JP2019017387A - Uv ozone-plasma composite treatment method and treatment apparatus - Google Patents

Abstract

To provide a surface modification method for culturing an adherent cell which is suitable for cell adhesion even more.SOLUTION: The present invention is a method for manufacturing a cell-carrying base material having a cell-carrying face which has non fluorine-based resin as a main component, in which the method includes: a UV irradiation process of irradiating a cell-carrying face of the base material with UV under humidifying environment in an oxygen and/or ozone feeding atmosphere; and a plasma irradiation process of irradiating the cell-carrying face of the base material with plasma, and a device to be used for the method is provided. The present invention provides a culture method for culturing a cell on the cell-carrying face of a cell-carrying base material which is obtained by the manufacturing method.SELECTED DRAWING: Figure 11

Description

  The present invention relates to a method for manufacturing a cell-supporting substrate having a surface suitable for cell support, adhesion, storage, culture, and / or proliferation, and a manufacturing apparatus.

  It is known that adherent cells hardly adhere to hydrophobic surfaces and surfaces having extremely high hydrophilicity, but take a form of adhering and developing on surfaces having appropriate hydrophilicity. In particular, it is known that cells adhere well to surfaces exhibiting moderate wettability with a water contact angle of 40 to 70 ° (Non-Patent Document 1) or 60 to 80 ° (Non-Patent Document 2). Therefore, means for obtaining an appropriate hydrophilic surface excellent in adhesion and proliferation of such adhesive cells has been developed. Until now, as a method for making the surface of a polymer material hydrophilic, atmospheric pressure plasma treatment (Patent Document 2) such as corona discharge treatment (Patent Document 1), grafting of a polymer chain containing a hydrophilic skeleton (Patent Document 3), A substrate having a contact angle of about 10 to 30 ° by binding aminopropylethylenemaleic anhydride on the surface (Patent Document 4) and a method of presenting hydrophilic groups of amphiphilic substances on the surface (Patent Document 5) Etc. have been reported. In addition, as a method of controlling the degree of hydrophilicity of the surface, a method of reducing the degree of hydrophilicity by oxidizing and / or decomposing the surface once made highly hydrophilic, A method of binding via a linker and adjusting the degree of hydrophilicity by the density of the linker has been reported (Patent Document 6). Of these, physical methods such as corona discharge treatment and atmospheric pressure plasma treatment are particularly simple, and have become major methods for modifying hydrophilic cell culture surfaces.

  In such a hydrophilic surface, it is considered that groups having an oxygen atom such as a hydroxyl group, a carbonyl group, and a carboxy group mainly contribute to the display of hydrophilicity. However, oxygen atoms introduced by corona discharge are rapidly removed, and the surface is liable to deteriorate. Furthermore, it is considered that the corona discharge treatment can provide only about 20% of surface oxygen on the base material, and there is a limit to imparting hydrophilicity. In addition, plasma discharge can achieve higher oxygen levels than corona discharge, but the substrate needs to be processed in vacuum, and the processing process is complicated. Furthermore, the plasma treatment has a problem that the surface is easily damaged.

  As another approach for making the surface of the polymer material hydrophilic, ultraviolet laser irradiation to a fluororesin substrate or the like (Patent Document 4), or a method of irradiating ultraviolet light having a wavelength that generates ozone (Non-Patent Documents 3 and 4) Has been reported. Also in these reports, it is reported that it is desirable that the ratio of surface oxygen atoms is medium for cell growth (Non-patent Document 4). In addition, in order to improve such a method using ozone / ultraviolet light, a combination of ozone / ultraviolet light treatment and a biomaterial (non-patent document 5), introduction of oxygen atoms into ultra-high molecular weight polyethylene (non-patent document) 6) etc. are being studied.

  In particular, when a hydrophilic surface is used as a substrate for undifferentiated cell culture, stem cells that are undifferentiated cells are difficult to culture, such as feeder cells, cytokines such as LIF, or Matrigel (registered trademark), collagen, etc. It required a special environment such as coating with extracellular matrix proteins. However, all of these technologies depend on biological materials, so they have low stability, resulting in lot-to-lot differences, potential contamination, and short storage life. It was.

  Thus, attempts have been made to more stably produce a cell culture surface capable of culturing stem cells, which are undifferentiated cells, by chemical modification. For example, it has been proposed to solve these problems by forming a swellable (meth) acrylate layer on the cell culture surface (Patent Document 7).

  In addition, the present inventors have found and reported that the surface of polystyrene or the like can be processed into a surface suitable for cultivation of adherent cells by performing UV treatment under humidification, oxygen or ozone supply (Patent Document 8). ).

JP-A-6-98756 International Publication No. WO2012 / 144624 JP 2009-17809 A JP 2012-527896 Gazette JP 2012-175893 A Special table 2011-510655 gazette JP 2010-68755 A International Publication No. WO2016 / 136251

Supervised by Yasuyuki Sakai and Eiichi Minutani, “Biomaterial Device for Animal Experiment Substitution”, CMC Publishing, 2014, 133-134 Yasushi Tamada et al., Journal of Biomedical Materials Research; 28: 783-789 (1994). D. O. H. Teare et al., Kanbmuir; 16: 2818-2824 (2000) S. A. Mitchell et al., Biomaterials; 25: 4079-4086 (2004). Fabio Formosa et al., Microvascular Research; 75: 330-342 (2008). Alexandra H. et al. C. Poulsson et al., Langmuir; 25: 3718-3727 (2009).

  However, the existing treatment methods have not yet obtained surface modification for supporting and culturing sufficient cells. Therefore, an object of the present invention is to provide a surface modification method for supporting and culturing selected cells.

  As a result of examining the relationship between cell growth and the surface of a resin substrate using various methods, the present inventors have found that both UV irradiation and plasma treatment in an oxygen and / or ozone atmosphere in a humidified environment. It was found that a surface more suitable for cell adhesion and cell growth was generated by performing the above-described treatment, and the present invention was completed. Furthermore, the present inventors have found that such a surface is superior to UV irradiation in an oxygen and / or ozone atmosphere in a humidified environment reported in the past, and completed the present invention.

Accordingly, the present invention includes the following inventions:
(1) A method for producing a cell-supporting substrate mainly comprising a non-fluorinated resin having a cell-supporting surface,
A method comprising: a UV irradiation step of irradiating UV to a cell carrying surface of the substrate in a humidified environment and an atmosphere of oxygen and / or ozone supply; and a plasma irradiation step of irradiating plasma to the cell carrying surface of the substrate.
(2) The cell support according to (1), wherein the non-fluorine resin is at least one resin selected from the group consisting of polyethylene, acrylic resin, ABS resin, polyethylene terephthalate, polypropylene, polycarbonate, and polystyrene. Method for manufacturing a substrate.
(3) The method for producing a cell-supporting substrate according to (1) or (2), wherein a plasma irradiation step is performed after the UV irradiation step.
(4) The method for producing a cell-supporting substrate according to (1) or (2), wherein a UV irradiation step is performed after the plasma irradiation step.
(5) The method for producing a cell-supporting substrate according to (1) or (2), wherein the UV irradiation step and the plasma irradiation step are performed simultaneously.
(6) The cell support according to any one of (1) to (5), wherein the UV irradiation is performed by irradiating UV having an average wavelength of 184.9 nm and 253.7 nm. A method for producing a substrate.
(7) The UV irradiation is performed until the water contact angle on the surface of the non-fluorine resin reaches 40 to 70 °, according to any one of (1) to (6), A method for producing a cell-supporting substrate.
(8) The manufacturing method according to any one of (1) to (7), further including a step of applying an ammonia solution to the cell-carrying surface before the UV irradiation step.
(9) A method for culturing adherent cells, comprising culturing cells on a cell-carrying surface of a cell-carrying substrate obtained by the production method according to any one of (1) to (8). A culture method comprising.
(10) The culture method according to (9), wherein the adherent cells are stem cells.
(11) The culture method according to (10), wherein the stem cells are mouse iPS cells or human iPS cells.
(12) The culture method according to (10) or (11), wherein the culture is performed in the absence of feeder cells.
(13) A storage unit for storing a cell-supporting substrate mainly composed of a non-fluorine resin having a cell-supporting surface, and irradiating the cell-supporting substrate stored in the storage unit with ultraviolet rays Possible ultraviolet irradiation means, plasma irradiation means capable of irradiating nitrogen plasma to the cell support substrate stored in the storage unit, and humidification means capable of controlling the humidity in the cell support substrate storage unit; , A cell-supporting substrate surface reforming device having oxygen or ozone supply means capable of controlling the ozone concentration in the cell-supporting substrate storage section.
(14) The cell-supporting substrate surface modifying apparatus according to (13), further comprising ammonia application means capable of applying an ammonia solution to the cell-supporting surface of the cell-supporting substrate.

  In the present specification, the “cell-supporting substrate” refers to a substrate used by supporting cells on the surface, and is a substrate intended for supporting, adhering, storing, culturing, and / or proliferating cells. It may be a material. For example, the cell-supporting base material includes a cell culture base material (for example, a cell culture container), a cell storage base material, or an implant base material. The cells carried by the cell-carrying substrate in the present specification are not particularly limited as long as they are adherent cells. For example, mammalian cells such as smooth muscle cells, endothelial cells, fibroblasts, osteoblasts, stem cells, etc. Preferably, it is a stem cell. In the present specification, the stem cells include iPS cells, ES cells, mesenchymal stem cells, and the like, and include mouse, rat, rabbit, dog, monkey, and human cells, preferably mouse iPS cells and human iPS. It is a cell. In addition, the shape of the cell-supporting substrate in the present specification can be appropriately selected according to the purpose, and for example, a plate shape, a sheet shape, a spherical shape, a dish shape, a chip shape, or a desired tissue (for example, , Artificial bone or the surface portion thereof). Preferably, the cell-supporting substrate is an adherent cell culture container or an adherent cell storage container, more preferably an iPS cell culture container or an iPS cell storage container.

The cell-supporting substrate in the present specification contains a non-fluorine resin as a main component, but may contain “other components” as necessary. For example, other substances that contribute to cell adhesion, such as Matrigel (registered trademark), laminin, collagen, or fibronectin, may be included as necessary. For example, cells carrying a substrate herein, 20~80μg ^/ cm 2 Matrigel, 30 to 70 / cm ^2, or 40~60μg / cm ² contains. Further, for example, cells carrying a substrate ^{herein, 0.1~1μg / cm 2, 0.3~0.8μg /} cm 2, or at a concentration of 0.5 [mu] g / cm ^2, containing laminin It may be.

  In the present specification, “non-fluorine-based resin” means a resin that does not contain fluorine. For example, polyethylene, ultra high molecular weight polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl alcohol, acrylic resin, polyethylene terephthalate, polyacetal. , Polycarbonate, polyamide, polyimide resin, phenol resin, amino resin, epoxy resin, polyester, and acrylonitrile-butadiene-styrene copolymer synthetic resin (ABS resin). Of these, polystyrene is preferable. Polystyrene has stereoregularity (tacticity), and there are isotactic (isotactic) type, syndiotactic type, and atactic type, but the type of polystyrene in the present invention is not limited, these Any of these types, or a mixture of two or more types among them may be used, and an atactic type is generally used. Since polystyrene is a high molecular compound, products with various degrees of polymerization (average molecular weight) are supplied, but the present invention is not limited thereto. For example, the degree of polymerization can be 10 to 100,000, 50 to 10,000. Accordingly, there may be differences in material and physical properties among polystyrene culture dishes supplied by various companies, but the invention is not limited and culture dishes from any manufacturer can be used. As an example, IWAKI (registered trademark) tissue culture dish (AGC Techno Glass Co., Ltd.) can be mentioned.

  The cell-carrying substrate of the present invention has a cell-carrying surface at least in part. In the present specification, the “cell-carrying surface” means a surface on which cells can be supported, adhered, stored, cultured, and / or proliferated, and the cells are not necessarily cultured or proliferated on the surface. It is not what you need. Further, the cell carrying surface may have any shape such as a flat surface, a curved surface, a wavy surface, and a spherical surface.

  In one embodiment, the cell-carrying surface of the cell-carrying substrate of the present invention has a C—C bond and / or a C—H bond causing chemical shift on the surface. In the present specification, “C—C bond and / or C—H bond causing chemical shift” means a structure in which the bonding state is changed by incorporating an oxygen atom into the molecular structure of polystyrene. It is a structure that does not contain. That is, the C—C bond and / or C—H bond causing a chemical shift means a C—C bond and / or C—H not contained in polystyrene, and C—H, C—C and C— This means a structure other than Ph. The cell-carrying surface of the present invention has a reduced contact angle despite a very small number of oxygen atom-introduced groups (OH, COOH, C═O, etc.). Therefore, it is considered that this decrease in the contact angle is caused by a C—C bond and / or a C—H bond causing a chemical shift.

  Carboxy groups (COOH groups) on the cell culture surface are thought to have a negative effect on the cells. The cell-carrying surface of the cell-carrying substrate of the present invention has an extremely small amount of carboxy groups as compared with the cell-carrying substrate that has been treated with UV / ozone under the same conditions under non-humidification. Therefore, the cell-supporting substrate of the present invention is substantially free of carboxy groups on the cell-supporting surface. Here, “substantially non-existent” does not mean that it does not exist at all, but means that it does not exist to the extent that it affects cell culture. Preferably, UV is used under the same conditions under non-humidification. / Means very little compared to the ozone-treated cell-carrying substrate, and more preferably means little or no detection in the C (1s) narrow scan XPS spectrum.

  The cell-supporting substrate of the present invention has a medium water contact angle on the cell-supporting surface, for example, 30 to 80 °, 30 to 70 °, 30 to 60 °, 40 to 80 °, 40 to 70 °, It is 40-60 degrees. Preferably, the water contact angle is 40 to 60 °. Preferably, the contact angle in the present specification is a contact angle measured by the θ / 2 method by dropping 1 μL of pure water onto a sample with an automatic contact angle meter. In addition, the cell-supporting substrate of the present invention is excellent in storage stability of the cell adhesive surface. For this reason, in the cell-supporting substrate of the present invention, preferably, the water contact angle on the surface of the sealed storage after 24 hours from UV irradiation and for 1 week is within the range of the contact angle. The water contact angle on the surface 24 hours after irradiation and for one month sealed storage is within the range of the contact angle. That is, the cell-supporting substrate of the present invention preferably maintains excellent cell adhesiveness even after one week of sealed storage or one month of sealed storage after UV irradiation.

  In one embodiment, the cell-carrying surface of the cell-carrying substrate of the present invention is a stem cell even in the absence of feeder cells (scaffold cells) and extracellular matrix protein coating or in the presence of a lower concentration of extracellular matrix protein coating. Can be carried or propagated in an undifferentiated state. For example, the cell-carrying surface of the cell-carrying substrate of the present invention can be applied to EB3 cells (Mol. Cell biol. (2002) which are mouse embryonic stem cells derived from the 129 / Ola lineage even without feeder cells and extracellular matrix protein coating. ) 22: 1526-36; Genes to Cell (2004) 9: 471-7) may be the surface on which it can grow. Alternatively, the cell-carrying surface may be 0.2 times the concentration of Matrigel (registered trademark) necessary for adhering stem cells on a base material mainly composed of the non-fluorinated resin that has not been surface-treated, It may be a surface on which stem cells (eg, human iPS cells or mouse iPS cells) can adhere or grow. Alternatively, the cell-carrying surface may be a stem cell (e.g., at a concentration of 0.5 times the concentration of laminin necessary for adhering the stem cell on a base material mainly composed of the non-fluorinated resin whose surface is not treated). It may be a surface to which human iPS cells or mouse iPS cells) can adhere or grow.

  In a specific aspect, the present invention relates to a cell culture vessel for adherent cells, comprising the cell-supporting substrate. Examples of the cell culture vessel include plate-like, sheet-like, spherical, dish-like, chip-like, fiber-like, or flask-like culture vessels.

  In another aspect, the present invention relates to an apparatus for realizing the above method, specifically, for storing a cell-supporting base material mainly composed of a non-fluorinated resin having a cell-supporting surface. Storage unit, ultraviolet irradiation means capable of irradiating the cell-supporting substrate stored in the storage unit with ultraviolet rays, and nitrogen plasma can be applied to the cell-supporting substrate stored in the storage unit A plasma irradiating means, a humidifying means capable of controlling the humidity in the cell-supporting substrate storage section, and an oxygen or ozone supply means capable of controlling the ozone concentration in the cell-supporting substrate storage section. The present invention also relates to a cell-supporting substrate surface modifying apparatus. In such a configuration, the ultraviolet irradiation means has a light source capable of generating ultraviolet light (electromagnetic wave in the range of 100 to 400 nm), such as a mercury lamp or LED. Preferably, the ultraviolet irradiation means has a light source capable of performing the UV irradiation process described herein. The plasma irradiation means consists of a decompression part that keeps the nitrogen gas pressure in a constant decompression state in the chamber, an electron acceleration part that accelerates electrons by an electric field by a DC voltage, a high-frequency voltage, a microwave, etc. applied between the electrodes, And a plasma generation unit that generates plasma using ionization caused by collision of gas molecules. The humidifying means may include a water holding unit, and may include a heating unit that generates water vapor by heating water supplied from the water holding unit as necessary. The oxygen or ozone supply means supplies oxygen gas or ozone gas into the storage unit. The ozone gas may be generated by first supplying oxygen gas and irradiating ultraviolet rays with the ultraviolet ray supply means, or supplying oxygen gas to ozone after being converted into ozone by a discharge type ozone generator. .

  The method for producing a cell-supporting substrate of the present invention can stably impart hydrophilicity suitable for cell adhesion by a simpler method. In addition, the cell-supporting substrate of the present invention does not require the use of a biological material, or adherent cells containing stem cells can be cultured in a smaller amount than in the past, so that the stability is high and the difference in results due to lot differences is different. In addition to being less likely to occur, it can be cultured at lower cost. In addition, the problem of potential contamination can be reduced because animal-derived components can be used or the amount used can be reduced.

It is drawing which shows the internal structure of a UV / ozone surface modification apparatus. It is a graph which shows the wide scan spectrum of a non-processed polystyrene. The vertical axis represents photoelectron intensity, and the horizontal axis represents binding energy (eV) (hereinafter, the same applies to FIGS. 3 to 9). It is a graph which shows the narrow scan spectrum of C1s of an untreated polystyrene. (A) It is a graph which shows the narrow scan spectrum of C1s of UV1min. (B) It is a graph which shows the narrow scan spectrum of C1s of UV3min. (C) It is a graph which shows the narrow scan spectrum of C1s of UV10min. (A) It is a graph which shows the narrow scan spectrum of C1s of Plasma5s. (B) It is a graph which shows the narrow scan spectrum of C1s of Plasma40s. (C) It is a graph which shows the narrow scan spectrum of C1s of Plasma160s. It is a graph which shows the narrow scan spectrum of O1s. It is a graph which shows the narrow scan spectrum of N1s. (A) It is a graph which shows the narrow scan spectrum of C1s of UV / zone3min-> plasma 40s. (B) It is a graph showing the narrow scan spectrum of C1s of plasma 40s-> UV / zone3min. (A) It is a graph showing the narrow scan spectrum of C1s of UV / zone10min-> plasma 160s. (B) It is a graph showing the narrow scan spectrum of C1s of plasma 160s-> UV / zone10min. It is a photograph of mouse ES cells (ES-D3 strain) cultured on cultured dish cells made of surface-modified polystyrene. (A) Untreated dish, (b) UV / zone single treatment 10 min, (c) Atmospheric pressure plasma 10 s, (d) Atmospheric pressure plasma 10 s → UV / zone 10 min, (e) UV / zone 10 min → Atmospheric pressure plasma 10 s . It is a graph which shows the total amount of DNA of the mouse | mouth ES cell (ES-B3 strain | stump | stock) culture | cultivated on the un-processed, gelatin coat, UV / zone or a plasma independent, UV / zone * plasma composite modified base material. It is a photograph of mouse iPS cells (APS0001 strain) cultured on cultured dish cells made of surface-modified polystyrene. The right side shows an image of GFP expression observed with a fluorescence microscope. (A) Normal culture on feeder cells, (b) UV / zone 3 min → atmospheric pressure plasma 40 s, (c) untreated dish. It is a block diagram of the base material surface modification apparatus for cell support concerning this invention. It is a block diagram of the base material surface modification apparatus for a cell support | carrier which is a multi-chamber cluster tool. It is a block diagram of the ultraviolet treatment part of the multi-chamber of FIG. It is a block diagram of the plasma processing part of the multi-chamber of FIG. It is a photograph of a human iPS cell (253G4 strain) cultured on an ammonia-treated surface modified polystyrene cultured dish cell. From left to right, recommended use amount (× 1.0) Matrigel addition group, 0.2 times (× 0.2) recommended use amount Matrigel addition group, UV30s treatment → atmospheric pressure plasma 80s treatment × 0.2 Matrigel An addition group, a group of × 0.2 matrigel added after UV treatment for 30 seconds after treatment with ammonium and then treated with atmospheric pressure plasma for 80 seconds. The scale bar represents 200 μm. The graph which counted the cell number of the Matrigel addition group of 0.2 times (x0.2) recommended use amount is represented. The graph shows, from the left, the results of dishing without surface modification, UV30s treatment, UV30s treatment → atmospheric pressure plasma 80s treatment, and after UV coating, UV30s treatment → atmospheric pressure plasma 80s treatment. The vertical axis represents the relative number of cells when the number of cells cultured in the recommended use amount (× 1.0) Matrigel-added dish is 1.0. The asterisk represents p <0.05 vs (number of cells without surface modification). It is a photograph of a human iPS cell (253G4 strain) cultured on an ammonia-treated surface modified polystyrene cultured dish cell. From the top left, the recommended use amount (× 1.0) laminin addition group, the recommended use amount 0.3 times (× 0.3) laminin addition group, UV30s treated × 0.3 laminin addition group, bottom left In this order, a group of x0.3 laminin added with UV30s treatment → atmospheric pressure plasma 60s treatment and a group of x0.3 laminin added with UV30s treatment → atmospheric pressure plasma 60s treatment after ammonium coating are shown. The scale bar represents 400 μm.

1. Method for Producing Cell-Supporting Substrate The method for producing a cell-supporting substrate of the present invention includes both a UV irradiation step and a plasma irradiation step.

(1) UV irradiation process The UV irradiation process humidifies the periphery of a substrate mainly composed of a non-fluorine resin, and the substrate is subjected to oxygen and / or during the humidification and / or after humidification, that is, in a humidified environment. Or it can carry out by irradiating UV in ozone atmosphere.

(Humidification)
The humidification may be performed by any method as long as it is a method capable of providing water vapor around the base material mainly composed of a non-fluorinated resin. Humidification is performed so that the surface of the substrate irradiated with UV is exposed to water vapor. That is, the next UV irradiation process is performed in a humidified environment. Also, it is not necessary for all surfaces of the substrate to be exposed to water vapor. For example, humidification can be performed by heating water in a container or device having a certain volume that is blocked from the outside world containing the resin. The humidity after humidification (for example, the humidity during UV irradiation) can be, for example, 20 to 60% RH, preferably 40 to 50% RH at 30 ° C., and 20 to 30% at 40 ° C. . Humidification may be performed in an environment where water vapor exists around the substrate during UV irradiation, or in an environment where the surface of the substrate irradiated with UV is exposed to water vapor, before UV irradiation and / or during UV irradiation. However, it is preferably performed before and during UV irradiation.

(UV irradiation)
The UV irradiation is performed by irradiating the substrate with UV in an oxygen and / or ozone atmosphere. The irradiation UV can have an average wavelength of 184.9 nm and 253.7 nm. The wavelength of UV can be measured using a spectroradiometer. UV, for example, as a UV illumination can be carried out in ^{2000~5000μW / cm 2, 2500~4500μW / cm} 2, 3000~4000μW / cm 2, 3200~3800μW / cm 2, or 3500μW / cm ^2. The distance from the UV lamp to each plate is 2-6 cm, 3-5 cm, 3.5-4.5 cm, 3.6-4.4 cm, 3.7-4.3 cm, 3.8-4.2 cm, It can be set to 3.9-4.1 cm or 4 cm. For the UV irradiation time, the water contact angle on the surface of the non-fluorinated resin is, for example, 40 to 90 °, 40 to 80 °, 40 to 70 °, 50 to 90 °, 50 to 80 °, 50 to 70 °, 55 to 55 °. It can be performed until the temperature reaches 65 ° C, 60 to 90 °, 60 to 80 °, 60 to 70 °, 70 to 90 °, or 70 to 80 °. Alternatively, the UV irradiation time is 1 to 20 minutes, 1 to 15 minutes, 1 to 10 minutes, 3 to 20 minutes, 3 to 15 minutes, 3 to 10 minutes, 5 to 20 minutes, 5 to 15 minutes, or 5 to 5 minutes. It can be 10 minutes.

  The oxygen atmosphere means that the oxygen concentration around the substrate is 80% or more (preferably 90% or more). An oxygen atmosphere can be obtained by supplying oxygen (for example, 99% oxygen) (for example, dry oxygen) to an environment in which the substrate is held for a certain time (for example, 5 minutes). The ozone atmosphere means that the ozone concentration is 400 ppm or more (preferably 450 ppm or more). By supplying oxygen to an environment in which oxygen is generated and supplied to an environment in which the substrate is held, oxygen atmosphere can be obtained. Although ozone is generated by UV irradiation even in an oxygen atmosphere, it is preferable because UV irradiation in an ozone atmosphere increases the C—C bond and / or C—H bond that causes a chemical shift on the substrate surface. Irradiates UV in an ozone atmosphere (including oxygen and ozone atmospheres).

  In the UV irradiation process, in addition to introducing a C—C bond and / or a C—H bond causing a chemical shift on the substrate surface, the water contact angle on the substrate surface is reduced to impart hydrophilicity. In addition, although the carboxy group renders the substrate surface hydrophilic but has high reactivity, it is not suitable for cell culture, but the UV irradiation step of the present invention introduces almost no carboxy group on the substrate surface. Provide a hydrophilic surface suitable for culturing.

(2) Plasma irradiation process The plasma generation source used in the plasma irradiation process is not particularly limited, but hydrogen, helium, rare gas, oxygen, nitrogen, halogen, ammonia, carbon dioxide, water vapor, or these A mixed gas of any two or more kinds of gases can be exemplified. Plasma irradiation can be performed one or more times. The pressure of the supply gas can be 0.001 to 10 MPa, 0.01 to 1 MPa, 0.05 to 0.15 MPa, or 0.1 to 0.2 MPa. The supply gas flow rate per plasma irradiation can be set to an arbitrary flow rate. For example, in order to stably generate plasma, 2 to 30 L / min, 3 to 20 L / min, and 5 to 15 L / min. , 8-12 L / min, or 10 L / min. The distance between the plasma irradiation port and the substrate surface can be 1 to 30 mm, 3 to 20 mm, 5 to 15 mm, 8 to 12 mm, or 10 mm. Further, the plasma irradiation time may be 1 to 500 seconds, and may be, for example, 2 to 300 seconds, 3 to 200 seconds, or 5 to 160 seconds.

(3) Ammonia solution coating step The production method of the present invention may include a step of coating an ammonia solution before UV irradiation. The application of the ammonia solution can be performed by bringing a liquid containing ammonia (NH ₃ ) into contact with the cell carrying surface of the substrate. Ammonia can be dissolved in a liquid such as water, PBS, or a medium and applied as an ammonia solution. The concentration of the ammonia solution is not particularly limited, but can be 1 to 50%, 5 to 40%, or 10 to 30%. Since ammonia is volatile, it is desirable to perform UV / plasma treatment immediately after application of ammonia.

2. Cell Culture Method Using Cell-Supporting Substrate In one aspect, the present invention relates to a method for culturing adhesive cells, comprising culturing cells on the cell-supporting surface of the cell culture container described above. Cell culture is usually performed by adding a medium to a cell culture vessel, seeding desired cells in the medium, and incubating a mixture of the medium and cells with an incubator (usually 5% CO ₂ , 37 (° C.). The culture can be performed for a period until the cells adhere or for a period until the cells divide to a desired number, for example, several hours to several weeks. When culturing is for a long time, it is desirable to change the medium as necessary.

  In the method for culturing cells in the present specification, the “medium” to be used can be appropriately selected from media known to those skilled in the art according to the type of cell used. For example, when culturing stem cells (for example, iPS cells), DMEM for mesenchymal cells, MSCBM for mesenchymal cells, medium for EC cells, medium for mesenchymal cells, medium for ES cells, medium for iPS cells, Medium for stem cells, iSTEM, Cellartis (registered trademark) DEF-CS 500 Xeno-Free Culture Medium, GS2-M (registered trademark), GS1-R (registered trademark) (above, Takara Bio Inc.), Poweredby 10, Plusoid- M, G031101, M06101, SODATT201 (Glyco Technica Co., Ltd.), ReproFF2, ReproNaive, RCHEMD001, RCHEMD001A, RCHEMD001B, ReproStem, ReproXF, ReproFF2, Rep A Stemfit medium (AJINOMOTO) such as roFF, NutriStem (above, Reprocell), StemFit (registered trademark) AK02N can be used.

  In the cell culture method in the present specification, the adherent cells to be cultured are not particularly limited, but are preferably stem cells (including iPS cells). Stem cells can be cultured in the absence of feeder cells and extracellular matrix protein coatings (laminin, Matrigel (registered trademark), etc.) or even at a lower concentration of extracellular matrix protein coatings by using the culture method of the present invention. Therefore, it is difficult to cause a decrease in stability due to the use of a biological material, a difference in results due to a lot difference, or contamination. In addition, when stem cells are cultured by the cell culture method of the present invention, the properties (differentiation ability and self-proliferation ability) as stem cells can be maintained. Therefore, since the cell culture method of the present invention can cultivate stem cells without using a biological material, a more stable and safe regenerative medical material can be prepared.

  For example, the culturing method of the present invention is a culturing method including culturing cells on the cell-carrying surface of the above-described cell culturing vessel, and culturing in the absence of feeder cells. There may be. In addition, the culture method of the present invention is 0.2 to 1 times the concentration of Matrigel (Registered Trademark), which is usually necessary for adhering stem cells on a base material mainly composed of the non-fluorinated resin that has not been surface-treated. Less than (e.g., 0.2 times to 0.9 times, 0.2 times to 0.8 times, 0.2 times to 0.7 times, 0.2 times to 0.6 times, 0.2 times to 0 times) .5 times, 0.2 times to 0.4 times, 0.2 times to 0.3 times, 0.2 times, 0.3 times to 0.9 times, 0.3 times to 0.8 times, 0 times .3 times to 0.7 times, 0.3 times to 0.6 times, 0.3 times to 0.5 times, 0.3 times to 0.4 times, 0.3 times, 0.4 times to 0 times .9 times, 0.4 times to 0.8 times, 0.4 times to 0.7 times, 0.4 times to 0.6 times, 0.4 times to 0.5 times, 0.4 times, 0 .5 times to 0.9 times, 0.5 times to 0.8 times, 0.5 times to 0.7 times, 0.5 times to 0.6 times, or 0.5 times) Characterized by culturing in the presence of Matrigel (TM), it may be the culture method. In the present specification, the “base material mainly composed of the non-fluorine resin with untreated surface” means a base material composed of only the non-fluorine resin whose surface is the main component of the base material. Further, in the present specification, Matrigel (registered trademark) having a concentration of Matrigel (registered trademark) usually required for adhering stem cells to a base material mainly composed of the non-fluorinated resin whose surface has not been treated is (1 time). The term "Matrigel (registered trademark) Growth factor reduced (Corning)" refers to a solution prepared by dissolving 10 ml of DMEM-F12 medium (Life Technologies) and coating the surface of the culture dish at room temperature for 1 hour.

Furthermore, the culture method of the present invention is not less than 0.2 times the concentration of laminin that is usually necessary for adhering stem cells on a base material mainly composed of the non-fluorinated resin that is untreated on the surface (for example, 0.2 times to 0.9 times, 0.2 times to 0.8 times, 0.2 times to 0.7 times, 0.2 times to 0.6 times, 0.2 times to 0.5 times, 0.2 times to 0.4 times, 0.2 times to 0.3 times, 0.2 times, 0.3 times to 0.9 times, 0.3 times to 0.8 times, 0.3 times to 0.7 times, 0.3 times to 0.6 times, 0.3 times to 0.5 times, 0.3 times to 0.4 times, 0.3 times, 0.4 times to 0.9 times, 0.4 times to 0.8 times, 0.4 times to 0.7 times, 0.4 times to 0.6 times, 0.4 times to 0.5 times, 0.4 times, 0.5 times to 0.9 times, 0.5 times to 0.8 times, 0.5 times to 0.7 times, 0.5 times to 0.6 times, or 0.5 times the concentration of laminin In characterized by culturing, it may be the culture method. In the present specification, the laminin concentration (1 time) usually required for adhering stem cells to the base material mainly composed of the non-fluorine-based resin untreated on the surface is finally 0.5 μg / cm ² . As shown, iMatrix (1 μg / μl) diluted with PBS, incubated at 37 ° C. in a 5% CO ₂ incubator for 1 hour, and coated. In this specification, laminin is typically laminin 511E8.

  One embodiment of a cell carrying substrate surface modifying apparatus according to the present invention will be described with reference to the accompanying drawings. FIG. 13 is a configuration diagram of a cell-supporting substrate surface modifying apparatus according to the present invention comprising a single storage unit 8. The nitrogen gas supplied from the nitrogen gas supply path 1 is converted into plasma by the plasma generator 2 having the high-frequency coil 3 connected to the high-frequency power source 4 and irradiated to the cell-supporting substrate 9 as nitrogen plasma. On the other hand, the ultraviolet rays 11 generated in the ultraviolet ray generator 6 are directly or reflected by the parabolic mirror 5 and applied to the cell-supporting substrate 9. The cell-supporting substrate surface modifying apparatus further includes a valve 7 for taking in and out the cell-supporting substrate 9 and an oxygen or ozone gas inlet 10. Although not shown in the drawing, the cell-supporting substrate surface modifying apparatus of this embodiment includes a humidifying means. The surface of the cell-supporting substrate inserted from the bulb 7 is modified by the plasma irradiated from the plasma generator 2 and the ultraviolet rays irradiated from the ultraviolet generator 6.

    FIG. 14 shows a cell-supporting substrate according to the present invention having a plurality of storage units 14, a storage unit 15, a storage unit 16, and a storage unit 17, wherein plasma processing and ultraviolet processing are performed in different storage units. It is a block diagram of a material surface modification apparatus. The cell-supporting substrate 9 installed in the cell-supporting substrate setting unit 18 is transported to the storage unit 14, the storage unit 15, the storage unit 16, and the storage unit 17 by the substrate transport mechanism 12 and the substrate transport mechanism 13. Is done. Each of the storage unit 14, the storage unit 15, the storage unit 16, and the storage unit 17 has a configuration of either the ultraviolet processing storage unit illustrated in FIG. 15 or the plasma processing storage unit illustrated in FIG. . Of the storage unit 14, storage unit 15, storage unit 16, and storage unit 17, at least one is a storage unit for ultraviolet processing, and at least one is a storage unit for plasma processing. The cell-supporting substrate 9 transported to the storage unit 14, the storage unit 15, the storage unit 16, and the storage unit 17 is subjected to ultraviolet irradiation from an ultraviolet generation unit installed in each storage unit or plasma irradiation by a plasma irradiation unit. Its surface is modified.

  FIG. 15 is a configuration diagram of the ultraviolet treatment storage unit 19 among the storage units in the cell-supporting substrate surface modifying apparatus shown in FIG. The ultraviolet rays generated in the ultraviolet ray generator 6 are directly or directly reflected by the parabolic mirror 5 and applied to the cell supporting substrate 9. The ultraviolet treatment storage unit 19 includes a valve 7 for taking in and out the cell-supporting base material 9 and an oxygen or ozone gas inlet 10. Although not shown in the figure, the ultraviolet ray processing storage unit 19 includes humidification means.

  FIG. 16 is a configuration diagram of the plasma processing storage unit 20 among the storage units in the cell-supporting substrate surface modifying apparatus illustrated in FIG. 14. The nitrogen gas supplied from the nitrogen gas supply path 1 is converted into plasma by the plasma generator 2 having the high-frequency coil 3 connected to the high-frequency power source 4 and irradiated to the cell-supporting substrate 9 as nitrogen plasma. On the other hand, the cell-supporting substrate surface modifying apparatus further includes a valve 7 for taking in and out the cell-supporting substrate 9.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples. It should be noted that all documents cited throughout the present application are incorporated herein by reference as they are.

(Example 1) UV / zone / atmospheric pressure plasma composite modification treatment (1) UV / zone surface modification UV / zone surface modification is performed using an ozone generation UV exposure device (Cell Array, EKBIO-1100, Sugawara Business). (Fig. 1). Two low-pressure mercury lamps that can irradiate two types of UV light having wavelengths of 185 nm and 254 nm are installed inside the apparatus. In addition, the apparatus can be purged with oxygen from an oxygen cylinder to fill the apparatus with oxygen, and can be filled with oxygen by removing the air in the tank before irradiating UV. Oxygen irradiation causes oxygen molecules to dissociate, and ozone and active oxygen are generated on the culture substrate surface, so that oxygen atoms can be introduced to the substrate surface. In this embodiment, oxygen purge is performed with an oxygen supply pressure of 0.1 MPa, a supply flow rate of 4 L / min, and a purge time of 5 min, and the atmospheric temperature in the tank and the water temperature of the humidifying tank are both set to 25 ° C. The UV irradiation distance was fixed at 40 mm.

(2) Atmospheric pressure plasma surface treatment Atmospheric pressure plasma surface treatment was performed using a pen-type atmospheric pressure plasma apparatus. By using nitrogen gas (N ₂ ) as a plasma generation source and generating atmospheric pressure low temperature plasma, surface modification can be carried out by introducing nitrogen atoms without causing heat or charge damage to the object to be processed. . In this example, the nitrogen gas supply pressure was 0.1 to 0.2 MPa, the supply flow rate was 10 L / min, and the irradiation distance was constant at 10 mm.

(3) Production of culture substrate by UV / zone / atmospheric pressure plasma combined modification treatment In this example, a culture dish made of polystyrene having a diameter of 60 mm (430589, Corning) was used. For surface modification, a sample irradiated with UV for 1 minute, 3 minutes, or 10 minutes was prepared in the single modification treatment of UV / zone surface modification. In addition, a sample irradiated with plasma for 5 seconds, 40 seconds, and 160 seconds was prepared for the single reforming treatment of the atmospheric pressure plasma treatment. In addition, an untreated sample not subjected to surface modification was also prepared as a control group. UV / zone surface modification and atmospheric pressure plasma treatment of the above UV irradiation time as a sample subjected to a combined process of UV / zone surface modification and atmospheric pressure plasma treatment, UV / zone surface modification The order of the atmospheric pressure plasma treatment was changed before and after. Detailed reforming conditions are shown in Table 1.

(Example 2) XPS analysis The sample prepared in Example 1 was analyzed for surface molecular structure by XPS, and the effect of surface modification according to the present invention on modification of surface properties was examined. Each plate of 8 mm was cut out from each dish, and surface analysis was performed using a photoelectron spectrometer (JEOL Ltd., JPS-9010). Each sample was affixed to the sample stage, the sample stage was placed in the preparation room and evacuated, and then the sample stage was inserted into the measurement room. Since the sample used in this analysis is made of polystyrene, the constituent elements are carbon and oxygen (hydrogen is also a constituent element, but since hydrogen has only one electron, it cannot be measured by XPS). Therefore, in this analysis, the wide scan was not performed, and the narrow scan spectrum of the carbon 1s orbital electron was acquired. The X-ray was an AlKa line (1486.6 eV), and the carbon narrow scan measurement range was 294.0 to 280.0 eV. The step width was 0.1 eV and the number of integrations was 10. After obtaining the spectrum, the spectrum was smoothed by smoothing at step number 5. The background of the spectrum caused by inelastically scattered electrons and noise was removed using Shirley background removal. Spectral waveform separation was performed by approximating the component waveform with a Gauss-Lorentz function normally distributed function.

  FIG. 2 shows a wide scan spectrum of samples of the Control group, and FIG. 3 shows a narrow scan spectrum of C (1s). The narrow scan spectrum and waveform separation of C (1s) of the processed sample are shown in FIGS. FIG. 6 shows a narrow scan spectrum of O (1s) in the analysis of the sample group subjected to each surface modification treatment alone. Further, FIG. 7 shows a narrow scan spectrum of N (1s) in the analysis of the sample group subjected to the atmospheric pressure plasma treatment alone. Further, FIG. 8 shows the narrow scan spectrum of C (1 s) of the sample subjected to the combined treatment of UV irradiation 3 min and plasma irradiation 40 s, and FIG. 9 shows the C of the sample subjected to the combined treatment of UV irradiation 10 min and plasma irradiation 160 s. The narrow scan spectrum of (1s) is shown.

  In the sample subjected to UV / zone surface modification, the amount of expressed oxygen increased as the UV irradiation time increased (FIG. 6). It was also found that when the UV irradiation time is short, only the carboxy group (COOH) is expressed, and when the UV irradiation time is long, a single bond of carbon and oxygen (CO) is expressed in addition to COOH. (FIG. 4). From the above results, it was shown that in the UV / zone surface modification, functional groups are introduced in the order of COOH and CO with increasing UV irradiation time.

  Next, in the sample subjected to the atmospheric pressure plasma treatment, the amount of oxygen increased as compared with the control group, and an increase in the amount of oxygen expressed was observed as the plasma irradiation time increased (FIG. 6). In addition, an increase in the amount of nitrogen was observed as the plasma irradiation time increased (FIG. 7). On the other hand, no bonding state other than the polystyrene-derived bond was observed from the sample with the plasma irradiation time of 5 s, but from the sample with the irradiation time of 40 s, the carbon and nitrogen single bond (CN) was the sample with the irradiation time of 160 s. From FIG. 5, a CN bond and a carboxy group were observed (FIG. 5C). Furthermore, an increase in the amount of CN bonds was observed as the plasma irradiation time increased (FIG. 5). From the above results, it was suggested that in the atmospheric pressure plasma treatment, the amount of CN bonds increased as the plasma irradiation time increased, and the carboxy group was introduced as the irradiation time increased.

  Finally, in the sample group subjected to UV / zone surface modification and atmospheric pressure plasma treatment as a combined process, —COOH was recognized as a functional group containing oxygen under all treatment conditions (FIGS. 8 and 9). On the other hand, for functional groups containing nitrogen, imino groups (C = N) are obtained from samples that have been subjected to UV / zone surface modification first, and amide bonds (NH--) from samples that have been previously subjected to atmospheric pressure plasma treatment. C = O) was observed (FIGS. 8 and 9). In addition, the sample subjected to the atmospheric pressure plasma treatment has a higher expression amount of functional groups containing nitrogen than the sample subjected to the UV / zone surface modification earlier, and the sample subjected to the atmospheric pressure plasma treatment first. From the sample, both C = N and NH-C = O were observed, and only C = N was observed from the sample that had been subjected to UV / zone surface modification first (FIGS. 8 and 9). From the above results, in the combined treatment of UV / zone surface modification and atmospheric pressure plasma treatment, C = N is easily formed when UV irradiation is performed first, and NH—C when plasma irradiation is performed first. It was suggested that ═O is easily formed. The imide group and amide group could not be formed by UV / zone alone or plasma alone, and could only be generated on the culture substrate by carrying out the present invention.

(Example 3) Cell culture (1) Cells Cells were used in two mouse ES cell lines and one mouse iPS cell line. As mouse ES cells, ES-B3 strain and ES-D3 strain were used. The ES-B3 strain is a cell line that does not require feeder cells for culture. The ES-B3 strain expresses the blasticinin S resistant gene by the transcriptional activity of the Oct3 / 4 gene because one of the Oct3 / 4 gene, which is a marker indicating an undifferentiated state, has been replaced with the ires-blasticin S resistant gene. Therefore, cells differentiated by culturing in the presence of Blasticidin S, which is a nucleoside antibiotic, are killed by Blasticidin S, and only undifferentiated ES cells can be purified and cultured. The ES-D3 strain is an ES cell strain in which there are many research examples and pluripotency is sufficiently secured by a differentiation induction test. The ES-D3 strain requires feeder cells for culture. The APS0001 strain provided by Riken Cell Bank was used in order to conduct a culture test using a mouse iPS cell line as a pluripotent stem cell. In this cell line, a GFP gene is introduced under the control of the promoter of Nanog, which is an undifferentiation marker, and cells in an undifferentiated form can be identified with a fluorescence microscope.

(2) Culture method B3 strain and D3 strain were used for mouse ES cells. The ES-B3 strain is composed of Glasgow Modified Minimum Essential Medium (GMEM), 10% fetal bovine serum (FBS), 1% antibacterial-antibiotic (antibiotic), 0.1% A solution containing essential amino acids (Non Essential Amino Acid, NEAA), 1 mM sodium pyruvate, and 0.1 mM mercaptoethanol (2-mercaptoethanol, 2ME) as a basic culture, 1000 μg / ml blasticidin S, 1000 / Ml Leukemia Inhibitory Factor (LIF) added The culture was performed in the prepared medium. The ES-D3 strain is in Dulbecco's Modified Eagle Medium high-glucose (DMEM high-glucose), 15% fetal bovine serum (FBS), antibacterial serum (FBS). -1000 μg / ml blasticinSin as a basic culture solution containing antibiotics (antibiotic-anticotic), 0.1 mM essential amino acid (Non Essential Amino Acid: NEAA), 0.1 mM mercaptoethanol (2-MEcaptoethanol: 2ME) , 1000 U / ml leukemia inhibitory factor (LIF) And culture was performed. Mouse iPS cells were obtained by using the iPS-MEF-Ng-20D-17 strain (APS0001 RIKEN Cell Bank) at a concentration of Dulbecco's Modified Eagle Medium (high glucose) (DMEM high-glucose). 15% fetal bovine serum (FBS), 1% antibacterial-antibiotic, 0.1 mM essential amino acid (NEAA), 0.1 mM mercaptoethanol (2-mercaptoethanol) 2ME) as a basic culture solution, 1000 μg / ml blasticin S, 1000 U / ml leukemia inhibitory factor Culturing was performed in a medium supplemented with pups (Leukemia Inhibitory Factor, LIF). In the ES-B3 strain, the culture substrate is an untreated polystyrene culture dish (control), UV / zone alone 10 min, plasma alone 10 s, 40 s, and the respective reforming treatments are sequentially performed in order. A polystyrene culture dish subjected to a combined process treatment was used. In ES-D3 strain, untreated substrate (control), UV / zone alone 10 min, plasma alone 10 s, and polystyrene-treated culture that has been subjected to a combined process treatment in which the respective reforming treatments are sequentially performed before and after. A dish was used.

  For the culture of mouse iPS cells, a polystyrene culture dish seeded with feeder cells, a polystyrene culture dish treated with UV / zone 3 m and then plasma 14 s, and an untreated polystyrene culture dish were used. The culture period was 72 hours after cell seeding, and cell adhesion and proliferation were evaluated with a digital camera for microscope (DP73, Olympus) equipped with an inverted phase contrast microscope (CKX41, Olympus) at a magnification of 100 times. This was done by taking an image. In mouse iPS cell culture, Nanog marker expression was performed by confirming green fluorescence under B excitation with an inverted phase contrast microscope (CKX41, Olympus) equipped with an epifluorescence tube. The number of cells was determined by separating and recovering all adherent cells with 0.25% trypsin 72 hours after cell seeding and quantifying the total amount of DNA. Since the total amount of DNA has a proportional relationship with the number of cells, the number of cells can be quantitatively evaluated. The collected cell suspension was quantified using a fluorescence spectrophotometer (Qubit (registered trademark) 2.0 Fluorometer, Life Technologies) and the attached Qubit Buffer, Quit Reagent. did.

(3) Results In the culture of the ES-D3 strain, compared to the control and single treatment groups, the group subjected to the complex process showed higher cell adhesion. In particular, a good cell adhesion was shown in a composite process in which nitrogen plasma treatment was performed immediately after UV / zone modification (FIG. 10). In the culture of ES-D3 strain, compared with culture on plasma alone (10 s, 40 s), UV alone modified substrate, it is a complex process in which nitrogen plasma treatment is performed immediately after UV / zone modification is performed. , Cell adhesion was better than other treatment groups. In addition, the total DNA content, which is an index indicating the number of cells on the substrate, was also found to be significantly higher in the sample group subjected to nitrogen plasma treatment immediately after the UV / zone modification (Fig. 11). Also in mouse iPS cell culture, cell adhesion was improved by a good complex process, and GFP expression was observed by fluorescence microscopy using Nanog as an undifferentiated marker as a promoter (FIG. 12). It has also been shown that the modification treatment maintains the pluripotency of pluripotent stem cells.

(Example 4) Composite surface-modified ammonium treatment (1) Surface modification treatment A polystyrene tissue culture dish (# 3000-035, AGC Techno Glass) having a diameter of 35 mm was used. First, after the culture equipment was installed in the surface reforming apparatus, 180 μl of 28% ammonia water (# 02511-05, Nacalai Tesque) was dropped on the center of the substrate. Next, the device door was quickly closed, and the UV lamp and plasma generator were immediately turned on to start surface modification. The UV lamp was turned off 30 seconds after the start of the modification, and the plasma apparatus was turned off 60 seconds after the start of the modification to finish the surface modification.

(2) Matrigel 0.2 times the recommended amount in the polystyrene dish prepared in (1) from human iPS cell 253G4 strain (Nakagawa M et al., Nat Biotechnol. 26 (1): 101-6 (2008)) Alternatively, a Stem fit medium (AJINOMOTO) (Scientific Reports 4, Artistic number: 3594 (2014) doi: 10.1038 / srep03594) (containing Y27364) supplemented with 0.3 times the recommended amount of laminin is contained. Seeded at ⁴ cells / dish. The cells were cultured for 3 days under conditions of 37 ° C., 5% CO ₂ , and humidity of 100%. As a control, the cells were cultured in the same manner in dishes that were not surface-modified using the recommended amounts of matrigel or laminin, respectively. After culture, non-adherent cells were removed, and the number of adherent cells was evaluated by counting the number of cells with ViCell.

(3) Results The results are shown in FIGS. By applying ammonia water before UV / plasma surface treatment, cell growth equivalent to the recommended amount of laminin concentration added group was confirmed by using 0.3 times the recommended amount of laminin. In addition, by applying aqueous ammonia before the UV / plasma surface treatment, cell growth exceeding the recommended amount of Matrigel concentration was observed even at a Matrigel concentration of 0.2 times the recommended amount. The cell proliferation of this ammonia water application group was further enhanced than the UV / plasma surface treatment group to which the same amount of laminin / matrigel was added. From these results, it was shown that application of aqueous ammonia before UV / plasma surface treatment further enhances the improvement of iPS cell proliferation ability by the UV / plasma surface.

DESCRIPTION OF SYMBOLS 1 Nitrogen gas supply path 2 Plasma generating part 3 High frequency coil 4 High frequency power supply 5 Parabolic mirror 6 Ultraviolet generating part 7 Valve 8 Storage part 9 Cell-supporting base material 10 Ozone gas inlet 11 Ultraviolet light 12 Base material transport mechanism 13 Base material transport Mechanisms 14 to 17 Storage unit 18 Cell-supporting substrate installation unit 19 Ultraviolet processing storage unit 20 Plasma processing storage unit

Claims (14)

A method for producing a cell-supporting substrate mainly comprising a non-fluorinated resin having a cell-supporting surface,
A method comprising: a UV irradiation step of irradiating UV to a cell carrying surface of the substrate in a humidified environment and an atmosphere of oxygen and / or ozone supply; and a plasma irradiation step of irradiating plasma to the cell carrying surface of the substrate.   The cell-supporting substrate according to claim 1, wherein the non-fluorine-based resin is at least one resin selected from the group consisting of polyethylene, acrylic resin, ABS resin, polyethylene terephthalate, polypropylene, polycarbonate, and polystyrene. Manufacturing method.   The method for producing a cell-supporting substrate according to claim 1 or 2, wherein a plasma irradiation step is performed after the UV irradiation step.   The method for producing a cell-supporting substrate according to claim 1 or 2, wherein a UV irradiation step is performed after the plasma irradiation step.   The method for producing a cell-supporting substrate according to claim 1 or 2, wherein the UV irradiation step and the plasma irradiation step are performed simultaneously.   The said UV irradiation is performed by irradiating UV with an average wavelength of 184.9 nm and 253.7 nm, The base material for cell carrying | support of any one of Claims 1-5 characterized by the above-mentioned. Production method.   The cell carrying according to any one of claims 1 to 6, wherein the UV irradiation is performed until a water contact angle on the surface of the non-fluorinated resin reaches 40 to 70 °. Method for manufacturing a substrate.   Furthermore, the manufacturing method of any one of Claims 1-7 provided with the process of apply | coating ammonia solution to this cell support surface before UV irradiation process.   A method for culturing adherent cells, comprising culturing cells on a cell-supporting surface of a cell-supporting substrate obtained by the production method according to any one of claims 1 to 8. .   The culture method according to claim 9, wherein the adherent cells are stem cells.   The culture method according to claim 10, wherein the stem cells are mouse iPS cells or human iPS cells.   The culture method according to claim 10 or 11, wherein the culture is performed in the absence of feeder cells.   A storage unit for storing a cell-supporting substrate mainly composed of a non-fluorine resin having a cell-supporting surface, and an ultraviolet ray capable of irradiating the cell-supporting substrate stored in the storage unit with ultraviolet rays Irradiation means, plasma irradiation means capable of irradiating nitrogen plasma to the cell support substrate stored in the storage unit, humidification means capable of controlling humidity in the cell support substrate storage unit, and the cells A cell-supporting substrate surface modifying apparatus having oxygen or ozone supply means capable of controlling the ozone concentration in the supporting substrate storage section.   14. The cell-supporting substrate surface modifying apparatus according to claim 13, further comprising an ammonia solution coating means capable of coating an ammonia solution on a cell-supporting surface of the cell-supporting substrate.