WO2013140822A1 - Cell culture base material and cell culture method - Google Patents

Cell culture base material and cell culture method Download PDF

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Publication number
WO2013140822A1
WO2013140822A1 PCT/JP2013/001973 JP2013001973W WO2013140822A1 WO 2013140822 A1 WO2013140822 A1 WO 2013140822A1 JP 2013001973 W JP2013001973 W JP 2013001973W WO 2013140822 A1 WO2013140822 A1 WO 2013140822A1
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cell culture
culture substrate
substrate
carbon
cells
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PCT/JP2013/001973
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French (fr)
Japanese (ja)
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勝 堀
馬場　嘉信
行広 岡本
博基 近藤
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国立大学法人名古屋大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C01B32/18—Nanoonions; Nanoscrolls; Nanohorns; Nanocones; Nanowalls
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates

Definitions

  • the present invention relates to a cell culture substrate and a cell culture method. More specifically, the present invention relates to a cell culture substrate made of carbon and a cell culture method using the same.
  • Non-Patent Documents 1 and 2 Conventionally, flat culture using a flat bottom flask has been generally used for culturing cells. However, it has been found that the cells cannot fully exhibit properties such as enzyme activity and biosynthetic activity that are inherent to cells (see Non-Patent Documents 1 and 2).
  • Patent Document 1 discloses a cell culture container using carbon nanotubes and a method for manufacturing the same.
  • the present inventors have found that it is preferable to use carbon nanowalls as a cell culture substrate.
  • the carbon nanowall refers to a graphene sheet formed in a direction crossing the plate surface of the substrate.
  • each graphene sheet is disposed substantially perpendicular to the substrate. Therefore, the tip part (edge) of the graphene sheet is used as a scaffold for cell growth.
  • cell culture substrates having different graphene sheet intervals can be prepared.
  • the chemical property can be changed by substituting the atom couple
  • the present invention has been made in order to solve the problems of the conventional techniques described above. That is, the subject is to provide a cell culture substrate having a scaffold suitable for cell culture and a cell culture method using the same.
  • the cell culture substrate in the first embodiment has a substrate and carbon nanowalls formed on the substrate.
  • the tip of the carbon nanowall graphene sheet is a scaffold for culturing cells.
  • This cell culture substrate can control the chemical properties of the tip of the graphene sheet. It can be continuously changed from hydrophilicity to water repellency. It is also possible to control the physical properties of the scaffold, such as the structure of the wall spacing. That is, this cell culture substrate has a suitable scaffold adapted to the cells to be cultured.
  • the graphene sheet is formed in a direction intersecting the plate surface of the substrate.
  • the average wall interval of the graphene sheet is in the range of 10 nm to 1000 nm.
  • the cell culture substrate according to the fourth aspect at least some of the carbon atoms at the tip of the graphene sheet are bonded to atoms other than carbon atoms. Thereby, a cell culture substrate in which various chemical species are terminal groups is realized.
  • the cell culture substrate in the fifth aspect at least some of the carbon atoms at the tip of the graphene sheet are bonded to oxygen atoms or nitrogen atoms.
  • This cell culture substrate has hydrophilicity. Therefore, it is possible to observe cells when grown on a hydrophilic scaffold.
  • the carbon atoms at the tip of the graphene sheet are bonded to fluorine atoms. Therefore, it is possible to observe cells when grown on a water-repellent scaffold.
  • the average wall spacing of the graphene sheet is in the range of 10 nm to 500 nm. In this case, the cultured cells grow well.
  • the average wall interval of the graphene sheet is in the range of 80 nm to 120 nm. In this case, the cultured cells grow well.
  • the average wall interval of the graphene sheet is in the range of 120 nm to 200 nm. A minimally invasive recovery of cells can be performed.
  • At least some of the carbon atoms at the tip of the graphene sheet are bonded to hydrogen atoms.
  • the contact angle with water at the scaffold is in the range of 1 ° to 170 °. This is because cells can be cultured in various scaffold environments.
  • the cell culture substrate in the twelfth aspect has a coating film that coats the tip of the graphene sheet.
  • the coating film has been subjected to a collagen coating treatment. In addition to promoting cell proliferation, it also promotes cell differentiation.
  • the cell culture method according to the fourteenth aspect is a method of culturing cells on a cell culture substrate. And as a cell culture substrate, a substrate in which carbon nanowalls are formed is used. The tip of the carbon nanowall graphene sheet is used as a scaffold for culturing cells. The cells can be cultured while controlling the physical and chemical properties of the scaffold.
  • a cell culture substrate in which the graphene sheet is formed in a direction intersecting the plate surface of the substrate is used.
  • a cell culture substrate having a graphene sheet in which at least some of the carbon atoms at the tip are bonded to atoms other than carbon atoms is used.
  • a cell culture substrate having a graphene sheet in which at least some of the carbon atoms at the tip are bonded to oxygen atoms or nitrogen atoms is used. Cells can be observed when grown on a hydrophilic scaffold.
  • a cell culture substrate having a graphene sheet in which at least some of the carbon atoms at the tip are bonded to fluorine atoms or hydrogen atoms is used. Therefore, it is possible to observe cells when grown on a water-repellent scaffold.
  • a cell culture substrate having a contact angle with water at the scaffold in the range of 1 ° to 170 ° is used. This is because cells can be cultured in various scaffold environments.
  • a cell culture substrate having a coating film for coating the tip of the graphene sheet is used.
  • the coating film has been subjected to a collagen coating treatment. In addition to promoting cell proliferation, it also promotes cell differentiation.
  • a cell culture substrate having a scaffold suitable for cell culture and a cell culture method using the same are provided.
  • FIG. 1 It is a perspective view for demonstrating the time of use of the 1st cell culture substratum concerning an embodiment.
  • Is a photograph showing a contact angle (10 ° or less) with water in the carbon nano-wall a CH 4 gas cell culture media were prepared by performing atmospheric pressure plasma treatment after creating with material in experiments A.
  • the carbon nano-wall is a photograph showing a contact angle (50 °) with water in a cell culture substrate was prepared by CH 4 gas in the experiment A.
  • the carbon nano-wall is a photograph showing a contact angle (105 °) with water in a cell culture substrate was prepared by C 2 F 6 gas in experiment A.
  • 6 is a table showing production conditions and properties of high-density, medium-density, and low-density carbon nanowalls produced in Experiment E. It is a graph which shows the density dependence and chemical species dependence of the contact angle with water measured in Experiment F. 6 is a graph showing a Raman spectrum corresponding to the density measured in Experiment G. 6 is a graph showing a spectrum of a chemical composition around the tip of a carbon nanowall measured in Experiment H. It is a graph which shows the contact angle dependence with water and density dependence in the cell number of the HeLa cell measured in Experiment I. 4 is a graph showing the relationship between the density of carbon nanowalls measured in Experiment J and the density of fluorine atoms or oxygen atoms.
  • FIG. 1 is a schematic configuration diagram showing a first cell culture substrate 100 according to the embodiment.
  • the cell culture substrate 100 is a cell culture substrate used for culturing animal cells, particularly human-derived cells. As shown in FIG. 1, the cell culture substrate 100 includes a substrate 110 and a carbon nanowall CNW1.
  • the substrate 110 has a support substrate 111 and a metal layer 112.
  • the support substrate 111 is a semiconductor substrate such as Si, Ge, or GaAs or an oxide substrate such as SiO 2 , TiO 2 , or Al 2 O 3 .
  • the metal layer 112 is a layer made of a metal such as Ti, Ta, Ni, Co, Al, W, Fe, Pt, or TiN.
  • the metal layer 112 acts as a catalyst for generating initial growth nuclei of the carbon nanowall. Therefore, the metal layer 112 is preferably provided. However, it is not always necessary.
  • the carbon nanowall CNW1 is formed on the substrate 110.
  • the base portion R1 is on the substrate 110 side, and the tip portion E1 is on the opposite side of the substrate 110.
  • the root portion R ⁇ b> 1 is a fixed portion that is fixed to the substrate 110.
  • the tip E1 is a scaffold that serves as a scaffold for culturing cells.
  • FIG. 2 is a diagram schematically showing the structure of the carbon nanowall CNW1.
  • the graphene sheet is formed in a direction intersecting the plate surface of the substrate 110.
  • the graphene sheet and the substrate 110 are substantially vertical. Therefore, there is a tip E1 at the tip of the graphene sheet.
  • the tip E1 is a location located at the tip of the graphene sheet.
  • tip part E1 becomes a scaffold for culturing a cell.
  • the carbon atom C1 at the tip E1 is bonded to a hydrogen atom. That is, the terminal group of the carbon nanowall CNW1 is a hydrogen atom. And this termination
  • the carbon nanowall CNW1 is formed by laminating a plurality of graphene sheets. Actually, the graphene sheets of each other do not extend in parallel. Since the graphene sheets grow in different directions in each initial growth nucleus, the graphene sheets are actually stacked at random. Details will be described later. And as shown in FIG. 2, let the distance between adjacent graphene sheets be the wall space
  • Table 1 shows numerical values indicating the structure of the carbon nanowall CNW1 including the wall interval D1. However, these numerical values are merely examples, and are not limited to these values.
  • the average wall interval that is the average value of the wall interval D1 is related to the density of the carbon nanowall CNW1. That is, the wider the average wall interval, the lower the density of the carbon nanowall CNW1. Conversely, the smaller the average wall interval, the higher the density of the carbon nanowall CNW1.
  • FIG. 3 is a schematic configuration diagram showing the second cell culture substrate 200.
  • the cell culture substrate 200 includes a substrate 110 and a carbon nanowall CNW2.
  • a replacement portion SP is formed on the tip end portion E2 side of the carbon nanowall CNW2.
  • the replacement part SP is obtained by replacing the atom bonded to the carbon atom of the tip part E2 of the carbon nanowall CNW2 with another atom.
  • the carbon atom at the tip E2 of the carbon nanowall CNW2 is bonded to an atom other than the carbon atom.
  • properties such as hydrophilicity and hydrophobicity of the cell culture substrate 200 change. That is, the chemical nature of the scaffold for culturing cells can be selected.
  • an oxygen atom can be used as an atom to be substituted. Therefore, at least some carbon atoms C1 of the tip E2 of the carbon nanowall CNW2 are bonded to oxygen atoms. The remaining carbon atoms of the tip E2 remain bonded to the hydrogen atoms. Therefore, the terminal group of the front-end
  • the degree of replacement varies depending on various conditions such as the processing time of the replacement process.
  • FIG. 4 is a schematic configuration diagram showing a third cell culture substrate 300.
  • the cell culture substrate 300 includes a substrate 110 and a carbon nanowall CNW3.
  • a coating film CM is formed on the tip E3 side of the carbon nanowall CNW3.
  • the coating film CM is obtained by coating the tip of a graphene sheet with a coating material having affinity with cells. Examples of the material of the coating film CM include collagen.
  • Other configurations of the cell culture substrate 300 except for the coating film CM are the same as those of the first cell culture substrate 100.
  • a coating film CM may be formed on the second cell culture substrate 200.
  • FIG. 5 is a schematic configuration diagram showing the configuration of the manufacturing apparatus 1.
  • the manufacturing apparatus 1 includes a plasma generation chamber 46 and a reaction chamber 10.
  • the plasma generation chamber 46 is for generating plasma inside and generating radicals to be supplied to the reaction chamber 10.
  • the reaction chamber 10 is for forming the carbon nanowall CNW1 using radicals generated in the plasma generation chamber 46.
  • the manufacturing apparatus 1 includes a waveguide 47, a quartz window 48, and a slot antenna 49.
  • the waveguide 47 is for introducing the microwave 39.
  • the slot antenna 49 is for introducing the microwave 39 from the quartz window 48 to the plasma generation chamber 46.
  • the plasma generation chamber 46 is for generating surface wave plasma (SWP) by the microwave 39.
  • the plasma generation chamber 46 is provided with a radical source inlet 42.
  • the radical source inlet 42 is for supplying a gas serving as a radical source into the plasma 61 generated in the plasma generation chamber 46.
  • a partition wall 44 is provided between the plasma generation chamber 46 and the reaction chamber 10.
  • the partition 44 is for partitioning the plasma generation chamber 46 and the reaction chamber 10. Further, as will be described later, it also serves as an electrode for applying a voltage.
  • a through hole is formed in the partition wall 44. This is for supplying radicals generated in the plasma generation chamber 46 to the reaction chamber 10.
  • the reaction chamber 10 is for generating capacitively coupled plasma (CCP). It is also for forming carbon nanowalls on the substrate 50.
  • the reaction chamber 10 includes a second electrode 24, a heater 25, a raw material introduction port 12, and an exhaust port 16. As will be described later, the second electrode 24 is for applying a voltage between the second electrode 24 and the first electrode 22.
  • the heater 25 is for heating the substrate 50 and controlling the temperature of the substrate 50.
  • the raw material inlet 12 is for supplying a carbon-based gas 32 that is a raw material of the carbon nanowall.
  • the exhaust port 16 is connected to a vacuum pump or the like. The vacuum pump is for adjusting the pressure inside the reaction chamber 10.
  • the partition wall 44 also serves as the first electrode 22 for applying a voltage between the second electrode 24.
  • a power source and a circuit are connected to the first electrode 22. This is for controlling the potential of the first electrode 22 in terms of time.
  • the second electrode 24 is for applying a voltage between the first electrode 22 and the second electrode 24.
  • the second electrode 24 is also a mounting table for mounting the substrate 50.
  • the second electrode 24 is grounded.
  • the distance between the first electrode 22 and the second electrode 24 is about 5 cm. Of course, it is not limited to this value.
  • the substrate 50 before the carbon nanowall CNW1 is formed is set inside the manufacturing apparatus 1.
  • the microwave 39 is introduced into the waveguide 47.
  • the microwave 39 is introduced into the plasma generation chamber 46 from the quartz window 48 by the slot antenna 49. Thereby, high-density plasma 60 is generated.
  • the high-density plasma 60 is diffused inside the plasma generation chamber 46 to become plasma 61.
  • This plasma 61 contains radical source ions supplied from the radical source inlet 42. Hydrogen is used as a radical source. Or oxygen, nitrogen, and other gas may be sufficient. Most of the ions in the plasma 61 collide with the partition walls 44 and are neutralized to become radicals.
  • the radical 38 passes through the through hole of the partition wall 44 and enters the reaction chamber 10.
  • a carbon-based gas 32 is supplied from the raw material inlet 12 into the reaction chamber 10.
  • the carbon-based gas 32 is, for example, CH 4 or C 2 F 6 . Of course, it may be other than that.
  • a voltage is applied between the first electrode 24 and the second electrode 22. As a result, plasma 34 is generated inside the reaction chamber 10.
  • the second cell culture substrate 200 is manufactured by subjecting the first cell culture substrate 100 to plasma treatment. Therefore, plasma is generated in the first cell culture substrate 100 using a gas containing atoms to be replaced as a plasma gas.
  • a plasma generator is placed inside the chamber.
  • Ar + O 2 gas is turned into plasma while purging the inside of the chamber with Ar gas. Thereby, radicals derived from oxygen atoms are generated in the plasma generation region.
  • the generated oxygen atoms react at the tip E1 of the cell culture substrate 100 to produce a tip E2 to which oxygen atoms are bonded.
  • other gas may be used.
  • the third cell culture substrate 300 is produced by subjecting the first cell culture substrate 100 to a collagen coating treatment. Therefore, a method for coating the cell culture substrate 100 with collagen will be described. First, the first cell culture substrate 100 is placed in a container containing an acidic collagen solution. Thereby, a thin film of collagen is formed at the tip E1. At this time, the cell culture substrate 100 is acidic. Next, the cell culture substrate 100 immersed in the collagen acidic solution is dried and then immersed in the medium. Therefore, the cell culture substrate 100 is neutral. Thereby, the front-end
  • FIG. 6 is a photomicrograph of the structure of the carbon nanowall formed as described above as viewed from the tip side. As shown in FIG. 6, the carbon nanowalls are growing randomly. However, the intervals are uniform to some extent.
  • FIG. 7 is a photomicrograph showing a cross section of the structure of the formed carbon nanowall viewed from the side of the substrate. As shown in FIG. 7, the carbon nanowall is formed substantially perpendicular to the substrate.
  • FIG. 8 is a diagram showing a state where cells are cultured using the cell culture substrate 100.
  • the cell culture substrate 100 is placed on the bottom surface of the petri dish 500.
  • the carbon nanowall CNW1 faces upward. Therefore, the tip E1 of the cell culture substrate 100 does not contact the bottom surface of the petri dish 500.
  • the culture solution is poured into the petri dish 500 on which the cell culture substrate 100 is placed.
  • a general solution used for cell culture may be used.
  • the cells to be cultured are supplied to the tip E1 of the cell culture substrate 100.
  • a pipette can be used.
  • the cells can be cultured similarly.
  • Experiment A (hydrophilic and water repellent) 5-1. Control of hydrophilicity and water repellency As described above, in the cell culture substrate 100 of the present embodiment, atoms or molecules bonded to the carbon atom C1 of the tip E1 can be replaced. Thereby, in carbon nanowall CNW1 of this embodiment, hydrophilicity or water repellency can be provided.
  • This control can be performed by supplying radicals after forming the carbon nanowall CNW1.
  • the chemical species of the carbon atom C1 can be replaced by irradiating the carbon nanowall CNW1 with plasma.
  • an oxygen atom can be bonded to the carbon atom C1 by introducing oxygen as a radical source.
  • bonded with the oxygen atom can be produced
  • This graphene sheet in which hydrogen atoms are replaced with oxygen atoms has hydrophilicity, as will be described later.
  • hydrophilicity or water repellency also changes depending on the degree of substitution of the chemical species (B) (for example, 50% substitution).
  • This control can be adjusted by changing the plasma irradiation time.
  • the hydrophilicity or water repellency slightly changes depending on the structure of carbon nanowalls (for example, wall interval D1). This control can be adjusted by changing other conditions such as the substrate temperature, source gas, and pressure in the reaction chamber 10. By combining these (A) to (C), the hydrophilicity or water repellency at the tip can be changed almost continuously. These (A) to (C) change not only the chemical properties such as hydrophilicity or water repellency, but also other chemical properties at the tip portion that becomes a scaffold for cells.
  • FIG. 9 to 12 show the contact angle with water.
  • FIG. 9 is a photograph showing a contact angle with water in a cell culture substrate produced by performing atmospheric pressure plasma treatment after creating a carbon nanowall using the raw material carbon-based gas 32 as CH 4 gas. The contact angle is 10 ° or less.
  • FIG. 10 is a photograph showing a contact angle with water in a cell culture substrate in which carbon nanowalls are made of CH 4 gas. The contact angle is about 50 °.
  • FIG. 11 is a photograph showing a contact angle with water in a cell culture substrate in which carbon nanowalls are prepared with C 2 F 6 gas.
  • the contact angle is approximately 105 °.
  • FIG. 12 is a photograph showing a contact angle with water in a cell culture substrate produced by producing a carbon nanowall with CH 4 gas and then performing a fluorine treatment. This fluorine treatment was performed by plasma irradiation. The supply gas at that time is a fluorocarbon gas. The contact angle is approximately 135 °. Note that this fluorine treatment may be performed by immersing in a hydrogen fluoride (HF) solution.
  • HF hydrogen fluoride
  • the contact angle with water in the scaffold is in the range of 1 ° to 170 °.
  • carbon nanowalls can replace chemical species that bind to the carbon atoms at the tip of the cell.
  • the side of the graphene sheet (for example, S1 in FIG. 2) serves as a scaffold for cells.
  • ⁇ electrons exist in the carbon nanotube.
  • the carbon nanotube does not have the tip E1 unlike the carbon nanowall CNW1. Therefore, in the carbon nanotube, although the surface structure can be changed by substitution of chemical species or formation of lattice defects, the degree of change is very small compared to the change amount of the wall interval D1 in the carbon nanowall CNW1. I can say that.
  • FIG. 13 to FIG. 16 show micrographs of cells when contact angles with water are different. These are all HeLa cells. And it is a microscope picture 4 days after starting culture
  • FIG. 13 is a photomicrograph of cells when the contact angle with water is 10 ° or less.
  • FIG. 14 is a photomicrograph of cells when the contact angle with water is approximately 50 °.
  • FIG. 15 is a photomicrograph of cells when the contact angle with water is approximately 105 °.
  • FIG. 16 is a photomicrograph of cells when the contact angle with water is approximately 135 °. From these results, as the contact angle with water increases, the cell changes to a shape close to a sphere. As described above, as shown with a specific example, the cell culture substrate 100 having a contact angle with water in the scaffold portion in the range of 1 ° to 170 ° can be produced.
  • FIG. 17 is a graph showing the relationship between the contact angle with water on the scaffold of the cell culture substrate and the number of cells after 4 days from the start of culture.
  • the cultured cells are HeLa cells.
  • the number of cells cultured on the cell culture substrate of this embodiment is compared with the number of cells cultured on a glass substrate.
  • the horizontal axis in FIG. 17 is the contact angle with water.
  • the vertical axis in FIG. 17 is the number of cells.
  • a cell culture substrate having a larger number of cells is suitable for culturing cells.
  • the number of cells cultured on the cell culture substrate of this embodiment is greater than the number of cells cultured on the glass substrate.
  • the cell culture substrate of this embodiment can culture cells at about 1 ⁇ 10 4 cells / cm 2 even at a contact angle of 135 ° with strong water repellency. In the case of a contact angle of 135 °, a minimally invasive recovery of cells could be performed.
  • FIG. 18 is a graph showing the relationship between the contact angle with water in the scaffold for cell culture substrate and the amount of protein adsorbed.
  • the cultured cells are also HeLa cells.
  • the horizontal axis in FIG. 18 is the contact angle with water.
  • the vertical axis in FIG. 18 represents the amount of protein adsorbed on the cell culture substrate.
  • the amount of protein adsorbed on the cell culture substrate of this embodiment is about twice the amount of protein adsorbed on the glass substrate. That is, the cell culture substrate of the present embodiment having the carbon nanowall CNW1 is more suitable for cell culture than the glass substrate.
  • alkaline phosphatase activity was measured and compared for cells cultured by four methods.
  • the four types are as follows.
  • FIG. 19 shows the result.
  • the vertical axis represents the measurement result of alkaline phosphatase activity based on undifferentiated cells. It is considered that the greater the measured value compared to the undifferentiated cells, the more differentiated into osteoblasts. As shown in FIG. 19, the measured values are all about 1.1 times the measured value of alkaline phosphatase activity in undifferentiated cells. The measured value when a glass substrate is used is slightly lower than other cases.
  • FIG. 20 shows the result of culturing cells using a cell culture substrate in which each substrate is coated with collagen.
  • the cell culture substrate (collagen-coated) using carbon nanowalls is considered to be more differentiated into osteoblasts than those using a culture dish or glass substrate.
  • FIG. 21 is a table showing what kind of carbon nanowalls are produced when the production conditions of carbon nanowalls such as pressure, growth time, and CCPPpower are changed. is there. As shown in FIG. 21, by changing the manufacturing conditions, it is possible to manufacture from a high density carbon nanowall to a low density carbon nanowall. Note that conditions other than those shown in FIG. 21 are the same as in Experiment A.
  • FIG. 21 (a) shows a high-density carbon nanowall.
  • the wall interval of the high-density carbon nanowall (a) was 95 nm.
  • the internal pressure of the manufacturing apparatus 1 was set to 1 Pa
  • the CCPP power was set to 100 W
  • the growth time was set to 80 minutes.
  • FIG. 21 (b) shows a medium density carbon nanowall.
  • the wall interval of the medium density carbon nanowall in (b) was 131 nm.
  • the internal pressure of the manufacturing apparatus 1 was set to 1 Pa
  • the CCPP power was set to 300 W
  • the growth time was set to 15 minutes.
  • FIG. 21 (c) shows a low-density carbon nanowall.
  • the wall interval of the low density carbon nanowall (c) was 313 nm.
  • the internal pressure of the manufacturing apparatus 1 was set to 5 Pa
  • the CCPP power was set to 500 W
  • the growth time was set to 8 minutes.
  • FIG. 21A what is shown in FIG. 21A will be referred to as a high density carbon nanowall, and what is shown in FIG. 21B will be referred to as a medium density carbon nanowall.
  • the material shown in 21 (c) is called a low density carbon nanowall.
  • the following experiment was conducted by culturing cells in these three types of samples (a) to (c).
  • the contact angle with water strongly depends on the type of atoms bonded at the tip E2.
  • the contact angle with water is about 5 °.
  • the contact angle with water is about 5 °.
  • the contact angle with water is about 5 °.
  • the contact angle with water is about 50 °.
  • the contact angle with water is approximately 130 ° to 150 °.
  • the contact angle with water hardly depended on the wall interval of the carbon nanowall. In other words, even if the wall interval is changed, the contact angle with water hardly changes.
  • FIG. 23 is a graph showing the results of measuring Raman shifts of low-density carbon nanowalls, medium-density carbon nanowalls, and high-density carbon nanowalls. These terminal groups are in the case of hydrogen. In other words, this is the result when the terminal group is not substituted.
  • the peak of the G band near 1590 cm -1, the peak of the D band near 1350 cm -1, and the peak of the D 'band near 1620 cm -1 were observed.
  • the spectra of these carbon nanowalls are slightly different from each other. For example, the higher the density, the larger the D band component.
  • FIG. 24 shows the chemical composition ratio when the terminal group of the medium density carbon nanowall is changed.
  • the horizontal axis in FIG. 24 is the binding energy.
  • the vertical axis in FIG. 24 is intensity.
  • FIG. 24A shows the result of replacing part of the terminal group of the graphene sheet with an oxygen atom.
  • FIG. 24B shows the result of replacing part of the terminal group of the graphene sheet with a nitrogen atom.
  • FIG.24 (c) shows the result of what did not substitute a part of terminal group of a graphene sheet. However, it was confirmed that a component of C—O bond was mixed in part.
  • FIG. 24 (d) shows the result of replacing part of the terminal group of the graphene sheet with a fluorine atom.
  • FIG. 25 is a graph showing the relationship between the scaffold part of the cell culture substrate and the number of cultured HeLa cells cultured on the scaffold part.
  • the horizontal axis in FIG. 25 is the contact angle with water.
  • the vertical axis in FIG. 25 is the number of HeLa cells.
  • the initial number of HeLa cells was about 10,000 cells / cm 2 .
  • the number of cells after 4 days is plotted.
  • the number of cells cultured on a glass substrate is also plotted.
  • the number of cells when cultured on a cell culture substrate having medium density carbon nanowalls is approximately the same as the number of cells when cultured on a glass substrate.
  • the glass substrate has a contact angle with water of about 40,000 cells / cm 2 and a contact angle with water of 60 °. In the case of culturing at 4 000 cells / cm 2, it was about 40,000 cells / cm 2 .
  • the cell culture When cultured on a cell culture substrate having medium density carbon nanowalls with a water contact angle of 5 °, the cell culture is about 31000 cells / cm 2 and is cultured on a glass substrate with a water contact angle of 10 °. In this case, it was about 25000 cells / cm 2 .
  • the glass substrate when cultured on a cell culture substrate having a medium density carbon nanowall having a contact angle with water of 135 °, the glass substrate has a contact angle with water of about 10,000 cells / cm 2 and a contact angle with water of 105 °. In the case of culturing at 10000 cells / cm 2, it was about 10,000 cells / cm 2 . However, in these cases, the contact angle with water is different. Note that the glass substrate can have a contact angle with water of only about 105 °.
  • the contact angle with water In the region where the contact angle with water is about 0 ° to 10 °, the number of cells when cultured on a cell culture substrate having low-density carbon nanowalls is about 55000 cells / cm 2 , and the number of cells is very high. There were many. In the region where the contact angle with water was about 0 ° to 10 °, the number of cells in other cases was between about 25000 cells / cm 2 and about 35000 cells / cm 2 .
  • the number of cells is different between when cultured on a cell culture substrate having medium density carbon nanowalls and when cultured on a glass substrate. It was about 40,000 cells / cm 2 . In other cases, it was about 25000 cells / cm 2 .
  • the contact angle with water was about 130 ° to 140 °
  • a large difference appeared depending on the difference in wall spacing. That is, when cultured on a cell culture substrate having high-density carbon nanowalls, the number of cells was about 60000 cells / cm 2 and the number of cells was very large. On the other hand, when cultured on a cell culture substrate having low-density carbon nanowalls, the number of cells was about 25000 cells / cm 2 . In addition, when cultured on a cell culture substrate having medium density carbon nanowalls, the number of cells was about 10,000 cells / cm 2 .
  • the number of cells was large when cultured on a cell culture substrate having a large contact angle with water and having high-density carbon nanowalls. That is, the contact angle with water is preferably in the range of 120 ° to 150 °, and the average wall interval is preferably in the range of 80 nm to 120 nm.
  • the contact angle with water falls within the range of 120 ° to 150 ° when the terminal group of the graphene sheet is a fluorine atom.
  • the number of cells was large when cultured on a cell culture substrate having a small contact angle with water and having low-density carbon nanowalls. That is, the contact angle with water is preferably in the range of 3 ° to 10 °, and the average wall interval is preferably in the range of 10 nm to 500 nm. In particular, the average wall interval is preferably in the range of 200 nm to 500 nm.
  • the contact angle with water is in the range of 3 ° to 10 ° when the terminal group of the graphene sheet is an oxygen atom or a nitrogen atom.
  • FIG. 26 is a graph showing the relationship between the density of carbon nanowalls and the oxygen density or fluorine density.
  • oxygen or fluorine was used as a plasma gas
  • the terminal group of the graphene sheet was an oxygen atom or a fluorine atom.
  • the horizontal axis of FIG. 26 is the density of the carbon nanowall.
  • the vertical axis on the left side of FIG. 26 represents the ratio of fluorine atoms to carbon atoms in the terminal group.
  • the vertical axis on the right side of FIG. 26 represents the ratio of oxygen atoms to carbon atoms in the terminal group.
  • the ratio of fluorine atoms to carbon atoms in the terminal group is about 1.0.
  • the ratio of fluorine atoms to carbon atoms in the terminal group is about 1.7.
  • the ratio of fluorine atoms to carbon atoms in the terminal group is about 1.5.
  • the ratio of oxygen atoms to carbon atoms in the terminal group is about 0.22.
  • the ratio of oxygen atoms to carbon atoms in the terminal group is about 0.32.
  • the ratio of oxygen atoms to carbon atoms in the terminal group is about 0.18.
  • FIG. 27 is a graph showing the relationship between the density of carbon nanowalls and the number of cells when the terminal group is a fluorine atom.
  • all of the terminal groups are not fluorine atoms, but have a ratio to carbon atoms as shown in FIG.
  • the ratio of fluorine atoms to carbon atoms is in the range of 1.0 to 1.5.
  • FIG. 28 corresponds to a combination of a high-density or low-density carbon nanowall and an atom of the terminal group, and a photomicrograph showing the shape of the cell on the cell culture substrate by the combination. And FIG. 28 image
  • the terminal group is a hydrogen atom
  • the shape of the HeLa cell is slightly circular or nearly spherical.
  • FIG. 29 is a graph showing the elongation of HeLa cells. The larger this value, the longer the length of one side of the cell. That is, it deviates from a circle. Conversely, the smaller this value is, the closer it is to a circle. As shown in FIG. 29, the value is particularly small when the terminal group is a hydrogen atom. That is, when the terminal group is a hydrogen atom, the shape of the HeLa cell is closer to a circle.
  • FIG. 30 is a photomicrograph of HeLa cells.
  • FIG. 31 is a photomicrograph of stained HeLa cells. A mercury lamp was used as a light source, and a 652 nm FITC was used as a filter. In FIG. 31, the stained cell is a living HeLa cell.
  • the tip part E1 of the graphene sheet can be made conductive by supporting metal fine particles on the carbon nanowall CNW1. Moreover, it is good also as driving a radical into carbon nanowall CNW1 after formation using the manufacturing apparatus 1.
  • FIG. Thereby, carbon nanowall CNW1 can also be made into a semiconductor. And carbon nanowall CNW1 can also be made into an n-type semiconductor by introduce
  • the third cell culture substrate 300 in which the tip of the carbon nanowall is coated with collagen is used.
  • other coating materials may be used.
  • the coating material other than collagen include collagen peptide, polyethylene glycol, dextran, polyacrylamide, and polymethacryloyloxyethyl phosphorylcholine.
  • the carbon nanowall CNW1 is formed.
  • the tip E1 of the carbon nanowall CNW1 serves as a cell scaffold. Fine adjustments can be made to the chemical properties and structure of the tip E1. Therefore, cell culture substrates 100, 200, and 300 suitable for culturing cells are realized.
  • cultivate a cell suitably is implement
  • SYMBOLS 1 Manufacturing apparatus 100, 200, 300 ... Cell culture base material 110 ... Substrate 111 ... Supporting substrate 112 ... Metal layer CNW1, CNW2, CNW3 ... Carbon nanowall E1, E2, E3 ... Tip part R1 ... Root part C1 ... Carbon atom D1 Wall spacing

Abstract

[Problem] To provide a cell culture base material having a scaffold suited to culturing cells and a cell culture method using the cell culture base material. [Solution] A cell culture base material (100) has a substrate (110) and a carbon nanowall (CNW1). The substrate (110) has a support substrate (111) and a metallic layer (112). The carbon nanowall (CNW1) is disposed so as to intersect the surface of the substrate (100). A distal end (E1) of a graphene sheet in the carbon nanowall (CNW1) is a scaffold for culturing cells. The chemical species bonded to at least some of the carbon atoms of the distal end (E1) can be selected.

Description

細胞培養基材および細胞培養方法Cell culture substrate and cell culture method
　本発明は、細胞培養基材および細胞培養方法に関する。さらに詳細には、カーボンを素材とする細胞培養基材およびそれを用いた細胞培養方法に関するものである。 The present invention relates to a cell culture substrate and a cell culture method. More specifically, the present invention relates to a cell culture substrate made of carbon and a cell culture method using the same.
　従来、細胞を培養するのに、平底フラスコを用いた平面培養が一般的であった。しかし、それでは、細胞が本来備える酵素活性や生合成活性等の性質を十分に発揮しきれないことが分かっている（非特許文献１、２参照）。 Conventionally, flat culture using a flat bottom flask has been generally used for culturing cells. However, it has been found that the cells cannot fully exhibit properties such as enzyme activity and biosynthetic activity that are inherent to cells (see Non-Patent Documents 1 and 2).
　ところで、一部の細胞、特にヒト由来の細胞は、何かに接着した状態で培養する必要がある。生体外で浮遊している状態では、長期間にわたって生存することが困難であるからである。つまり、細胞の培養には、その成長の基礎とするための足場が必要である。そのため、細胞が接着するための基材が開発されてきている。例えば、特許文献１には、カーボンナノチューブを利用した細胞培養容器およびその製造方法が開示されている。 By the way, some cells, especially human-derived cells, need to be cultured while adhering to something. This is because it is difficult to survive over a long period of time in a floating state outside the living body. In other words, cell culture requires a scaffold to serve as a basis for its growth. Therefore, a base material for cell adhesion has been developed. For example, Patent Document 1 discloses a cell culture container using carbon nanotubes and a method for manufacturing the same.
特開２００９－１４２２１８号公報JP 2009-142218 A
　カーボンナノチューブを足場にする場合には、細胞は、グラフェンシートのシート面（側面）を足場とすることとなる。そのため、細胞を培養するための好適性は、グラフェンシートの物理的性質および化学的性質に拘束される。したがって、カーボンナノチューブのシート面（側面）に化学修飾や孔を形成する研究がなされてきている。しかしそれでもなお、カーボンナノチューブのシート面（側面）の物理的性質および化学的性質を変化させる自由度は、それほど高くない。つまり、足場の性質を著しく変えるには不十分である。 When carbon nanotubes are used as a scaffold, cells use the sheet surface (side surface) of the graphene sheet as a scaffold. Therefore, the suitability for culturing cells is bound by the physical and chemical properties of graphene sheets. Therefore, studies have been made to form chemical modifications and pores on the sheet surface (side surface) of carbon nanotubes. However, the degree of freedom to change the physical properties and chemical properties of the carbon nanotube sheet surface (side surface) is still not so high. That is, it is not enough to significantly change the nature of the scaffold.
　本発明者らは、鋭意研究の結果、カーボンナノウォールを細胞培養基材とすると好適であることを発見した。ここで、カーボンナノウォールとは、基板の板面に交差する向きに形成されたグラフェンシートのことをいう。実際には、後述するように、各グラフェンシートは、基板に対してほぼ垂直に配置されている。そのため、グラフェンシートの先端部（エッジ）を、細胞の成長の足場とする。この場合には、グラフェンシートの間隔の異なる細胞培養基材を作成することができる。また、グラフェンシートの先端部（エッジ）の炭素原子と結合している原子を置換することにより、その化学的性質を変えることができる。 As a result of intensive studies, the present inventors have found that it is preferable to use carbon nanowalls as a cell culture substrate. Here, the carbon nanowall refers to a graphene sheet formed in a direction crossing the plate surface of the substrate. Actually, as will be described later, each graphene sheet is disposed substantially perpendicular to the substrate. Therefore, the tip part (edge) of the graphene sheet is used as a scaffold for cell growth. In this case, cell culture substrates having different graphene sheet intervals can be prepared. Moreover, the chemical property can be changed by substituting the atom couple | bonded with the carbon atom of the front-end | tip part (edge) of a graphene sheet.
　本発明は、前述した従来の技術が有する問題点を解決するためになされたものである。すなわちその課題とするところは、細胞の培養に適した足場を有する細胞培養基材およびそれを利用した細胞培養方法を提供することである。 The present invention has been made in order to solve the problems of the conventional techniques described above. That is, the subject is to provide a cell culture substrate having a scaffold suitable for cell culture and a cell culture method using the same.
　第１の態様における細胞培養基材は、基板と、基板に形成されたカーボンナノウォールとを有するものである。そして、カーボンナノウォールのグラフェンシートの先端部が、細胞を培養するための足場部である。 The cell culture substrate in the first embodiment has a substrate and carbon nanowalls formed on the substrate. The tip of the carbon nanowall graphene sheet is a scaffold for culturing cells.
　この細胞培養基材では、グラフェンシートの先端部の化学的性質を制御することができる。親水性から撥水性まで、連続的に変えることができる。また、ウォール間隔等の構造など、足場の物理的性質をも制御することができる。つまり、この細胞培養基材は、培養しようとする細胞に合わせた好適な足場を有している。 This cell culture substrate can control the chemical properties of the tip of the graphene sheet. It can be continuously changed from hydrophilicity to water repellency. It is also possible to control the physical properties of the scaffold, such as the structure of the wall spacing. That is, this cell culture substrate has a suitable scaffold adapted to the cells to be cultured.
　第２の態様における細胞培養基材では、グラフェンシートは、基板の板面に交差する向きに形成されている。 In the cell culture substrate in the second aspect, the graphene sheet is formed in a direction intersecting the plate surface of the substrate.
　第３の態様における細胞培養基材では、グラフェンシートの平均ウォール間隔は、１０ｎｍ以上１０００ｎｍ以下の範囲内である。 In the cell culture substrate in the third aspect, the average wall interval of the graphene sheet is in the range of 10 nm to 1000 nm.
　第４の態様における細胞培養基材では、グラフェンシートの先端部の少なくとも一部の炭素原子は、炭素原子以外の原子と結合している。これにより、種々の化学種が終端基である細胞培養基材が実現されている。 In the cell culture substrate according to the fourth aspect, at least some of the carbon atoms at the tip of the graphene sheet are bonded to atoms other than carbon atoms. Thereby, a cell culture substrate in which various chemical species are terminal groups is realized.
　第５の態様における細胞培養基材では、グラフェンシートの先端部の少なくとも一部の炭素原子は、酸素原子または窒素原子と結合している。この細胞培養基材は、親水性を有している。そのため、親水性の足場で生育した場合の細胞を観察することができる。 In the cell culture substrate in the fifth aspect, at least some of the carbon atoms at the tip of the graphene sheet are bonded to oxygen atoms or nitrogen atoms. This cell culture substrate has hydrophilicity. Therefore, it is possible to observe cells when grown on a hydrophilic scaffold.
　第６の態様における細胞培養基材では、グラフェンシートの先端部の少なくとも一部の炭素原子は、フッ素原子と結合している。そのため、撥水性の足場で生育した場合の細胞を観察することができる。 In the cell culture substrate in the sixth aspect, at least some of the carbon atoms at the tip of the graphene sheet are bonded to fluorine atoms. Therefore, it is possible to observe cells when grown on a water-repellent scaffold.
　第７の態様における細胞培養基材では、グラフェンシートの平均ウォール間隔は、１０ｎｍ以上５００ｎｍ以下の範囲内である。この場合に、培養する細胞がよく増殖する。 In the cell culture substrate according to the seventh aspect, the average wall spacing of the graphene sheet is in the range of 10 nm to 500 nm. In this case, the cultured cells grow well.
　第８の態様における細胞培養基材では、グラフェンシートの平均ウォール間隔は、８０ｎｍ以上１２０ｎｍ以下の範囲内である。この場合に、培養する細胞がよく増殖する。 In the cell culture substrate according to the eighth aspect, the average wall interval of the graphene sheet is in the range of 80 nm to 120 nm. In this case, the cultured cells grow well.
　第９の態様における細胞培養基材では、グラフェンシートの平均ウォール間隔は、１２０ｎｍ以上２００ｎｍ以下の範囲内である。細胞の低侵襲性回収を行うことができる。 In the cell culture substrate according to the ninth aspect, the average wall interval of the graphene sheet is in the range of 120 nm to 200 nm. A minimally invasive recovery of cells can be performed.
　第１０の態様における細胞培養基材では、グラフェンシートの先端部の少なくとも一部の炭素原子は、水素原子と結合している。 In the cell culture substrate according to the tenth aspect, at least some of the carbon atoms at the tip of the graphene sheet are bonded to hydrogen atoms.
　第１１の態様における細胞培養基材では、足場部での水との接触角が、１°以上１７０°以下の範囲内である。種々の足場の環境で、細胞を培養することができるからである。 In the cell culture substrate according to the eleventh aspect, the contact angle with water at the scaffold is in the range of 1 ° to 170 °. This is because cells can be cultured in various scaffold environments.
　第１２の態様における細胞培養基材では、グラフェンシートの先端部をコーティングするコーティング膜を有する。 The cell culture substrate in the twelfth aspect has a coating film that coats the tip of the graphene sheet.
　第１３の態様における細胞培養基材では、コーティング膜は、コラーゲンコート処理がなされたものである。細胞の増殖を促進するとともに、細胞の分化をも促進する。 In the cell culture substrate according to the thirteenth aspect, the coating film has been subjected to a collagen coating treatment. In addition to promoting cell proliferation, it also promotes cell differentiation.
　第１４の態様における細胞培養方法は、細胞培養基材の上に細胞を培養する方法である。そして、細胞培養基材として、基板にカーボンナノウォールが形成されたものを用いる。また、カーボンナノウォールのグラフェンシートの先端部を、細胞を培養するための足場とする。足場の物理的性質および化学的性質を制御した上で、細胞の培養を行うことができる。 The cell culture method according to the fourteenth aspect is a method of culturing cells on a cell culture substrate. And as a cell culture substrate, a substrate in which carbon nanowalls are formed is used. The tip of the carbon nanowall graphene sheet is used as a scaffold for culturing cells. The cells can be cultured while controlling the physical and chemical properties of the scaffold.
　第１５の態様における細胞培養方法では、細胞培養基材として、グラフェンシートが基板の板面に交差する向きに形成されているものを用いる。 In the cell culture method according to the fifteenth aspect, a cell culture substrate in which the graphene sheet is formed in a direction intersecting the plate surface of the substrate is used.
　第１６の態様における細胞培養方法では、細胞培養基材として、先端部の少なくとも一部の炭素原子は、炭素原子以外の原子と結合しているグラフェンシートを有するものを用いる。 In the cell culture method according to the sixteenth aspect, a cell culture substrate having a graphene sheet in which at least some of the carbon atoms at the tip are bonded to atoms other than carbon atoms is used.
　第１７の態様における細胞培養方法では、細胞培養基材として、先端部の少なくとも一部の炭素原子が、酸素原子または窒素原子と結合しているグラフェンシートを有するものを用いる。親水性の足場で生育した場合の細胞を観察することができる。 In the cell culture method according to the seventeenth aspect, a cell culture substrate having a graphene sheet in which at least some of the carbon atoms at the tip are bonded to oxygen atoms or nitrogen atoms is used. Cells can be observed when grown on a hydrophilic scaffold.
　第１８の態様における細胞培養方法では、細胞培養基材として、先端部の少なくとも一部の炭素原子が、フッ素原子または水素原子と結合しているグラフェンシートを有するものを用いる。そのため、撥水性の足場で生育した場合の細胞を観察することができる。 In the cell culture method according to the eighteenth aspect, a cell culture substrate having a graphene sheet in which at least some of the carbon atoms at the tip are bonded to fluorine atoms or hydrogen atoms is used. Therefore, it is possible to observe cells when grown on a water-repellent scaffold.
　第１９の態様における細胞培養方法では、細胞培養基材として、足場部での水との接触角が、１°以上１７０°以下の範囲内であるものを用いる。種々の足場の環境で、細胞を培養することができるからである。 In the cell culture method according to the nineteenth aspect, a cell culture substrate having a contact angle with water at the scaffold in the range of 1 ° to 170 ° is used. This is because cells can be cultured in various scaffold environments.
　第２０の態様における細胞培養方法では、細胞培養基材として、グラフェンシートの先端部をコーティングするコーティング膜を有するものを用いる。 In the cell culture method according to the twentieth aspect, a cell culture substrate having a coating film for coating the tip of the graphene sheet is used.
　第２１の態様における細胞培養方法では、コーティング膜は、コラーゲンコート処理がなされたものである。細胞の増殖を促進するとともに、細胞の分化をも促進する。 In the cell culture method according to the twenty-first aspect, the coating film has been subjected to a collagen coating treatment. In addition to promoting cell proliferation, it also promotes cell differentiation.
　本発明によれば、細胞の培養に適した足場を有する細胞培養基材およびそれを利用した細胞培養方法が提供されている。 According to the present invention, a cell culture substrate having a scaffold suitable for cell culture and a cell culture method using the same are provided.
実施形態に係る第１の細胞培養基材を示す概略構成図である。It is a schematic block diagram which shows the 1st cell culture substratum which concerns on embodiment. 実施形態に係る細胞培養基材におけるカーボンナノウォールを説明するための模式図である。It is a schematic diagram for demonstrating the carbon nanowall in the cell culture substratum which concerns on embodiment. 実施形態に係る第２の細胞培養基材を示す概略構成図である。It is a schematic block diagram which shows the 2nd cell culture substratum which concerns on embodiment. 実施形態に係る第３の細胞培養基材を示す概略構成図である。It is a schematic block diagram which shows the 3rd cell culture substratum which concerns on embodiment. 実施形態に係る細胞培養基材を製造する製造装置を説明するための概略構成図である。It is a schematic block diagram for demonstrating the manufacturing apparatus which manufactures the cell culture substratum which concerns on embodiment. 実施形態に係る細胞培養基材におけるカーボンナノウォールを上方から見たＳＥＭ写真である。It is the SEM photograph which looked at carbon nanowall in the cell culture substrate concerning an embodiment from the upper part. 実施形態に係る細胞培養基材におけるカーボンナノウォールの断面を見たＳＥＭ写真である。It is the SEM photograph which looked at the section of carbon nanowall in the cell culture substrate concerning an embodiment. 実施形態に係る第１の細胞培養基材の使用時を説明するための斜視図である。It is a perspective view for demonstrating the time of use of the 1st cell culture substratum concerning an embodiment. 実験ＡにおいてカーボンナノウォールをＣＨ4 ガスで作成した後に大気圧プラズマ処理を行うことにより製造された細胞培養基材における水との接触角（１０°以下）を示す写真である。Is a photograph showing a contact angle (10 ° or less) with water in the carbon nano-wall a CH 4 gas cell culture media were prepared by performing atmospheric pressure plasma treatment after creating with material in experiments A. 実験ＡにおいてカーボンナノウォールをＣＨ4 ガスで作成した細胞培養基材における水との接触角（５０°）を示す写真である。The carbon nano-wall is a photograph showing a contact angle (50 °) with water in a cell culture substrate was prepared by CH 4 gas in the experiment A. 実験ＡにおいてカーボンナノウォールをＣ2 Ｆ6 ガスで作成した細胞培養基材における水との接触角（１０５°）を示す写真である。The carbon nano-wall is a photograph showing a contact angle (105 °) with water in a cell culture substrate was prepared by C 2 F 6 gas in experiment A. 実験ＡにおいてカーボンナノウォールをＣＨ4 ガスで作成した後にフッ素処理を行って製造された細胞培養基材における水との接触角（１３５°）を示す写真である。Is a photograph showing a contact angle (135 °) with water in a cell culture substrate prepared by performing the fluorine treatment after creating a carbon nano-wall at CH 4 gas in the experiment A. 実験Ｂにおいて接触角１０°以下の細胞培養基材に培養した細胞を示す写真である。It is a photograph which shows the cell cultured on the cell culture substratum whose contact angle is 10 degrees or less in Experiment B. 実験Ｂにおいて接触角５０°の細胞培養基材に培養した細胞を示す写真である。It is a photograph which shows the cell cultured on the cell-culture base material of contact angle 50 degrees in experiment B. FIG. 実験Ｂにおいて接触角１０５°の細胞培養基材に培養した細胞を示す写真である。It is a photograph which shows the cell cultured on the cell-culture base material of contact angle 105 degrees in experiment B. FIG. 実験Ｂにおいて接触角１３５°の細胞培養基材に培養した細胞を示す写真である。It is a photograph which shows the cell cultured on the cell-culture base material of contact angle 135 degrees in experiment B. FIG. 実験Ｂにおいて培養を開始してから４日経過後の細胞数を実施形態の細胞培養基材とガラス基板とで比較したグラフである。It is the graph which compared the cell number after progress for 4 days after starting culture | cultivation in Experiment B with the cell culture substratum and glass substrate of embodiment. 実験ＣにおいてＢＣＡ法を用いたタンパク質の吸着量を実施形態の細胞培養基材とガラス基板とで比較したグラフである。It is the graph which compared the adsorption amount of the protein using BCA method in Experiment C with the cell culture substratum and glass substrate of embodiment. 実験Ｄにおいて各培養方法で培養した細胞における未分化細胞に対する活性比を示すグラフ（コラーゲンコート無し）である。It is a graph (no collagen coat) which shows the activity ratio with respect to an undifferentiated cell in the cell cultured by Experiment D in Experiment D. 実験Ｄにおいて各培養方法で培養した細胞における未分化細胞に対する活性比を示すグラフ（コラーゲンコート有り）である。6 is a graph (with a collagen coat) showing an activity ratio of cells cultured by each culture method in Experiment D to undifferentiated cells. 実験Ｅにおいて製造した高密度および中密度および低密度のカーボンナノウォールの製造条件および性質を示す表である。6 is a table showing production conditions and properties of high-density, medium-density, and low-density carbon nanowalls produced in Experiment E. 実験Ｆにおいて測定した水との接触角の密度依存性および化学種依存性を示すグラフである。It is a graph which shows the density dependence and chemical species dependence of the contact angle with water measured in Experiment F. 実験Ｇにおいて測定した密度に応じたラマンスペクトルを示したグラフである。6 is a graph showing a Raman spectrum corresponding to the density measured in Experiment G. 実験Ｈにおいて測定したカーボンナノウォールの先端部周辺の化学組成のスペクトルを示すグラフである。6 is a graph showing a spectrum of a chemical composition around the tip of a carbon nanowall measured in Experiment H. 実験Ｉにおいて測定したＨｅＬａ細胞の細胞数における水との接触角依存性および密度依存性を示すグラフである。It is a graph which shows the contact angle dependence with water and density dependence in the cell number of the HeLa cell measured in Experiment I. 実験Ｊにおいて測定したカーボンナノウォールの密度とフッ素原子密度もしくは酸素原子密度との関係を示すグラフである。4 is a graph showing the relationship between the density of carbon nanowalls measured in Experiment J and the density of fluorine atoms or oxygen atoms. 実験Ｊにおいて測定したカーボンナノウォールの密度とＨｅＬａ細胞の細胞数との関係を示すグラフである。It is a graph which shows the relationship between the density of the carbon nanowall measured in Experiment J, and the cell number of a HeLa cell. 実験Ｋにおいて測定したＨｅＬａ細胞の形状の密度依存性および終端基依存性を示す表である。10 is a table showing density dependency and terminal group dependency of the shape of HeLa cells measured in Experiment K. 実験Ｋにおいて測定したＨｅＬａ細胞の伸びを示すグラフである。3 is a graph showing the elongation of HeLa cells measured in Experiment K. 実験Ｌにおいて観察したＨｅＬａ細胞の顕微鏡写真である。2 is a photomicrograph of HeLa cells observed in Experiment L. FIG. 実験Ｌにおいて観察した染色後のＨｅＬａ細胞の顕微鏡写真である。It is a microscope picture of the HeLa cell after dyeing observed in Experiment L.
　以下、具体的な実施形態について、細胞培養基材および細胞培養方法を例に挙げて図を参照しつつ説明する。 Hereinafter, specific embodiments will be described with reference to the drawings, taking a cell culture substrate and a cell culture method as examples.
１．細胞培養基材
１－１．第１の細胞培養基材
１－１－１．第１の細胞培養基材の構造
　図１は、実施形態に係る第１の細胞培養基材１００を示す概略構成図である。細胞培養基材１００は、動物細胞、特にヒト由来細胞を培養するために使用される細胞培養基材である。図１に示すように、細胞培養基材１００は、基板１１０と、カーボンナノウォールＣＮＷ１と、を有している。 1. Cell culture substrate 1-1. First cell culture substrate 1-1-1. Structure of First Cell Culture Substrate FIG. 1 is a schematic configuration diagram showing a first cell culture substrate 100 according to the embodiment. The cell culture substrate 100 is a cell culture substrate used for culturing animal cells, particularly human-derived cells. As shown in FIG. 1, the cell culture substrate 100 includes a substrate 110 and a carbon nanowall CNW1.
　基板１１０は、支持基板１１１と、金属層１１２とを有している。支持基板１１１は、Ｓｉ、Ｇｅ、ＧａＡｓなどの半導体基板またはＳｉＯ2 、ＴｉＯ2 、Ａｌ2 Ｏ3 などの酸化物基板である。金属層１１２は、Ｔｉ、Ｔａ、Ｎｉ、Ｃｏ、Ａｌ、Ｗ、Ｆｅ、Ｐｔ、ＴｉＮなどの金属から成る層である。金属層１１２は、カーボンナノウォールの初期成長核を発生させる触媒として作用するものである。そのため、金属層１１２を設けることが好ましい。しかし、必ずしも必要であるわけではない。 The substrate 110 has a support substrate 111 and a metal layer 112. The support substrate 111 is a semiconductor substrate such as Si, Ge, or GaAs or an oxide substrate such as SiO 2 , TiO 2 , or Al 2 O 3 . The metal layer 112 is a layer made of a metal such as Ti, Ta, Ni, Co, Al, W, Fe, Pt, or TiN. The metal layer 112 acts as a catalyst for generating initial growth nuclei of the carbon nanowall. Therefore, the metal layer 112 is preferably provided. However, it is not always necessary.
　カーボンナノウォールＣＮＷ１は、基板１１０の上に形成されている。カーボンナノウォールＣＮＷ１において、基板１１０の側には根元部Ｒ１があり、基板１１０の反対側には、先端部Ｅ１がある。根元部Ｒ１は、基板１１０に固定されている固定部である。先端部Ｅ１は、細胞を培養するための足場となる足場部である。 The carbon nanowall CNW1 is formed on the substrate 110. In the carbon nanowall CNW1, the base portion R1 is on the substrate 110 side, and the tip portion E1 is on the opposite side of the substrate 110. The root portion R <b> 1 is a fixed portion that is fixed to the substrate 110. The tip E1 is a scaffold that serves as a scaffold for culturing cells.
１－１－２．第１の細胞培養基材の微細構造
　図２は、カーボンナノウォールＣＮＷ１の構造を模式的に表した図である。カーボンナノウォールＣＮＷ１において、グラフェンシートは、基板１１０の板面に交差する向きに形成されている。図２では、グラフェンシートと、基板１１０とは、ほぼ垂直である。そのため、グラフェンシートの先端には、先端部Ｅ１がある。先端部Ｅ１は、グラフェンシートの先端に位置する箇所である。そして、前述したように、先端部Ｅ１が細胞を培養するための足場となる。なお、先端部Ｅ１における炭素原子Ｃ１は、水素原子と結合している。つまり、カーボンナノウォールＣＮＷ１の終端基は、水素原子である。そして、この終端基が主に足場部としての役割を担うこととなる。 1-1-2. FIG. 2 is a diagram schematically showing the structure of the carbon nanowall CNW1. In the carbon nanowall CNW1, the graphene sheet is formed in a direction intersecting the plate surface of the substrate 110. In FIG. 2, the graphene sheet and the substrate 110 are substantially vertical. Therefore, there is a tip E1 at the tip of the graphene sheet. The tip E1 is a location located at the tip of the graphene sheet. And as above-mentioned, the front-end | tip part E1 becomes a scaffold for culturing a cell. The carbon atom C1 at the tip E1 is bonded to a hydrogen atom. That is, the terminal group of the carbon nanowall CNW1 is a hydrogen atom. And this termination | terminus group will mainly play the role as a scaffold part.
　また、図２に示すように、カーボンナノウォールＣＮＷ１は、グラフェンシートを多数枚積層したものである。実際には、互いのグラフェンシートが平行に延びているわけではない。各々の初期成長核で異なる方向にグラフェンシートが成長するため、実際には、グラフェンシートがランダムに重ね合わせられた形状となっている。詳しくは、後述する。そして、図２に示すように、隣り合うグラフェンシート間の距離をウォール間隔Ｄ１ということとする。ウォール間隔Ｄ１は、細胞の足場部における足場間の距離を決めるものである。また、ウォール間隔Ｄ１を含めたカーボンナノウォールＣＮＷ１の構造を示す数値を表１に示す。ただし、これらの数値はあくまで例示であり、これらの値に限るものではない。 Further, as shown in FIG. 2, the carbon nanowall CNW1 is formed by laminating a plurality of graphene sheets. Actually, the graphene sheets of each other do not extend in parallel. Since the graphene sheets grow in different directions in each initial growth nucleus, the graphene sheets are actually stacked at random. Details will be described later. And as shown in FIG. 2, let the distance between adjacent graphene sheets be the wall space | interval D1. The wall interval D1 determines the distance between the scaffolds in the cell scaffold. In addition, Table 1 shows numerical values indicating the structure of the carbon nanowall CNW1 including the wall interval D1. However, these numerical values are merely examples, and are not limited to these values.
　このウォール間隔Ｄ１の平均値である平均ウォール間隔は、カーボンナノウォールＣＮＷ１の密度と関連している。つまり、平均ウォール間隔が広いほど、カーボンナノウォールＣＮＷ１の密度は低い。逆に、平均ウォール間隔が狭いほど、カーボンナノウォールＣＮＷ１の密度は高い。 The average wall interval that is the average value of the wall interval D1 is related to the density of the carbon nanowall CNW1. That is, the wider the average wall interval, the lower the density of the carbon nanowall CNW1. Conversely, the smaller the average wall interval, the higher the density of the carbon nanowall CNW1.
［表１］
　　ウォール間隔　　　１０ｎｍ～１０００ｎｍ
　　ウォールの厚さ　　０．３ｎｍ～５０ｎｍ
　　ウォールの高さ　　２０ｎｍ～３０００ｎｍ [Table 1]
Wall spacing 10nm-1000nm
Wall thickness 0.3nm-50nm
Wall height 20nm to 3000nm
１－２．第２の細胞培養基材
１－２－１．第２の細胞培養基材の構造
　図３は、第２の細胞培養基材２００を示す概略構成図である。図３に示すように、細胞培養基材２００は、基板１１０と、カーボンナノウォールＣＮＷ２と、を有している。カーボンナノウォールＣＮＷ２における根本部Ｒ１の反対側には、先端部Ｅ２がある。カーボンナノウォールＣＮＷ２の先端部Ｅ２の側には、置換部ＳＰが形成されている。 1-2. Second cell culture substrate 1-2-1. Structure of Second Cell Culture Substrate FIG. 3 is a schematic configuration diagram showing the second cell culture substrate 200. As shown in FIG. 3, the cell culture substrate 200 includes a substrate 110 and a carbon nanowall CNW2. On the opposite side of the root portion R1 in the carbon nanowall CNW2, there is a tip E2. A replacement portion SP is formed on the tip end portion E2 side of the carbon nanowall CNW2.
　置換部ＳＰは、カーボンナノウォールＣＮＷ２の先端部Ｅ２の炭素原子と結合する原子を、別の原子で置き換えたものである。この置換により、カーボンナノウォールＣＮＷ２の先端部Ｅ２の炭素原子は、炭素原子以外の原子と結合している。そして、この置換により、細胞培養基材２００の親水性、疎水性といった性質が変化する。すなわち、細胞を培養するための足場部の化学的性質を選ぶことができる。 The replacement part SP is obtained by replacing the atom bonded to the carbon atom of the tip part E2 of the carbon nanowall CNW2 with another atom. By this substitution, the carbon atom at the tip E2 of the carbon nanowall CNW2 is bonded to an atom other than the carbon atom. And by this substitution, properties such as hydrophilicity and hydrophobicity of the cell culture substrate 200 change. That is, the chemical nature of the scaffold for culturing cells can be selected.
　置換する原子として、例えば、酸素原子を用いることができる。そのため、カーボンナノウォールＣＮＷ２の先端部Ｅ２の少なくとも一部の炭素原子Ｃ１は、酸素原子と結合する。そして、先端部Ｅ２の残部の炭素原子は、水素原子と結合したままである。そのため、グラフェンシートの先端部Ｅ２の終端基は、酸素原子もしくは水素原子である。これらの置換の度合いは、置換処理の処理時間等、種々の条件により変わる。また、置換する原子として、窒素原子、フッ素原子等種々の原子が挙げられる。 As an atom to be substituted, for example, an oxygen atom can be used. Therefore, at least some carbon atoms C1 of the tip E2 of the carbon nanowall CNW2 are bonded to oxygen atoms. The remaining carbon atoms of the tip E2 remain bonded to the hydrogen atoms. Therefore, the terminal group of the front-end | tip part E2 of a graphene sheet is an oxygen atom or a hydrogen atom. The degree of replacement varies depending on various conditions such as the processing time of the replacement process. Moreover, various atoms, such as a nitrogen atom and a fluorine atom, are mentioned as an atom to substitute.
１－３．第３の細胞培養基材
１－３－１．第３の細胞培養基材の構造
　図４は、第３の細胞培養基材３００を示す概略構成図である。図４に示すように、細胞培養基材３００は、基板１１０と、カーボンナノウォールＣＮＷ３と、を有している。カーボンナノウォールＣＮＷ３における根元部Ｒ１の反対側には、先端部Ｅ３がある。カーボンナノウォールＣＮＷ３の先端部Ｅ３の側には、コーティング膜ＣＭが形成されている。コーティング膜ＣＭは、グラフェンシートの先端部を細胞と親和性のあるコーティング材でコーティングしたものである。コーティング膜ＣＭの材質として、例えば、コラーゲンが挙げられる。細胞培養基材３００におけるコーティング膜ＣＭを除くその他の構成は、第１の細胞培養基材１００と同様である。また、第２の細胞培養基材２００にコーティング膜ＣＭを形成してもよい。 1-3. Third cell culture substrate 1-3-1. Structure of Third Cell Culture Substrate FIG. 4 is a schematic configuration diagram showing a third cell culture substrate 300. As shown in FIG. 4, the cell culture substrate 300 includes a substrate 110 and a carbon nanowall CNW3. On the opposite side of the root portion R1 in the carbon nanowall CNW3, there is a tip E3. A coating film CM is formed on the tip E3 side of the carbon nanowall CNW3. The coating film CM is obtained by coating the tip of a graphene sheet with a coating material having affinity with cells. Examples of the material of the coating film CM include collagen. Other configurations of the cell culture substrate 300 except for the coating film CM are the same as those of the first cell culture substrate 100. In addition, a coating film CM may be formed on the second cell culture substrate 200.
２．細胞培養基材の製造装置
　続いて、細胞培養基材１００を製造する製造装置について説明する。 2. Next, a manufacturing apparatus for manufacturing the cell culture substrate 100 will be described.
　図５は、製造装置１の構成を示す概略構成図である。製造装置１は、プラズマ生成室４６と、反応室１０とを有している。プラズマ生成室４６は、その内部でプラズマを発生させるとともに、反応室１０に供給するラジカルをも発生させるためのものである。反応室１０は、プラズマ生成室４６で生じたラジカルを利用して、カーボンナノウォールＣＮＷ１を形成するためのものである。 FIG. 5 is a schematic configuration diagram showing the configuration of the manufacturing apparatus 1. The manufacturing apparatus 1 includes a plasma generation chamber 46 and a reaction chamber 10. The plasma generation chamber 46 is for generating plasma inside and generating radicals to be supplied to the reaction chamber 10. The reaction chamber 10 is for forming the carbon nanowall CNW1 using radicals generated in the plasma generation chamber 46.
　また、製造装置１は、導波路４７と、石英窓４８と、スロットアンテナ４９とを、有している。導波路４７は、マイクロ波３９を導入するためのものである。スロットアンテナ４９は、石英窓４８からプラズマ生成室４６にマイクロ波３９を導入するためのものである。 In addition, the manufacturing apparatus 1 includes a waveguide 47, a quartz window 48, and a slot antenna 49. The waveguide 47 is for introducing the microwave 39. The slot antenna 49 is for introducing the microwave 39 from the quartz window 48 to the plasma generation chamber 46.
　プラズマ生成室４６は、マイクロ波３９により表面波プラズマ（ＳＷＰ）を発生させるためのものである。プラズマ生成室４６には、ラジカル源導入口４２が設けられている。ラジカル源導入口４２は、プラズマ生成室４６に発生するプラズマ６１の内部にラジカル源となるガスを供給するためのものである。 The plasma generation chamber 46 is for generating surface wave plasma (SWP) by the microwave 39. The plasma generation chamber 46 is provided with a radical source inlet 42. The radical source inlet 42 is for supplying a gas serving as a radical source into the plasma 61 generated in the plasma generation chamber 46.
　プラズマ生成室４６と、反応室１０との間には、隔壁４４が設けられている。隔壁４４は、プラズマ生成室４６と、反応室１０とを仕切るためのものである。また、後述するように、電圧を印加するための電極も兼ねている。そして、隔壁４４には、貫通孔が形成されている。プラズマ生成室４６で生成されたラジカルを反応室１０に供給するためである。 A partition wall 44 is provided between the plasma generation chamber 46 and the reaction chamber 10. The partition 44 is for partitioning the plasma generation chamber 46 and the reaction chamber 10. Further, as will be described later, it also serves as an electrode for applying a voltage. A through hole is formed in the partition wall 44. This is for supplying radicals generated in the plasma generation chamber 46 to the reaction chamber 10.
　反応室１０は、容量結合型プラズマ（ＣＣＰ）を発生させるためのものである。また、基板５０にカーボンナノウォールを形成するためのものでもある。反応室１０は、第２電極２４と、ヒーター２５と、原料導入口１２と、排気口１６とを有している。第２電極２４は、後述するように、第１電極２２との間に電圧を印加するためのものである。ヒーター２５は、基板５０を加熱して、基板５０の温度を制御するためのものである。原料導入口１２は、カーボンナノウォールの原料となる炭素系ガス３２を供給するためのものである。排気口１６は、真空ポンプ等に接続されている。真空ポンプは、反応室１０の内部の圧力を調整するためのものである。 The reaction chamber 10 is for generating capacitively coupled plasma (CCP). It is also for forming carbon nanowalls on the substrate 50. The reaction chamber 10 includes a second electrode 24, a heater 25, a raw material introduction port 12, and an exhaust port 16. As will be described later, the second electrode 24 is for applying a voltage between the second electrode 24 and the first electrode 22. The heater 25 is for heating the substrate 50 and controlling the temperature of the substrate 50. The raw material inlet 12 is for supplying a carbon-based gas 32 that is a raw material of the carbon nanowall. The exhaust port 16 is connected to a vacuum pump or the like. The vacuum pump is for adjusting the pressure inside the reaction chamber 10.
　ここで、隔壁４４は、第２電極２４との間に電圧を印加するための第１電極２２を兼ねている。第１電極２２には、電源および回路が接続されている。第１電極２２の電位を時間的に制御するためである。第２電極２４は、第１電極２２との間に電圧を印加するためのものである。そして、第２電極２４は、基板５０を載置するための載置台でもある。第２電極２４は、接地されている。第１電極２２と第２電極２４との間の距離は約５ｃｍである。もちろん、この値に限らない。 Here, the partition wall 44 also serves as the first electrode 22 for applying a voltage between the second electrode 24. A power source and a circuit are connected to the first electrode 22. This is for controlling the potential of the first electrode 22 in terms of time. The second electrode 24 is for applying a voltage between the first electrode 22 and the second electrode 24. The second electrode 24 is also a mounting table for mounting the substrate 50. The second electrode 24 is grounded. The distance between the first electrode 22 and the second electrode 24 is about 5 cm. Of course, it is not limited to this value.
３．細胞培養基材の製造方法
３－１．第１の細胞培養基材の製造方法
　まず、製造装置１の内部に、カーボンナノウォールＣＮＷ１を形成する前の基板５０をセットする。次に、マイクロ波３９を導波路４７に導入する。マイクロ波３９は、スロットアンテナ４９により、石英窓４８から、プラズマ生成室４６に導入される。これにより、高密度プラズマ６０が発生する。 3. 3. Method for producing cell culture substrate 3-1. First Method for Manufacturing Cell Culture Substrate First, the substrate 50 before the carbon nanowall CNW1 is formed is set inside the manufacturing apparatus 1. Next, the microwave 39 is introduced into the waveguide 47. The microwave 39 is introduced into the plasma generation chamber 46 from the quartz window 48 by the slot antenna 49. Thereby, high-density plasma 60 is generated.
　そして、この高密度プラズマ６０がプラズマ生成室４６の内部で拡散して、プラズマ６１となる。このプラズマ６１は、ラジカル源導入口４２から供給されるラジカル源のイオンを含んでいる。ラジカル源として、水素を用いる。もしくは、酸素、窒素、その他の気体であってもよい。プラズマ６１中の大部分のイオンは、隔壁４４に衝突して中性化して、ラジカルとなる。ラジカル３８は、隔壁４４の貫通孔を通過して、反応室１０に入る。 The high-density plasma 60 is diffused inside the plasma generation chamber 46 to become plasma 61. This plasma 61 contains radical source ions supplied from the radical source inlet 42. Hydrogen is used as a radical source. Or oxygen, nitrogen, and other gas may be sufficient. Most of the ions in the plasma 61 collide with the partition walls 44 and are neutralized to become radicals. The radical 38 passes through the through hole of the partition wall 44 and enters the reaction chamber 10.
　反応室１０の内部には、ラジカル３８の他に、原料導入口１２から炭素系ガス３２が供給される。炭素系ガス３２とは、例えば、ＣＨ4 やＣ2 Ｆ6 である。もちろん、それ以外のものであってもよい。そして、第１電極２４と、第２電極２２との間に電圧を印加する。これにより、反応室１０の内部にプラズマ３４が発生する。 In addition to the radicals 38, a carbon-based gas 32 is supplied from the raw material inlet 12 into the reaction chamber 10. The carbon-based gas 32 is, for example, CH 4 or C 2 F 6 . Of course, it may be other than that. A voltage is applied between the first electrode 24 and the second electrode 22. As a result, plasma 34 is generated inside the reaction chamber 10.
　プラズマ３４の雰囲気中には、原料である炭素系ガス３２と、ラジカル３８とが混在している。そして、このプラズマ３４の雰囲気中で基板５０の表面にカーボンナノウォールが成長する。なお、反応室１０の内部の圧力は、５～２０００ｍＴｏｒｒ（０．６５Ｐａ～２６７Ｐａ）の範囲内である。また、基板５０の温度は、１００～８００℃の範囲内である。もちろん、これらは例示であり、これらの数値範囲に限らない。 In the atmosphere of plasma 34, carbon-based gas 32 as a raw material and radical 38 are mixed. Then, carbon nanowalls grow on the surface of the substrate 50 in the atmosphere of the plasma 34. The pressure inside the reaction chamber 10 is in the range of 5 to 2000 mTorr (0.65 Pa to 267 Pa). The temperature of the substrate 50 is in the range of 100 to 800 ° C. Of course, these are examples, and are not limited to these numerical ranges.
３－２．第２の細胞培養基材の製造方法
　第２の細胞培養基材２００は、第１の細胞培養基材１００にプラズマ処理を施すことにより製造される。そのため、第１の細胞培養基材１００に、置換しようとする原子を含むガスをプラズマガスとしてプラズマを発生させる。ここでは、まず、チャンバーの内部にプラズマ発生装置を置く。そして、チャンバーの内部をＡｒガスでパージしながら、Ａｒ＋Ｏ2 ガスをプラズマ化する。これにより、酸素原子に由来するラジカル等がプラズマ発生領域に発生する。そして、発生した酸素原子は、細胞培養基材１００の先端部Ｅ１で反応して、酸素原子の結合した先端部Ｅ２が製造される。先端部Ｅ２の炭素原子にその他の原子を結合させるには、その他のガスを用いればよい。 3-2. Method for Manufacturing Second Cell Culture Substrate The second cell culture substrate 200 is manufactured by subjecting the first cell culture substrate 100 to plasma treatment. Therefore, plasma is generated in the first cell culture substrate 100 using a gas containing atoms to be replaced as a plasma gas. Here, first, a plasma generator is placed inside the chamber. Then, Ar + O 2 gas is turned into plasma while purging the inside of the chamber with Ar gas. Thereby, radicals derived from oxygen atoms are generated in the plasma generation region. The generated oxygen atoms react at the tip E1 of the cell culture substrate 100 to produce a tip E2 to which oxygen atoms are bonded. In order to bond other atoms to the carbon atom of the tip E2, other gas may be used.
３－３．第３の細胞培養基材の製造方法
　第３の細胞培養基材３００は、第１の細胞培養基材１００にコラーゲンコート処理を施すことにより製造される。そのため、細胞培養基材１００へのコラーゲンのコーティング方法について説明する。まず、コラーゲン酸性溶液の入っている容器に、第１の細胞培養基材１００を入れる。これにより、先端部Ｅ１にコラーゲンの薄い膜が形成される。このとき、細胞培養基材１００は、酸性となる。次に、コラーゲン酸性溶液に浸けた後の細胞培養基材１００を乾燥後、培地に浸す。そのため、細胞培養基材１００は、中性となる。これにより、先端部Ｅ１は、コラーゲンコート処理がなされた先端部Ｅ３となる。以上により、先端部Ｅ３にコーティング膜ＣＭを有する細胞培養基材が作製される。 3-3. Method for Producing Third Cell Culture Substrate The third cell culture substrate 300 is produced by subjecting the first cell culture substrate 100 to a collagen coating treatment. Therefore, a method for coating the cell culture substrate 100 with collagen will be described. First, the first cell culture substrate 100 is placed in a container containing an acidic collagen solution. Thereby, a thin film of collagen is formed at the tip E1. At this time, the cell culture substrate 100 is acidic. Next, the cell culture substrate 100 immersed in the collagen acidic solution is dried and then immersed in the medium. Therefore, the cell culture substrate 100 is neutral. Thereby, the front-end | tip part E1 turns into the front-end | tip part E3 to which the collagen coat process was made | formed. As described above, the cell culture substrate having the coating film CM at the tip E3 is produced.
３－２．製造されたカーボンナノウォール
　図６は、上記のように形成されたカーボンナノウォールの構造を先端部の側から見た顕微鏡写真である。図６に示すように、カーボンナノウォールは、ランダムに成長している。ただし、その間隔は、ある程度均一である。図７は、形成されたカーボンナノウォールの構造を基板側面から見た断面を示す顕微鏡写真である。図７に示すように、カーボンナノウォールは、基板に対してほぼ垂直に形成されている。 3-2. Manufactured Carbon Nanowall FIG. 6 is a photomicrograph of the structure of the carbon nanowall formed as described above as viewed from the tip side. As shown in FIG. 6, the carbon nanowalls are growing randomly. However, the intervals are uniform to some extent. FIG. 7 is a photomicrograph showing a cross section of the structure of the formed carbon nanowall viewed from the side of the substrate. As shown in FIG. 7, the carbon nanowall is formed substantially perpendicular to the substrate.
４．細胞培養基材における細胞培養方法
　図８は、細胞培養基材１００を用いて細胞を培養しているところを示す図である。図８に示すように、シャーレ５００の底面に細胞培養基材１００を置く。その向きはもちろん、カーボンナノウォールＣＮＷ１が上となる向きである。したがって、細胞培養基材１００の先端部Ｅ１は、シャーレ５００の底面と接触することはない。そして、細胞培養基材１００を置いたシャーレ５００に、培養液を注ぐ。その培養液としては、細胞の培養に用いられる一般的なものを用いればよい。そして、細胞培養基材１００の先端部Ｅ１に、培養したい細胞を供給する。例えば、ピペットを用いることができる。また、細胞培養基材２００、３００を用いる場合にも、同様に細胞を培養することができる。 4). Cell Culture Method in Cell Culture Substrate FIG. 8 is a diagram showing a state where cells are cultured using the cell culture substrate 100. As shown in FIG. 8, the cell culture substrate 100 is placed on the bottom surface of the petri dish 500. Of course, the carbon nanowall CNW1 faces upward. Therefore, the tip E1 of the cell culture substrate 100 does not contact the bottom surface of the petri dish 500. Then, the culture solution is poured into the petri dish 500 on which the cell culture substrate 100 is placed. As the culture solution, a general solution used for cell culture may be used. Then, the cells to be cultured are supplied to the tip E1 of the cell culture substrate 100. For example, a pipette can be used. Further, when the cell culture substrates 200 and 300 are used, the cells can be cultured similarly.
５．実験Ａ（親水性および撥水性）
５－１．親水性および撥水性の制御
　前述したように、本実施形態の細胞培養基材１００では、先端部Ｅ１の炭素原子Ｃ１と結合している原子もしくは分子を置換することができる。これにより、本実施形態のカーボンナノウォールＣＮＷ１では、親水性または撥水性を備えるようにすることができる。 5. Experiment A (hydrophilic and water repellent)
5-1. Control of hydrophilicity and water repellency As described above, in the cell culture substrate 100 of the present embodiment, atoms or molecules bonded to the carbon atom C1 of the tip E1 can be replaced. Thereby, in carbon nanowall CNW1 of this embodiment, hydrophilicity or water repellency can be provided.
　所望の親水性または撥水性を備えるカーボンナノウォールを形成するには、次の３点が主に重要である。
　　　（Ａ）先端部の炭素原子と結合する化学種
　　　（Ｂ）化学種の置換の度合い
　　　（Ｃ）カーボンナノウォールの構造 In order to form a carbon nanowall having desired hydrophilicity or water repellency, the following three points are mainly important.
(A) Chemical species bonded to the carbon atom at the tip (B) Degree of substitution of chemical species (C) Structure of carbon nanowall
　ここで、（Ａ）先端部の炭素原子と結合する化学種（例えば、Ｈ、Ｆ等）により、カーボンナノウォールＣＮＷ１の先端部Ｅ１の親水性または撥水性は、もちろん変化する。この制御は、カーボンナノウォールＣＮＷ１を形成後に、ラジカルを供給することにより行うことができる。 Here, (A) The hydrophilicity or water repellency of the tip E1 of the carbon nanowall CNW1 changes, of course, depending on the chemical species (for example, H, F, etc.) bonded to the carbon atom at the tip. This control can be performed by supplying radicals after forming the carbon nanowall CNW1.
　カーボンナノウォールＣＮＷ１にプラズマを照射することにより、炭素原子Ｃ１の化学種を置換することができる。例えば、ラジカル源として酸素を導入することで、炭素原子Ｃ１に酸素原子を結合させることができる。これにより、先端部Ｅ２の炭素原子Ｃ１が酸素原子と結合している酸化グラフェンシートを生成することができる。つまり、複数の炭素原子Ｃ１のうちの少なくとも一部は、酸素原子と結合している。ただし、先端部Ｅ２に位置する全ての炭素原子を酸素原子に結合させることは困難である。つまり、終端基のすべてを酸素原子とすることは困難である。水素原子を酸素原子で置換したこのグラフェンシートは、後述するように、親水性を有する。 The chemical species of the carbon atom C1 can be replaced by irradiating the carbon nanowall CNW1 with plasma. For example, an oxygen atom can be bonded to the carbon atom C1 by introducing oxygen as a radical source. Thereby, the graphene oxide sheet | seat in which the carbon atom C1 of the front-end | tip part E2 has couple | bonded with the oxygen atom can be produced | generated. That is, at least some of the plurality of carbon atoms C1 are bonded to oxygen atoms. However, it is difficult to bond all the carbon atoms located at the tip E2 to oxygen atoms. That is, it is difficult to make all of the terminal groups oxygen atoms. This graphene sheet in which hydrogen atoms are replaced with oxygen atoms has hydrophilicity, as will be described later.
　また、ラジカル源として水素を導入することで、炭素原子Ｃ１により多くの水素を結合させることができる。これにより、先端部Ｅ１の炭素原子Ｃ１が水素と結合しているグラフェンシートを生成することができる。このグラフェンシートは、撥水性を有する。また、ラジカル源としてフッ素を導入することで、先端部Ｅ１の炭素原子Ｃ１がフッ素原子と結合しているグラフェンシートを生成することができる。このグラフェンシートも、撥水性を有する。 Further, by introducing hydrogen as a radical source, more hydrogen can be bonded to the carbon atom C1. Thereby, the graphene sheet in which the carbon atom C1 of the tip E1 is bonded to hydrogen can be generated. This graphene sheet has water repellency. In addition, by introducing fluorine as a radical source, it is possible to generate a graphene sheet in which the carbon atom C1 of the tip E1 is bonded to the fluorine atom. This graphene sheet also has water repellency.
　そして、その（Ｂ）化学種の置換の度合い（例えば、５０％置換）によっても、親水性または撥水性は、変化する。この制御は、プラズマの照射時間を変えることで、調整することができる。 And the hydrophilicity or water repellency also changes depending on the degree of substitution of the chemical species (B) (for example, 50% substitution). This control can be adjusted by changing the plasma irradiation time.
　また、（Ｃ）カーボンナノウォールの構造（例えば、ウォール間隔Ｄ１）によっても、親水性または撥水性は、わずかに変化する。この制御は、反応室１０における基板温度、原料ガス、圧力など、その他の条件を変えることで調整することができる。これら（Ａ）～（Ｃ）の組み合わせにより、先端部における親水性または撥水性を、ほぼ連続的に変えることができる。なお、これら（Ａ）～（Ｃ）により、親水性または撥水性といった化学的性質のみならず、細胞の足場となる先端部におけるその他の化学的性質も変化する。 (C) The hydrophilicity or water repellency slightly changes depending on the structure of carbon nanowalls (for example, wall interval D1). This control can be adjusted by changing other conditions such as the substrate temperature, source gas, and pressure in the reaction chamber 10. By combining these (A) to (C), the hydrophilicity or water repellency at the tip can be changed almost continuously. These (A) to (C) change not only the chemical properties such as hydrophilicity or water repellency, but also other chemical properties at the tip portion that becomes a scaffold for cells.
５－２．親水性および撥水性についての実験
　続いて、親水性および撥水性について行った実験について説明する。実験条件は次の通りである。マイクロ波３９を発生させるための電力として４００Ｗを用いた。その周波数として２．４５ＧＨｚを用いた。容量結合型プラズマを発生させるための電力として３００Ｗを用いた。その周波数として１００ＭＨｚを用いた。基板温度を５４０℃とした。成長時間を１５分とした。反応室１０の内部の圧力を１Ｐａとした。原料ガスとして、
　１）ＣＨ4 ガス（１００ｓｃｃｍ）、
　２）Ｃ2 Ｈ6 ／Ｈ2 ガス（１００ｓｃｃｍ／５０ｓｃｃｍ）
のいずれかを用いた。 5-2. Experiments on hydrophilicity and water repellency Next, experiments conducted on hydrophilicity and water repellency will be described. The experimental conditions are as follows. 400 W was used as electric power for generating the microwave 39. 2.45 GHz was used as the frequency. 300 W was used as power for generating capacitively coupled plasma. 100 MHz was used as the frequency. The substrate temperature was 540 ° C. The growth time was 15 minutes. The pressure inside the reaction chamber 10 was 1 Pa. As source gas,
1) CH 4 gas (100 sccm),
2) C 2 H 6 / H 2 gas (100 sccm / 50 sccm)
Any one of was used.
　図９から図１２に水との接触角を示す。図９は、原料である炭素系ガス３２をＣＨ4 ガスとしてカーボンナノウォールを作成した後に、大気圧プラズマ処理を行って製造された細胞培養基材における水との接触角を示す写真である。その接触角は１０°以下である。図１０は、カーボンナノウォールをＣＨ4 ガスで作成した細胞培養基材における水との接触角を示す写真である。その接触角は約５０°である。 9 to 12 show the contact angle with water. FIG. 9 is a photograph showing a contact angle with water in a cell culture substrate produced by performing atmospheric pressure plasma treatment after creating a carbon nanowall using the raw material carbon-based gas 32 as CH 4 gas. The contact angle is 10 ° or less. FIG. 10 is a photograph showing a contact angle with water in a cell culture substrate in which carbon nanowalls are made of CH 4 gas. The contact angle is about 50 °.
　図１１は、カーボンナノウォールをＣ2 Ｆ6 ガスで作成した細胞培養基材における水との接触角を示す写真である。その接触角はおよそ１０５°である。図１２は、カーボンナノウォールをＣＨ4 ガスで作成した後にフッ素処理を行って製造された細胞培養基材における水との接触角を示す写真である。このフッ素処理は、プラズマ照射により行った。その際の供給ガスは、フルオロカーボンガスである。その接触角はおよそ１３５°である。なお、このフッ素処理は、フッ化水素（ＨＦ）溶液に浸漬することにより行ってもよい。 FIG. 11 is a photograph showing a contact angle with water in a cell culture substrate in which carbon nanowalls are prepared with C 2 F 6 gas. The contact angle is approximately 105 °. FIG. 12 is a photograph showing a contact angle with water in a cell culture substrate produced by producing a carbon nanowall with CH 4 gas and then performing a fluorine treatment. This fluorine treatment was performed by plasma irradiation. The supply gas at that time is a fluorocarbon gas. The contact angle is approximately 135 °. Note that this fluorine treatment may be performed by immersing in a hydrogen fluoride (HF) solution.
　以上説明したように、足場部における水との接触角は、１°以上１７０°以下の範囲内である。 As described above, the contact angle with water in the scaffold is in the range of 1 ° to 170 °.
５－３．カーボンナノチューブとの比較
　以上説明したように、カーボンナノウォールでは、細胞の足場となる先端部の炭素原子に結合する化学種を置換することができる。これに対して、カーボンナノチューブでは、細胞の足場となるのは、グラフェンシートの側面（例えば、図２のＳ１）である。このグラフェンシートの側面には、π電子が存在する。しかし、カーボンナノチューブには、カーボンナノウォールＣＮＷ１のように、先端部Ｅ１が存在するわけではない。したがって、カーボンナノチューブでは、化学種の置換や、格子欠陥の形成による表面構造の変化も可能であるものの、カーボンナノウォールＣＮＷ１におけるウォール間隔Ｄ１の変化量に比べると、その変化の度合いは極めて小さいといえる。 5-3. Comparison with Carbon Nanotubes As described above, carbon nanowalls can replace chemical species that bind to the carbon atoms at the tip of the cell. In contrast, in carbon nanotubes, the side of the graphene sheet (for example, S1 in FIG. 2) serves as a scaffold for cells. On the side surface of the graphene sheet, π electrons exist. However, the carbon nanotube does not have the tip E1 unlike the carbon nanowall CNW1. Therefore, in the carbon nanotube, although the surface structure can be changed by substitution of chemical species or formation of lattice defects, the degree of change is very small compared to the change amount of the wall interval D1 in the carbon nanowall CNW1. I can say that.
６．実験Ｂ（親水性または撥水性と培養する細胞との関係）
　図１３から図１６までに、水との接触角の異なる場合における細胞の顕微鏡写真を示す。これらは、いずれもＨｅＬａ細胞である。そして、培養を開始してから４日経過後の顕微鏡写真である。 6). Experiment B (Relationship between hydrophilicity or water repellency and cells to be cultured)
FIG. 13 to FIG. 16 show micrographs of cells when contact angles with water are different. These are all HeLa cells. And it is a microscope picture 4 days after starting culture | cultivation.
　図１３は、水との接触角が１０°以下の場合における細胞の顕微鏡写真である。図１４は、水との接触角がおよそ５０°の場合における細胞の顕微鏡写真である。図１５は、水との接触角がおよそ１０５°の場合における細胞の顕微鏡写真である。図１６は、水との接触角がおよそ１３５°の場合における細胞の顕微鏡写真である。これらの結果から、水との接触角が大きくなるほど、細胞は球形に近い形状に変化している。以上、具体例を挙げて示したように、足場部での水との接触角が、１°以上１７０°以下の範囲内の細胞培養基材１００を製造することができる。 FIG. 13 is a photomicrograph of cells when the contact angle with water is 10 ° or less. FIG. 14 is a photomicrograph of cells when the contact angle with water is approximately 50 °. FIG. 15 is a photomicrograph of cells when the contact angle with water is approximately 105 °. FIG. 16 is a photomicrograph of cells when the contact angle with water is approximately 135 °. From these results, as the contact angle with water increases, the cell changes to a shape close to a sphere. As described above, as shown with a specific example, the cell culture substrate 100 having a contact angle with water in the scaffold portion in the range of 1 ° to 170 ° can be produced.
　図１７は、細胞培養基材の足場における水との接触角と、培養を開始してから４日経過後の細胞数との関係を示すグラフである。培養した細胞はＨｅＬａ細胞である。図１７では、本実施形態の細胞培養基材で培養した細胞数と、ガラス基板で培養した細胞数とを比較している。図１７の横軸は、水との接触角である。図１７の縦軸は、細胞数である。ここで、細胞数が多い細胞培養基材ほど、細胞の培養に適していることを示している。全体的に、本実施形態の細胞培養基材で培養した細胞数は、ガラス基板で培養した細胞数よりも多い。 FIG. 17 is a graph showing the relationship between the contact angle with water on the scaffold of the cell culture substrate and the number of cells after 4 days from the start of culture. The cultured cells are HeLa cells. In FIG. 17, the number of cells cultured on the cell culture substrate of this embodiment is compared with the number of cells cultured on a glass substrate. The horizontal axis in FIG. 17 is the contact angle with water. The vertical axis in FIG. 17 is the number of cells. Here, a cell culture substrate having a larger number of cells is suitable for culturing cells. Overall, the number of cells cultured on the cell culture substrate of this embodiment is greater than the number of cells cultured on the glass substrate.
　また、本実施形態の細胞培養基材では、撥水性の強い接触角１３５°であっても細胞を１×１０4 細胞／ｃｍ2 程度は培養できることが明らかとなった。また、接触角１３５°の場合には、細胞の低侵襲性回収を行うことができた。 In addition, it has been clarified that the cell culture substrate of this embodiment can culture cells at about 1 × 10 4 cells / cm 2 even at a contact angle of 135 ° with strong water repellency. In the case of a contact angle of 135 °, a minimally invasive recovery of cells could be performed.
７．実験Ｃ（タンパク質の吸着量）
　図１８は、細胞培養基材の足場における水との接触角と、タンパク質の吸着量との関係を示すグラフである。培養した細胞は、同じくＨｅＬａ細胞である。図１８の横軸は、水との接触角である。図１８の縦軸は、細胞培養基材におけるタンパク質の吸着量である。図１８に示すように、本実施形態の細胞培養基材におけるタンパク質の吸着量は、ガラス基板におけるタンパク質の吸着量の２倍程度である。すなわち、ガラス基板に比べてカーボンナノウォールＣＮＷ１を有する本実施形態の細胞培養基材のほうが、細胞の培養に適している。 7). Experiment C (Adsorption amount of protein)
FIG. 18 is a graph showing the relationship between the contact angle with water in the scaffold for cell culture substrate and the amount of protein adsorbed. The cultured cells are also HeLa cells. The horizontal axis in FIG. 18 is the contact angle with water. The vertical axis in FIG. 18 represents the amount of protein adsorbed on the cell culture substrate. As shown in FIG. 18, the amount of protein adsorbed on the cell culture substrate of this embodiment is about twice the amount of protein adsorbed on the glass substrate. That is, the cell culture substrate of the present embodiment having the carbon nanowall CNW1 is more suitable for cell culture than the glass substrate.
８．実験Ｄ（骨芽細胞への分化（アルカリフォスファターゼ活性））
　ここで、骨芽細胞への分化について行った実験結果について説明する。本実験では、間葉系幹細胞を用いた。培養に用いた溶液は、ＤＭＥＭにＦＢＳおよびペニシリン－ストレプトマイシンを混合した混合溶液である。ＦＢＳの混合比は１０％である。ペニシリン－ストレプトマイシンの混合比は、１％である。培養を開始してから１０日経過後に、その間葉系幹細胞が骨芽細胞に分化しているか調べた。 8). Experiment D (differentiation into osteoblasts (alkaline phosphatase activity))
Here, a description will be given of the results of experiments conducted on differentiation into osteoblasts. In this experiment, mesenchymal stem cells were used. The solution used for the culture is a mixed solution in which FMEM and penicillin-streptomycin are mixed in DMEM. The mixing ratio of FBS is 10%. The penicillin-streptomycin mixing ratio is 1%. After 10 days from the start of culture, it was examined whether the mesenchymal stem cells were differentiated into osteoblasts.
　本実験では、４種類の方法で培養した細胞に対してアルカリフォスファターゼ活性を測定して比較した。４種類とは、次のようなものである。
　　　Ａ．培養ディッシュ（接触角７０°）
　　　Ｂ．ガラス基板（接触角６０°）
　　　Ｃ．カーボンナノウォール（接触角１０°以下）
　　　Ｄ．カーボンナノウォール（接触角５０°） In this experiment, alkaline phosphatase activity was measured and compared for cells cultured by four methods. The four types are as follows.
A. Culture dish (contact angle 70 °)
B. Glass substrate (contact angle 60 °)
C. Carbon nanowall (contact angle of 10 ° or less)
D. Carbon nanowall (contact angle 50 °)
　図１９は、その結果である。縦軸は、未分化細胞を基準としたアルカリフォスファターゼ活性の測定結果である。未分化細胞に比べて、この測定値が大きいほど、骨芽細胞への分化が進んでいると考えられる。図１９に示すように、測定値はいずれも未分化細胞におけるアルカリフォスファターゼ活性の測定値の１．１倍程度である。ガラス基板を用いた場合の測定値が他の場合と比べてやや低い。 FIG. 19 shows the result. The vertical axis represents the measurement result of alkaline phosphatase activity based on undifferentiated cells. It is considered that the greater the measured value compared to the undifferentiated cells, the more differentiated into osteoblasts. As shown in FIG. 19, the measured values are all about 1.1 times the measured value of alkaline phosphatase activity in undifferentiated cells. The measured value when a glass substrate is used is slightly lower than other cases.
　図２０は、各基板にコラーゲンコートした細胞培養基材を用いて細胞を培養した結果である。カーボンナノウォールを用いた細胞培養基材（コラーゲンコート済）のほうが、培養ディッシュやガラス基板を用いたものに比べて、骨芽細胞への分化が進んでいると考えられる。 FIG. 20 shows the result of culturing cells using a cell culture substrate in which each substrate is coated with collagen. The cell culture substrate (collagen-coated) using carbon nanowalls is considered to be more differentiated into osteoblasts than those using a culture dish or glass substrate.
９．実験Ｅ（密度制御）
９－１．実験に用いる細胞培養基材の種類
　図２１は、圧力、成長時間、ＣＣＰＰｏｗｅｒ、というカーボンナノウォールの製造条件を変えた場合に、どのような性質のカーボンナノウォールが製造されるかを示す表である。図２１に示すように、製造条件を変えることにより、高密度のカーボンナノウォールから低密度のカーボンナノウォールまで製造することができる。なお、図２１に示した条件以外の条件については、実験Ａと同様である。 9. Experiment E (density control)
9-1. Types of Cell Culture Substrates Used in Experiments FIG. 21 is a table showing what kind of carbon nanowalls are produced when the production conditions of carbon nanowalls such as pressure, growth time, and CCPPpower are changed. is there. As shown in FIG. 21, by changing the manufacturing conditions, it is possible to manufacture from a high density carbon nanowall to a low density carbon nanowall. Note that conditions other than those shown in FIG. 21 are the same as in Experiment A.
　図２１の（ａ）に、高密度のカーボンナノウォールを示す。（ａ）の高密度のカーボンナノウォールのウォール間隔は、９５ｎｍであった。（ａ）の高密度のカーボンナノウォールを製造するために、製造装置１の内圧を１Ｐａとし、ＣＣＰＰｏｗｅｒを１００Ｗとし、成長時間を８０分とした。 FIG. 21 (a) shows a high-density carbon nanowall. The wall interval of the high-density carbon nanowall (a) was 95 nm. In order to manufacture the high density carbon nanowall (a), the internal pressure of the manufacturing apparatus 1 was set to 1 Pa, the CCPP power was set to 100 W, and the growth time was set to 80 minutes.
　図２１の（ｂ）に、中密度のカーボンナノウォールを示す。（ｂ）の中密度のカーボンナノウォールのウォール間隔は、１３１ｎｍであった。（ｂ）の中密度のカーボンナノウォールを製造するために、製造装置１の内圧を１Ｐａとし、ＣＣＰＰｏｗｅｒを３００Ｗとし、成長時間を１５分とした。 FIG. 21 (b) shows a medium density carbon nanowall. The wall interval of the medium density carbon nanowall in (b) was 131 nm. (B) In order to manufacture medium density carbon nanowalls, the internal pressure of the manufacturing apparatus 1 was set to 1 Pa, the CCPP power was set to 300 W, and the growth time was set to 15 minutes.
　図２１の（ｃ）に、低密度のカーボンナノウォールを示す。（ｃ）の低密度のカーボンナノウォールのウォール間隔は、３１３ｎｍであった。（ｃ）の低密度のカーボンナノウォールを製造するために、製造装置１の内圧を５Ｐａとし、ＣＣＰＰｏｗｅｒを５００Ｗとし、成長時間を８分とした。 FIG. 21 (c) shows a low-density carbon nanowall. The wall interval of the low density carbon nanowall (c) was 313 nm. In order to manufacture the low density carbon nanowall (c), the internal pressure of the manufacturing apparatus 1 was set to 5 Pa, the CCPP power was set to 500 W, and the growth time was set to 8 minutes.
　このように、これ以降、便宜上、図２１（ａ）に示すものを高密度のカーボンナノウォールと呼ぶこととし、図２１（ｂ）に示すものを中密度のカーボンナノウォールと呼ぶこととし、図２１（ｃ）に示すものを低密度のカーボンナノウォールと呼ぶこととする。これら（ａ）～（ｃ）の３種類のサンプルに細胞を培養することとして、以下の実験を実施した。 Thus, hereinafter, for the sake of convenience, what is shown in FIG. 21A will be referred to as a high density carbon nanowall, and what is shown in FIG. 21B will be referred to as a medium density carbon nanowall. The material shown in 21 (c) is called a low density carbon nanowall. The following experiment was conducted by culturing cells in these three types of samples (a) to (c).
１０．実験Ｆ（水との接触角の密度依存性）
　本実験では、（ａ）～（ｃ）のそれぞれのサンプルに、酸素原子置換、窒素原子置換、水素原子置換、フッ素原子置換を行い、計１２種類のサンプルを得た。そして、それぞれのサンプルで接触角を測定した。図２２にその結果を示す。 10. Experiment F (Density dependence of contact angle with water)
In this experiment, each of the samples (a) to (c) was subjected to oxygen atom substitution, nitrogen atom substitution, hydrogen atom substitution, and fluorine atom substitution to obtain a total of 12 types of samples. And the contact angle was measured with each sample. FIG. 22 shows the result.
　図２２に示すように、水との接触角は、先端部Ｅ２で結合する原子の種類に強く依存することが分かった。先端部Ｅ２の原子が酸素原子である場合には、水との接触角は、５°程度である。先端部Ｅ２の原子が窒素原子である場合には、水との接触角は、５°程度である。先端部Ｅ２の原子が水素原子である場合には、水との接触角は、５０°程度である。先端部Ｅ２の原子がフッ素原子である場合には、水との接触角は、およそ１３０°～１５０°である。 As shown in FIG. 22, it was found that the contact angle with water strongly depends on the type of atoms bonded at the tip E2. When the atom of the tip E2 is an oxygen atom, the contact angle with water is about 5 °. When the atom of the tip E2 is a nitrogen atom, the contact angle with water is about 5 °. When the atom of the tip E2 is a hydrogen atom, the contact angle with water is about 50 °. When the atom of the tip E2 is a fluorine atom, the contact angle with water is approximately 130 ° to 150 °.
　一方、酸素原子置換、窒素原子置換、水素原子置換、フッ素原子置換のいずれを行った場合であっても、水との接触角はカーボンナノウォールのウォール間隔にほとんど依存していなかった。つまり、ウォール間隔を変えても、水との接触角は、ほとんど変わらない。 On the other hand, even when oxygen atom substitution, nitrogen atom substitution, hydrogen atom substitution, or fluorine atom substitution was performed, the contact angle with water hardly depended on the wall interval of the carbon nanowall. In other words, even if the wall interval is changed, the contact angle with water hardly changes.
１１．実験Ｇ（結晶構造）
　図２３は、低密度のカーボンナノウォールと、中密度のカーボンナノウォールと、高密度のカーボンナノウォールとで、ラマンシフトを測定した結果を示すグラフである。これらの終端基は、水素の場合である。つまり、終端基の置換を施していない場合の結果である。図２３に示すように、１５９０ｃｍ-1付近のＧバンドのピークと、１３５０ｃｍ-1付近のＤバンドのピークと、１６２０ｃｍ-1付近のＤ’バンドのピークとが観測された。これらのカーボンナノウォールのスペクトルは、互いにやや異なっている。例えば、高密度になるほど、Ｄバンドの成分が大きくなる。 11. Experiment G (Crystal structure)
FIG. 23 is a graph showing the results of measuring Raman shifts of low-density carbon nanowalls, medium-density carbon nanowalls, and high-density carbon nanowalls. These terminal groups are in the case of hydrogen. In other words, this is the result when the terminal group is not substituted. As shown in FIG. 23, the peak of the G band near 1590 cm -1, the peak of the D band near 1350 cm -1, and the peak of the D 'band near 1620 cm -1 were observed. The spectra of these carbon nanowalls are slightly different from each other. For example, the higher the density, the larger the D band component.
１２．実験Ｈ（化学組成比）
　図２４は、中密度のカーボンナノウォールの終端基を変化させた場合における化学組成比を示すものである。図２４の横軸は、結合エネルギーである。図２４の縦軸は、強度である。図２４（ａ）は、グラフェンシートの終端基の一部を酸素原子で置換したものの結果を示す。図２４（ｂ）は、グラフェンシートの終端基の一部を窒素原子で置換したものの結果を示す。図２４（ｃ）は、グラフェンシートの終端基の一部を置換しなかったものの結果を示す。しかし、一部に、Ｃ－Ｏ結合の成分が混じっていることが確認された。図２４（ｄ）は、グラフェンシートの終端基の一部をフッ素原子で置換したものの結果を示す。 12 Experiment H (chemical composition ratio)
FIG. 24 shows the chemical composition ratio when the terminal group of the medium density carbon nanowall is changed. The horizontal axis in FIG. 24 is the binding energy. The vertical axis in FIG. 24 is intensity. FIG. 24A shows the result of replacing part of the terminal group of the graphene sheet with an oxygen atom. FIG. 24B shows the result of replacing part of the terminal group of the graphene sheet with a nitrogen atom. FIG.24 (c) shows the result of what did not substitute a part of terminal group of a graphene sheet. However, it was confirmed that a component of C—O bond was mixed in part. FIG. 24 (d) shows the result of replacing part of the terminal group of the graphene sheet with a fluorine atom.
１３．実験Ｉ（足場部と細胞数との関係）
　図２５は、細胞培養基材の足場部と、その足場部で培養したＨｅＬａ細胞の培養数との関係を示すグラフである。図２５の横軸は、水との接触角である。図２５の縦軸は、ＨｅＬａ細胞の細胞数である。図２５に示すように、初期のＨｅＬａ細胞の細胞数は、１００００細胞／ｃｍ2 程度であった。そして、図２５には、４日後の細胞数をプロットした。なお、比較のために、ガラス基板で培養した細胞数もプロットしてある。 13. Experiment I (Relationship between scaffold and cell number)
FIG. 25 is a graph showing the relationship between the scaffold part of the cell culture substrate and the number of cultured HeLa cells cultured on the scaffold part. The horizontal axis in FIG. 25 is the contact angle with water. The vertical axis in FIG. 25 is the number of HeLa cells. As shown in FIG. 25, the initial number of HeLa cells was about 10,000 cells / cm 2 . In FIG. 25, the number of cells after 4 days is plotted. For comparison, the number of cells cultured on a glass substrate is also plotted.
　図２５に示すように、中密度のカーボンナノウォールを有する細胞培養基材で培養した場合の細胞数と、ガラス基板で培養した場合の細胞数とは、ほぼ同程度である。例えば、水との接触角が５５°の中密度のカーボンナノウォールを有する細胞培養基材で培養した場合には、４００００細胞／ｃｍ2 程度であり、水との接触角が６０°のガラス基板で培養した場合も、４００００細胞／ｃｍ2 程度であった。水との接触角が５°の中密度のカーボンナノウォールを有する細胞培養基材で培養した場合には、３１０００細胞／ｃｍ2 程度であり、水との接触角が１０°のガラス基板で培養した場合には、２５０００細胞／ｃｍ2 程度であった。 As shown in FIG. 25, the number of cells when cultured on a cell culture substrate having medium density carbon nanowalls is approximately the same as the number of cells when cultured on a glass substrate. For example, when cultured on a cell culture substrate having a medium density carbon nanowall having a contact angle with water of 55 °, the glass substrate has a contact angle with water of about 40,000 cells / cm 2 and a contact angle with water of 60 °. In the case of culturing at 4 000 cells / cm 2, it was about 40,000 cells / cm 2 . When cultured on a cell culture substrate having medium density carbon nanowalls with a water contact angle of 5 °, the cell culture is about 31000 cells / cm 2 and is cultured on a glass substrate with a water contact angle of 10 °. In this case, it was about 25000 cells / cm 2 .
　また、水との接触角が１３５°の中密度のカーボンナノウォールを有する細胞培養基材で培養した場合には、１００００細胞／ｃｍ2 程度であり、水との接触角が１０５°のガラス基板で培養した場合には、１００００細胞／ｃｍ2 程度であった。ただし、これらの場合には、水との接触角が異なっている。なお、ガラス基板では、水との接触角を１０５°程度までしかとることができない。 Further, when cultured on a cell culture substrate having a medium density carbon nanowall having a contact angle with water of 135 °, the glass substrate has a contact angle with water of about 10,000 cells / cm 2 and a contact angle with water of 105 °. In the case of culturing at 10000 cells / cm 2, it was about 10,000 cells / cm 2 . However, in these cases, the contact angle with water is different. Note that the glass substrate can have a contact angle with water of only about 105 °.
　水との接触角が０°～１０°程度の領域では、低密度のカーボンナノウォールを有する細胞培養基材で培養した場合の細胞数が５５０００細胞／ｃｍ2 程度であり、細胞数が非常に多かった。水との接触角が０°～１０°程度の領域では、それ以外の場合の細胞数は、２５０００細胞／ｃｍ2 程度から３５０００細胞／ｃｍ2 程度までの間であった。 In the region where the contact angle with water is about 0 ° to 10 °, the number of cells when cultured on a cell culture substrate having low-density carbon nanowalls is about 55000 cells / cm 2 , and the number of cells is very high. There were many. In the region where the contact angle with water was about 0 ° to 10 °, the number of cells in other cases was between about 25000 cells / cm 2 and about 35000 cells / cm 2 .
　水との接触角が５０°～６０°程度の領域では、前述したように、中密度のカーボンナノウォールを有する細胞培養基材で培養した場合とガラス基板で培養した場合とで、細胞数が４００００細胞／ｃｍ2 程度であった。それ以外の場合には、２５０００細胞／ｃｍ2 程度であった。 In the region where the contact angle with water is about 50 ° to 60 °, as described above, the number of cells is different between when cultured on a cell culture substrate having medium density carbon nanowalls and when cultured on a glass substrate. It was about 40,000 cells / cm 2 . In other cases, it was about 25000 cells / cm 2 .
　水との接触角が１３０°～１４０°程度の領域では、ウォール間隔の違いに応じて大きな差異が表れた。つまり、高密度のカーボンナノウォールを有する細胞培養基材で培養した場合には、細胞数が６００００細胞／ｃｍ2 程度であり、細胞数が非常に多かった。一方、低密度のカーボンナノウォールを有する細胞培養基材で培養した場合には、細胞数は２５０００細胞／ｃｍ2 程度であった。また、中密度のカーボンナノウォールを有する細胞培養基材で培養した場合には、細胞数は１００００細胞／ｃｍ2 程度であった。 In the region where the contact angle with water was about 130 ° to 140 °, a large difference appeared depending on the difference in wall spacing. That is, when cultured on a cell culture substrate having high-density carbon nanowalls, the number of cells was about 60000 cells / cm 2 and the number of cells was very large. On the other hand, when cultured on a cell culture substrate having low-density carbon nanowalls, the number of cells was about 25000 cells / cm 2 . In addition, when cultured on a cell culture substrate having medium density carbon nanowalls, the number of cells was about 10,000 cells / cm 2 .
　このように、細胞数という観点からは、水との接触角が大きく、高密度のカーボンナノウォールを有する細胞培養基材で培養した場合に、細胞数が多かった。つまり、水との接触角が１２０°以上１５０°以下の範囲内であって、平均ウォール間隔が８０ｎｍ以上１２０ｎｍ以下の範囲内であるとよい。ここで、水との接触角が１２０°以上１５０°以下の範囲内となるのは、グラフェンシートの終端基がフッ素原子の場合である。 Thus, from the viewpoint of the number of cells, the number of cells was large when cultured on a cell culture substrate having a large contact angle with water and having high-density carbon nanowalls. That is, the contact angle with water is preferably in the range of 120 ° to 150 °, and the average wall interval is preferably in the range of 80 nm to 120 nm. Here, the contact angle with water falls within the range of 120 ° to 150 ° when the terminal group of the graphene sheet is a fluorine atom.
　また、細胞数という観点からは、水との接触角が小さく、低密度のカーボンナノウォールを有する細胞培養基材で培養した場合に、細胞数が多かった。つまり、水との接触角が３°以上１０°以下の範囲内であって、平均ウォール間隔が１０ｎｍ以上５００ｎｍ以下の範囲内であるとよい。特に平均ウォール間隔が２００ｎｍ以上５００ｎｍ以下の範囲内であるとよい。ここで、水との接触角が３°以上１０°以下の範囲内となるのは、グラフェンシートの終端基が酸素原子または窒素原子の場合である。 Also, from the viewpoint of the number of cells, the number of cells was large when cultured on a cell culture substrate having a small contact angle with water and having low-density carbon nanowalls. That is, the contact angle with water is preferably in the range of 3 ° to 10 °, and the average wall interval is preferably in the range of 10 nm to 500 nm. In particular, the average wall interval is preferably in the range of 200 nm to 500 nm. Here, the contact angle with water is in the range of 3 ° to 10 ° when the terminal group of the graphene sheet is an oxygen atom or a nitrogen atom.
１４．実験Ｊ（化学組成と細胞数）
１４－１．化学組成
　図２６は、カーボンナノウォールの密度と、酸素密度もしくはフッ素密度との関係を示すグラフである。ここでは、酸素もしくはフッ素をプラズマガスとして用いて、グラフェンシートの終端基を酸素原子もしくはフッ素原子とした。図２６の横軸は、カーボンナノウォールの密度である。図２６の左側の縦軸は、終端基における炭素原子に対するフッ素原子の割合である。図２６の右側の縦軸は、終端基における炭素原子に対する酸素原子の割合である。 14 Experiment J (Chemical composition and number of cells)
14-1. Chemical Composition FIG. 26 is a graph showing the relationship between the density of carbon nanowalls and the oxygen density or fluorine density. Here, oxygen or fluorine was used as a plasma gas, and the terminal group of the graphene sheet was an oxygen atom or a fluorine atom. The horizontal axis of FIG. 26 is the density of the carbon nanowall. The vertical axis on the left side of FIG. 26 represents the ratio of fluorine atoms to carbon atoms in the terminal group. The vertical axis on the right side of FIG. 26 represents the ratio of oxygen atoms to carbon atoms in the terminal group.
　図２６に示すように、低密度では、終端基における炭素原子に対するフッ素原子の割合は、１．０程度である。中密度では、終端基における炭素原子に対するフッ素原子の割合は、１．７程度である。高密度では、終端基における炭素原子に対するフッ素原子の割合は、１．５程度である。 As shown in FIG. 26, at a low density, the ratio of fluorine atoms to carbon atoms in the terminal group is about 1.0. At medium density, the ratio of fluorine atoms to carbon atoms in the terminal group is about 1.7. At high density, the ratio of fluorine atoms to carbon atoms in the terminal group is about 1.5.
　また、低密度では、終端基における炭素原子に対する酸素原子の割合は、０．２２程度である。中密度では、終端基における炭素原子に対する酸素原子の割合は、０．３２程度である。高密度では、終端基における炭素原子に対する酸素原子の割合は、０．１８程度である。 Further, at a low density, the ratio of oxygen atoms to carbon atoms in the terminal group is about 0.22. At medium density, the ratio of oxygen atoms to carbon atoms in the terminal group is about 0.32. At high density, the ratio of oxygen atoms to carbon atoms in the terminal group is about 0.18.
１４－２．細胞数
　図２７は、終端基をフッ素原子とした場合におけるカーボンナノウォールの密度と細胞数との関係を示すグラフである。ただし、終端基のすべてがフッ素原子になったわけではなく、図２６に示したような炭素原子に対する割合をもったものである。図２６に示したように、炭素原子に対するフッ素原子の割合は、１．０から１．５までの範囲内である。それに比べて、図２７に示すように、培養後の細胞数には大きな差異がある。中密度と高密度とでは、フッ素原子の割合に差異はほとんどないのに対して、細胞数では６倍程度異なっている。これは、水との接触角と、ウォール間隔とが、培養する細胞の数に大きく影響を与えることを示すとともに、終端基の化学種の影響はそれほど大きくないことを示唆している。 14-2. FIG. 27 is a graph showing the relationship between the density of carbon nanowalls and the number of cells when the terminal group is a fluorine atom. However, all of the terminal groups are not fluorine atoms, but have a ratio to carbon atoms as shown in FIG. As shown in FIG. 26, the ratio of fluorine atoms to carbon atoms is in the range of 1.0 to 1.5. In comparison, as shown in FIG. 27, there is a large difference in the number of cells after culture. There is almost no difference in the proportion of fluorine atoms between medium density and high density, but the number of cells differs by about 6 times. This indicates that the contact angle with water and the wall interval greatly affect the number of cells to be cultured, and suggest that the influence of the chemical species of the terminal group is not so great.
１５．実験Ｋ（細胞の形状）
　図２８は、高密度もしくは低密度のカーボンナノウォールと、その終端基の原子との組み合わせと、その組み合わせによる細胞培養基材での細胞の形状を示す顕微鏡写真を対応させたものである。そして、図２８は、細胞を培養して１日後の様子を撮影したものである。図２８に示すように、終端基が水素原子の場合に、ＨｅＬａ細胞の形状がやや円形もしくは球形に近い形状となっている。 15. Experiment K (cell shape)
FIG. 28 corresponds to a combination of a high-density or low-density carbon nanowall and an atom of the terminal group, and a photomicrograph showing the shape of the cell on the cell culture substrate by the combination. And FIG. 28 image | photographs the mode one day after culture | cultivating a cell. As shown in FIG. 28, when the terminal group is a hydrogen atom, the shape of the HeLa cell is slightly circular or nearly spherical.
　図２９は、ＨｅＬａ細胞の伸びを示すグラフである。この値が大きいほど、細胞の一辺の長さが長い。つまり、円形からずれている。逆に、この値が小さいほど、円形に近い。図２９に示すように、終端基が水素原子の場合に、特に値が小さい。すなわち、終端基が水素原子の場合に、ＨｅＬａ細胞の形状は、より円形に近い。 FIG. 29 is a graph showing the elongation of HeLa cells. The larger this value, the longer the length of one side of the cell. That is, it deviates from a circle. Conversely, the smaller this value is, the closer it is to a circle. As shown in FIG. 29, the value is particularly small when the terminal group is a hydrogen atom. That is, when the terminal group is a hydrogen atom, the shape of the HeLa cell is closer to a circle.
１６．実験Ｌ（細胞の回収）
　図３０は、ＨｅＬａ細胞の顕微鏡写真である。そして、図３１は、ＨｅＬａ細胞を染色した顕微鏡写真である。光源として、水銀ランプを用いるとともに、フィルターとして、６５２ｎｍのＦＩＴＣを用いた。図３１では、染色されている細胞が生存しているＨｅＬａ細胞である。 16. Experiment L (cell recovery)
FIG. 30 is a photomicrograph of HeLa cells. FIG. 31 is a photomicrograph of stained HeLa cells. A mercury lamp was used as a light source, and a 652 nm FITC was used as a filter. In FIG. 31, the stained cell is a living HeLa cell.
　そして、これらの写真からは明らかではないが、本実施形態の細胞培養基材から浮遊している細胞が生存していることを確認した。一般に、基板から遊離して浮遊している細胞は、死滅している。これに対して、本実施形態は、生存しているＨｅＬａ細胞を遊離させることができる。そのため、細胞にダメージを与えないで細胞を回収することができる。ただし、遊離しており、かつ、生存している細胞は、水との接触角が１３５°であって、中密度のカーボンナノウォールを有する細胞培養基材についてのみ、発見された。つまり、水との接触角が１２０°以上１５０°以下の範囲内であって、平均ウォール間隔が、１２０ｎｍ以上２００ｎｍ以下の範囲内であると、低侵襲性回収を行うことができる。ここで水との接触角が１２０°以上１５０°以下の範囲内となるのは、グラフェンシートの終端基がフッ素原子の場合である。 And although not clear from these photographs, it was confirmed that cells floating from the cell culture substrate of this embodiment were alive. In general, cells floating free from the substrate are dead. In contrast, this embodiment can release living HeLa cells. Therefore, the cells can be collected without damaging the cells. However, free and surviving cells were found only for cell culture substrates having a contact angle with water of 135 ° and having medium density carbon nanowalls. That is, minimally invasive recovery can be performed when the contact angle with water is in the range of 120 ° to 150 ° and the average wall interval is in the range of 120 nm to 200 nm. Here, the contact angle with water falls within the range of 120 ° or more and 150 ° or less when the terminal group of the graphene sheet is a fluorine atom.
１７．変形例
１７－１．導電性
　また、カーボンナノウォールＣＮＷ１に、金属微粒子を担持させることにより、グラフェンシートの先端部Ｅ１を導電性にすることもできる。また、製造装置１を用いて、形成後のカーボンナノウォールＣＮＷ１に、ラジカルを打ち込むこととしてもよい。これにより、カーボンナノウォールＣＮＷ１を半導体とすることもできる。そして、反応室１０の内部に窒素を導入して窒素雰囲気中でプラズマを発生させることにより、カーボンナノウォールＣＮＷ１をｎ型半導体とすることもできる。 17. Modification 17-1. Conductivity Further, the tip part E1 of the graphene sheet can be made conductive by supporting metal fine particles on the carbon nanowall CNW1. Moreover, it is good also as driving a radical into carbon nanowall CNW1 after formation using the manufacturing apparatus 1. FIG. Thereby, carbon nanowall CNW1 can also be made into a semiconductor. And carbon nanowall CNW1 can also be made into an n-type semiconductor by introduce | transducing nitrogen inside the reaction chamber 10 and generating a plasma in nitrogen atmosphere.
１７－２．コーティング材の種類
　本実施形態では、カーボンナノウォールの先端にコラーゲンコートした第３の細胞培養基材３００を用いた。しかし、その他のコーティング材を用いてもよい。例えば、コラーゲン以外のコーティング材として、コラーゲンペプチド、ポリエチレングリコール、デキストラン、ポリアクリルアミド、ポリメタクリロイルオキシエチルホスホリルコリンなどが挙げられる。 17-2. Type of Coating Material In the present embodiment, the third cell culture substrate 300 in which the tip of the carbon nanowall is coated with collagen is used. However, other coating materials may be used. Examples of the coating material other than collagen include collagen peptide, polyethylene glycol, dextran, polyacrylamide, and polymethacryloyloxyethyl phosphorylcholine.
１８．本実施形態のまとめ
　以上詳細に説明したように、本実施形態に係る細胞培養基材１００では、カーボンナノウォールＣＮＷ１を形成することとした。カーボンナノウォールＣＮＷ１の先端部Ｅ１が、細胞の足場となる。先端部Ｅ１の化学的性質および構造について、細かい調整を行うことが可能である。したがって、細胞を培養するのに好適な細胞培養基材１００、２００、３００が実現されている。また、その細胞培養基材１００、２００、３００を利用することにより、好適に細胞を培養することのできる細胞培養方法が実現されている。 18. Summary of the present embodiment As described in detail above, in the cell culture substrate 100 according to the present embodiment, the carbon nanowall CNW1 is formed. The tip E1 of the carbon nanowall CNW1 serves as a cell scaffold. Fine adjustments can be made to the chemical properties and structure of the tip E1. Therefore, cell culture substrates 100, 200, and 300 suitable for culturing cells are realized. Moreover, the cell culture method which can culture | cultivate a cell suitably is implement | achieved by utilizing the cell culture substratum 100,200,300.
　なお、本実施形態は単なる例示にすぎない。したがって当然に、その要旨を逸脱しない範囲内で種々の改良、変形が可能である。例えば、カーボンナノウォールＣＮＷ１を形成する基板１１０の表面に予め凹凸を形成しておいてもよい。 Note that this embodiment is merely an example. Therefore, naturally, various improvements and modifications can be made without departing from the scope of the invention. For example, unevenness may be formed in advance on the surface of the substrate 110 on which the carbon nanowall CNW1 is formed.
１…製造装置
１００、２００、３００…細胞培養基材
１１０…基板
１１１…支持基板
１１２…金属層
ＣＮＷ１、ＣＮＷ２、ＣＮＷ３…カーボンナノウォール
Ｅ１、Ｅ２、Ｅ３…先端部
Ｒ１…根元部
Ｃ１…炭素原子
Ｄ１…ウォール間隔 DESCRIPTION OF SYMBOLS 1 ... Manufacturing apparatus 100, 200, 300 ... Cell culture base material 110 ... Substrate 111 ... Supporting substrate 112 ... Metal layer CNW1, CNW2, CNW3 ... Carbon nanowall E1, E2, E3 ... Tip part R1 ... Root part C1 ... Carbon atom D1 Wall spacing

Claims (21)

  1. 基板と、
    前記基板に形成されたカーボンナノウォールとを有する細胞培養基材において、
    　前記カーボンナノウォールのグラフェンシートの先端部が、
    　　細胞を培養するための足場部であること
    を特徴とする細胞培養基材。     A substrate,
    In a cell culture substrate having carbon nanowalls formed on the substrate,
    The tip of the graphene sheet of the carbon nanowall is
    A cell culture substrate, which is a scaffold for culturing cells.
  2. 請求項１に記載の細胞培養基材において、
    　前記グラフェンシートは、
    　　前記基板の板面に交差する向きに形成されていること
    を特徴とする細胞培養基材。     In the cell culture substrate according to claim 1,
    The graphene sheet is
    A cell culture substrate characterized by being formed in a direction intersecting the plate surface of the substrate.
  3. 請求項２に記載の細胞培養基材において、
    　前記グラフェンシートの平均ウォール間隔は、
    　　１０ｎｍ以上１０００ｎｍ以下の範囲内であること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 2,
    The average wall interval of the graphene sheet is
    A cell culture substrate characterized by being in the range of 10 nm or more and 1000 nm or less.
  4. 請求項１から請求項３までのいずれか１項に記載の細胞培養基材において、
    　前記グラフェンシートの前記先端部の少なくとも一部の炭素原子は、
    　　炭素原子以外の原子と結合していること
    を特徴とする細胞培養基材。     In the cell culture substrate according to any one of claims 1 to 3,
    At least some of the carbon atoms at the tip of the graphene sheet are
    A cell culture substrate characterized by being bonded to an atom other than a carbon atom.
  5. 請求項４に記載の細胞培養基材において、
    　前記グラフェンシートの前記先端部の少なくとも一部の炭素原子は、
    　　酸素原子または窒素原子と結合していること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 4,
    At least some of the carbon atoms at the tip of the graphene sheet are
    A cell culture substrate characterized by being bonded to an oxygen atom or a nitrogen atom.
  6. 請求項４に記載の細胞培養基材において、
    　前記グラフェンシートの前記先端部の少なくとも一部の炭素原子は、
    　　フッ素原子と結合していること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 4,
    At least some of the carbon atoms at the tip of the graphene sheet are
    A cell culture substrate characterized by being bonded to a fluorine atom.
  7. 請求項５に記載の細胞培養基材において、
    　前記グラフェンシートの平均ウォール間隔は、
    　　１０ｎｍ以上５００ｎｍ以下の範囲内であること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 5,
    The average wall interval of the graphene sheet is
    A cell culture substrate characterized by being in the range of 10 nm or more and 500 nm or less.
  8. 請求項６に記載の細胞培養基材において、
    　前記グラフェンシートの平均ウォール間隔は、
    　　８０ｎｍ以上１２０ｎｍ以下の範囲内であること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 6,
    The average wall interval of the graphene sheet is
    A cell culture substrate characterized by being in the range of 80 nm or more and 120 nm or less.
  9. 請求項６に記載の細胞培養基材において、
    　前記グラフェンシートの平均ウォール間隔は、
    　　１２０ｎｍ以上２００ｎｍ以下の範囲内であること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 6,
    The average wall interval of the graphene sheet is
    A cell culture substrate characterized by being in the range of 120 nm or more and 200 nm or less.
  10. 請求項１から請求項９までのいずれか１項に記載の細胞培養基材において、
    　前記グラフェンシートの前記先端部の少なくとも一部の炭素原子は、
    　　水素原子と結合していること
    を特徴とする細胞培養基材。     The cell culture substrate according to any one of claims 1 to 9,
    At least some of the carbon atoms at the tip of the graphene sheet are
    A cell culture substrate characterized by being bonded to a hydrogen atom.
  11. 請求項１から請求項１０までのいずれか１項に記載の細胞培養基材において、
    　前記足場部での水との接触角が、
    　　１°以上１７０°以下の範囲内であること
    を特徴とする細胞培養基材。     The cell culture substrate according to any one of claims 1 to 10,
    The contact angle with water at the scaffold is
    A cell culture substrate characterized by being within a range of 1 ° to 170 °.
  12. 請求項１から請求項１１までのいずれか１項に記載の細胞培養基材において、
    　前記グラフェンシートの前記先端部をコーティングするコーティング膜を有すること
    を特徴とする細胞培養基材。     The cell culture substrate according to any one of claims 1 to 11,
    A cell culture substrate comprising a coating film that coats the tip of the graphene sheet.
  13. 請求項１２に記載の細胞培養基材において、
    　前記コーティング膜は、
    　　コラーゲンコート処理がなされたものであること
    を特徴とする細胞培養基材。     The cell culture substrate according to claim 12,
    The coating film is
    A cell culture substrate characterized by being subjected to a collagen coating treatment.
  14. 細胞培養基材の上に細胞を培養する細胞培養方法において、
    　前記細胞培養基材として、
    　　基板にカーボンナノウォールが形成されたものを用い、
    　前記カーボンナノウォールのグラフェンシートの先端部を、
    　　細胞を培養するための足場とすること
    を特徴とする細胞培養方法。     In a cell culture method for culturing cells on a cell culture substrate,
    As the cell culture substrate,
    Using carbon nanowalls formed on the substrate,
    The tip of the carbon nanowall graphene sheet,
    A cell culture method characterized by being used as a scaffold for culturing cells.
  15. 請求項１４に記載の細胞培養方法において、
    　前記細胞培養基材として、
    　　前記グラフェンシートが前記基板の板面に交差する向きに形成されているものを用いること
    を特徴とする細胞培養方法。     The cell culture method according to claim 14, wherein
    As the cell culture substrate,
    A method for culturing cells, wherein the graphene sheet is formed in a direction intersecting the plate surface of the substrate.
  16. 請求項１４または請求項１５に記載の細胞培養方法において、
    　前記細胞培養基材として、
    　　前記先端部の少なくとも一部の炭素原子は、炭素原子以外の原子と結合しているグラフェンシートを有するものを用いること
    を特徴とする細胞培養方法。     The cell culture method according to claim 14 or 15,
    As the cell culture substrate,
    A cell culture method comprising using a graphene sheet in which at least some of the carbon atoms in the tip are bonded to atoms other than carbon atoms.
  17. 請求項１６に記載の細胞培養方法において、
    　前記細胞培養基材として、
    　　前記先端部の少なくとも一部の炭素原子が、酸素原子または窒素原子と結合しているグラフェンシートを有するものを用いること
    を特徴とする細胞培養方法。     The cell culture method according to claim 16, wherein
    As the cell culture substrate,
    A cell culture method comprising using a graphene sheet in which at least some of the carbon atoms at the tip are bonded to oxygen atoms or nitrogen atoms.
  18. 請求項１６に記載の細胞培養方法において、
    　前記細胞培養基材として、
    　　前記先端部の少なくとも一部の炭素原子が、フッ素原子または水素原子と結合しているグラフェンシートを有するものを用いること
    を特徴とする細胞培養方法。     The cell culture method according to claim 16, wherein
    As the cell culture substrate,
    A cell culture method comprising using a graphene sheet in which at least some of the carbon atoms at the tip are bonded to fluorine atoms or hydrogen atoms.
  19. 請求項１４から請求項１８までのいずれか１項に記載の細胞培養方法において、
    　前記細胞培養基材として、
    　　前記足場部での水との接触角が、１°以上１７０°以下の範囲内であるものを用いること
    を特徴とする細胞培養方法。     The cell culture method according to any one of claims 14 to 18,
    As the cell culture substrate,
    A method for culturing cells, wherein the scaffold has a contact angle with water in the range of 1 ° to 170 °.
  20. 請求項１４から請求項１９までのいずれか１項に記載の細胞培養方法において、
    　前記細胞培養基材として、
    　　前記グラフェンシートの前記先端部をコーティングするコーティング膜を有するものを用いること
    を特徴とする細胞培養方法。     The cell culture method according to any one of claims 14 to 19,
    As the cell culture substrate,
    A cell culture method comprising using a coating film that coats the tip of the graphene sheet.
  21. 請求項２０に記載の細胞培養方法において、
    　前記コーティング膜は、
    　　コラーゲンコート処理がなされたものであること
    を特徴とする細胞培養方法。     The cell culture method according to claim 20,
    The coating film is
    A cell culture method characterized by being subjected to a collagen coating treatment.
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