Culture substrate and method for expansion culture of stem cells

Abstract

To provide a culture substrate capable of expansion culture while safely maintaining the mitochondrial activity of stem cells low.SOLUTION: The present invention relates to a culture substrate comprising a metal film on a substrate, wherein the RMS roughness of the surface of the culture substrate comprising the metal film is 250 nm or less, and the metal film contains a metal having a standard electrode potential of 0.35 E0 / V or more at 25°C and 1 atm.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a culture substrate and a method for expanding stem cells. [Background technology]

[0002] In recent years, research into regenerative medicine using stem cells as a transplant cell type has been actively conducted. Stem cells, one of the most commonly used cell types in current cell transplantation regenerative medicine, must be collected from patients and then expanded to a sufficient number for transplantation. However, there is a problem in the process of collecting and expanding stem cells from patients or animals. If stem cells are cultured in an inappropriate environment, mitochondrial activity increases, causing the stem cells to lose their undifferentiated state. In response to this, research has been conducted into reducing mitochondrial activity by inducing mitophagy through genetic modification (Non-Patent Document 1) and by adding drugs to the culture medium (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Lin et al.Stem Cell Res Ther(2021)12:452 [Non-patent document 2] Sepideh et al.Front.Cell Dev. Biol.,14 October 2021,Sec.Cellular Biochemistry Volume 9-2021 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the above-mentioned methods involve genetic modification and the addition of drugs such as Wnt inhibitors to the culture medium, which poses numerous issues, such as safety concerns regarding the return of genetically engineered cells to the body and their subsequent engraftment, the complexity of drug addition, and the impact on costs.

[0005] Therefore, an object of the present invention is to provide a culture substrate that allows stem cells to be safely expanded while maintaining low mitochondrial activity, and a method for expanding stem cells using the culture substrate. [Means for solving the problem]

[0006] As a result of extensive research, the inventors discovered that a culture substrate having a metal film on the substrate, in which the surface roughness of the culture substrate is within a specific range and the metal film contains a specific metal, allows for safe expansion of stem cells while maintaining low mitochondrial activity, and thus completed the present invention.

[0007] That is, the present invention is as follows. 1. A culture substrate having a metal film on a substrate, the RMS roughness of the surface of the culture substrate having the metal film is 250 nm or less; The metal film has a standard electrode potential of 0.35 E at 25°C and 1 atmosphere. 0 Contains metals above / V, Culture substrate. 2. The culture substrate according to item 1 above, wherein the metal is at least one metal selected from the group consisting of Au, Ag, Pt, Pd, Rh, Ru, and Ir. 3. The culture substrate according to claim 1, wherein the RMS roughness is 200 nm or less. 4. The culture substrate according to the above item 1, wherein the metal film has a thickness of 5 to 200 nm. 5. The culture substrate according to item 1 above, wherein the substrate comprises at least one selected from the group consisting of polystyrene, glass, polyethylene terephthalate, polyethylene, polyvinyl alcohol, polyvinyl chloride, polypropylene, and polycarbonate. 6. A method for expanding stem cells by seeding the stem cells on the culture substrate according to any one of 1 to 5 above. [Effects of the Invention]

[0008] According to the culture substrate of the embodiment of the present invention, stem cells can be safely expanded while maintaining low mitochondrial activity. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a cross-sectional view of a culture substrate 10 of this embodiment. [Figure 2] FIG. 2 is a graph showing the relationship between the RMS roughness of the surface of the Au culture substrate and the thickness of the Au film. [Figure 3] Figure 3 shows atomic force microscope images of the surface of an Au culture substrate. Figure 3(a) is an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 0 nm, Figure 3(b) is an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 20 nm, Figure 3(c) is an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 50 nm, Figure 3(d) is an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 100 nm, and Figure 3(e) is an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 200 nm. [Figure 4] FIG. 4 is a graph showing the number of cells cultured using the culture substrates of Comparative Example 1 and Examples 1 to 5. [Figure 5] FIG. 5 is a graph showing the number of cells cultured using the culture substrates of Comparative Example 1, Example 2, Example 6, and Example 7. [Figure 6] FIG. 6 is a graph showing the MTT activity per cell when cells were cultured using the culture substrates of Comparative Example 1 and Examples 1 to 5. [Figure 7] FIG. 7 is a graph showing the MTT activity per cell when cells were cultured using the culture substrates of Comparative Example 1, Example 2, Example 6, and Example 7. [Figure 8] FIG. 8 is a micrograph of cells cultured on the culture substrates of Examples 2, 6, and 7 on the second day. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below, but the present invention is not limited to the following embodiments and can be implemented in any modified form without departing from the gist of the present invention. In this specification, the use of "to" to indicate a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0011] [Culture substrate] A culture substrate according to one embodiment of the present invention (hereinafter also referred to as the culture substrate of this embodiment) is a culture substrate having a metal film on a substrate, wherein the RMS roughness of the surface of the culture substrate having the metal film is 250 nm or less, and the metal film has a standard electrode potential of 0.35 E at 25° C. and 1 atmosphere. 0 It is characterized by containing a metal of 0.15 or more.

[0012] The culture substrate of this embodiment allows for safe expansion of stem cells while maintaining low mitochondrial activity. The reason for this is unclear, but is speculated as follows. Specifically, metals with a standard electrode potential within the above-mentioned specific range have a low tendency to ionize, resulting in almost no elution of metal ions. Therefore, adhesion of proteins in serum is highly stable. However, because metals with a standard electrode potential within the above-mentioned specific range have a high affinity for thiol groups, proteins containing cysteine, i.e., thiol groups, adhere to the precious metal surface with a different orientation than conventional plastic materials, providing a moderately unstable scaffold for cells. This design results in significant energy consumption for adhesion, preventing excess energy consumption by mitochondria. Therefore, it is believed that expansion of stem cells while maintaining low mitochondrial activity is possible. On the other hand, although this creates an unstable scaffold for cells, designing the substrate surface roughness within the above-mentioned specific range allows for integrin-mediated mechanotransduction, altering the localization of the transcriptional regulator YAP, resulting in proliferation comparable to that achieved with conventional culture vessels such as plastic or glass. As a result, it is presumed that cells proliferate while mitochondrial activity remains low. Note that the mechanism of action of the culture substrate of this embodiment is not to be construed as being limited to the above-mentioned mechanism.

[0013] As shown in Fig. 1, the culture substrate 10 of this embodiment includes a metal film 12 on a substrate 11. The surface of the culture substrate 10 of this embodiment on which the metal film 12 is provided serves as a cell culture surface, and cells isolated from tissue or the like are seeded on the surface. Each element will be described in detail below.

[0014] (base material) The material of the substrate 11 in this embodiment is not particularly limited, and any material that is conventionally used as a culture substrate can be used, for example, a substrate containing resin or glass. Examples of the substrate containing a resin include substrates containing a resin such as polystyrene, polyethylene terephthalate, polyethylene, polyvinyl alcohol, polyvinyl chloride, polypropylene, and polycarbonate. Among these, a substrate containing polystyrene is preferred from the viewpoints of processability and cost. Examples of the substrate containing glass include substrates containing glass such as quartz glass, bioceramics, bioglass, etc. Among these, substrates containing quartz glass are preferred from the viewpoints of transparency and cost. In this embodiment, the material of the substrate 11 preferably contains at least one selected from the group consisting of polystyrene, glass, polyethylene terephthalate, polyethylene, polyvinyl alcohol, polyvinyl chloride, polypropylene, and polycarbonate.

[0015] The shape of the substrate 11 in this embodiment is not particularly limited. From the viewpoint of facilitating cell observation, the substrate 11 may be a flat plate having a desired shape in a plan view, such as a rectangle or a circle, or from the viewpoint of reducing the number of culture steps, the number of workers, and the installation space, the substrate 11 may be a three-dimensional body made by joining together small pieces with flat or curved surfaces, such as a sphere or an oval sphere. The substrate 11 in this embodiment may be a dish-type container or a flask-type container, which allows one cell population to be cultured per container, or may be a well plate, which allows multiple cell populations to be cultured per container. The substrate 11 can have any thickness. From the viewpoint of having the strength to support the metal film 12, the thickness of the substrate 11 is preferably, for example, 0.1 mm or more, more preferably 0.15 mm or more, and even more preferably 0.3 mm or more. Furthermore, from the viewpoint of ease of cell observation, the thickness is preferably 2 mm or less, more preferably 1.5 mm or less, and even more preferably 1 mm or less.

[0016] In the present embodiment, the substrate 11 preferably has an RMS roughness of 250 nm or less on the surface provided with the metal film 12, more preferably 220 nm or less, even more preferably 200 nm or less, even more preferably 150 nm or less, even more preferably 100 nm or less, even more preferably 50 nm or less, and particularly preferably 25 nm or less. As described below, this makes it easier to keep the RMS roughness of the surface 12s of the culture substrate 10, which includes the metal film 12 on the substrate 11, to 250 nm or less. The method for measuring the RMS roughness is the same as the method for measuring the RMS roughness of the surface 12s of the culture substrate 10, described below. In this specification, the surface 12s of the culture substrate 10 refers to the surface provided with the metal film 12, and the RMS roughness of the surface 12s refers to the roughness of the surface of the metal film 12 opposite to the bonding surface with the substrate 11.

[0017] (metal film) In this embodiment, the metal film 12 is provided on a substrate 11. In this embodiment, the metal film 12 has a standard electrode potential of 0.35 E at 25° C. and 1 atmosphere. 0It is important that the substrate contains a metal with a standard electrode potential of 1 / V or more. As mentioned above, metals with a standard electrode potential within the specified range have a low ionization tendency, resulting in almost no elution of metal ions. This results in highly stable adhesion of proteins in serum. However, because metals with a standard electrode potential within the specified range have a high affinity for thiol groups, proteins containing cysteine, i.e., thiol groups, adhere to the precious metal surface in a different orientation than conventional plastic materials, creating a moderately unstable scaffold for cells. This design results in a high energy consumption for adhesion, which prevents excess energy consumption by mitochondria. This is thought to enable expansion of stem cells while maintaining low mitochondrial activity. On the other hand, although this creates an unstable scaffold for cells, designing the substrate surface roughness within the specified range induces integrin-mediated mechanotransduction, altering the localization of the transcription factor YAP, resulting in proliferation comparable to that achieved with conventional culture vessels such as plastic or glass. As a result, it is speculated that cells proliferate while maintaining low mitochondrial activity.

[0018] The metal film 12 has a standard electrode potential of 0.35 E at 25°C and 1 atmosphere. 0 It is preferable that the metal content is 0.38E / V or more. 0 It is more preferable that the metal content is 0.40E / V or more. 0 It is more preferable that the metal content is 1 / V or more.

[0019] Examples of metals having a standard electrode potential in the above specific range include Au (standard electrode potential: 1.498 E 0 / V), Ag(0.7996E 0 / V), Pt(1.18E 0 / V), Pd(0.951E 0 / V), Rh(0.758E 0 / V), Ru(0.455E 0 / V), Ir(1.156E 0 / V), Os(0.838E 0 / V). The metal film 12 in this embodiment preferably contains at least one metal selected from the group consisting of Au, Ag, Pt, Pd, Rh, Ru, and Ir, and particularly preferably contains at least one metal selected from the group consisting of Au, Pd, and Ru.

[0020] The thickness of the metal film 12 is preferably 5 to 200 nm. By keeping the thickness within this range, film growth occurs not only in the vertical direction but also in the horizontal direction during film formation, making it easier to keep the RMS roughness of the surface 12s of the culture substrate (described later) to 250 nm or less, thereby achieving proliferation equivalent to that of conventional culture vessels. The thickness of the metal film 12 is more preferably 150 nm or less, even more preferably 120 nm or less, and particularly preferably 100 nm or less, and more preferably 10 nm or more, even more preferably 15 nm or more, and particularly preferably 20 nm or more.

[0021] The method for forming the metal film 12 on the substrate 11 is not particularly limited, and any known method can be used. For example, wet plating such as electroplating and electroless plating may be used, or dry plating such as vacuum deposition, ion plating, and sputtering may be used. Among these, from the viewpoint of film density and film thickness controllability, it is preferable to form the metal film 12 on the substrate 11 by sputtering. Examples of sputtering include magnetron sputtering, pulse sputtering, AC sputtering, and digital sputtering.

[0022] (RMS roughness) It is important that the RMS roughness (root mean square roughness) of the surface 12s of the culture substrate 10 of this embodiment is 250 nm or less. When the RMS roughness of the surface 12s of the culture substrate 10 of this embodiment is 250 nm or less, integrin-mediated mechanotransduction occurs, the localization of the transcriptional regulator YAP changes, and proliferation equivalent to that of conventional culture vessels made of plastic, glass, etc. is achieved. The RMS roughness of the surface 12s of the culture substrate 10 of this embodiment is more preferably 200 nm or less, even more preferably 100 nm or less, even more preferably 20 nm or less, particularly preferably 15 nm or less, and may be, for example, 0.01 nm or more.

[0023] The RMS roughness of the surface 12s of the culture substrate 10 of this embodiment can be measured, for example, using an atomic force microscope (e.g., NanoWizard4XP, manufactured by Bruker) by preparing a 1 cm square measurement sample from the culture substrate and fixing it to a glass plate, and then using force curve-based imaging (QI) mode.

[0024] In order to make the RMS roughness of the surface 12s of the culture substrate 10 of this embodiment 250 nm or less, for example, the surface of the substrate 11 before the metal film is formed, or the surface 12s of the culture substrate 10 after the metal film is formed, may be polished or treated with dilute acid.

[0025] The metal film may have a surface that is chemically or physically modified to enhance cell seeding efficiency, proliferation efficiency, or differentiation efficiency, or may be patterned, such as with metal islands arranged on the substrate.Furthermore, the metal film usually has a structure that provides a gap between the plate and lid, as seen in general culture substrates, to ensure efficient circulation of dissolved gases in the cell culture solution and the gas inside the incubator.

[0026] [Method for expanding stem cells] The method for expanding stem cells according to one embodiment of the present invention (hereinafter also referred to as the method for expanding stem cells according to this embodiment) involves expanding stem cells using the culture substrate according to this embodiment described above. As used herein, "expansion" refers to a process in which cells are maintained under conditions suitable for the cells, such as medium, growth factors, nutrients, temperature, and gas composition, thereby increasing the number of cells through cell division while preserving their phenotypes as much as possible. The cells used here are not limited to those collected from a living organism, those already established as cell lines, or those that have been genetically modified. Hereinafter, the method for expanding stem cells of this embodiment will be described using examples, but the method for expanding stem cells of this embodiment should not be construed as being limited to this embodiment.

[0027] In the method for expanding stem cells of this embodiment, stem cells are seeded on the culture substrate of this embodiment described above and then expanded. In the method for expanding stem cells of this embodiment, the stem cells are cultured using the culture substrate of this embodiment described above, so that the stem cells can be safely expanded while maintaining low mitochondrial activity.

[0028] In the method for expanding stem cells of this embodiment, cells are seeded on the surface of the metal film of the culture substrate of this embodiment described above, and allowed to adhere to the surface of the metal film.

[0029] The cells that can be used are not particularly limited as long as they are stem cells, and examples thereof include mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and nerve cells. In the method for expanding stem cells of this embodiment, it is particularly preferable to use mesenchymal stem cells. The animal species of stem cells is not particularly limited, and examples include mammals such as humans, pigs, mice, rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats.

[0030] In the method for expanding stem cells of this embodiment, the culture conditions are not particularly limited and can be changed depending on the cell type, the use of the cultured cells, etc. Preferably, stem cells are seeded on the culture substrate of this embodiment and expanded, and then the stem cells are replanted (passaged) before reaching confluence. Furthermore, in the method for expanding stem cells of this embodiment, the total culture period from seeding the stem cells on the culture substrate of this embodiment to replacing (passaging) the stem cells before they reach confluence can be set appropriately, but may be, for example, 2 to 14 days, or 2 to 5 days. In the method for expanding stem cells of this embodiment, when the stem cells are replanted (passaged) before they reach confluence, a cell detachment agent or a scraper is preferably used. The cell detachment agent is not particularly limited as long as it is a detachment agent commonly used in cell culture, and examples thereof include enzymes such as trypsin.

[0031] In the expansion culture of stem cells of this embodiment, the culture substrate of this embodiment is used. As described above, metals having a standard electrode potential within the above-mentioned specific range have a low ionization tendency, resulting in almost no elution of metal ions. Therefore, adhesion of proteins in serum is highly stable. However, because metals having a standard electrode potential within the above-mentioned specific range have a high affinity for thiol groups, proteins containing cysteine, i.e., thiol groups, adhere to the precious metal surface in an orientation different from that of conventional plastic materials, providing a moderately unstable scaffold for cells. This design results in a high consumption of energy for adhesion, thereby preventing excess energy consumption by mitochondria. Therefore, it is believed that expansion culture of stem cells can be achieved while maintaining low mitochondrial activity. On the other hand, although this creates an unstable scaffold for cells, designing the substrate surface roughness within the above-mentioned specific range allows integrin-mediated mechanotransduction, changing the localization of the transcriptional regulator YAP, resulting in proliferation comparable to that achieved with conventional culture vessels such as plastic or glass. As a result, it is believed that cells proliferate while maintaining low mitochondrial activity.

[0032] [Cell sheet manufacturing method] In one embodiment of the present invention, a cell sheet is produced using the culture substrate of the present embodiment. The cell sheet production method of the present embodiment will be described below using examples, but the cell sheet production method of the present embodiment should not be construed as being limited to these examples.

[0033] The method for producing a cell sheet of this embodiment involves seeding cells on the culture substrate of this embodiment described above, culturing the cells to form a cell sheet, and then detaching and collecting the cell sheet. In the method for producing a cell sheet of this embodiment, since the culture substrate of this embodiment described above is used for culturing, stem cells can be safely expanded while maintaining low mitochondrial activity, and the formed cell sheet can be detached and collected. When the formed cell sheet is detached and collected, it is preferable to use a cell detachment agent. The cell detachment agent is not particularly limited as long as it is a detachment agent commonly used in cell culture, and examples thereof include enzymes such as trypsin.

[0034] As described above, the present specification discloses the following: [1] A culture substrate having a metal film on a substrate, the RMS roughness of the surface of the culture substrate having the metal film is 250 nm or less; The metal film has a standard electrode potential of 0.35 E at 25°C and 1 atmosphere. 0 Contains metals above / V, Culture substrate. [2] The culture substrate according to [1] above, wherein the metal is at least one metal selected from the group consisting of Au, Ag, Pt, Pd, Rh, Ru, and Ir. [3] The culture substrate according to [1] or [2] above, wherein the RMS roughness is 200 nm or less. [4] The culture substrate according to any one of the above [1] to [3], wherein the metal film has a thickness of 5 to 200 nm. [5] The culture substrate according to any one of [1] to [4] above, wherein the substrate comprises at least one selected from the group consisting of polystyrene, glass, polyethylene terephthalate, polyethylene, polyvinyl alcohol, polyvinyl chloride, polypropylene, and polycarbonate. [6] A method for expanding stem cells by seeding them on the culture substrate according to any one of [1] to [5] above.

[0035] Although the embodiments of the present invention have been described above, the present invention is not limited to the embodiments disclosed in this specification and can be appropriately modified and implemented without departing from the spirit of the invention. The embodiments disclosed in this specification can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. [Example]

[0036] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0037] [Preparation of culture substrate] (Comparative Example 1, Examples 1 to 5) Au films were formed by magnetron sputtering on polystyrene-containing substrates (thickness 1 mm, RMS roughness 2 nm, product name Nunc (registered trademark) Cell-Culture Treated Multidishes, manufactured by ThermoFisher). The standard electrode potential of Au at 25°C and 1 atmosphere was 1.498 E. 0 The target was pure Au (>99.9%). The vacuum chamber was maintained at 2×10 -3 The pressure was reduced to below 100 Pa, and Ar was introduced into the chamber. After cleaning the target by pre-sputtering, sputtering was performed for a time calculated in advance from the film thickness rate. Au films were formed on the substrates with the target film thicknesses listed in Table 1 below (Comparative Example 1: 0 nm, Example 1: 20 nm, Example 2: 50 nm, Example 3: 100 nm, Example 4: 200 nm, Example 5: 500 nm), thereby producing the culture substrates of Comparative Example 1 and Examples 1 to 5 (hereinafter also referred to as Au culture substrates). These Au culture substrates were cleaned with 70% ethanol to ensure they could withstand cell culture and were stored until cell culture. Note that the target film thickness of 0 nm in Comparative Example 1 means that no Au film was formed.

[0038] Example 6 Except for using pure Pd (>99.9%) as the target and a film thickness of 50 nm, a culture substrate (hereinafter also referred to as Pd culture substrate) was prepared in the same manner as in Example 1. This Pd culture substrate was cleaned with 70% ethanol so that it could withstand cell culture, and was stored until cell culture.

[0039] Example 7 A culture substrate (hereinafter also referred to as Ru culture substrate) was prepared in the same manner as in Example 1, except that the target was pure Ru (>99.9%) and the film thickness was 50 nm. This Ru culture substrate was cleaned with 70% ethanol so that it could withstand cell culture, and was stored until cell culture.

[0040] [Measurement of RMS roughness of the surface of the culture substrate] The surface roughness of each culture substrate was measured using an atomic force microscope (NanoWizard4XP, manufactured by Bruker). Measurement samples measuring 1 cm square were prepared from the culture substrate and fixed to a glass plate. Measurements were performed using force curve-based imaging (QI) mode. A graph showing the relationship between the RMS roughness of the surface of the Au culture substrate and the Au film thickness is shown in Figure 2. An atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 0 nm (Comparative Example 1) is shown in Figure 3(a), an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 20 nm (Example 1) is shown in Figure 3(b), an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 50 nm (Example 2) is shown in Figure 3(c), an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 100 nm (Example 3) is shown in Figure 3(d), and an atomic force microscope image of the surface of an Au culture substrate with an Au film thickness of 200 nm (Example 4) is shown in Figure 3(e).

[0041] [Cell culture] For each of the prepared culture substrates (Comparative Example 1, Examples 1 to 5), human adipose-derived mesenchymal stem cells were inoculated onto each cleaned culture substrate at a density of 5,000 cells / cm. 2 The cells were seeded so that the cells were 100% saturation, and cultured in a CO2 incubator (37°C, CO2 concentration 5.0%). The medium was changed every two days, starting one day after seeding. After culturing for the specified period, the cell count and MTT activity were measured, as described below. The results are shown in Table 1. In addition, for each culture substrate (Comparative Example 1, Examples 2, 6, and 7), human bone marrow-derived mesenchymal stem cells were inoculated into each cleaned culture substrate at a density of 5,000 cells / cm. 2 The cells were seeded so that the cell density was 100% and cultured in a CO2 incubator (37°C, CO2 concentration 5.0%). The medium was changed every two days, starting one day after seeding. After culturing for the specified period, the cell count and MTT activity were measured as described below. The results are shown in Table 2.

[0042] [Measurement of cell number] When cells were cultured using the culture substrates of Comparative Example 1 and Examples 1 to 5, the scaffolds on days 1, 3, 4, and 7 of culture were washed with PBS and immersed in a 2 mM SDS solution for 24 hours. The amount of DNA in the recovered solution was quantified using a nuclear staining dye (Hoechst 33288, Dojindo Laboratories), and the number of cells was measured. The results are shown in Table 1 and Figure 4. In addition, when cells were cultured using the culture substrates of Comparative Example 1, Example 2, Example 6, and Example 7, the scaffolds on days 1, 2, 3, and 7 of culture were washed with PBS, immersed in 2 mM SDS solution, and held overnight. The amount of DNA in the recovered solution was quantified using a nuclear staining dye (Hoechst 33288, Dojindo Laboratories), and the number of cells was measured. The results are shown in Table 2 and Figure 5.

[0043] [Measurement of MTT activity] The MTT activity of the cells was measured using a viable cell count reagent (Nacalai Tesque, Cell Count Reagent SF, 07553-15). The MTT activity per cell was calculated using the MTT activity measured by the above method and the cell count according to the following formula: MTT activity per cell = MTT activity / number of cells Table 1 and Figure 6 show the MTT activity per cell on days 1, 3, 4, and 7 after the start of culture when cells were cultured using the culture substrates of Comparative Example 1 and Examples 1 to 5. Table 2 and Figure 7 show the MTT activity per cell on days 1, 2, 3, and 7 after the start of culture when cells were cultured using the culture substrates of Comparative Example 1, Example 2, Example 6, and Example 7.

[0044] [Table 1]

[0045] [Table 2]

[0046] When adipose-derived mesenchymal stem cells were cultured using the culture substrates of Examples 1 to 5, as shown in Table 1 and Figure 6, it was found that the MTT activity per cell was kept low up to day 7 of cell culture compared to when the cells were cultured using the culture substrate of Comparative Example 1. Furthermore, when bone marrow-derived mesenchymal cells were cultured using the culture substrates of Examples 2, 6, and 7, it was found that the MTT activity per cell was kept low up to day 3 of cell culture, as compared with the case of cell culture using the culture substrate of Comparative Example 1, as shown in Table 2 and Figure 7. On day 7 of cell culture, which corresponds to the later stage of culture, overgrowth of the cells occurred, i.e., the cells had already reached confluence, resulting in increased MTT activity, as shown in Figure 5. Figure 8 shows micrographs of cells on day 2 of cell culture using the culture substrates of Examples 2, 6, and 7.

[0047] As can be seen from the above results, the culture substrate having a metal film on the substrate, the RMS roughness of the surface of the culture substrate having the metal film is 250 nm or less, and the metal film has a standard electrode potential of 0.35 E at 25°C and 1 atmosphere. 0When stem cells were cultured using the culture substrates of Examples 1 to 7 containing metals at or above 1 / V, MTT activity per cell was kept low in all cases at the early stage of culture. Furthermore, in some cases where confluence was reached at the later stage of culture, MTT activity per cell increased. These results indicate that the majority of MTT activity is due to mitochondrial activity, and therefore, by using the culture substrate of this embodiment, stem cells can be expanded while maintaining low mitochondrial activity. Furthermore, since mitochondrial activity is thought to be involved in the loss of stem cell potential and cellular senescence, it was suggested that the use of the culture substrate of this embodiment allows stem cells to be expanded while maintaining low mitochondrial activity and suppressing the loss of stem cell potential and cellular senescence. The RMS roughness of the culture substrate surface in Examples 1 to 7 is 0.54 nm to 4.36 nm, but even if the surface roughness is increased to 250 nm, it is estimated that the roughness in the depth direction increases relative to the distance accessible by cells, and conversely, the number of adhesion points decreases, so it is thought that more energy is consumed for adhesion and no excess energy is consumed by mitochondria. Therefore, if the surface roughness is 250 nm or less, it is thought that stem cells can be expanded while maintaining low mitochondrial activity. On the other hand, when the culture substrate of Comparative Example 1 was used, MTT activity was high in the early stage of culture, and the stem cells could not be expanded while maintaining low mitochondrial activity. [Explanation of symbols]

[0048] 10 Culture substrate 11 Base material 12 Metal Film 12s culture substrate surface

Claims

1. A culture substrate having a metal film on a substrate, The RMS roughness of the surface of the culture substrate having the metal film is 250 nm or less; The metal film has a standard electrode potential of 0.35 E at 25° C. and 1 atmosphere. ０ / V or more metals, Culture substrate.

2. The culture substrate according to claim 1, wherein the metal is at least one metal selected from the group consisting of Au, Ag, Pt, Pd, Rh, Ru, and Ir.

3. The culture substrate of claim 1 , wherein the RMS roughness is 200 nm or less.

4. The culture substrate according to claim 1, wherein the metal film has a thickness of 5 to 200 nm.

5. The culture substrate according to claim 1, wherein the substrate comprises at least one selected from the group consisting of polystyrene, glass, polyethylene terephthalate, polyethylene, polyvinyl alcohol, polyvinyl chloride, polypropylene, and polycarbonate.

6. A method for expanding stem cells by seeding the stem cells on the culture substrate according to any one of claims 1 to 5.

Images