JP2007267673A - Cell culture substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture substrate inexpensive and capable of carrying out various sterilization treatments without trouble. <P>SOLUTION: Zeta-potential of the surface of a polystyrene which is the cell culture substrate, is usually zero to positive charge in 10 mM of aqueous NaCl solution with 5.0-7.5 of pH, and the ζ-potential of the polystyrene is changed into negative charge by a UV irradiation treatment, etc. In culturing cells by using the cell culture substrate with ζ-potential modified into negative charge, the cells are clearly proliferated as compared to the cell culture using the cell culture substrate with a positive ζ-potential. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a polystyrene cell culture substrate that has been surface-treated so as to be suitable for cell culture.

  Cell culture techniques are used for various purposes such as elucidation of biochemical phenomena and properties of cells and production of useful substances. There have been many reports on cell culture, with a focus on the development of material production technology using cells in the progress of biotechnology, centering on genetic engineering. Has been tried. For example, a prosthetic material for a living lesion part or a defect part typified by regenerative medicine, evaluation of a drug, and the like can be mentioned. In addition to floating cells such as lymphocytes, cell types include adhesion-dependent cells that grow by attaching to something, and various culturing methods have been attempted according to the nature of each cell.

  Adhesion-dependent cells cannot survive for a long time in a floating state in vitro. The nature of the culture substrate is remarkably related to cell function, activity, or expression of the product, as well as cell growth, the need for serum proteins during culture, in attachment-dependent cells. In cell culture, it is almost impossible to maintain the activity and function of cultured cells over a long period of time only by efficient supply of nutrients such as oxygen and efficient removal of cell waste products. In recent years, attempts have been actively made to improve maintenance of cell activity and function by variously modifying the properties of a culture substrate for cell culture.

  Polystyrene is widely used as a material for cell culture vessels for adhesion-dependent cells in terms of optical transparency, non-toxicity, good mechanical properties and moldability, and low cost. However, the polystyrene surface has a serious drawback that cell adhesion is remarkably inhibited due to its strong hydrophobicity and maintenance of cell activity. Accordingly, various attempts have been made on the surface treatment of polystyrene used for cell culture.

  As a general surface treatment method for improving cell adhesion performance, a cell adhesion protein such as collagen, gelatin or casein is applied. These cell adhesion proteins are known to act on cultured cells to facilitate cell attachment and to affect cell morphology. However, although the above-mentioned adhesive protein can be obtained naturally or synthetically, it is not economical because it has a high purity obtained through complicated purification steps using an expensive protein raw material. In addition, these adhesive proteins (especially collagen) have a drawback that they cannot be sterilized after being coated on a culture device because they are unstable.

As another method for improving cell adhesion performance, a plasma-treated surface such as polystyrene is commercially available. Plasma treatment increases the hydrophilicity of the polystyrene surface and makes it easier for cells to adhere (Patent Documents 1 and 2).
JP-A-5-64579 JP-T-2001-507218

  An object of the present invention is to provide a cell culture substrate suitable for cell culture that is inexpensive and can be subjected to various sterilization treatments without problems.

  As a result of intensive studies, the present inventors have found that the zeta potential of polystyrene as a cell culture substrate is usually from zero to around a positive charge in a 10 mM NaCl aqueous solution having a pH of 5.0 to 7.5. As a result of the modification, cell proliferation was found to be effective, and the present invention was completed.

That is, this invention consists of the following.
1. A cell culture substrate made of polystyrene having a negative zeta potential in a 10 mM NaCl aqueous solution having a pH of 5.0 to 7.5.
2. 2. The cell culture substrate according to item 1, wherein the zeta potential on the surface is −130 to −15 mV.
3. 3. The cell culture substrate according to 1 or 2 above, which is made of polystyrene irradiated with ultraviolet rays having a wavelength of 180 to 400 nm.
4). 4. The cell culture substrate according to 3 above, comprising polystyrene irradiated with ultraviolet rays having an illuminance of ^{2 to 20} mW / cm ² for 10 seconds to 120 minutes.

  When cells are cultured using a cell culture substrate made of polystyrene having a negative zeta potential according to the present invention, cell proliferation is observed more effectively than when cultured on a cell culture substrate positively charged. It was.

Hereinafter, the present invention will be described in detail.
Since polystyrene is a synthetic polymer material that is inexpensive, strong, and easy to handle, cell culture materials made of polystyrene material are often used. However, if the cell culture substrate of polystyrene is not treated at all, its cell adhesion is low. This is because the surface of polystyrene is hydrophobic. In addition, it is said that those that have not been treated with polystyrene tend to have a positive surface charge from zero.

  The polystyrene of the present invention has a degree of polymerization of about 1000 to 5000, and preferably about 3000 to 4000 from the viewpoint of strength after substrate formation. The polystyrene used as the cell culture substrate of the present invention can be formed and treated by a method known per se. For example, polystyrene formed by spin coating on a substrate such as glass can be produced as a cell culture substrate. By further processing the formed cell culture substrate, a cell culture substrate in which the zeta potential on the surface of the present invention is negatively charged can be obtained.

  The zeta potential on the polystyrene surface needs to be negatively charged. Specifically, when measured in a 10 mM NaCl aqueous solution having a pH of 5.0 to 7.5, −130 to −0.1 mV, Preferably, it may be −80 to −15 mV. In the present invention, the zeta potential is used in the meaning that is commonly used by those skilled in the art. The treatment method is not particularly limited as long as polystyrene having such a surface potential can be obtained. For example, discharge treatment such as corona treatment, ultraviolet irradiation treatment, electron beam / radiation treatment, chemical treatment, and the like can be performed. it can. In particular, since the operation is simple, ultraviolet irradiation treatment is preferable. By such treatment, the polystyrene surface can be modified to a moderately negatively charged surface, giving the cell adhesion to the surface while retaining the bulk properties such as the inherent toughness of polystyrene. Can do.

For example, as a method of ultraviolet irradiation treatment, a method of applying hydrophilicity by irradiating an excimer laser as shown in Japanese Patent No. 3340501 can be applied as long as the zeta potential shown above can be shown. In addition, a low-pressure mercury lamp can be used instead of an excimer laser as an ultraviolet ray source. The wavelength of ultraviolet rays is 180 to 400 nm, preferably 200 to 380 nm, and the illuminance of ultraviolet rays is ² to ²⁰ mW / cm ² , preferably 5 to 10 mW / cm ² for 10 seconds to 120 minutes, preferably 30 seconds. Irradiation for ~ 20 minutes.

The cell culture substrate obtained by the above treatment preferably further has a water static contact angle in the range of 90 ° to 40 °. The contact angle in this case refers to the contact angle in a planar state. Furthermore, the center line surface average roughness (Ra) defined by JIS surface roughness JIS-B-0601 is preferably in the range of 1.0 to 5.0 nm. The surface roughness in this case is an average roughness within 100 μm ² . Such a property of polystyrene is considered to increase hydrophilicity and further improve cell adhesion.

The cell culture substrate obtained by the above treatment shifts to the negative charge side, or the hydrophilization represented by the decrease in the water static contact angle is caused by hydrogen atoms of C—H bonds on the polymer surface of polystyrene. Is considered to be dissociated from the carbon atom by ultraviolet rays and substituted with a hydroxyl group or a hydrogen atom. In particular, from the viewpoint of cell culture properties, C—OH, C═O, and the like are preferable as the type of covalent bond substituted from the C—H bond.
By introducing the substituents as described above, it is considered that the protein forming the extracellular matrix is easily attached to the cell culture substrate and the culturing property is improved.

  Hereinafter, the present invention will be described with reference to examples. However, it is needless to say that the present example is for the purpose of better understanding the contents of the present invention, and the present invention is not limited to this example.

Example 1
(Preparation of base material)
Quartz glass was placed on a polystyrene substrate having a polymerization degree of 3000 produced by spin coating, and irradiated with a low-pressure mercury lamp through the quartz glass for 15 minutes. The zeta potential of the polystyrene surface after the treatment was −17.2 mV in a 10 mM NaCl aqueous solution at pH 5.6, and the water static contact angle was 45.6 °.

(Cell culture)
Next, the obtained treated polystyrene base material was sterilized with ethylene oxide gas. Next, L6 cells (mouse skeletal muscle-derived cell line) were seeded on the treated surface of the substrate so as to be 40 cells / mm ^2, and in DMEM (1% penicillin / streptomycin, 10% fetal bovine serum) for 4 days. Cultured.
The number of cells was measured using a WST assay kit (DOJINDO).

(Example 2)
(Preparation of base material)
A polystyrene base material having a polymerization degree of 3000 produced by spin coating was set in a plasma processing apparatus. Depressurized argon gas was introduced, a voltage of 150 W was applied at 0.03 Torr, and the treatment was performed for 4 minutes. The surface zeta potential of the polystyrene-treated substrate surface after treatment was -62. In 10 mM NaCl aqueous solution at pH 5.6 of the polystyrene substrate treated surface after surface zeta potential treatment in pH 5.6. The water static contact angle was 65.7 °.

(Cell culture)
Next, the obtained treated polyethylene substrate was sterilized with ethylene oxide gas in the same manner as in Example 1. Similarly, L6 cells were seeded and cultured for 4 days, and the cells were counted. The measurement procedure was the same as in Example 1.
(Comparative Example 1)
Cell culture was carried out in the same manner as in Examples 1 and 2, except that the surface-treated polystyrene having a polymerization degree of 3000 produced by spin coating was not subjected to cell measurement.

(Experimental example)
The results when L6 cells were cultured using the cell culture substrates of Example 1, Example 2 and Comparative Example 1 are shown in Table 1 and FIG. As a result, it was confirmed that the number of L6 cells on the fourth day was larger when cultured using a substrate having a negative zeta potential, which was more suitable for cell culture.

  As described above, when the cultured cell substrate of the present invention was used, the cells clearly proliferated compared to the cell culture substrate having a positive zeta potential. Thereby, it was confirmed that the base material for cultured cells of the present invention is significant when cell culture is performed.

It is a figure which shows the grade of the proliferation of a cell when it culture | cultivates using the cell culture substratum of Examples 1, 2 and Comparative Example 1.

Explanation of symbols

□ L6 cell count (cell / mm ² ) 4th day ◆ Zeta potential (mV)

Claims (4)

A cell culture substrate made of polystyrene in which the zeta potential on the surface in a 10 mM NaCl aqueous solution of pH 5.0 to 7.5 is negatively charged. The cell culture substrate according to claim 1, wherein the surface has a zeta potential of -130 to -0.1 mV. The cell culture substrate according to claim 1 or 2, comprising polystyrene irradiated with ultraviolet rays having a wavelength of 180 to 400 nm. The cell culture substrate according to claim 3, comprising a polystyrene irradiated with ultraviolet rays having an illuminance of ^{2 to 20} mW / cm ² for 10 seconds to 120 minutes.

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