Method for inducing and culturing nerve cells
阅读说明：本技术 一种对神经细胞进行诱导培养的方法 (Method for inducing and culturing nerve cells ) 是由 陈刚 王国航 栗国贝 孙冰冰 于 2019-12-12 设计创作，主要内容包括：本发明公开了一种对神经细胞进行诱导培养的方法,包括如下步骤：1)制备神经细胞悬浮液；2)准备石墨烯薄膜,石墨烯薄膜的表面具有沟槽状结构的图案；3)以步骤2)获得的石墨烯薄膜为培养基底材料,将步骤1)获得的神经细胞悬浮液涂布于所述步骤2)获得的石墨烯薄膜具有沟槽状结构图案的表面,以获得装载神经细胞的石墨烯薄膜；4)将步骤3)获得的装载神经细胞的石墨烯薄膜置于培养箱中培养。本申请提供的神经细胞的诱导培养方法,通过将表面具有沟槽状结构石墨烯薄膜作为神经细胞的培养基底材料,使得神经细胞能够被诱导沿其若干个沟槽线性定向的生长,提高神经细胞培养效率。(The invention discloses a method for carrying out induction culture on nerve cells, which comprises the following steps: 1) preparing a neural cell suspension; 2) preparing a graphene film, wherein the surface of the graphene film is provided with a pattern with a groove-shaped structure; 3) coating the neural cell suspension obtained in the step 1) on the surface of the graphene film obtained in the step 2) with a groove-shaped structure pattern by taking the graphene film obtained in the step 2) as a culture substrate material to obtain a graphene film loaded with neural cells; 4) placing the graphene film loaded with the nerve cells obtained in the step 3) into an incubator for culture. According to the induction culture method of the nerve cells, the graphene film with the groove-shaped structure on the surface is used as a culture substrate material of the nerve cells, so that the nerve cells can be induced to grow in a linear orientation mode along a plurality of grooves of the nerve cells, and the culture efficiency of the nerve cells is improved.)
1. A method for inducing culture of neural cells, comprising the steps of:
1) preparing a neural cell suspension;
2) preparing a graphene film, wherein the surface of the graphene film is provided with a pattern with a groove-shaped structure;
3) coating the neural cell suspension obtained in the step 1) on the surface of the graphene film obtained in the step 2) with a groove-shaped structure pattern by taking the graphene film obtained in the step 2) as a culture substrate material to obtain a graphene film loaded with neural cells;
4) placing the graphene film loaded with the nerve cells obtained in the step 3) into an incubator for culture;
the graphene film in the step 2) is prepared by the following method: taking graphene oxide dispersion liquid with the sheet diameter of 2 mu m as a raw material, and performing suction filtration to prepare a graphene oxide film; preparing a silica gel template with a groove-shaped surface by using a reverse mold method, flatly laying the dried graphene oxide film on the silica gel template wetted by water, standing, airing and forming; and (3) reducing the formed graphene oxide film by using a reducing solution prepared from a hydrogen iodide solution and absolute ethyl alcohol, taking the film down, and cleaning.
2. The method according to claim 1, wherein the preparation method of the neural cell suspension in the step 1) comprises: the neural cells were collected in a culture solution, treated with an enzyme solution at 37 ℃ for 30 minutes, ground and centrifuged, and the neural cells were resuspended in the culture solution to obtain a neural cell suspension.
3. The method of claim 2, wherein the culture medium comprises 90% medium, 10% fetal bovine serum, 100U/ml penicillin, 100 μ g/ml streptomycin; the enzyme solution is phosphate buffered saline containing 5mg/ml protease and 1mg/mlI type collagenase.
4. The method according to claim 1, wherein the groove-like structure on the surface of the graphene film in step 2) comprises a plurality of linear grooves, and the width of each linear groove is 5-200 μm and the depth of each linear groove is 10-50 μm.
5. The method of claim 4, wherein the single linear grooves are adjacent to each other with linear protrusions therebetween, and the linear protrusions are hollow and pass through.
6. The method of claim 1, wherein the step 3) further comprises: applying an electrical stimulus to the graphene thin film loaded with neural cells.
7. The method according to claim 6, wherein the graphene film loaded with the nerve cells is externally connected with a constant current source of 0.1 mA.
8. The method of claim 1, wherein the surface of the graphene thin film with the groove-like pattern in the step 3) is further coated with a cell adhesion growth promoter.
9. The method of claim 1, wherein the step 4) further comprises: adding nerve growth factor and glial cell line-derived neurotrophic factor to the graphene film loaded with nerve cells.
10. The method of any one of claims 1-9, wherein the neural cells comprise dorsal root ganglion cells.
Technical Field
The invention belongs to the technical field of biological cell culture, and particularly relates to a method for carrying out induction culture on nerve cells.
Background
The directional differentiation and growth of nerve cells are of great significance for wound healing, nerve repair and the like. In the prior art, a cell culture dish is mostly adopted for cell culture, and when the cell culture dish is used, a cell suspension is injected into the culture dish and then placed in a carbon dioxide incubator for standing culture, so that adherent growth of cells is promoted. However, the cells grown by the above culture method are disordered and the growth speed can only depend on the cells, so that the method is obviously not suitable for experimental research on nerve cell culture and directional growth.
Graphene has a structure formed by passing sp through carbon atoms2The unique structure endows the graphene with unique physical and electrical properties, excellent mechanical properties, electrical conductivity, larger specific surface area, good biocompatibility and the like, and can be applied as a nerve repair material.
Currently, graphene has been used for cell culture in the prior art, such as CN208748112U and CN208776741U both utilize the thermal conductivity of graphene, and graphene is used as a heating film in a cell culture or observation system; CN10243304A provides a graphene substrate, which can be suitable for the growth of tumor cells, primary nerve cells and nerve stem cells; CN207877745U provides a biological cell culture vessel, the interface of which contacting cultured cells is provided with graphene glass. Patent documents CN110467177A and document [1] also provide a two-sided three-dimensional graphene scaffold, which can be used for differentiation of nerve cells, but the preparation method is very complicated and cumbersome, and is not easy to operate, and the obtained graphene scaffold has a very thin wall thickness, only a single layer or a few layers of graphene are present, and the precision is too small, which undoubtedly increases the difficulty of practical application (1 [ rapid, biological research on neural progenitor cells by controllable graphene scaffold [ D ].2019.5 ]).
Although all be used for cell culture with graphite alkene in above-mentioned scheme, still do not provide a quick convenient, the practicality is strong, can also impel the problem of nerve cell directional growth simultaneously.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide an induction culture method capable of promoting rapid growth of nerve cells, improving culture efficiency of nerve cells, and also capable of performing directional differentiation and linear growth of nerve cells, the method comprising the steps of:
1) preparing a neural cell suspension;
2) preparing a graphene film, wherein the surface of the graphene film is provided with a pattern with a groove-shaped structure;
3) coating the neural cell suspension obtained in the step 1) on the surface of the graphene film obtained in the step 2) with a groove-shaped structure pattern by taking the graphene film obtained in the step 2) as a culture substrate material to obtain a graphene film loaded with neural cells;
4) placing the graphene film loaded with the nerve cells obtained in the step 3) into an incubator for culture;
the graphene film in the step 2) is prepared by the following method: taking graphene oxide dispersion liquid with the sheet diameter of 2 mu m as a raw material, and performing suction filtration to prepare a graphene oxide film; preparing a silica gel template with a groove-shaped surface by using a reverse mold method, flatly laying the dried graphene oxide film on the silica gel template wetted by water, standing, airing and forming; and (3) reducing the formed graphene oxide film by using a reducing solution prepared from a hydrogen iodide solution and absolute ethyl alcohol, taking the film down, and cleaning.
According to the induction culture method of the nerve cells, the culture substrate made of the graphene material is non-toxic and harmless to the nerve cells, and plays a certain role in promoting the growth and culture of the nerve cells; meanwhile, the groove-shaped structure on the surface of the graphene film is beneficial to inducing cells to grow along the linear orientation of the groove structure. The preparation method of the graphene film utilizes the surface interface effect, realizes the controllable custom pattern manufacturing of the graphene film under the mesoscopic scale, and is simple and easy to operate.
Further, the preparation method of the neural cell suspension in the step 1) comprises the following steps: the neural cells were collected in a culture solution, treated with an enzyme solution at 37 ℃ for 30 minutes, ground and centrifuged, and the neural cells were resuspended in the culture solution to obtain a neural cell suspension.
Further, the culture medium comprises 90% of culture medium, 10% of fetal bovine serum, 100U/ml of penicillin, and 100. mu.g/ml of streptomycin; the enzyme solution is phosphate buffered saline containing 5mg/ml protease and 1mg/ml collagenase type I.
Further, the groove-like structure on the surface of the graphene film in step 2) includes a plurality of grooves, the width of each groove is 5-200 μm, preferably 10-100 μm, more preferably 15-100 μm, more preferably 20-50 μm, and the depth of each groove is 10-50 μm, preferably 20-50 μm, more preferably 20 μm.
Further, the thickness of the graphene film is 8-12 μm.
Further, linear protrusions are arranged between the adjacent single grooves, and the inner parts of the linear protrusions are hollow and penetrate through. Preferably, the height of the linear protrusions is 10 to 50 μm, preferably 15 to 40 μm, more preferably 20 to 30 μm, more preferably 20 to 25 μm, more preferably 20 μm; the width of the linear protrusions is 20 to 100. mu.m, preferably 20 to 80 μm, more preferably 20 to 60 μm, and still more preferably 20 to 50 μm.
The linear protruding structure can simplify the preparation method of the groove-shaped structure, for example, part of a lamellar layer of lamellar graphene with a certain thickness can extend in a protruding manner along the thickness direction of the film without etching the lamellar layer, and on the other hand, the linear protruding structure can be used for loading cell growth promoting factors or glial cell line-derived neurotrophic factors such as NGF, NT-3, NT-4, BDNF and the like, so that the growth and culture efficiency of nerve cells is improved. In addition, the size range of the groove-shaped structure is matched with the size of most cells, so that the nerve cells are favorably induced to directionally grow along the set direction of the groove.
Further, the step 3) further comprises: applying an electrical stimulus to the graphene thin film loaded with neural cells.
Further, the graphene film loaded with the nerve cells is externally connected with a constant current power supply of 0.1mA for applying electric stimulation.
The arrangement utilizes the conductivity of graphene, so that nerve cells placed in the graphene can be directly electrically stimulated to promote growth and differentiation of the nerve cells.
Further, the surface of the graphene film with the groove-shaped pattern in the step 3) is also coated with a cell adherence growth promoter so as to promote the adherence growth of biological cells on the surface of the graphene film.
Still further, the cell adhesion growth promoter includes poly-D-lysine and laminin.
Further, the step 4) further comprises: adding nerve growth factor and glial cell line-derived neurotrophic factor to the graphene film loaded with nerve cells.
Further, the neural cells include dorsal root ganglion cells. Among them, the dorsal root ganglion cells may be derived from a human source or a murine source, preferably from a murine source, more preferably from 3 to 6 weeks old C57BL/6 mice.
In one embodiment, the graphene thin film may be prepared by the following method: preparing a graphene oxide film with the thickness of 10 microns by using a suction filtration method by taking 2mg/ml graphene oxide dispersion liquid with the sheet diameter of 2 microns as a raw material; preparing a silica gel template with a micron-scale patterned surface by using a reverse mold method, flatly paving the dried graphene oxide film on the wetted silica gel template, standing, airing and forming; and (3) reducing the formed graphene oxide film by using a reducing solution prepared from a hydrogen iodide solution and absolute ethyl alcohol, taking the film down, and cleaning to obtain the graphene film with the groove-shaped structure pattern on the surface.
The invention has the beneficial effects that:
according to the induction culture method of the nerve cells, the graphene film with the groove-shaped structure on the surface is used as a culture substrate material of the nerve cells, so that the nerve cells can be induced to grow along a plurality of grooves in a linear orientation mode, in addition, the electric stimulation is applied to the induction culture of the nerve cells by utilizing the conductivity of the graphene, the rapid growth of the cells is further promoted, and the cell culture efficiency is improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a schematic structural diagram of an apparatus used in the method for inducing culture of nerve cells provided in the present application;
fig. 2 is a schematic view of a surface structure of a graphene film;
fig. 3 is a confocal laser scanning microscope image of the growth effect of dorsal root ganglion cells on different graphene films, wherein a is the confocal laser scanning microscope image obtained in comparative example 1, b is the confocal laser scanning microscope image obtained in comparative example 2, c is the confocal laser scanning microscope image obtained in example 1, and d is the confocal laser scanning microscope image obtained in example 2;
in the figure: 1. a cover; 2. a base plate; 3. culture chamber walls; 4. a graphene film; 41. a linear trench; 42. a linear protrusion; 5. a constant current power supply.
Detailed Description
In order to more clearly explain the overall concept of the present application, the following detailed description is given by way of example. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. It will be apparent, however, to one skilled in the art, that the present application may be practiced without one or more of these specific details. In other instances, well-known features of the art have not been described in order to avoid obscuring the present application.
Unless otherwise specified, the reagents and equipment in the following examples were commercially available, wherein the C57BL/6 mice were provided by the third Hospital of Beijing university; the biological reagents are all supplied by Invitrogen corporation; confocal laser scanning microscopy was supplied by Leica, germany under the model SP8 light.
In one embodiment, the method for inducing nerve cells provided herein uses a graphene thin film prepared by the following method: preparing a graphene oxide film with the thickness of 10 microns by using a suction filtration method by taking the graphene oxide dispersion liquid as a raw material; preparing a silica gel template with a micron-scale patterned surface by using a reverse mold method, flatly paving the dried graphene oxide film on the wetted silica gel template, standing, airing and forming; and (3) reducing the formed graphene oxide film by using a reducing solution prepared from a hydrogen iodide solution and absolute ethyl alcohol, taking the film down, and cleaning to obtain the graphene film with the groove-shaped structure pattern on the surface.
In the method for inducing nerve cells provided in the following examples, the graphene thin film obtained by the following method is used, and the specific steps are as follows:
s1, preparing a graphene oxide film: the graphene oxide dispersion with the concentration of 2mg/ml and the sheet diameter of 2 mu m is mixed according to the proportion of 0.8ml/cm2The application ratio of (1) is that 10ml of dispersion liquid is filtered on a filter membrane with the diameter of 4 cm; filtering with 0.45 μm cellulose acetate water filter membrane under-0.8 atmosphere for about 8 hr; taking down the filter membrane with the graphene oxide film, and naturally and completely airing at room temperature; stripping the graphene oxide film from the filter membrane;
s2, manufacturing a parallel-arranged groove-shaped pattern silicon wafer, wherein the specification size of the groove pattern is 50 microns in width, 50 microns in interval and 20 microns in depth, manufacturing a polydimethylsiloxane silica gel template by utilizing a reverse mold of the silicon wafer, processing the surface of the pattern of the silica gel template by using a plasma cleaner, and processing for 90S at 150W power; wetting the surface of the silica gel pattern with pure water, then flatly paving the dried graphene oxide film prepared in the
In the method for inducing nerve cells provided in the following examples, a cell culture apparatus as shown in fig. 1 is used for culturing, wherein a