[0081] Therefore, the hydrophobic polystyrene surface should have a stronger affinity for cells that do not express DsRed. When mixed cell spheroids were incubated on the hydrophobic polystyrene surface, cells that do not express DsRed attached to the surface faster than DsRed-expressing OECs. As a result, cells that do not express DsRed migrated out of the cell spheroids, while DsRed-expressing OECs remained in the mixed cell spheroids during the 3-day incubation.
[0082] It can be seen that the affinity of hydrophobic surface polystyrene to DsRed-expressing olfactory ensheathing cells is lower than that of negatively charged polystyrene surface. After the mixed cell spheroids were transferred to the hydrophobic surface polystyrene plate, the DsRed-expressing olfactory ensheathing cells were able to remain in the cell spheroids ( Figure 6 In contrast, cells that do not express DsRed adhere to hydrophobic surfaces ( Figure 5 ). It can be seen that the hydrophobic surface of polystyrene increases the attachment difference between the two types of cells.
[0083] Example 5 Size and shape changes of mixed cell spheroids
[0084] To further evaluate the morphological changes of the mixed cell spheroids, the present example used Imaris software to construct 3D reconstructions of the mixed cell spheroids before and after incubation with a low-attachment polystyrene surface.
[0085] The samples and specific 3D culture purification process used in this example are the same as those in Example 1, and the primary olfactory ensheathing cells are also genetically modified to express fluorescence through the S100β-DsRed promoter.
[0086] Figure 7 Characteristic images of mixed cell spheroids before and after low attachment incubation. The scale bar of the image is, and the side length α of the regular hexahedron is 50 μm. Data are expressed as mean ± standard error. n = 9, ****p < 0.0001, student T-test.
[0087] Representative images of cell spheroids are shown in Figures a and b, which vary in size and shape. The average and vertical diameters of the cell spheroids are approximately 483 μm, which is shorter than the 715 μm before incubation with low-attachment hydrophobic polystyrene ( Figure 7 c and d). The thickness and cell density of the spheroids did not change during the low attachment incubation period ( Figure 7 e and 7f).
[0088] Therefore, cells that do not express DsRed may migrate in the horizontal direction (x-axis) and vertical direction (y-axis). In addition, the separation of cells that do not express DsRed and OECs that express DsRed did not affect the cell density of the cell spheres.
[0089] Example 6 Expression of p75 neurotrophic factor receptor by olfactory ensheathing cells purified from 3D culture
[0090] p75 neurotrophin receptor (p75NTR) is the most commonly used olfactory ensheathing cell marker. In order to determine whether the DsRed-expressing olfactory ensheathing cells normally express p75NTR, this example also evaluates the p75 expression of olfactory ensheathing cells that migrated from the cell sphere after 7 days of culture. The samples and specific 3D culture purification process used in this example are the same as those in Example 1, and the primary olfactory ensheathing cells are also genetically modified to express fluorescence through the S100β-DsRed promoter.
[0091] The cells were fixed and immunolabeled for p75 7 days after primary OECs were isolated and cultured.
[0092] Figure 8 The colocalization of p75NTR and DsRed fluorescence is shown. Figure 8 Figure a 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 8 Middle b is a representative image of olfactory ensheathing cells purified by 3D culture of the present invention.
[0093] It can be seen that after 3D purification and culture, the OECs did not dedifferentiate during the culture, indicating that the OECs separated and purified by the method of the present invention maintained good biological activity and could be used in 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 principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for 3D culture and purification of olfactory ensheathing cells, characterized in that: The mixed solution containing primary olfactory ensheathing cells is separated and cultured to obtain mixed cell spheres; the mixed cell spheres are incubated on a low-adsorption culture plate to obtain purified olfactory ensheathing cell spheres; the surface of the low-adsorption culture plate is a hydrophobic surface.
2. The method for 3D culture and purification of olfactory ensheathing cells according to claim 1, characterized in that: The mixed solution includes the primary olfactory ensheathing cells and fibroblasts, and the cell number ratio of the primary olfactory ensheathing cells to the fibroblasts is 20:
80.
3. A 3D culture and purification method for 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 culture is to inoculate the mixed solution on a low-adsorption 3D cell culture plate for culture.
5. A method for 3D culture and purification of olfactory ensheathing cells according to any of claims 2 to 4, characterized in that: The culture medium for separation and culture and the culture medium for incubation are both complete culture medium, and the components of the complete culture medium include fetal bovine 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 are: culturing at 37° C. in an incubator containing 5% by volume carbon dioxide for 24 to 48 hours.
7. The method for 3D culture and purification of olfactory ensheathing cells according to claim 5, characterized in that: The incubation condition is to move the mixed cell spheroids to the low-adsorption culture plate for culturing in an incubator at 37° C. and containing 5% by volume of carbon dioxide for 36 to 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 spheres, the number of olfactory ensheathing cells accounts for 70% to 80% of the total number of cells.
9. An olfactory ensheathing cell spheroid, characterized in that: The 3D culture and purification method is used to prepare the 3D culture and purification method according to any one of claims 1 to 8.
10. A nerve damage repair preparation, characterized in that: Comprising the olfactory ensheathing cell spheroid as described in claim 9.