JP2607989B2 - Culture substrate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

2. Description of the Related Art It is an important technology in the fields of life science, medicine, pharmacy, agriculture, and the like and in industries that apply them to take out and culture cells in a living body outside the body. At present, as a result of research and development of various media, growth factors, and culture substrates, many cell types have been transferred to culture systems. However, there are still many cells for which it is difficult to maintain a sufficient function or to take the same form as in a living body. A typical cell is a nerve cell. Nerve cells have many unclear points due to the high complexity of their functions.
The establishment of a stable culture system that is easy to assemble with an experimental system has been desired.

[0002]

2. Description of the Related Art Nerve cells have fibrous thin neurites whose morphology is significantly different from other cells. The elongated form of the neurite is the original form of the nerve cell, and maintaining this state is the basis for examining the function of the nerve cell extracted from the living body in vitro. In order to favorably extend thin neurites of several μm or less, adhesion of nerve cells to a culture substrate becomes a problem.

[0003] As a method for enhancing cell adhesion, a cell adhesion protein such as laminin or collagen, or a cationic polyamine such as polylysine, polyornithine, polyallylamine, or a mixture thereof is glass- or surface-treated. Coated plastic substrate for culture. In many nerve cells, if these coatings are not performed, cell adhesion and neurite extension are insufficient, and good culture cannot be performed.

[0004]

The cell adhesion protein is:
It cannot be obtained in large quantities from living organisms, and has poor stability. That is, although it is high in function, it is difficult to use from a practical viewpoint. Polyamine-based substances are toxic to the substance itself, and may be rather a negative factor when culturing nerve cells under serum-free conditions, and cannot be used in all cases. Substrates that do not require careful coating of unstable cell adhesion proteins and toxic polyamines,
A substrate showing high stability and good cultivation properties can be said to have practically great utility.

The present inventors have made intensive studies to develop such a substrate, and as a result, they have found that transparent plastic treated with low-temperature plasma of fluorocarbon has the desired effect, and completed the present invention. It has been reached.

That is, the present invention does not require an operation of carefully coating an unstable cell adhesive protein or a toxic polyamine as in the prior art, and exhibits high stability and good cultivation. It is an object of the present invention to provide a novel culture substrate. Another object of the present invention is to provide a novel culture substrate having good adhesion to a substrate and excellent neurite extension when culturing neurons.

[0007]

The structure of the present invention for achieving the above object comprises the following technical means (1) to (4). (1) A culture substrate for nerve cells, wherein transparent plastic is treated with low-temperature plasma of fluorocarbon. (2) A culture substrate for nerve cells, wherein the transparent plastic is subjected to low-temperature plasma treatment of fluorocarbon and then to low-temperature plasma treatment of ammonia. (3) The culture substrate for nerve cells according to (1) or (2), wherein the fluorocarbon is selected from tetrafluoromethane, hexafluoroethane, or a mixture thereof. (4) The culture substrate for neural cells according to (1) or (2), wherein the transparent plastic is selected from polystyrene, polymethylpentene, methyl methacrylate polysulfone, polyether sulfone, polyethylene terephthalate, polycarbonate, and polyvinylidene fluoride.

[0008] The shape of the culture substrate may be a film, a plate,
Any type of cell culture, such as a dish, is possible. Observation of the cultured cells under a microscope is usually performed, and the culture substrate needs to be transparent. For special cells, such as light-sensitive cells, an opaque substrate may be appropriate. A typical transparent material is glass, which is still used in some cases. However, it is almost being replaced by plastic because of its ease of use, that is, lightness and ease of use, and low disposable price.

Generally, plastics are not composed of a single composition, but various additives are added to improve moldability and stability. These can be toxic to cells and not all plastics can be used. The characteristics of the cultured cells are also identified by immunostaining or other methods, but in this case, alcohols, amines, weak acids, weak alkalis, etc. are used, so some degree of chemical resistance is required. It is. For these reasons, transparent plastics include polystyrene,
Polymethylpentene, methyl methacrylate, polysulfone, polyethersulfone, and polyethylene terephthalate are preferred. Polycarbonate, polyvinylidene fluoride, and the like can also be used unless a wide variety of applications are considered.

In the low-temperature plasma treatment of fluorocarbon, a chamber containing a sample is depressurized, and then a fluorocarbon gas is introduced. Sample surface treatment low-temperature plasma treatment is generated by applying a high-frequency (e.g. 13,56MH _Z is used) to the two electrodes was placed between the is performed. The amount of the fluorocarbon to be introduced depends on the size of the high-frequency generator, but can be arbitrarily set up to several hundred mTorr. There are various gas species that can be used, but tetrafluoromethane, hexafluoroethane and a mixture thereof are preferable because they are easy to use. Fluorine gas can also be used, but handling is somewhat difficult. The processing time is preferably between tens of seconds to tens of minutes. Gas purity must be high. A low-purity one causes a decrease in surface uniformity. The ammonia plasma treatment is performed in the same manner, but the treatment time is preferably between several seconds and several minutes. A high ammonia purity is required.

[0011]

When the nerve cells are cultured using the substrate of the present invention, good adhesion to the substrate and extension of the neurite are observed. In other words, since it is not necessary to use cell adhesive proteins that have been used so far, it is possible to more clearly examine the effects and effects of proteins, peptides, hormones, and the like produced by various cells. The culture substrate of the present invention is very useful and has industrial convenience. Hereinafter, the present invention will be described more specifically with reference to examples.

[0012]

【Example】

Embodiment 1 FIG. Polystyrene, polymethylpentene diameter 3
A 5-mm plastic tissue (manufactured by Sumitomo Bakelite Co., Ltd.) was treated with low-temperature plasma of tetrafluoromethane.
After placing the sample in a chamber of a plasma polymerization apparatus (manufactured by Samco), the pressure was reduced to 0.05 Torr, and tetrafluoromethane (manufactured by Showa Denko KK, purity: 99.999% was 0.1%).
It was introduced until it reached 2 Torr, and was treated at 50 W for 5 minutes. The sample was subjected to surface analysis by XPS (X-ray photoelectron spectroscopy). As a result, the elemental ratio of fluorine / carbon on the surface was 1.0 with polystyrene and 0.8 with polymethylpentene.

[0013] Serum-free culture of hippocampal neurons of rat embryos (day 18 embryos) was performed as follows. Cut out the hippocampus from 5 fetuses and immediately papain (Worthington)
The solution was treated with the enzyme (10 units / ml). The medium is DM
Using E / F-12 (manufactured by Sigma, hereinafter referred to as DF medium), inulin (added to Sigma with an equivalent of 5 μg / ml),
Transferrin (manufactured by Sigma, 5 μg / ml) and prodiesterone (manufactured by Sigma, 5 μg / ml) were used. After the enzyme-treated cell dispersion was centrifuged, 2 × 10 ⁴ cells / ml of cell solution was added to each dish in an amount of 2 ml in DF medium. As a comparative product, a cell culture dish of the same shape (manufactured by Sumitomo Bakelite Co., Ltd.) was used. 3
The cells were cultured in a carbon dioxide incubator (CO ₂ : 5%) at 7 ° C. for 2 days. Observation of the cells under a microscope showed that both the adhesion of the nerve cells and the extension of the neurites were in a better condition than the comparative product.

Embodiment 2 FIG. The polystyrene dish prepared in Example 1 was further subjected to an ammonia plasma treatment. Ammonia (manufactured by Showa Denko KK, purity 99.999%) was added in an amount of 0.
0.2To in the chamber depressurized to 005 Torr
Introduced up to rr. The cells were treated at 50 W for 2 minutes, and it was confirmed by the XPS method that amine and amide groups were introduced into the surface in addition to fluorine, and a cell culture test was performed. Fetal bovine serum (Hyclone was added to DF medium (same as in Example 1) (1
0%). A dorsal root ganglion of a rat newborn was cut out, centrifuged at 37 ° C. for 45 minutes with 0.1% trypsin (Sigma), and centrifuged. The neurotrophic factor (NGF, Sigma) was added to the above medium. (5
0 ng / ml) to prepare a cell dispersion, 3 × 10 ³ cells /
ml of the dispersion was added at 2 ml / date. The same comparative product was used as in Example 1 and cultured for 2 days. It was found that the neurite outgrowth was superior to the comparative product in the preparation of the present invention.

Claims (4)

(57) [Claims] 1. A culture substrate for nerve cells, wherein a transparent plastic is treated with low-temperature plasma of fluorocarbon. 2. A culture substrate for nerve cells, wherein the transparent plastic is subjected to low-temperature plasma treatment of fluorocarbon and then to low-temperature plasma treatment of ammonia. 3. The culture substrate for nerve cells according to claim 1, wherein the fluorocarbon is selected from tetrafluoromethane, hexafluoroethane, and a mixture thereof. 4. The culture substrate for nerve cells according to claim 1, wherein the transparent plastic is selected from polystyrene, polymethylpentene, methyl methacrylate, polysulfone, polyethersulfone, polyethylene terephthalate, polycarbonate, and polyvinylidene fluoride. [0001]