WO2018092670A1 - Culture substrate and method for producing culture substrate - Google Patents

Abstract

The present invention addresses the problem of constructing a culture substrate capable of accelerating differentiation induction of stem cells. The present invention also addresses the problem of constructing a culture substrate capable of accelerating differentiation induction in all of the differentiation directions in which stem cells have been determined. The present invention pertains to: a culture substrate which has a nanometer-order periodic protrusion structure on the surface thereof and which cultures stem cells on said surface; and a method for producing the culture substrate.

Description

Culture substrate and method for producing culture substrate

The present application relates to a culture substrate, and more particularly, to a culture substrate having a periodic projection structure of nanometer order on the surface and culturing stem cells on the surface, and a method for producing the culture substrate. This application is based on Japanese Patent Application No. 2016-222732 filed on November 15, 2016, and claims that the entire contents of this application are incorporated by reference.

In recent years, research on regenerative medicine has been actively conducted and is expected to play a part in conventional medicine. Regenerative medicine is a treatment that regenerates tissues and organs that have been dysfunctional or dysfunctional due to illness or injury and restores their functions. Stem cells play an important role in the realization of regenerative medicine. Stem cells have self-renewal ability and differentiation ability. By utilizing the differentiation ability of stem cells, it becomes possible to artificially create tissues and organs necessary for healing diseases and injuries. Stem cells are also responsible for drug discovery screening such as the use of biological cells that have been induced to differentiate from stem cells for pharmacology and toxicity assessment of drug candidates, and for understanding the mechanisms of development, differentiation and disease, and for biological functions that can be used as protein drugs. It can be used for production technology of useful substances such as proteins. Therefore, stem cell utilization technology is expected to be applied to a wide range of technical fields such as medicine and drug discovery.

However, in order to regenerate enough tissues and organs that can be used for transplantation therapy and drug discovery screening, it is necessary to appropriately control the proliferation and differentiation of stem cells. In other words, in order to apply stem cells to regenerative medicine, there is a need for solutions such as the establishment of a culture technique that can safely and stably cultivate and proliferate stem cells while maintaining undifferentiation and efficiently induce differentiation into desired cells. There is a big problem.

For example, Patent Document 1 describes that N-cadherin is a differentiation control factor from pluripotent stem cells to nerve cells and plays an important function in the generation of the nervous system. It has been confirmed that selective differentiation of pluripotent stem cells into neurons occurs by immobilizing such N-cadherin or a homologous substance on the surface of a culture substrate. Patent Document 2 discloses that insulin-like growth factor binding protein (hereinafter abbreviated as “IGFBP”) is a differentiation regulator of pluripotent stem cells into cardiomyocytes, and strongly promotes induction into cardiomyocytes. Is described. It has been confirmed that selective differentiation into cardiomyocytes occurs by immobilizing such IGFBP or homologous substance on the surface of the culture substrate.

However, the differentiation control factors described in Patent Document 1 and Patent Document 2 are both organic substances having high biochemical activity. Therefore, advanced knowledge, technology, and equipment are required, such as the need to store in a sterilized environment until the culture is performed after immobilization on the culture substrate.

Stem cell culture also has problems associated with feeder cells. When culturing stem cells, co-culture with feeder cells has been common. Feeder cells provide the factors necessary for stem cell survival, proliferation, and maintenance of undifferentiation, as well as provide a scaffold for cell adhesion. However, in co-culture with feeder cells, the components derived from feeder cells are mixed, so there is a problem in safety when applying to living organisms such as regenerative medicine, and stable supply of high-quality feeder cells. It was not easy. Although the technique of the said patent document 1 is a culture system of the stem cell which does not use a feeder cell, as above-mentioned, advanced knowledge, technique, and equipment were required.

When culturing stem cells, the influence of the fine structure on the surface of the culture substrate on the survival, proliferation, division process, undifferentiated retention, and cell adhesion of stem cells has been studied. Patent Document 3 describes the possibility that differentiation of pluripotent stem cells is caused by surface microstructure. In the technique described in Patent Document 3, topographical projections (circular, star-shaped, rectangular, crescent-shaped, etc.) are provided at lattice points on the culture substrate surface.

Patent Document 3 presents 1 to 2 μm and 1 to 8 μm, respectively, as the protrusion spacing and the cross-sectional diameter that affect stem cell differentiation. In addition to photolithography, electron beam lithography, hot embossing, nanoimprint, laser ablation, chemical etching, plasma spray coating, spray grinding, engraving, scratching, and microfabrication are presented as methods for creating this. Yes. However, it has been difficult to precisely produce the shape presented in Patent Document 3 by laser ablation, in which the size of the focused spot is usually several to several tens of μm. As a result, it is considered that a costly processing means such as photolithography must be selected as a method that is actually sufficient for manufacturing.

Further, in Patent Document 4, by irradiation with high-intensity femtosecond laser pulses, a micrometer order hemispherical ridge having a groove around it, and a large number of nanometer order on the entire surface of the surrounding groove and hemispherical ridge. Surface-treated titanium having a fine surface structure composed of fine spherical protrusions and fine recesses is described. Titanium has become the mainstream of implant materials such as artificial joints and artificial tooth roots because it rarely causes an immune response even when implanted in a living body, but to the titanium surface that is originally a foreign body for living bodies However, there are still problems to be solved such as poor cell adhesion and tendency of tissue to be hardly regenerated. In contrast, Patent Document 4 improves the adhesion of osteoblasts to the titanium surface by microfabrication of the titanium surface, and is an osteoblast system that is a progenitor cell of osteoblasts separated from the bone marrow. It has been reported to promote cell proliferation and induce differentiation into osteoblasts.

However, the technique of Patent Document 4 has been confirmed to promote the proliferation of osteoblast cells and differentiation into osteoblasts, but application to undifferentiated stem cells has not been studied. It was. Further, in Patent Document 4, a hemispherical 2-20 μm bulge surrounded by a groove is formed by irradiation with a femtosecond laser pulse at 800 μJ on the titanium surface, and a fine spherical protrusion of 100-300 nm and a fine fit are formed. It is described that a concave surface structure is formed, and in view of such description, it can be said that the nanometer-order fine structure is formed accompanying the production of a micrometer-order fine structure.

As a culture substrate, it is simple and low-cost, has high biocompatibility and biological cell affinity, and can safely and stably cultivate and proliferate stem cells while maintaining undifferentiation, and There is a demand for those capable of efficiently inducing differentiation of stem cells. Therefore, the present inventors have a culture having on its surface a hybrid periodic groove structure formed on the same plane so that the periodic groove structure of the micrometer order is overwritten with the periodic groove structure of the nanometer order and arranged in parallel to each other. A base material was constructed (Non-patent document 1 and Non-patent document 2). By culturing stem cells on the surface of such a culture substrate, it was reported that special improvement in biocompatibility and affinity for living cells was observed, and the stem cells were proliferated safely and stably while maintaining their undifferentiation. In addition, stem cells can be efficiently induced to differentiate into specific cell types. In addition, by appropriately controlling the periodic fine structure formed on the surface of the culture substrate, stem cells can be induced to differentiate into different tissues on the same plane.

The technologies of Non-Patent Document 1 and Non-Patent Document 2 are closely related to the periodic fine structure formed on the surface of the culture substrate and the direction in which stem cells can be induced to differentiate, and future generations with a view to tailored medicine This technique is suitable for the preparation of implants. However, in the research stage where searching for the present generation and differentiation mechanisms is a major issue, the overall differentiation induction time is required to be shorter than the acceleration of induction of stem cells in a specific differentiation direction.

JP 2014-82956 JP 2013-223446 A Japanese Patent Laid-Open No. 2014-138605 JP 2010-227551 A

Therefore, an object of the present invention is to construct a culture substrate capable of accelerating differentiation induction of stem cells. Another object of the present invention is to construct a culture substrate that can accelerate differentiation induction in all differentiation directions in which stem cells are determined.

As a result of repeated studies to solve the above-mentioned problems, the present inventors have found that stem cells can be efficiently induced to differentiate by forming fine periodic protrusion structures of nanometer order on the surface of the culture substrate. It was. Such differentiation-inducing effect was recognized without selecting tissue cells for the differentiation of bone cells, chondrocytes, nerve cells, and adipocytes. Based on these findings, the present invention has been completed.

That is, the present application provides the following inventions [1] to [4] in order to achieve the above object.
[1] A culture substrate having a periodic protrusion structure of nanometer order on the surface and culturing stem cells on the surface.
[2] The culture substrate according to [1], wherein the nanometer-order periodic protrusion structure has a diameter of 0.1 to 1 μm, a height of 0.01 to 0.5 μm, and a pitch of 0.1 to 1 μm.
[3] The culture substrate according to the above [1] or [2], wherein the material is titanium.
[4] The culture substrate according to any one of the above [1] to [3], which has a step of forming the periodic protrusion structure of the nanometer order on the substrate surface by forming a nano periodic structure by circular polarization of an ultrashort pulse laser. Manufacturing method.

According to the configuration of [1] above, it is possible to provide a culture substrate for stem cell culture having a fine periodic protrusion structure on the order of nanometers on the surface. The culture substrate of the present invention can efficiently induce differentiation of stem cells by culturing stem cells on the surface thereof, and can accelerate differentiation induction into more mature cells. Such an effect of accelerating differentiation induction of stem cells is recognized not only in a specific direction but also in all the differentiation directions in which the stem cells are determined. The culture substrate of the present invention having such characteristics is expected as a culture substrate for producing a large amount of differentiated cells, and can be used for searching for generation and differentiation mechanisms using stem cells. As a result, it can contribute to the development of stem cell utilization technologies such as regenerative medicine and drug discovery screening.

According to the configuration of [2] above, it is possible to provide a culture substrate for stem cell culture in which the dimensions of the periodic protrusion structure on the nanometer order are optimized. The culture substrate of the present invention has a nanometer-order periodic protrusion structure of a suitable dimension formed on the surface, and the presence of a minute periodic protrusion structure of a specific dimension can induce differentiation of stem cells more efficiently and become more mature. Can accelerate the differentiation induction into cells. Such an effect of accelerating differentiation induction of stem cells is observed in all the differentiation directions in which the stem cells are determined.

According to the configuration of [3] above, the culture substrate of the present invention can be produced by forming a fine periodic projection structure on a titanium material having high biocompatibility and biocell affinity. Affinity is high, and further efficiency in induction of stem cell differentiation can be achieved.

According to the configuration of [4] above, the formation of a fine periodic protrusion structure in the nanometer order of the culture substrate of the present invention can be easily formed, for example, by scanning with an ultrashort pulse laser, which has a thermal effect. Since there are few, there exists an advantage that there are few restrictions to manufacture, such as the process in air | atmosphere being possible. Thereby, the culture substrate of the present invention can be produced easily and at low cost.

The result of Example 1 which performed production examination of a culture substrate is shown, and the section SEM image of the produced culture substrate of the present invention is shown. The graph which shows the result of Example 2 which performed differentiation induction analysis (confirmation with a differentiation marker) of MSC on a culture substrate.

Hereinafter, the present invention will be described in detail.

[Culture substrate 1]
The culture substrate 1 of the present invention has a periodic projection structure 2 of nanometer order formed on the surface. Hereinafter, the nanometer order periodic protrusion structure 2 of the culture substrate 1 of the present invention may be abbreviated as “nano periodic protrusion 2”.

As the material of the culture substrate 1 of the present invention, it is preferable to select a substance that is chemically stable, and has good biocompatibility and cell affinity. Here, chemically stable means having required strength, durability, and wear resistance. For example, when the culture substrate 1 is used as an implant material, it is necessary to have mechanical compatibility according to the place to be implanted. Biocompatibility is a property that does not affect the living body and components derived from living bodies such as cells, tissues, organs, blood, etc., and is not affected by these living bodies and components derived from living bodies. It means a property that is difficult to recognize. Biological cell affinity means, in particular, that it does not affect living cells and components derived from living cells, and is not affected by living cells and components derived from living cells. It means a property that is difficult to inhibit. Specifically, biocompatibility and biological cell affinity are exemplified by having no toxicity, carcinogenicity, and antigenicity, and not causing blood coagulation, hemolysis, or metabolic abnormality. Preferably, a material having a required level of biocompatibility and biological cell affinity is selected according to the use of the culture substrate 1.

Specifically, among metal materials, ceramic materials, synthetic polymer materials, etc., known materials can be used as long as they have the above properties. Examples of the metal material include titanium, titanium alloy and oxide, stainless steel, niobium, niobium alloy and oxide, tantalum, tantalum alloy and oxide, nickel-chromium alloy, chromium-cobalt alloy and the like. An alloy indicates a metallic property composed of a plurality of metallic elements or metallic elements and nonmetallic elements. For example, as the titanium alloy, one in which one or more other elements such as nickel, niobium, tantalum, molybdenum, zirconium, and platinum are added to titanium and the composition is adjusted can be used. Examples of the ceramic material include alumina and its oxide, zirconium and its oxide, and hydroxyapatite. Use ceramic materials that are molded with other additives, those that are coated with a ceramic material that melts the metal material surface, and those that are coated with a metal material such as an alloy. You can also. Examples of the synthetic polymer material include silicone and polyurethane.

The shape and size of the culture substrate 1 of the present invention are not particularly limited, and can be appropriately selected depending on the application, such as plate, cube, column, rod, fiber, sphere, granule, and lump. The nano-periodic protrusion 2 may be formed on all surfaces of the culture substrate 1, or may be formed on a part of the surface or a part of the surface. Depending on the intended use of the culture substrate 1 of the present invention, its shape and size can be set as appropriate. For example, when using the culture substrate 1 of the present invention as an implant, the shape and size can be appropriately set according to the implantation site and the tissue desired to be regenerated.

The nano-periodic protrusion 2 formed on the surface of the culture substrate 1 of the present invention means a structure provided with fine protrusions regularly in the order of nanometers at regular intervals. That is, the nano-periodic protrusion 2 means a structure in which a plurality of fine protrusions formed with dimensions that are appropriate to display in nm units are periodically arranged. For example, the structure is formed by laser irradiation or the like. It means a fine structure with dimensions that can be understood as a phenomenon. Specifically, it is 10 to 1000 nm. Here, the dimensions of the nano-periodic protrusion 2, for example, the length, height, and pitch of one side in the case of a rectangular protrusion, the diameter, the height, and the pitch in the case of a circular protrusion. , Etc., are formed with dimensions on the order of nanometers.

The shape of each protrusion of the nano periodic protrusion 2 can be formed as a pyramid such as a triangular pyramid, a quadrangular pyramid, a hexagonal pyramid, a conical shape, a cylindrical shape, a hemispherical shape, a wave shape, a bell shape, or the like, and can also be referred to as a dot. Examples in which a plurality of protrusions are arranged in parallel, concentric, lattice, spiral, random, etc. As for the protrusion, the area of the cross section orthogonal to the height direction may or may not change from the bottom to the top, and if it changes, even if it has a shape that gradually decreases, Even if it is a shape which increases, the shape which combined increase and decrease may be sufficient.

The dimensions of each protrusion of the nano periodic protrusion 2 are preferably set to a diameter of 0.1 to 1 μm (100 to 1000 nm), a height of 0.01 to 0.5 μm (10 to 500 nm), and a pitch of 0.1 to 1 μm (100 to 1000 nm). In particular, the projection diameter is set to 0.5 μm (500 nm), the height is 0.2 μm (200 nm), the pitch is 0.5 to 0.8 μm (500 to 800 nm), and particularly preferably 0.7 μm (700 nm). The density of the nano periodic protrusions 2 can be preferably 10,000 to 30,000 per 1 mm ² .

Here, the diameter means a distance from one end of the protrusion to the other, and is a distance of one side in the case of a rectangular protrusion, and is a distance of a diameter in the case of a circular protrusion. Even when the distance changes in the height direction of the protrusion, the distance on the surface of the culture substrate 1 is meant. The height of the protrusion means the distance from the average height of the bottom surface of the protrusion to the average height of the top surface. The pitch of the protrusions means the distance between the closest protrusions and is a distance corresponding to one cycle of unevenness on the surface of the culture substrate 1. The dimensions such as the diameter, height, and pitch of the protrusions are preferably calculated as an average of measured values of the dimensions of the protrusions existing in a certain region, for example, a region of 1 mm ² .

The nano-periodic protrusions 2 in the culture substrate 1 of the present invention are isotropic, that is, the arrangement of the nano-periodic protrusions 2 is nondirectional regardless of the direction.

The nano-periodic protrusion 2 of the culture substrate 1 of the present invention can be formed by a known method. For example, it can be formed by using an ultrashort pulse laser having a pulse width of several femtoseconds to several picoseconds. As the ultrashort pulse laser, a processing laser device having a near infrared region with a wavelength of 800 nm to 1500 nm, a pulse time width of 10 ps or less, and an output of 1 W or more is preferable, and a femtosecond pulse laser is particularly preferable. As the femtosecond pulse laser, FCPA μJewel D-1000 manufactured by Imla America, Inc. can be preferably used.

For example, a periodic groove structure can be formed in a direction perpendicular to the polarization direction by linearly polarized light of an ultrashort pulse laser, a granular structure can be formed by circularly polarized light, and a cage structure can be formed by elliptically polarized light. The periodic protrusions 2 are preferably formed by circularly polarized light. When the nano periodic protrusion 2 is formed on the titanium material, the wavelength is preferably 800 to 1500 nm, the fluence 0.5 to 1.5 J / cm ² , the pulse line density 100 to 1000 pulses / mm, and the number of scans 1 to 10 times. Particularly preferably, the fluence is 0.8 J / cm ² , the pulse linear density is 200 pulses / mm, the number of scans is once, and the polarization is circularly polarized.

It is formed by irradiating a laser beam so that the laser beam draws a desired shape on the substrate surface. Laser scanning can be performed using any of raster scanning, vector scanning, spot scanning, and the like, but the raster scanning method is preferable.

[Cell culture using culture substrate 1 of the present invention]
The culture substrate 1 of the present invention is a substrate for culturing stem cells, and cultures stem cells using the surface on which the nanoperiodic protrusions 2 are formed as the culture surface. In the realization of regenerative medicine, stem cells are proliferated in an undifferentiated state (hereinafter sometimes referred to as “undifferentiated proliferation stage”), and then the differentiated stem cells are induced to become target cells. Differentiation (hereinafter sometimes referred to as “differentiation induction stage”). In order to regenerate tissues and organs that can be transplanted, stem cells with an extremely large number of cells are required, and it is necessary to efficiently induce differentiation into the intended cells. According to the culture substrate 1 of the present invention, the proliferation and differentiation of stem cells can be appropriately controlled.

Stem cells are undifferentiated cells having differentiation ability and self-replication ability. Here, the differentiation ability means the ability to change into various cells having specific functions constituting tissues and organs. In other words, cells existing in the body have a certain function and shape, but stem cells have the ability to change into a cell having a certain function and shape. From the viewpoint of differentiation potential, a stem cell may have pluripotency capable of changing into all cells present in the body, or can differentiate into only some cells. Also good. Here, undifferentiated means a state that has not differentiated into somatic cells or germ cells having a specific function or shape. Self-replicating ability means the ability of a cell to make the same cell as itself while repeating cell division.

Stem cells are cells that have not finally differentiated, and include all cells that have differentiation ability and self-replication ability. Therefore, a cell having differentiation ability that occurs in the process until the stem cell is finally differentiated is also included in the stem cell as long as it has differentiation ability and self-replication ability. Stem cells include artificial pluripotent stem cells (hereinafter abbreviated as “iPS cells”), embryonic stem cells (hereinafter abbreviated as “ES cells”), nuclear transfer embryonic stem cells (hereinafter abbreviated as “ntES cells”). ), Somatic stem cells, umbilical cord blood stem cells, and the like, but are not limited thereto. Stem cells are hierarchical, and iPS cells and ES cells at the top have high self-replicating ability and can differentiate into various cell lineages. As it turns out, it can only differentiate into specific cell lineages.

An iPS cell is a pluripotent stem cell that is artificially induced by introducing a specific gene into a somatic cell that has originally lost differentiation potential. ES cells are pluripotent stem cells obtained by culturing the inner cell mass in embryos at the blastocyst stage of fertilized eggs, and ntES cells make embryos by putting somatic cell nuclei into eggs excluding nuclei. It is a pluripotent stem cell obtained by culturing an inner cell mass in an embryo like ES cells. iPS cells are described in Takahashi K. et al., “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.”, Cell, 126 (4), 663-676.
(2006), Takahashi K. et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131 (5), 861-872 (2007), Yu J. et al., “Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells ”, Science, 318 (5858), 1917-1920 (2007),“ Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts ”, Nakagawa M. et al., Nat Biotechnol., 26 (1) , 101-106, (2008), and the like. ES cells are described in MJ Evans et al., “Establishment in culture of pluripotential cells from mouse embryos”, Nature, 292, 154-156 (1981), Thomson JA et al., “Embryonic stem cell lines derived from human blastocysts.”, Science , 282 (5391), 1145-1147 (1988), Amit, M., et al., “Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.”, Dev. Biol. 227 (2) , 271-278 (2000), etc. Further, iPS cells and ES cells may be obtained from commercial products or cell banks.

IPS cells and ES cells are pluripotent stem cells having the ability to differentiate into ectoderm, mesoderm, endoderm three germ layers, and all types of cells produced by differentiation of the three germ layers. Pluripotent stem cells can differentiate into cells that make up all tissues and organs except placenta and amniotic membrane. For example, the ectoderm differentiates into nerve cells of the nervous system, axons, myelin sheaths, oral epithelium of the digestive system, tongue, tooth enamel, sensory organ skin, cornea, retina, inner ear, outer ear, etc. The germ layers are skeletal systems such as bone and cartilage, heart, vascular endothelial cells, blood cells such as leukocytes, platelets and erythrocytes, circulatory systems such as spleen and bone marrow, nervous system microglia, urinary kidneys, urine It differentiates into ducts, genital ovaries, uterus, testis, connective tissue, etc., and endoderm is digestive esophageal epithelium, stomach epithelium, liver, pancreas, endocrine thyroid, thymus, sensory organ canal Differentiates into the tympanic chamber, respiratory tonsils, pharyngeal epithelium, laryngeal epithelium, tracheal epithelium, lungs, etc.

Somatic stem cells are cells that are not terminally differentiated in the living body, and are mesenchymal stem cells (hereinafter sometimes abbreviated as “MSC”), neural stem cells, hematopoietic stem cells, hepatic stem cells, vascular endothelial stem cells, There are various types of epithelial stem cells. Somatic stem cells can also be generated in the process of terminal differentiation of iPS cells and ES cells. Unlike pluripotent stem cells, somatic stem cells can be differentiated only into cells that constitute a specific tissue or organ.

MSC is a type of somatic stem cell that is mesoderm-derived stromal cell (bone marrow), osteoblast (bone cell), chondroblast (chondrocyte), muscle cell, adipocyte, fibroblast (tendon, Ligament) and the ability to differentiate into vascular endothelial cells. In addition, it has been reported that it has the ability to differentiate into ectoderm cells, endoderm cells, and pancreatic cells across germ layers.

MSC can be obtained from various tissues such as bone marrow, adipose tissue and muscle, but preferably bone marrow mesenchymal stem cells can be obtained from bone marrow. Bone marrow mesenchymal stem cells are contained in bone marrow stromal cells. For example, bone marrow fluid collected by bone marrow puncture is seeded on a petri dish, and fibroblast-like cells that grow on the bottom of the petri dish are proliferated by subculture. Can be obtained by MSCs can also be induced to differentiate from pluripotent stem cells such as iPS cells and ES cells. For example, it is possible to obtain MSC by culturing ES cells in the presence of retinoic acid and selecting SOX1-positive cells, and culturing ES cells, stromal cell-like morphology, PDGFRα-positive and FLK1-negative, expressing Mesp2 It has been reported that MSCs can be obtained by sorting cells that do not (see JP 2005-304443, WO 2004/106502). MSCs may be obtained from commercial products or cell banks.

The origin of cells cultured on the culture substrate 1 of the present invention is not limited. Therefore, cells derived from mammals such as humans, monkeys, mice, rats, hamsters, rabbits, cows, horses, pigs, dogs, cats, goats, sheep, etc., birds, reptiles, and preferably cells derived from mammals. Can be used for culture.

Stem cell culture in the undifferentiated growth stage is performed on the surface of the culture substrate 1 of the present invention on which the nano-periodic protrusions 2 are formed. In the undifferentiated growth stage in which differentiation induction is not performed, by culturing using the culture substrate 1 of the present invention, the appearance of differentiated cells due to spontaneous differentiation or the like is suppressed, and the stem cells remain undifferentiated. Can proliferate. That is, on the culture substrate 1 of the present invention, the stem cells do not differentiate into any cell unless induced to differentiate, and are not accompanied by karyotypic abnormalities. On the other hand, stem cells can sufficiently exhibit self-replicating ability to replicate cells having the same properties as self. Thereby, it is possible to efficiently proliferate stem cells while maintaining undifferentiation, and it is possible to stably provide a high-quality stem cell population. This makes it possible to provide an amount of stem cells that can provide a sufficient amount of differentiated cells that can be used for regenerative medicine, drug discovery screening, and the like.

The culture of stem cells in the undifferentiated growth stage is not particularly limited as long as the stem cells can be maintained. Therefore, it can be performed based on a method known in the art, and cells can be seeded in a liquid medium for primary culture and cultured under appropriate conditions. The exchange and passage of the liquid medium can be performed in the same manner as a known method.

Specifically, in the culture of iPS cells and ES cells, the culture medium and culture culture conditions are not particularly limited as long as iPS cells and ES cells can be maintained, and can be performed based on known culture media and culture conditions. As the medium, a known medium used for culturing normal iPS cells and ES cells can be used. For example, a medium in which a cell growth factor such as basic fibroblast growth factor (bFGF) is added to a serum-free medium can be used, and can be appropriately selected according to the cells to be cultured. Moreover, commercially available culture media for iPS cells and ES cells can be used. For example, StemPro (registered trademark) hESC (Life Technologies), ReproFF2 (Reprocell) and the like can be used.

In the culture of MSC, the culture medium and culture culture conditions are not particularly limited as long as MSC can be maintained, and can be performed based on known culture media and culture conditions. As the medium, a medium usually used for MSC culture can be used. Examples include MEM medium, DMEM medium, and the like, which can be appropriately selected according to the cells to be cultured. Commercially available MSC growth media and kits can be used, and for example, MSCGM ^™ BulletKit ^™ (Lonza, catalog number PT-3001) can be used.

The culture conditions can also be appropriately selected according to the cells to be cultured. For example, the initial seed density is 5000 to 6000 cells / cm ² and the cells can be cultured in an incubator set at 37 ° C. and 5% CO ₂ .

Confirmation of whether or not stem cells maintain differentiation potential can be performed by observation of cell morphology, confirmation of differentiation ability, confirmation of undifferentiated markers, and the like. An undifferentiated marker is a molecule that is specifically expressed in undifferentiated stem cells and plays a very important role in the expression of differentiation ability. The expression or expression level of such a molecule is detected by a known method. be able to. For example, Real time RT-PCR can be used to detect marker gene expression, and protein marker expression can be detected by immunostaining or enzyme staining using marker-specific polyclonal or monoclonal antibodies. An activity measurement method or the like can be used. As an immunostaining method using an antibody, an antibody fluorescence staining method can be preferably used. Here, the antibody fluorescence staining method is a method in which an antibody labeled with a fluorescent dye is incorporated into a sample by using an antigen-antibody reaction and stained, and has high specificity for an antigen substance (undifferentiation marker). Dyeing can be performed.

As an undifferentiated marker, in the case of pluripotent stem cells such as iPS cells and ES cells, Nanog, SRY (sex determining region Y) -box 2 (SOX2), SSEA-1, SSEA-3, SSEA-4 , TRA-1-60, TRA-1-81, OCT3 / 4, etc. In the case of MSC, CD29, CD44, CD71, CD73 (SH3 / 4), CD90 (Thy-1), CD105 (SH2), CD106, CD166, Stro-1, etc. are exemplified. Negative markers can also be used, and examples of MSC negative markers include CD11b, CD14, CD19, CD31, CD18, CD34, CD45, CD56, CD79α, HLA-DR, and the like.

The differentiation induction of stem cells in the differentiation induction stage is performed on the surface of the culture substrate 1 of the present invention on which the nano-periodic protrusions 2 are formed. In the differentiation induction stage, differentiation is induced using the culture substrate 1 of the present invention, so that on the culture substrate 1 of the present invention, the stem cells are efficiently generated while suppressing the generation of stem cells that maintain undifferentiation. Differentiate into mature cells. The culture substrate 1 of the present invention has high differentiation induction efficiency and can reduce the time required for differentiation induction. Such high differentiation induction efficiency is not for differentiation in a certain direction but high differentiation in all directions. Shows induction efficiency. Therefore, a large amount of differentiated cells can be obtained from the stem cells.

As a method for inducing differentiation, any method known in the art can be used as long as a stem cell can be differentiated into a target cell. For example, it can be performed by culturing cells on the culture substrate 1 of the present invention using a differentiation-inducing medium containing a differentiation-inducing factor, and after the above-described undifferentiated growth stage on the culture substrate 1 of the present invention. The desired differentiated cells can be obtained by switching the medium to a differentiation-inducing medium. In addition, only one of the undifferentiated growth stage and the differentiation induction stage can be performed using the culture substrate 1 of the present invention.

The differentiation-inducing factor can be appropriately selected according to the type of cells desired to be differentiated, the differentiation hierarchy of stem cells to be differentiated, and the like. The induction conditions such as the contact time with the differentiation-inducing factor are not limited as long as differentiation induction into the target cell occurs. Commercially available differentiation induction reagents and kits can also be used.

For example, in the case of pluripotent stem cells such as iPS cells and ES cells, differentiation differentiation factors are differentiated into cells of a specific lineage via the germ layers by contacting differentiation inducers at a specific time, order and concentration. Can do. For example, by adding activin and basic fibroblast growth factor (bFGF) to pluripotent stem cells, it is possible to differentiate the mesoderm and endoderm, and the mesoderm by adding bone morphogenetic protein (BMP).

Insulin, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, 3,3,5-triiodothyronine (T3), etc. can be used for induction of differentiation from MSC to adipocytes. For differentiation into bone cells, dexamethasone, L-glutamine, ascorbic acid, β-glucerophosphate, and the like can be used. For differentiation into chondrocytes, dexamethasone, ascorbic acid, ITS (insulin, transferrin, selenium) and the like can be used. For differentiation into nerve cells, β-mercaptoethanol, dimethyl sulfoxide (DMSO), forskolin and bFGF can be used. For the differentiation into skeletal muscle cells, 5-azacytidine and the like can be used. For differentiation into hepatocytes, ITS, dexamethasone, hepatocyte growth factor (HGF), oncostatin and the like can be used. For differentiation into cardiomyocytes, Dickkoph-1 (Dkk1), insulin-like growth factor binding protein 4 (IGFBP-4), or the like can be used.

Specifically, when differentiation induction from MSC to adipocytes is performed, differentiation induction can be started when MSC is preferably 80 to 90% confluent. As the adipocyte differentiation induction medium, hMSC-BulletKit ^™ -for adipocyte differentiation (Lonza, catalog number PT-3004) can be used to induce differentiation into adipocytes according to the manufacturer's instructions. This kit consists of basal medium, L-glutamine, Mesenchymal cell growth supplement (MCGS), dexamethasone, indomethacin, 3-isobutyl-1-methyl-xanthine (IBMX), GA- Containing 1000 (gentamicin, amphotericin B). The initial cell seed density is preferably 2.1 × 10 ⁴ cells / cm ² .

When differentiation from MSC to chondrocytes is performed, differentiation induction can be started when MSC is preferably 100% confluent. As the chondrocyte differentiation induction medium, hMSC-BulletKit ^™ -for cartilage differentiation (Lonza, catalog number PT-3003) can be used to induce differentiation into chondrocytes according to the manufacturer's instructions. This kit comprises basal medium, L-glutamine, dexamethasone, ascorbic acid, ITS + supplement, sodium pyruvate, proline, GA-1000 (gentamicin, amphotericin B). The initial cell seed density is preferably 5 × 10 ⁵ cells / cm ² .

When differentiation from MSC to bone cells is performed, differentiation induction can be started when MSC is preferably 100% confluent. As an osteoblast differentiation medium, hMSC-BulletKit ^™ -for osteoblast differentiation (Lonza, catalog number PT-3002) can be used to induce differentiation into bone cells according to the manufacturer's instructions. This kit consists of basal medium, L-glutamine, dexamethasone, ascorbic acid, ITS + supplement, sodium pyruvate, proline, mesenchymal cell growth supplement (MCGS), β-glycerophosphate, penicillin / streptomycin . The initial cell seed density is preferably 3.1 × 10 ⁵ cells / cm ² .

When differentiation from MSC to nerve cells is performed, differentiation induction can be started when MSC is preferably 80-90% confluent. The neural cell differentiation induction medium can be induced to differentiate into neural cells using Mesenchymal Stem Cell Neurogenic Differentiation Medium (PromoCell, Catalog No. C-28015) according to the manufacturer's instructions. it can. This kit comprises a basic medium, Supplement Mix (PromoCell, catalog number C-39815). The initial cell seed density is preferably 5000 cells / cm ² .

Whether a stem cell has differentiated into a target cell can be determined by observing the morphology of the cell and confirming the expression of a differentiation marker for confirmation of differentiation unique to the target differentiated cell. A method known in the art can be used for confirming the expression of the differentiation marker. For example, Real time RT-PCR can be used to detect marker gene expression, and protein marker expression can be detected by immunostaining or enzyme staining using marker-specific polyclonal or monoclonal antibodies. An activity measurement method or the like can be used. As an immunostaining method using an antibody, an antibody fluorescence staining method can be preferably used. Here, the antibody fluorescence staining method is a method in which an antibody labeled with a fluorescent dye is incorporated into a sample and stained using an antigen-antibody reaction, and has high specificity for an antigen substance (differentiation marker). Dyeing can be performed.

Specifically, differentiation into adipocytes is confirmed by peroxisome proliferator-activated receptor γ (PPARγ, (also referred to as NR1C3, PPARG)), CCAAT / enhancer binding protein β (C / EBPβ), fatty acid binding protein (FABP (Also referred to as aP2)), lipoprotein lipase (LPL) and the like can be used. For confirmation of differentiation into chondrocytes, Sex determining region Y-type high mobility group box protein 9 (SOX9), aggrecan, etc. can be used. Confirmation of differentiation into bone cells uses secreted phosphate protein 1 (SPP1, (also known as osteopontin: OPN)), bone sialoprotein (BSP), osteocalcin (OCN), alkaline phosphatase (ALP), and calcification ability. be able to. Confirmation of differentiation to nerve cells microtubule-associated protein 2 (MAP2), can be used nestin, class III beta-tubulin (β _III -tubulin) or the like.

Here, PPARγ is a protein belonging to the nuclear receptor superfamily, functions also as a transcription factor, is mainly distributed in adipose tissue, and is involved in induction of adipocyte differentiation from preadipocytes. SOX9 plays an essential role in the aggregation of undifferentiated mesenchymal cells and the subsequent differentiation process of chondrocytes. On the other hand, SOX5 and SOX6 are induced by SOX9, and the three cooperate to induce the transcription of cartilage-specific genes such as type II collagen and determine differentiation into chondrocytes. SPP1 is involved in the adhesion of osteoclasts to calcified bone matrix, and its expression level is increased before and during osteoblast differentiation. MAP2 is a microtubule-associated protein that is abundant in vertebrate neurons. MAP2 begins to express when differentiated from neural progenitor cells, and in mature neurons it is almost absent from axons and is almost specifically localized to dendrites and cell bodies.

The culture substrate 1 of the present invention is a culture substrate 1 for culturing stem cells on which nano-periodic protrusions 2 are formed, can efficiently induce differentiation of stem cells, and can accelerate differentiation induction into more mature cells. . Such an effect of accelerating differentiation induction of stem cells is observed in all the differentiation directions in which the stem cells are determined. Therefore, it does not selectively induce differentiation into a specific cell type. The culture substrate 1 of the present invention having such properties is expected as a culture substrate 1 for producing a large amount of differentiated cells, rather than making a tissue from stem cells.

Formation of the nano-periodic protrusions 2 of the culture substrate 1 of the present invention can be easily formed, for example, by scanning with an ultrashort pulse laser, and since it is less affected by heat, it can be processed in the atmosphere. There is an advantage that there are few restrictions to manufacture. Thereby, the culture base material 1 of this invention can be produced simply and at low cost. In addition, since the culture substrate 1 of the present invention can be produced by forming the nano-periodic protrusion 2 on a titanium material having high biocompatibility and biocell affinity, coupled with the effect of improving biocompatibility and biocell affinity, Further acceleration of differentiation induction can be achieved.

The culture substrate 1 of the present invention having such characteristics can be suitably used in the technical field using stem cells. In particular, it can be used to search for development and differentiation mechanisms. For example, it can be suitably used for elucidating the developmental pathways of tissues and organs, cell-cell interactions in the process of organ formation, differentiation induction pathways, and the molecular mechanisms that control differentiation induction ability and differentiation direction. It can contribute to the establishment of basic technology for practical application including clinical application. Furthermore, by culturing stem cells on the culture substrate 1 of the present invention, the created cells, tissues, and organs can be used for pharmacological tests and drug discovery such as drug efficacy evaluation, pharmacokinetic evaluation, safety evaluation, etc. It is expected to contribute to drug discovery, life sciences, and medical care, such as screening, regenerative medicine that restores damaged organs and organ functions, and cell therapy. In the field of regenerative medicine, for example, differentiation into bone cells can be used for implants such as refractory fractures, artificial joints, and artificial tooth roots, and so on, which can contribute to the development of tailor-made medicine.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. In the examples, the culture substrate 1 having a substantially circular protrusion formed as the nano-periodic protrusion 2 is prepared, and differentiation induction into bone cells, chondrocytes, nerve cells, and adipocytes is induced using mesenchymal stem cells. An example of what was done is disclosed. However, the present invention is not limited to this.

[Example 1] Preparation of culture substrate 1 Outline In this example, in order to construct the culture substrate 1 showing the MSC differentiation induction acceleration effect, the production of the culture substrate 1 having nano-periodic protrusions 2 formed on the surface thereof was examined.

2. 2. Materials and methods 2-1. Laser A femtosecond laser was used as a laser for forming the nano periodic protrusions 2 on the substrate surface. As a femtosecond laser, FCPA μJewel D-1000 (hereinafter referred to as “D-1000”) manufactured by Imra America Co., Ltd. was used. The details of the laser are summarized in Table 1 below.

2-2. Substrate As a substrate for preparing the culture substrate 1, a mirror-polished medical titanium plate (φ14 mm × 1 mm single-side polished product) was used. Titanium material was purchased from T.D.C. Co., Ltd. (24-15, Iimai, Chofu-cho, Miyagi-gun, Miyagi-ken) from a grade 2 pure titanium round bar with a mirror surface.

Titanium is widely used as an implant material for artificial joints and artificial tooth roots because it rarely causes an immune reaction when implanted in a living body. However, the compatibility of titanium materials with soft tissues is an area that has not yet been studied, and it has been reported that implants are extracted due to incompatibility. Therefore, in this example, the production of the culture substrate 1 having the periodic fine structure formed on the surface by laser processing was examined in order to investigate the possibility of improving the biocompatibility by forming the periodic fine structure.

2-2-1. Culture substrate (comparative example) with micrometer-order periodic grooves formed on the substrate surface
A micrometer-order periodic groove (hereinafter sometimes referred to as a “microperiodic groove”) in which a plurality of micrometer-order grooves are arranged in parallel on the surface of the substrate, and femtosecond laser light is applied to the surface of the medical titanium plate. It was formed by scanning while irradiating (fluence: 0.7 J / cm ² , scanning speed: 300 mm / second, scanning frequency: 14 times, polarized light: circularly polarized light or linearly polarized light). As a result, periodic grooves having a width of 6 μm, a depth of 0.6 μm, and a pitch of 12 μm were formed ((1)).

Here, the width of the groove means a distance from the end of the groove to the end, and is a distance in a direction perpendicular to the longitudinal direction of the groove. Even when the distance changes in the height direction of the groove, it means the distance on the surface of the culture substrate. The height of the groove means the distance from the average height of the bottom surface of the groove to the average height of the top surface. The pitch of the grooves means the distance between the closest grooves, and is the distance between the closest grooves in the direction perpendicular to the longitudinal direction of the grooves, and is a distance corresponding to one cycle of the unevenness in the direction perpendicular to the longitudinal direction of the grooves. .

2-2-2. A culture substrate with periodic grooves of nanometer order formed on the substrate surface (comparative example)
A nanometer ordano periodic groove (hereinafter sometimes referred to as “nanoperiodic groove”) in which a plurality of nanometer-order grooves are arranged in parallel on the surface of the substrate, and femtosecond laser light is applied to the surface of the medical titanium plate. by scanning while irradiating the, was formed (fluence: 0.8 J / cm ^2, scanning speed: 500 mm / sec, the number of scans: 1 times, polarization: linear polarization). As a result, a periodic groove having a depth of 0.2 μm and a pitch of 0.7 μm was formed ((2)).

2-2-3. Culture substrate with hybrid periodic grooves formed on the substrate surface (comparative example)
The culture substrate is a culture substrate in which a hybrid periodic groove formed by the inventors described in the section of the prior art is arranged on the substrate surface in parallel with the micro periodic grooves and the nano periodic grooves arranged in parallel with each other ( Non-Patent Documents 1 and 2). It was formed by scanning while irradiating the surface of the titanium plate for medical use with femtosecond laser light. The above 2-2-1 except that the number of scans is 20 times. After the micro periodic groove is formed by the procedure described in the above item 2-2-2. The nanoperiod groove was overwritten by the procedure described in. In the nano periodic groove, the polarization direction was orthogonal to the scanning direction. As a result, a culture substrate having a micro periodic groove having a groove width of 6 μm, a depth of 0.6 μm, and a pitch of 12 μm and a hybrid periodic groove in which a nano periodic groove having a depth of 0.2 μm and a pitch of 0.7 μm was formed ( (3)).

2-2-4. Culture substrate 1 having nano-periodic protrusions 2 formed on the substrate surface (Example)
The nano periodic protrusion 2 was performed with a fluence of 0.8 J / cm ² , a scanning speed of 500 mm / second, a scanning frequency of once, and a polarization of circularly polarized light. As a result, a culture substrate 1 having nano-periodic protrusions 2 having a diameter of 0.5 μm, a height of 0.2 μm, and a pitch of 0.7 μm formed on the surface was produced ((4)). FIG. 1 shows a scanning electron microscope (SEM) image of the processed surface of the culture substrate 1 in which the produced nanoperiodic protrusions 2 are formed on the substrate surface.

[Example 2] Analysis of differentiation induction of MSC on culture substrate 1 (confirmation with differentiation marker)
1. Outline In this example, differentiation induction of MSC on the culture substrate 1 on which the nanoperiodic protrusions 2 produced in Example 1 were formed was examined. In this example, differentiation induction of MSC into bone cells, chondrocytes, nerve cells, and adipocytes was examined, and the differentiation induction state was confirmed using a differentiation marker.

2. 2. Material and test method 2-1. Culture substrate 2-2-1 of Example 1 above. 2-2-4. The culture substrates of Comparative Examples and Examples prepared in the above were used. A mirror-polished medical titanium plate that was used as it was without laser processing was used as a control ((5)).

2-2. Cells Human MSCs (hMSC mesenchymal stem cells, Lonza, catalog number PT-2501) were used.

2-3. Differentiation induction Each of the above culture substrates was immersed in 70% ethanol for 20 minutes for sterilization, and then washed three times with distilled water. After washing, the culture substrate was allowed to stand on the bottom of the well of a 12-well cell culture plate, a medium was added, and the culture substrate was immersed in the medium. Each culture substrate immersed in the medium was seeded with MSC and cultured for 6 hours.

At this time, the initial seed density of the cells was 5000 / cm ² . As the culture medium, MSCGM ^™ BulletKit ^™ (Lonza, catalog number PT-3001) that had been prepared for use was used.

The MSCs cultured on each of the above-mentioned culture substrates were treated for 72 hours (the desired confluence described above (100% confluent in the case of differentiation into bone cells and chondrocytes, 80 in the case of differentiation into nerve cells and adipocytes). Cultivated in growth medium until ˜90% confluent). Thereafter, differentiation was induced by culturing in a differentiation induction medium for 72 hours. Here, the culture medium of Example 3 was used as the growth medium, and MSC at the 4th passage was induced to differentiate. The details of the differentiation induction method are as follows.

2-1-1. Induction of differentiation into bone cells When differentiation into bone cells is performed, differentiation induction is started when MSC is preferably 100% confluent. As an osteoblast differentiation medium, hMSC-BulletKit ^™ -for osteoblast differentiation (Lonza, catalog number PT-3002) can be used to induce differentiation into bone cells according to the manufacturer's instructions. This kit includes basal medium, L-glutamine, dexamethasone, ascorbic acid, ITS + supplement (included in hMSC-BulletKit ^TM -cartilage differentiation (Lonza, Cat. No. PT-3003), sodium pyruvate, proline, mesenchyme Lineage cell growth supplement (MCGS), β-glycerophosphate, penicillin / streptomycin. The initial cell seed density is preferably 3.1 × 10 ⁵ cells / cm ² .

2-1-2. Induction of differentiation into chondrocytes When differentiation into chondrocytes is performed, differentiation induction is started when MSC is preferably 100% confluent. As the chondrocyte differentiation induction medium, hMSC-BulletKit ^™ -for cartilage differentiation (Lonza, catalog number PT-3003) can be used to induce differentiation into chondrocytes according to the manufacturer's instructions. This kit comprises basal medium, L-glutamine, dexamethasone, ascorbic acid, ITS + supplement, sodium pyruvate, proline, GA-1000 (gentamicin, amphotericin B). The initial cell seed density is preferably 5 × 10 ⁵ cells / cm ² .

2-1-3. Differentiation into nerve cells When differentiation into nerve cells is performed, differentiation induction can be started when MSCs are preferably 80-90% confluent. The neural cell differentiation induction medium can be induced to differentiate into neural cells using Mesenchymal Stem Cell Neurogenic Differentiation Medium (PromoCell, Catalog No. C-28015) according to the manufacturer's instructions. it can. This kit comprises a basic medium, Supplement Mix (PromoCell, catalog number C-39815). The initial cell seed density is preferably 5000 cells / cm ² .

2-1-4. Differentiation into adipocytes When differentiation into adipocytes is induced, differentiation induction can be started when MSC is preferably 80-90% confluent. As the adipocyte differentiation induction medium, hMSC-BulletKit ^™ -for adipocyte differentiation (Lonza, catalog number PT-3004) can be used to induce differentiation into adipocytes according to the manufacturer's instructions. This kit consists of basal medium, L-glutamine, Mesenchymal cell growth supplement (MCGS), dexamethasone, indomethacin, 3-isobutyl-1-methyl-xanthine (IBMX), GA- Containing 1000 (gentamicin, amphotericin B). The initial cell seed density is preferably 2.1 × 10 ⁴ cells / cm ² .

2-2. Confirmation of differentiation induction After culture, differentiation induction was confirmed by analyzing differentiation markers that are not expressed in MSC but expressed specifically in bone cells, chondrocytes, nerve cells, and adipocytes. As differentiation markers, SPP1 was used to confirm differentiation into bone cells, SOX9 was used to confirm differentiation into chondrocytes, MAP2 was used to confirm differentiation into nerve cells, and PPARG was used to confirm differentiation into adipocytes. . The experiment was performed at N = 3.

The expression analysis of the cell differentiation marker was performed by Real-time RT-PCR gene analysis. Specifically, total RNA was extracted from the cells after differentiation induction, and cDNA was synthesized from the RNA by reverse transcription reaction. Subsequently, PCR was performed using the synthesized cDNA as a template. Similarly, the expression level of GAPDH was measured, and the relative expression level with respect to GAPDH was calculated. In addition, for tissue culture polystyrene (TCPS), differentiation induction into each cell was confirmed in the same manner.

Here, gene-specific primers were designed using the Primer Bank (http://pga.mgh.harvard.edu/primerbank/) of Harvard Medical School, USA. The designed primer was commissioned to Greiner bio-one. Table 2 summarizes the sequence information of the gene specific primers used in this example. As a control, the expression level of GAPDH was measured.

3. Results The results are shown in the graph of FIG. In the figure, (1) is a culture substrate in which the micro periodic grooves of the comparative example are formed on the substrate surface, (2) is a culture substrate in which the nano periodic grooves of the comparative example are formed on the substrate surface, and (3) is a hybrid of the comparative example. The culture base material in which the periodic grooves are formed on the substrate surface, (4) is the culture base material 1 in which the nano-periodic protrusions 2 of the example are formed on the substrate surface, and (5) is a control.

The culture substrate 1 in which the nano-periodic protrusions 2 are formed on the substrate surface shows a high acceleration effect for induction of differentiation into any of the examined bone cells, chondrocytes, nerve cells, and adipocytes. ((4)). This effect was particularly remarkable in bone cells. On the other hand, when the hybrid periodic grooves constructed previously by the present inventors were formed on the substrate surface, a significant acceleration effect was observed for induction of differentiation into bone and chondrocytes ((3)). When micro periodic grooves and nano periodic grooves are independently formed on the substrate surface, and in the control, the degree of differentiation is not significantly different from that in the case of culturing in an environment without a substrate, and it can be said that there is an effect of accelerating differentiation induction. ((1), (2), (5)).

From this result, the previously constructed hybrid periodic groove can exert a differentiation induction accelerating effect on differentiation in a specific direction of differentiation induction into bone and chondrocytes. In contrast, the culture substrate 1 on which the nanoperiodic protrusions 2 of the present invention are formed has a high differentiation induction acceleration effect in any direction of the differentiation induction direction in which the stem cells are destined. It can be understood that it is possible.

The present invention relates to a culture substrate and can be used particularly in all fields where stem cell culture is required, in particular, in industrial fields such as drug discovery, life science, and medicine. For example, it can be suitably used for elucidation of development, differentiation and disease mechanism, and can contribute to the establishment of basic technology for practical application including clinical application of stem cell utilization technology. In addition, since the culture substrate of the present invention has a remarkable acceleration effect on the induction of differentiation into bone cells, a specific application is a future generation implant having a bone forming function on the surface. It is done.

1 culture substrate 2 nanometer order periodic protrusion structure (nanoperiodic protrusion)

Claims (4)

1. A culture substrate having a periodic projection structure on the order of nanometers on which the stem cells are cultured on the surface.
2. The culture substrate according to claim 1, wherein the nanometer-order periodic protrusion structure has a diameter of 0.1 to 1 µm, a height of 0.01 to 0.5 µm, and a pitch of 0.1 to 1 µm.
3. The culture substrate according to claim 1 or 2, wherein the material is titanium.
4. The method for producing a culture substrate according to any one of claims 1 to 3, further comprising a step of forming the nanometer-order periodic protrusion structure on the substrate surface by forming a nano-periodic structure by circular polarization of an ultrashort pulse laser. .
   
   
   

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