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WO2013140822A1 - Matériau support pour culture cellulaire et procédé de culture cellulaire - Google Patents

Matériau support pour culture cellulaire et procédé de culture cellulaire Download PDF

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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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Prior art keywords
cell culture
culture substrate
substrate
carbon
cells
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Japanese (ja)
Inventor
勝 堀
馬場　嘉信
行広 岡本
博基 近藤
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Nagoya University NUC
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Nagoya University NUC
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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

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JP2016163988A (ja) * 2015-01-29 2016-09-08 ダイキン工業株式会社 基材及びその用途
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JP2019504290A (ja) * 2015-10-07 2019-02-14 ザ・リージェンツ・オブ・ザ・ユニバーシティー・オブ・カリフォルニアＴｈｅ Ｒｅｇｅｎｔｓ Ｏｆ Ｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃａｌｉｆｏｒｎｉａ グラフェン系マルチモーダルセンサー
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JP2022526508A (ja) * 2019-03-26 2022-05-25 ユナイテッド キングダム リサーチ アンド イノベーション グラフェンの官能化方法、装置、及び官能化されたグラフェン生成物
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JP2021098624A (ja) * 2019-12-20 2021-07-01 国立大学法人東海国立大学機構 カーボンナノウォールとその製造方法および気相成長装置
WO2021125334A1 (fr) * 2019-12-20 2021-06-24 国立大学法人東海国立大学機構 Nanoparois de carbone, leur procédé de production et dispositif de croissance en phase vapeur
JP7274747B2 (ja) 2019-12-20 2023-05-17 国立大学法人東海国立大学機構 カーボンナノウォールの製造方法
US12469670B2 (en) 2020-03-24 2025-11-11 United Kingdom Research And Innovation Electron microscopy support
CN112430521A (zh) * 2020-11-27 2021-03-02 无锡费曼科技有限公司 用于观察微生物活性的流体观察皿
CN114164180A (zh) * 2021-12-08 2022-03-11 苏州博奥龙科技有限公司 一种单克隆抗体制备用hat半固体筛选培养基
CN118086048A (zh) * 2024-03-27 2024-05-28 苏州大学 一种三维细胞球培养装置及其培养方法

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