JP5780530B2 - Cell culture method by removing cell adhesion region of substrate - Google Patents

Description

  In the present invention, the surface of a substrate for arraying a sample, particularly a biological sample such as a cell, is imparted with a function of inhibiting the adhesion of a cell or the like, and more specifically, selectively ozone treatment is performed. In particular, the present invention relates to a method for producing an array substrate for cells and the like and culturing cells thereon by suppressing adhesion.

  As represented by DNA chips and protein chips, a technique for immobilizing biomolecules on the chip and analyzing with high throughput is required. Furthermore, in recent years, cell chips have also appeared, and high-throughput functional analysis of genes and proteins is being applied to medical diagnosis at the cellular level. In addition, it is expected that evaluation of patients' own cells will lead to tailor-made diagnosis. In order to analyze the function evaluation of cells with high throughput, it is necessary to adhere cells to the surface of a small chip device as in other biochips (see, for example, Patent Document 1).

JP 2004-65087 A

  Looking at immobilizing cells on the chip device surface, the cells adhere to a specific chemical state surface and proliferate, so the surface treatment to which the cells adhere is very important. Since cell adhesion greatly affects cell function, chemical treatment of the substrate surface to which cells adhere is necessary to truly evaluate cell function.

  Patent Document 1 describes that the surface of a sheet is treated with ozone to improve cell adhesion and proliferation. However, these improve the adhesion uniformly by hydrophilizing the surface of the sheet material itself such as lactic acid, and do not control the adhesion and non-adhesion regions of cells on the substrate.

  The problem to be solved is simple and highly accurate that can control the quality and quantity of the adhesion area and non-adhesion area of a sample, particularly a biological sample such as a cell, on the substrate. There is no excellent versatile substrate processing method and cell culture method.

  The main feature of the present invention is that the substrate is treated with ozone and a mask in order to control the adhesion region and non-adhesion region of a biological sample such as a cell in the substrate.

  Preferably, in order to create an adhesion region and a non-adhesion region, an organic molecular layer modified on the substrate as a cell adhesion region is chemically dissociated from the substrate by exposure to ozone.

  In vivo, cells bind to proteins that constitute the extracellular matrix and maintain normal life activity. Therefore, the present inventors form a non-cell-adhesive organic molecular layer on the substrate, locally expose the ozone by using a mask or the like, and dissociate the organic molecular layer from the substrate. By such ozone exposure, the substrate surface can control the cell adhesion region.

  Moreover, a cell adhesion protein layer is formed on a board | substrate, and a cell adhesion protein is remove | excluded from a board | substrate by performing ozone exposure locally using a mask etc. By using such an ozone method, the substrate surface can also control the cell adhesion region.

  Cell non-adhesive protein and cell adhesion protein can be adhered to the region where the organic molecular layer is removed by ozone exposure.

  The method of the present invention makes it possible to create an adhesion region and a non-adhesion region of cells by ozone exposure, and repeat these as necessary to control their quality and quantity. According to this method, it is possible to provide an excellent substrate processing method and cell culture method that are simple and highly accurate.

  By applying this method, protein arrays, cell arrays, and single cell array devices are possible, and application to highly efficient chip analysis is possible. With respect to cell arrays, cell responses can also be measured in combination with sensors.

It is the schematic diagram explaining the cell adhesion region control board | substrate preparation method concerning this invention. (Reference Example 1) It is the figure which observed the thing which seeded and cultured HUVEC (human umbilical vein endothelial cell) concerning the present invention on the substrate with the microscope. It is the figure which observed with a microscope what dye | stained HUVEC seed | inoculated on the board | substrate concerning this invention with Calcein (calcein) -AM2. It is the schematic diagram explaining the cell non-adhesion area | region control board | substrate production method of this invention. (Example 1)

  The present invention aims to control a biological sample, for example, an adhesion region and a non-adhesion region of a cell or the like on a substrate, by an excellent substrate processing method that is simple and highly accurate. Realized without being affected by the type of non-bonded area.

  In the previous Patent Document 1, it is specified in claims 9 and 10 that “the surface of the sheet is treated with ozone to improve cell adhesion and proliferation”. However, these are completely different from the contents examined by the present inventors in terms of applications, processes, academic or technical meanings and contents.

  1) This Patent Document 1 is an invention relating to “a three-dimensional structure in which cells are cultured at a high density”, and “the surface of the single body is treated with one of ozone treatment, corona treatment, and plasma treatment to improve cell adhesion. Is written in the claim. By the way, in the embodiment described in the specification of Patent Document 1, the process using ozone or the like is not performed, and what is “chemically” necessary for those “treatments”? I don't know what is going on.

  2) The surface of the carrier proposed in Patent Document 1 is a polymer such as lactic acid, and these polymer materials are also known to form a hydrophobic surface. Therefore, what is called ozone treatment, corona treatment, or plasma treatment is simply to hydrophilize the surface, that is, although there is no description in the examples, hydrogen removal and hydroxyl groups on the material surface by plasma treatment. It is only thought that we are promoting the introduction of the material and thereby imparting the wettability of the material.

  On the other hand, the inventors' invention is that the chemical layer on the surface of the material is degraded by ozone or ozone + UV (ultraviolet light), and the ozone treatment according to the present invention is chemically completely different from Patent Document 1. Do something different (or vice versa).

  Samples, particularly biological samples, include cells, as well as various biomolecules such as DNA, RNA, proteins, carbohydrates, lipids, glycoproteins, glycolipids, and complexes thereof. Includes animals, microorganisms, etc. Preferred biological samples are cells, proteins, glycoproteins. The reason for the preference is that the adhesion of proteins and glycoproteins largely depends on the material and state of the substrate. The method of the present invention can chemically remove the organic molecular layer modified on the substrate by ozone exposure. By using ozone and a mask, the adhesion and non-adhesion regions of proteins and glycoproteins can be easily controlled. The cells are adhered via cell adhesive proteins. As described above, since the adhesion and non-adhesion regions of proteins can be easily controlled when the method of the present invention is used, the adhesion of cells can also be easily controlled.

For the substrate, a material such as glass, metal thin film, quartz, silicon wafer, silica, resin, or the like can be used.
Preferred types of substrates are glass, metal thin films, and silicon wafers in relation to biological samples, in relation to formation of bonded or non-bonded areas, and in relation to ozone treatment. The reason why they are preferred is that glass is a common substrate, is easily available, has good processability, has a surface that itself binds to non-cell-adhesive organic molecules, cell-adhesion proteins, etc. When the non-adhesive organic molecules are partially detached by exposure to ozone and the cell adhesion protein is coated on the exfoliated portion, the cell non-adhesion region and the cell adhesion region can be obtained in a desired pattern. This is because the metal thin film is generally used as a sensor device substrate and itself has a surface that binds to a thiol organic molecule or the like. Further, since the organic molecular layer can be partially peeled by exposure to ozone, the adhesion and non-adhesion regions of the sample can be easily controlled. This is because the silicon wafer is made of silicon dioxide, and it is easy to form a region that binds to protein adhesion-inhibiting organic molecules and cell adhesion proteins.

  Formation of the adhesion region or the non-adhesion region of the biological sample is not particularly limited, and is performed by, for example, forming a non-adhesion layer on the substrate, forming an adhesion layer, or dissociating these layers from the substrate by ozone treatment. be able to. These can also be improved by various known methods and means.

Non-adhesion regions such as cells include organic molecule layers such as cell non-adhesion proteins, and adhesion regions include structures having organic molecule layers such as cell adhesion proteins.
As organic molecules such as cell adhesion proteins used here, fibronectin, collagen, proteoglycan, hyaluronic acid, laminin, tenascin, entactin, elastin, fibrillin, and other cell adhesion molecules can be used.
Examples of organic molecules such as cell non-adhesive proteins include cell non-adhesive proteins such as bovine serum albumin (BSA), cell non-adhesive organic molecules such as polyethylene glycol (PEG), and other cells and proteins. A method that can modify and immobilize molecules and the like that inhibit or suppress adhesion and / or adsorption at high density on the substrate surface can be widely used.

  For the dissociation, control, and maintenance of the cell adhesion region or the non-cell adhesion region, means for selectively performing ozone exposure, ozone blocking, and ozone exposure quality and quantity can be used. For example, a mask can be used. For the material and shape of the mask, stainless steel, glass, polymer film, cellulose film and the like can be used. A preferred method of using the mask is to adhere the mask to the substrate. Depending on the material properties of the substrate, better adhesion can be obtained by dropping a solution or gel solution on the substrate and placing a mask thereon.

  The principle of dissociation and control of the adhesion and non-adhesion areas due to ozone exposure is that the oxygen atom radical generated by ozone and ozone self-decomposition works as an adhesion layer or non-adhesion area of the structure where the adhesion area or non-adhesion area works. This is considered to be due to dissociation of the bond between the non-adhesive layer and the substrate. The bond between the adhesive layer and the substrate includes chemical bonds such as hydrogen bonds, ionic bonds, covalent bonds, and coordinate bonds. Preferred types of bonds are hydrogen bonds, ionic bonds, coordination bonds, and bonds by hydrophobic interaction. These bonds are preferable because they are bonds that can be decomposed by ozone and oxygen atom radicals.

  The ozone exposure can be performed by placing the substrate to be processed, the site to be processed, and the region in the presence of ozone. Ozone can be generated by filling an ozone generator with oxygen and irradiating UV. The concentration of ozone, processing temperature, processing time, etc. can be arbitrarily set. Preferred processing conditions vary depending on the type of substrate to be processed, the type, size, thickness, etc. of the chemical layer.

Although ozone exposure can be used with ozone alone, the ozone exposure time can be shortened by the reaction of active species generated by using ozone and UV in combination.
The UV wavelength range to be used can be widely used, but a higher energy UV wavelength having a wavelength of about 200 nm or less is preferable.

The non-adhesion region or the adhesion region can be made an adhesion region or a non-adhesion region by dissociating from the substrate in a predetermined pattern by exposure to ozone. The surface of the adhesion region of the substrate thus formed can be made into a non-adhesion region by coating with a cell non-adhesion protein or the like. This non-adhesion region can be formed in a pattern different from the previous adhering region.
Non-adhesion or adhesion criteria can be relative. That is, when it is referred to as non-adhesive or adhesive, it may have a lower or higher adhesiveness in itself or in its region than in an adhesive or non-adhered region.

  An intermediate layer may be present between the substrate and the adhesive layer or the non-adhesive layer. In this case, the bond dissociated by exposure to ozone may be a bond between an adhesive layer or the like and the intermediate layer.

  For the types of substrates, biological samples, non-adhesive areas, adhesive areas, masks, patterns, etc., selection, design, creation, array formation, etc., use the conventional method for creating a single cell array substrate, etc. Can do. For example:

(1) Micro contact printing method A cell array pattern stamp is created using polydimethylsiloxane (PDMS) and photolithography. The cell array substrate is formed by attaching an alkanethiol solution to a pattern stamp and stamping the substrate having a gold thin film.
Reference 1: Langmuir, 18, 3645-3649 (2002)
Reference 2: Experimental Cell Research, 235, 305-313 (1997)

(2) Laser method Cell adhesion by controlling the hydrophilicity and hydrophobicity of PIPAAm by grafting a temperature-responsive polymer polyisopropylacrylamide (PIPAAm) that changes its structure in response to temperature and controlling the temperature. And control desorption. The polymer layer grafted with the temperature-responsive polymer layer is aligned with cell capture domains in an array using a UV excimer laser.
Reference 3: Polymer Science Series A, 46 (4), 405-410 (2004)

(3) Photomask method A self-organized film of alkanethiol having a terminal methyl group is formed on the entire surface of the gold thin film. Next, a photomask is placed on the film, and ultraviolet light is irradiated for 5 hours to photooxidize and decompose a part of the self-organized film of alkanethiol. The oxide surface is washed away to expose the gold surface in a pattern. After that, it is immersed in an alkanethiol solution having a desired terminal functional group to embed new alkanethiol in the pattern portion.
Reference 4: Biomolecular Engineering, 19, 161-170 (2002)

(4) Microelectrode method Proteins having a blocking function, such as albumin and heparin, are lost by the oxidizing agent of hypobromite (HOBr). Microelectrodes are placed near the surface of the substrate to generate HOBr. The blocking layer is desorbed by the oxidizing action.
Reference 5: Langmuir, 22, 10784-10787 (2006)

Reference example 1
Cell adhesion array substrate fabrication method by ozone exposure (Fig. 1)

1. Creation of cell non-adhesive glass substrate
(1) A cell adheres to a substrate via a cell adhesion protein. Therefore, the cell non-adhesive substrate is a substrate in which polyethylene glycol (PEG) that inhibits adhesion of cell adhesive proteins is immobilized on the substrate. The cell non-adherent substrate was subjected to hydrophilic treatment by immersing the glass substrate in an 8M aqueous sodium hydroxide solution for 10 minutes and then ultrasonically washing with ultrapure water.
(2) PEG was immobilized in two ways. Each method is shown below.
(2-1)
The hydrophilic glass substrate was immersed in 50 ml of a 2% 2-methoxy (polyethyleneoxy) propyltrichlorosilane / chloroform solution for 2 hours. The cell non-adherent glass substrate was then ultrasonically washed with chloroform and dried with nitrogen gas.
(2-2)
A 2-Methoxy (polyethyleneoxy) propyl trichlorosilane / toluene solution was sandwiched between two hydrophilic glass substrates. The glass substrate was allowed to stand in an 80 ° C. atmosphere for 2 hours. The cell non-adherent glass substrate was then subjected to ultrasonic cleaning with toluene, ethanol, and ultrapure water, and dried with nitrogen gas.

2. Method of ozone exposure
(1) Ozone exposure was performed by filling the ozone generator with oxygen and irradiating UV. The cell adhesion region was controlled by covering the mask with a cell non-adhesive substrate.
(2) Water was dropped on the cell non-adhesive substrate prepared in 1. and a mask was placed on it. The feature of this research method is that the shape, location, and number of cells can be easily controlled by changing the mask.
(3) Ozone was generated by oxygen gas inflow for 10 minutes and UV irradiation for 5 minutes.
(4) The substrate of 2. (2) was left in an ozone atmosphere generated under the conditions of 2. (3). It is considered that the ozone radicals dissociate the bond between PEG and the substrate at the site not covered with the mask on the surface of the cell non-adherent substrate.
(5) The cell array substrate prepared in 2. (4) was immersed in a 10 mM fibronectin solution for 1 hour. Fibronectin, which is a cell adhesion protein, is adsorbed at the site dissociated by ozone.
(6) The cell array substrate prepared in 2. (5) was seeded with 5 × 10 ⁴ cells and cultured in a 37 ° C., 5% CO ₂ incubator. One hour later, the cells were washed with PBS and cultured for 24 hours.
(7) The cell non-adherent glass substrate was successfully exposed to ozone and coated with the cell adhesion protein to control the cell adhesion region. Moreover, a cell adhesion area | region can be created according to mask patterning by using a mask.
(8) Since this cell array substrate uses a glass substrate, the cell array substrate is excellent in permeability, and the fluorescent probe can be evaluated with a microscope.

  As shown in FIGS. 2 and 3, cells adhere in a number that depends on the size of the mask. In addition, it can be seen that single cells are in a state of maintaining cell activity and adhere. From this, it was found that the cell adhesion region by ozone can be easily controlled to a single cell size by using a mask.

Example 1
Method for making cell non-adhesive array substrate by ozone exposure (Figure 4)

1. Creation of cell adhesion substrate A cell adhesion substrate is a substrate in which adhesion of cell adhesion protein is immobilized on the substrate. The cell adhesion substrate was subjected to a hydrophilic treatment by immersing the glass substrate in an 8M aqueous sodium hydroxide solution for 10 minutes. The hydrophilic glass substrate was immersed in a 100 mg / ml poly-L-lysine / PBS solution and incubated overnight at 37 ° C. in a 5% CO ₂ atmosphere. Thereafter, the glass substrate is immersed in a 1 mg / ml fibronectin / PBS solution and incubated for 1 hour to obtain a cell adhesion glass substrate.

2. Method of ozone exposure
(1) The ozone treatment was performed by filling the ozone generator with oxygen and irradiating UV. The cell non-adhesion region was controlled by covering the cell adhesion substrate with a mask.
(2) Water was dropped on the cell adhesion substrate prepared in 1. and a mask was placed thereon. The feature of this research method is that the shape, location, and number of cells can be easily controlled by changing the mask.
(3) Ozone was generated by oxygen gas inflow for 10 minutes and UV irradiation for 5 minutes.
(4) The substrate of 2. (2) was allowed to stand in an ozone atmosphere generated under the conditions of 2. (3). It is considered that fibronectin is dissociated from the substrate at the site not covered with the mask on the cell adhesion substrate surface.
(5) The cell array substrate prepared in 2. (4) was immersed in a 5 mg / ml BSA solution, which is a cell non-adhesion protein, for 1 hour. 2. The site treated with O _{3 in} (4) adsorbs BSA, which is a non-cell adhesion protein.
(6) The cell array substrate prepared in 2. (5) was seeded with 5 × 10 ⁴ cells and cultured in a 37 ° C., 5% CO ₂ incubator. One hour later, the cells were washed with PBS and cultured for 24 hours.
(7) The cell adhesion glass substrate was successfully exposed to ozone and coated with the cell non-adhesion protein to control the cell non-adhesion region. Further, by using a mask, the cell non-adhesion region can be created in accordance with mask patterning.

By applying the method of the present invention, a protein array, a cell array, and a single cell array device are possible, and application to highly efficient chip analysis is possible. With respect to cell arrays, cell responses can also be measured in combination with sensors.

Claims (5)

Hydrophilizing the substrate,
Forming a cell adhesion region on the substrate by binding cell adhesive organic molecules on the substrate;
Use a water, solution or gel solution on the substrate so that the mask is in close contact and ozone does not enter between the mask and the substrate .
Covering the cell adhesion region with a mask formed with a pattern for forming a cell non-adhesion region;
Dissociating the bond between the cell-adhesive organic molecule and the substrate by selective exposure with ozone according to the patterning of the mask;
Forming a cell non-adhesion region on the substrate by binding the cell non-adhesion organic molecule on the substrate in the region where the cell adhesion organic molecule is dissociated, and culturing the cell on the cell adhesion region A cell culture method comprising:   The cell culture method according to claim 1, wherein the cell-adhesive organic molecule is chemically dissociated from the substrate by exposure to ozone and ultraviolet rays.   The cell culture method according to claim 1 or 2, wherein the cell adhesion organic molecule is fibronectin.   The cell culture method according to any one of claims 1 to 3, wherein the cell non-adhesive organic molecule is bovine serum albumin (BSA). The cell culture method according to any one of claims 1 to 3, wherein the cell non-adhesive organic molecule is polyethylene glycol.