Cell stimulation methods

Abstract

The objective is to provide a novel method for improving the efficiency of osteogenic differentiation induction based on immobilized BMP-2. [Solution] A method for stimulating cells with ligand molecules, comprising a stimulation step of applying physical stimulation to the cells and / or ligand molecules, wherein the ligand molecule is bound to or interacts with a receptor protein expressed on the cells and is immobilized on a culture substrate and / or matrix molecule used for culturing the cells, the receptor protein mediates the transmission of extracellular signals to the cells based on its binding to the ligand molecule, and the extracellular signals are enhanced by the physical stimulation.

Description

[Technical Field]

[0001] This invention relates to a method for stimulating cells with ligand molecules, and a method for producing cells, etc. [Background technology]

[0002] In living tissues, cell survival, proliferation, migration, and differentiation are controlled by biochemical and biophysical signals.

[0003] Among these, biosignaling molecules have a particularly significant effect, and various biomacromolecules have been discovered to date. Furthermore, it has become clear that their effects can be enhanced by immobilizing them on materials to increase their local concentration or by suppressing their intracellular integration (Non-Patent Literature 1).

[0004] For example, bone morphogenetic protein 2 (BMP-2), which belongs to the TGF-β superfamily, is known to regulate cell migration and osteogenic differentiation involved in bone formation, playing an essential role in bone formation such as skeletal formation and fracture healing. Based on its bone-inducing activity, BMP-2 is being applied to bone regeneration therapy and is widely used in bone reconstruction, treatment of jaw deformities, and other applications. In the United States, BMP-2 is being used clinically in the field of orthopedics.

[0005] As clinical applications of BMP-2 progress, side effects associated with high-dose administration of soluble BMP-2 are becoming apparent. Specifically, while high concentrations of BMP are necessary to induce bone regeneration, side effects such as inflammatory reactions at the administration site and heterotopic ossification (bone formation in unintended areas) have been reported. Such side effects are a major obstacle to the clinical application of BMP-2.

[0006] To avoid the side effects of soluble BMP-2, methods have been developed to immobilize BMP-2 on the surface of biomaterials. For example, methods have been developed in the past to immobilize BMP-2 on biomaterial carriers such as metals, ceramics, and polymers, or to compound it with hydrogels or nanoparticles, and these are used in orthopedic surgeries such as spinal fusion.

[0007] The technology for immobilizing BMP-2 has been shown to be useful in in vitro cell differentiation into bone. Patent Document 1 discloses a method for linking growth factors such as BMP-2 and VEGF to a polypeptide containing 3,4-dihydroxyphenylalanine (DOPA), a type of tyrosine derivative, in order to bind them to the surface of a metal material such as titanium. For example, a fusion protein obtained by fusing DOPA with BMP-2 (DOPA-BMP-2 fusion protein) can be immobilized on the surface of a titanium plate. Furthermore, it has been disclosed that culturing human mesenchymal stem cells (hMSCs) in the presence of a titanium plate coated with DOPA-BMP-2 fusion protein induces differentiation of hMSCs and yields cells that have undergone a morphological change to a spherical shape.

[0008] There is a need for new methods to enhance signaling based on immobilized growth factor proteins, such as methods for more efficiently inducing osteogenic differentiation using immobilized BMP-2. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2017-43547 [Non-patent literature]

[0010] [Non-Patent Document 1] Takashi Kitajima, Seiichi Tada, Yoshihiro Ito, Polymer Papers, 69, 1-10 (2012) [Non-Patent Document 2] Heckman, JD et al., J Bone Joint Surg Am, 1994, 76(1), 26-34. [Non-Patent Document 3] Gebauer, D., et al., Ultrasound Med Biol, 2005, 31(10), 1391-1402. [Overview of the project]

Problems to be Solved by the Invention

[0011] An object of the present invention is to provide a new method for enhancing signals such as activation signals based on immobilized growth factor proteins.

Means for Solving the Problems

[0012] So far, it has been found that various ligand molecules can exhibit high activity when immobilized on a culture substrate. Specifically, in addition to the above-mentioned BMP-2, ligand molecules such as the TGF-β superfamily, epidermal growth factor (EGF) family, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) family, tumor necrosis factor (TNF), interleukin (IL), interferon (IFN), and colony-stimulating factor (CSF) can exhibit high activity when immobilized on a culture substrate.

[0013] It is known that various biological phenomena such as fractures and non-adhesive regeneration in vivo are promoted by ultrasonic irradiation such as low-intensity pulsed ultrasound (LIPUS) and mechanical stimulation (Non-Patent Documents 2 and 3). Based on these findings, the inventors of the present invention conceived that when mechanical stimulation such as ultrasonic irradiation is applied to stimulate cells using ligand molecules immobilized on a culture substrate, extracellular signals based on the ligand molecules can be enhanced.

[0014] To verify this hypothesis, the inventors of the present invention selected BMP-2 as an example of various ligand molecules that can exhibit activity when immobilized on a culture substrate, and verified whether the bone differentiation-inducing signal based on BMP-2 can be enhanced by ultrasonic irradiation. Specifically, ultrasonic waves were irradiated when inducing osteoblast differentiation using BMP-2 immobilized on the surface of the culture substrate. As a result, it was found that the induction effect of osteoblast differentiation was significantly enhanced under the condition of ultrasonic irradiation, and a synergistic effect was obtained between the ligand stimulation based on BMP-2 and ultrasonic irradiation.

[0015] The present invention is based on the above findings and provides the following: (1) A method of stimulating cells with ligand molecules, A stimulation step in which physical stimuli are applied to cells and / or ligand molecules. Includes, The ligand molecule binds to or interacts with the receptor protein expressed on the cell, and is immobilized on the culture substrate and / or matrix molecule used for culturing the cell. The method wherein the receptor protein mediates the transmission of an extracellular signal to the cell based on its binding to the ligand molecule, and the extracellular signal is enhanced by the physical stimulus. (2) The method according to (1), wherein the cells are arranged on the culture substrate or on a cell culture substrate coated with the matrix molecule. (3) The method according to (1), wherein the cells are contained in a three-dimensional matrix containing the matrix molecules. (4) The method according to any one of (1) to (3), wherein the receptor protein is an enzyme-coupled receptor and / or a G protein-coupled receptor. (5) The method according to any one of (1) to (4), wherein the ligand molecule is a secretory factor, an extracellular domain of a membrane protein, an artificial peptide, and / or a small molecule compound. (6) The method according to (5), wherein the secreted factor is selected from the group consisting of the TGF-β superfamily, epidermal growth factor (EGF) family, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) family, tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Wnt protein, SHH protein, interleukin (IL), interferon (IFN), and colony-stimulating factor (CSF). (7) The method according to (6), wherein the TGF-β superfamily is bone morphogenetic protein 2 (BMP-2). (8) The method according to any one of (1) to (7), wherein the physical stimulus is a mechanical stimulus and / or an electrical stimulus. (9) The method according to (8), wherein the mechanical stimulus is ultrasound, compression, extension, pressurization, depressurization, vibration, or change in gravity. (10) The intensity of the ultrasound is 1 mW / cm 2 ~500 mW / cm 2 The method according to (9), wherein the frequency of the ultrasound is 1 MHz to 3 MHz. (11) The method according to (10), wherein the ultrasound is applied in pulses. (12) The method according to any one of (9) to (11), wherein the ultrasound is applied for 10 seconds to 60 minutes per day. (13) The method according to any one of (1) to (12), further comprising a culture step of culturing the cells after the stimulation step. (14) A method for producing differentiated cells from undifferentiated cells, A stimulation step, which involves applying physical stimuli to undifferentiated cells and / or ligand molecules, A culture step in which the undifferentiated cells after the stimulation step are cultured. Includes, The ligand molecule binds to or interacts with a receptor protein expressed on the undifferentiated cells and is immobilized on a culture substrate and / or matrix molecule used for culturing the undifferentiated cells. The method wherein the receptor protein mediates the transmission of an extracellular signal that induces the differentiation of the undifferentiated cells into differentiated cells based on its binding to the ligand molecule, and the extracellular signal is enhanced by the physical stimulus. (15) The method according to (14), wherein the undifferentiated cell is an osteoblast and the ligand molecule is bone morphogenetic protein 2 (BMP-2). Apparatus for use in the method described in (16)(2) or (3), A mounting section for placing the culture substrate, the cell culture substrate, or the culture vessel containing the three-dimensional matrix, and A physical stimulus delivery unit that can deliver the aforementioned physical stimulus to the ligand molecules and / or cells on the culture substrate, the cell culture substrate, or the culture vessel, which is placed on the aforementioned mounting unit. The apparatus, including the above. (17) The apparatus according to (16), wherein the preceding section includes a temperature control means capable of controlling the temperature of the culture substrate, the cell culture substrate, or the culture vessel. (18) The apparatus according to (16) or (17), wherein the physical stimulation unit includes an ultrasonic irradiation means capable of providing ultrasonic-based mechanical stimulation to the ligand molecules and / or the cells. (19) A kit for stimulating cells with ligand molecules, Ligand molecules for immobilization on culture substrates and / or matrix molecules, or Culture substrates and / or matrix molecules immobilized with ligand molecules Includes, The ligand molecule is immobilized on the culture substrate and / or matrix molecule used for culturing the cells, and is intended to bind to or interact with the receptor protein expressed on the cells. A kit for enhancing extracellular signals transmitted to a cell based on the binding of a receptor protein to a ligand molecule, by applying a physical stimulus to the cell bound to the ligand molecule and / or to the ligand molecule bound to the receptor protein. [Effects of the Invention]

[0016] The present invention provides a novel method for improving the efficiency of osteogenic differentiation induction based on immobilized BMP-2. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the immobilization of BMP-2 on the substrate surface. Figure 1A schematically shows how DOPA-BMP-2 is immobilized on the substrate surface by applying a DOPA-BMP-2 solution to the TCPS substrate surface. Figure 1B shows the relationship between the amount of DOPA-BMP-2 applied (horizontal axis) and the amount of DOPA-BMP-2 bound to the substrate surface (vertical axis). [Figure 2]Figure 2 shows osteogenic differentiation based on soluble BMP-2 and immobilized BMP-2. Figure 2A schematically shows how cells are stimulated with soluble BMP-2 and immobilized BMP-2. Figure 2B shows the ALP activity of cells stimulated with soluble BMP-2 and immobilized BMP-2. [Figure 3] Figure 3 shows the ossification of cells in the control group (no BMP-2), the group treated with soluble BMP-2, and the group presented with immobilized BMP-2. Figure 3A shows the results of evaluating calcification (calcium deposition) of differentiated cells using Alizarin Red S staining. Figure 3B shows the quantitative results of Alizarin Red S staining. [Figure 4] Figure 4 schematically illustrates how irradiating osteoblasts with ultrasound while culturing them on a culture substrate immobilized with BMP-2 synergistically promotes osteogenic differentiation. [Figure 5] Figure 5 shows the results of evaluating osteogenic differentiation in the control group (no BMP-2), the group with soluble BMP-2 added, and the group presented with immobilized BMP-2. Figure 5A shows the results of evaluating ALP activity. Figure 5B shows the results of evaluating calcification (calcium deposition) of differentiated cells using alizarin red S staining. In the figures, * indicates p<0.05 and **** indicates p<0.0001. [Figure 6] Figure 6 shows the quantitative results of the number of focal adhesions in the following conditions: no BMP-2 (Control group), a group with soluble BMP-2 added, and a group with immobilized BMP-2 presented. In the figure, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. [Modes for carrying out the invention]

[0018] 1. Cell stimulation method 1-1. Overview A first aspect of the present invention is a method for stimulating cells with ligand molecules (hereinafter sometimes abbreviated as "cell stimulation method"). The cell stimulation method of the present invention can enhance extracellular signals transmitted to cells based on the application of physical stimuli to cells and / or ligand molecules. In the cell stimulation method of the present invention, enhanced extracellular signals can be transmitted to cells based on the synergistic effect between the binding of ligand molecules such as BMP-2 to receptor proteins and physical stimuli such as ultrasound, making it possible to efficiently induce cell differentiation, for example.

[0019] 1-2. Definitions of Terms The terms frequently used in this specification are defined below.

[0020] In this specification, "ligand molecule" refers to any molecule that binds to a receptor on the cell surface and thereby transmits a signal into the cell via the receptor. Ligand molecules may be either biomolecules or artificial substances. The organism from which biomolecules originate is not limited and may be, for example, a mammal such as a human, a vertebrate, or an invertebrate. Examples of ligand molecules include secretory protein factors that function as endocrines or paracrines, binding proteins that function as juxtacrines or matricrines, artificial peptides, and small molecule compounds.

[0021] In this specification, "secretory factor" refers to any factor secreted from inside a cell to outside the cell. Examples of secretory factors include secretory proteins and proteinaceous or non-proteinaceous hormones.

[0022] In this specification, "secreted protein" refers to a protein produced within a cell and secreted outside the cell. While not limited to these, examples include growth factors and cytokines. Examples of growth factors include the TGF-β superfamily (e.g., TGF-β, BMP, GDF, activin), the epidermal growth factor (EGF) family (e.g., EGF), platelet-derived growth factor (PDGF) (e.g., PDFFA, PDFBB, PDGFC, PDFD), the fibroblast growth factor (FGF) family (e.g., FGF1, FGF2), vascular endothelial growth factor (VEGF) (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF-1, PlGF-2), and nerve growth factor (NGF). Examples of cytokines include tumor necrosis factor (TNF) (e.g., TNFα, TNFβ, TNFγ, CD252, CD154, CD178, CD70, CD153, 4-1 BBL), interleukins (IL) (e.g., IL-1 to IL-18), interferons (IFN) (e.g., IFN-α, IFN-β, IFN-γ, IFN-ε, IFN-κ, IFN-λ), and colony-stimulating factors (CSF) (e.g., G-CSF).

[0023] In this specification, "bone morphogenetic proteins (BMPs)" refers to a group of proteins identified as molecules that induce and promote the differentiation of bone tissue and cartilage. BMPs belonging to the TGF-β superfamily are classified into the BMP2 / 4 group (BMP2, BMP4), the OP-1 group (BMP5, BMP6, BMP7, BMP8a, BMP8b), the BMP9 group (BMP9, BMP10), and the GDF5 group (GDF5, GDF6, GDF7). These BMPs are known to bind to dimers composed of type I / II receptors, and to transmit signals into the nucleus via phosphorylation of the transcription factor SMAD. For example, BMP-2 is known to bind to the BMPR1A receptor, inducing the differentiation of osteoblasts and stimulating bone formation.

[0024] In this specification, "extracellular domain of a membrane protein" refers to the extracellular domain of a membrane protein capable of binding to a receptor on the cell surface, including the binding domain responsible for binding to said receptor. Examples of such membrane protein-receptor combinations include those that bind between different cells to transmit intercellular signals.

[0025] In this specification, "artificial peptide" refers to any peptide other than naturally derived peptides and polypeptides. Examples include growth factor substitute peptides and functional peptides obtained by screening peptide libraries using receptor binding ability as an indicator.

[0026] In this specification, "receptor protein" refers to a protein that functions as a receptor for a ligand molecule. Receptor proteins are mainly classified into enzyme-coupled receptors, G protein-coupled receptors, ion channel receptors, and intracellular receptors. In this specification, enzyme-coupled receptors, G protein-coupled receptors (GPCRs), and ion channel receptors are preferred, and enzyme-coupled receptors and G protein-coupled receptors are more preferred. Enzyme-coupled receptors include receptor tyrosine kinases and serine / threonine kinase receptors, but in this specification, any of these may be referred to as enzyme-coupled receptors. Specific examples of receptors include growth factor receptors (e.g., TGF-β receptor, EGF receptor, PDGF receptor, FGF receptor) and cytokine receptors (e.g., TNF receptor, IL receptor, IFN receptor, CSF receptor), most of which are classified as enzyme-coupled receptors. For example, bone morphogenetic protein receptor 1A (BMPR1A), the receptor for BMP-2, belongs to the transmembrane serine / threonine kinase family among enzyme-coupled receptors. On the other hand, examples of G protein-coupled receptors include the Fizzled receptor, which functions as a receptor for the Wnt protein, and the Patched receptor, which functions as a receptor for the SHH protein.

[0027] In this specification, "extracellular signal" refers to any signal transmitted from outside the cell to inside the cell. In this specification, extracellular signals also include intercellular signals and autocrine signals. Within cells where extracellular signals are transmitted, proliferation, differentiation, survival, adhesion, and movement are controlled by these extracellular signals.

[0028] In this specification, the type of "cell" is not limited. Cells may be either prokaryotic or eukaryotic. Examples of prokaryotic cells include bacterial cells such as E. coli cells. Examples of eukaryotic cells include fungal cells (e.g., yeast cells), algal cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). Furthermore, cells may be either differentiated or undifferentiated. Undifferentiated cells may be either stem cells or progenitor cells, as described later. In addition, cells may be either adherent cells or suspension cells, but adherent cells are preferred.

[0029] In this specification, "osteoblast" refers to cells that exist on the bone matrix in vivo and are responsible for the production and calcification of the bone matrix. The BMP signaling pathway, mediated by the binding of bone morphogenetic protein 2 (BMP-2) to bone morphogenetic protein receptor 1A (BMPR1A), is known to differentiate mesenchymal stem cells into stromal cells and stromal cells into osteoblasts. In this specification, the differentiation into stromal cells and osteoblasts is referred to as "osteogenic differentiation."

[0030] In this specification, "culture substrate" or "cell culture substrate" means any substrate on which cells can survive or grow. The culture substrate preferably has flat areas to which cells can adhere. Specific examples of culture substrates include culture vessels such as multi-well plates, dishes, flasks, and petri dishes, as well as tubes, trays, and roller bottles. The material of the culture substrate is not particularly limited, but a material to which cells can adhere is preferred, such as plastics like polyethylene, polystyrene, polypropylene, and polyethylene terephthalate, glass, and resins like silicone resin and acrylic resin.

[0031] In this specification, "matrix molecule" refers to a molecule that constitutes a scaffold material for cell culture or tissue regeneration, and is a molecule that constitutes a material that enables the control of cell adhesion, proliferation, differentiation, etc., and the culture, formation, and regeneration of transplanted cells, transplanted tissues, or transplanted organs. Examples include biomolecules that constitute the extracellular matrix, non-natural artificial synthetic matrix molecules, and animal-free matrix molecules. Specific examples include collagen, fibronectin, laminin, heparan sulfate proteoglycan, cadherin, gelatin, fibrinogen, fibrin, poly-L-lysine, hyaluronic acid, and polyvinyl alcohol. Matrix molecules that constitute PEG-based hydrogels and cellulose nanofibers are also examples.

[0032] 1-3. Method The cell stimulation method of the present invention includes a stimulation step as an essential step and a pre-culture step, a culture step, and / or a harvesting step as optional steps. Each step will be described below.

[0033] (stimulation process) In the cell stimulation method of the present invention, the "stimulation step" is a step of applying a physical stimulus to cells and / or ligand molecules. In this step, the physical stimulus may be applied to cells only, to ligand molecules only, or to both cells and ligand molecules.

[0034] In this specification, "physical stimulus" refers to any stimulus that produces mechanical or electrical stimuli, excluding chemical stimuli such as those caused by chemical substances. Examples of mechanical stimuli include ultrasound, compression, stretching, pressurization, depressurization, vibration, and changes in gravity (e.g., weightlessness). Electrical stimulation refers to stimulation applied electrically using electric current, but it is preferable to use currents and voltages of an intensity that do not kill cells. These physical stimuli are used in physiotherapy and are well known to have effects such as pain relief, improved tissue extensibility, muscle strengthening, and wound healing. Therefore, they can exhibit effects similar to those of ultrasound stimulation, whose effects were demonstrated in the examples described later.

[0035] In this process, the ligand molecule binds to or interacts with a receptor protein expressed on the cell. This receptor protein can mediate the transmission of extracellular signals to the cell based on its binding to or interaction with the ligand molecule. In this process, the extracellular signal is enhanced based on the application of physical stimuli to the cell and / or the ligand molecule.

[0036] In this process, the ligand molecule is immobilized on the culture substrate and / or matrix molecule used for cell culture.

[0037] In this specification, "immobilization" means that the ligand molecule does not substantially separate from the culture substrate or matrix molecule while physical stimulation is applied in this process, and the binding state is maintained. Immobilization of the ligand molecule to the culture substrate or matrix molecule may be by covalent bonding or affinity interaction. Covalent bonding is not limited to and includes, for example, peptide bonds, disulfide bonds, etc. In this specification, "affinity interaction" means non-covalent interaction between molecules. Examples of affinity interactions include hydrogen bonding, hydrophobic interaction or hydrophobic bonding, electrostatic interaction or ionic bonding, salt bridges, coordination bonds, etc. Furthermore, as biomimetic immobilization, the ligand molecule may be immobilized on the culture substrate based on the high binding affinity between the catechol group and the substrate surface by introducing a catechol group such as DOPA into the ligand molecule. In addition, if immobilization is to matrix molecules, the ligand molecule and the matrix molecule may be immobilized by peptide bonding.

[0038] In this specification, "a ligand molecule is bound to or interacts with a receptor protein expressed on a cell" specifically means that a cell expressing a receptor protein on its cell membrane is in contact with a culture substrate and / or matrix molecule on which the ligand molecule is immobilized, and that the ligand molecule and / or matrix molecule are arranged in such a way that a signal mediated by the ligand molecule and / or receptor protein can be transmitted. In one embodiment, in this step, the cell is placed on a culture substrate on which the ligand molecule is immobilized, or on a cell culture substrate coated with a matrix molecule on which the ligand molecule is immobilized.

[0039] In one embodiment, the cells in this process are contained within a three-dimensional matrix containing matrix molecules. In this specification, "three-dimensional matrix" refers to a structure composed of a gel, polymer, or the like in which cells or tissues can survive or proliferate. The three-dimensional matrix may be biodegradable or non-biodegradable. Examples of three-dimensional matrices include those composed of proteinaceous collagen, fibrin, gelatin, Matrigel, polysaccharides (e.g., chitosan), alginic acid, hyaluronic acid, cellulose, synthetic polymers such as polyethylene glycol derivatives and polypeptide derivatives. PEG-based hydrogels and cellulose nanofibers are also examples.

[0040] In this specification, "enhanced extracellular signaling" means that a stronger extracellular signal is input to the cell compared to a state without physical stimulation. The enhancement of extracellular signaling and the degree of enhancement can be determined by detecting changes such as phosphorylation and other modifications or gene expression that occur downstream of the extracellular signaling, or by detecting changes in proliferation, differentiation, survival, adhesion, and motility controlled by the extracellular signaling. The degree of enhanced extracellular signaling may be 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 2.0 times or more, 3.0 times or more, 4 times or more, 5 times or more, or 6 times or more compared to a state without physical stimulation, based on the changes described above (e.g., proliferation rate, number of differentiated cells).

[0041] In this process, the physical stimuli applied to cells and / or ligand molecules are of any type and method, as long as they can enhance extracellular signals. For example, mechanical stimuli can include ultrasound, compression, stretching, pressurization, depressurization, vibration, gravity changes, and combinations thereof. There are no particular restrictions on the conditions such as stimulation time when using each stimulus, and the conditions can be the same as those for ultrasound irradiation described below.

[0042] When ultrasound is used as the mechanical stimulus in this process, the ultrasound intensity is 1 mW / cm². 2 ~500 mW / cm 2 5 mW / cm² 2~400 mW / cm 2 、10 mW / cm 2 ~300 mW / cm 2 、20 mW / cm 2 ~200 mW / cm 2 、30 mW / cm 2 ~100 mW / cm 2 、40 mW / cm 2 ~80 mW / cm 2 、 or 50 mW / cm 2 ~60 mW / cm 2 may also be used. In addition, the frequency of the ultrasonic wave may be 1 MHz to 3 MHz or 1.5 MHz to 2.5 MHz. The irradiation time of the ultrasonic wave can be 10 seconds to 60 minutes, 1 minute to 30 minutes, 2 minutes to 20 minutes, 3 minutes to 15 minutes, or 5 minutes to 10 minutes per day. Note that the type and intensity of the physical stimulus are preferably such that they do not impair the survival and proliferation of cells.

[0043] When using compression, stretching, pressurization, or decompression as mechanical stimuli, these mechanical stimuli can be applied to the three-dimensional matrix containing cells to efficiently stimulate the cells. The pressure conditions for compression, stretching, pressurization, or decompression are not limited and may be, for example, 1 Pa to 1000 kPa, 10 Pa to 700 kPa, 100 Pa to 100 kPa, or 1 kPa to 10 kPa. The application time of compression, stretching, pressurization, or decompression may be 1 minute or more, 5 minutes or more, 10 minutes or more, 1 hour or more, 2 hours or more, or 3 hours or more, and / or 4 weeks or less, 3 weeks or less, 2 weeks or less, 1 week or less, 2 days or less, 1 day or less, or 12 hours or less.

[0044] When using gravity changes as mechanical stimuli, the gravity changes can be zero gravity, low gravity (microgravity), or hypergravity. Examples of zero gravity include the use of outer space, and an example of low gravity is the use of the lunar surface. Centrifugal force can also be used as hypergravity. Examples of specific gravity conditions include 0 g to 300 g, 10 g to 150 g, or 50 g to 100 g. The application time of gravity changes can range from 1 second to 100 days, 10 seconds to 50 days, 20 seconds to 30 days, 30 seconds to 10 days, 1 minute to 1 day, or 1 hour to 12 hours for low gravity or zero gravity (less than 1 g), and from 1 minute to 100 days, 30 minutes to 50 days, 1 hour to 30 days, or 12 hours to 1 week for hypergravity (e.g., 2 g to 150 g). It is preferable that the application time of gravity changes becomes shorter as the gravity increases.

[0045] When electrical stimulation is used as physical stimulation in this process, it is sufficient to use currents and voltages of an intensity that do not kill cells. For example, the electrical stimulation current may be 0.1 μA to 1 A, 1 μA to 100 mA, 10 μA to 10 mA, or 100 μA to 1 mA. The electrical stimulation voltage may be 1 mV / cm to 20 V / cm, 10 mV / cm to 10 V / cm, 20 mV / cm to 6.6 V / cm, or 100 mV / cm to 1 V / cm. The electrical stimulation frequency may be, for example, 1 Hz to 100 kHz, 10 Hz to 10 kHz, or 100 Hz to 1 kHz. The application time of electrical stimulation may be 1 minute or more, 5 minutes or more, 10 minutes or more, 1 hour or more, 2 hours or more, or 3 hours or more, and / or 4 weeks or less, 3 weeks or less, 2 weeks or less, 1 week or less, 2 days or less, or 12 hours or less.

[0046] Other conditions such as the temperature used when applying physical stimuli to the cells in this process, and the culture medium used, can be the same as those described in the culture process later, so a detailed explanation is omitted here.

[0047] In one embodiment, the ligand molecule in this step is a growth factor or a cytokine. The growth factor is preferably selected from the group consisting of the TGF-β superfamily, epidermal growth factor (EGF) family, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) family, vascular endothelial growth factor (VEGF), and nerve growth factor (NGF). The cytokine is preferably selected from the group consisting of tumor necrosis factor (TNF), interleukin (IL), interferon (IFN), and colony-stimulating factor (CSF). In this embodiment, the receptor protein in this step is an enzyme-coupled receptor or a G protein-coupled receptor, preferably an enzyme-coupled receptor. Enzyme-coupled receptors include receptor tyrosine kinases or serine / threonine kinase receptors. The specific receptor protein should be one that binds to the above-mentioned ligand molecule and transmits extracellular signals. When the ligand molecule is a growth factor, the receptor protein is a growth factor receptor, and may be a TGF-β receptor, EGF receptor, PDGF receptor, FGF receptor, VEGR receptor, or NGF receptor, etc. When the ligand molecule is a cytokine, the receptor protein is a cytokine receptor, and may be a TNF receptor, IL receptor, IFN receptor, or CSF receptor.

[0048] In further embodiments, the ligand molecule and receptor protein in this process are, respectively, the following combinations: (a) Bone morphogenetic proteins (BMPs) and bone morphogenetic protein receptors (BMPRs), (b) epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR), (c) Platelet-derived growth factor (PDGF) and platelet-derived growth factor receptor (PDGFR), (d) Fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR), (e) Tumor necrosis factor (TNF) and tumor necrosis factor receptor (TNFR), (f) Interleukins (ILs) and interleukin receptors (ILRs), (g) Interferon (IFN) and interferon receptor (IFNR), (h) Colony-stimulating factor (CSF) and colony-stimulating factor receptor (CSFR), (i) Wnt protein and Fizzled receptor, (j) SHH protein and patched receptor, (k) Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR), or (l) Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) That is the case.

[0049] In (a) above, BMP may be bone morphogenetic protein 2 (BMP-2), and BMPR may be bone morphogenetic protein receptor 1A (BMPR1A). In this case, the cells to which physical stimulation is applied in this process may be mesenchymal stem cells, stromal cells, or osteoblasts. When extracellular signals mediated by BMP-2 and BMPR1A (BMP signals) are input to these cells, mesenchymal stem cells may differentiate into stromal cells, and stromal cells may differentiate into osteoblasts. When physical stimuli such as ultrasonic stimulation or vibration are applied to these cells in this process, BMP signals may be enhanced, and the differentiation of mesenchymal stem cells into stromal cells and the differentiation of stromal cells into osteoblasts may be promoted.

[0050] In (b) above, EGF may be epidermal growth factor and EGFR may be the epidermal growth factor receptor. In this case, the cells to which physical stimulation is applied in this process may be various EGFR-containing cells such as fibroblasts. When an extracellular signal mediated by epidermal growth factor and the epidermal growth factor receptor (EGF signal) is input to these cells, the growth of EGFR-containing cells can be promoted. When physical stimuli such as ultrasonic stimulation or vibration are applied to these cells in this process, the EGF signal is enhanced, and the growth of EGFR-containing cells can be promoted.

[0051] In (c) above, PDGF may be platelet-derived growth factor, and PDGFR may be platelet-derived growth factor receptor. In this case, the cells to which physical stimulation is applied in this process may be PDGFR-containing mesenchymal cells (fibroblasts, smooth muscle cells, glial cells, etc.). Upon binding with a ligand, the tyrosine residue of PDGFR undergoes autophosphorylation, becoming a binding site for signal transduction molecules having an SH2 domain (PLC-γ, Grb2, PI3K, etc.) and transmitting signals downstream. When physical stimuli such as ultrasonic stimulation or vibration are applied to these cells in this process, the PDGF signal is enhanced, and neurons and glial cells, which express PDGF and its receptor, can be promoted to differentiate and proliferate.

[0052] In (d) above, FGF may be fibroblast growth factor, and FGFR may be fibroblast growth factor receptor. In this case, the cells to which physical stimulation is applied in this process include a wide range of cells and tissues, and they play an important role in the processes of proliferation and differentiation. When an extracellular signal (FGF signal) is input to these cells, it stimulates the proliferation, migration, and differentiation of epithelial cells, thereby promoting the repair of damaged skin and mucous membrane tissue. It also directly affects chemotaxis in tissue reconstruction. FGF plays an important role in neurogenesis, axon growth, and differentiation during the development of the central nervous system, and is also important for maintaining the function of the adult brain. When physical stimuli such as ultrasound stimulation or vibration are applied to these cells in this process, FGF signaling may be enhanced, and the survival of neurons and other cells may be promoted.

[0053] In (e) above, TNF may be tumor necrosis factor, and TNFR may be a tumor necrosis factor receptor. In this case, the cells to which physical stimulation is applied in this process may be TNFR-containing cells, which are widely present in living cells excluding red blood cells. When an extracellular signal (TNF signal) is input to these cells, infection defense and antitumor effects may be promoted by increasing the expression of cell adhesion molecules, induction of apoptosis, and enhancement of antibody production by inflammatory mediators such as IL-1, IL-6, and prostaglandin E2, as well as plasma cells. However, in the present invention, effects other than the induction of apoptosis are preferred.

[0054] In (f) above, IL may be an interleukin and ILR may be an interleukin receptor. More than 30 types of interleukins have been discovered to date. Interleukins basically bind to interleukin receptors present in cells and activate signaling pathways. In this case, the cells to which physical stimulation is applied in this process may be various immune cells. When an extracellular signal (IL signal) is input to these cells, interleukin 3 stimulates bone marrow stem cells and activates differentiation signals. Other immune cells are differentiated by interleukins: T cells are induced by interleukins 2, 4, and 7; B cells by interleukins 7, 13, and 15; NK cells by interleukins 7 and 12; and mast cells by interleukin 4. When physical stimuli such as ultrasonic stimulation or vibration are applied to these cells in this process, IL signaling can be enhanced, and T cell differentiation, B cell differentiation, NK cell differentiation, and mast cell differentiation can be promoted.

[0055] In (g) above, IFN is interferon, and its extracellular receptor is a Toll-like receptor (TLR), specifically TLR3, TLR7, and TLR9, which are found in endosomes. When an extracellular signal (IFN signal) is input to these cells, one of the main functions of type I IFN is the inactivation of eukaryotic translation initiation factor 2a (eIF-2a), thereby inhibiting viral protein synthesis. Furthermore, type I IFN activates RNase L, cleaving cytoplasmic ssRNA and further inhibiting viral replication. In this case, the cells subjected to physical stimulation in this process increase the expression of MHC class I molecules, making it easier for activated killer T cells to function. Applying physical stimuli such as ultrasound or vibration to these cells in this process can enhance IFN signaling and promote the antiviral activity of immune cells.

[0056] In (h) above, CSF is a colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF), in cooperation with IL-3,5, etc., differentiates pluripotent hematopoietic stem cells into myeloid progenitor cells (CFU-GEMM), which then differentiate into early erythroid progenitor cells (BFU-E), granulocyte-monocyte colony-forming cells (CFU-GM), eosinophil colony-forming cells (CFU-Eo), basophil colony-forming cells (CFU-Ba), etc. Furthermore, it has the function of differentiating CFU-GM into neutrophils and monocytes, and CFU-Eo into eosinophils. Monocytes leave the circulatory system and migrate into tissues, where they mature into macrophages and dendritic cells. In this case, the cells that provide physical stimulation in this process can be macrophages. In macrophages, signals are sent via STAT3, activating the macrophages and inhibiting fungal survival. GM-CSF reduces free zinc in cells, increases the production of reactive oxygen species, and causes zinc starvation and toxicity in fungi. In this way, GM-CSF promotes the development of the immune system and enhances defense against infection. Applying physical stimuli such as ultrasonic stimulation or vibration to these cells in this process may enhance CSF signaling and promote the activation of immune cells.

[0057] In (i) above, the Wnt protein binds to Fizzled family receptors and transmits Wnt signaling through the classical Wnt pathway, the non-classical planar cell polarity pathway, and the Wnt / calcium pathway. Wnt signaling then induces differentiation of pluripotent stem cells into mesoderm and endoderm progenitor cells. These progenitor cells further differentiate into cell types such as endothelial cells, cardiomyocytes, and vascular smooth muscle cell lineages. It also induces the proliferation of embryonic stem cells and cell migration at later stages of development, controlling the migratory behavior of neuroblasts, neural crest cells, muscle cells, and tracheal cells. In this case, the cells to which physical stimulation is applied in this process may be cells containing the Wnt signaling pathway. When Wnt signaling is input to these cells, stem cells can differentiate into mesoderm and endoderm progenitor cells. Applying physical stimuli such as ultrasound or vibration to these cells in this process can enhance Wnt signaling and promote differentiation into stem cell progenitor cells. Furthermore, pattern formation can be controlled based on the concentration gradient of Wnt protein and the SHH protein described below.

[0058] In (j) above, the SHH protein may also be a Sonic Hedgehog signaling pathway protein. It promotes the proliferation of somatic stem cells in various tissues, such as hematopoietic stem cells and stem cells of the mammary gland and nervous system. It is also necessary when hair follicles transition from the resting phase to the growth phase. In this case, the cells to which physical stimulation is applied in this process may be mouse fibroblasts (NIH3T3 cells), human keratinocytes, chicken or mouse neural progenitor cells, etc. When physical stimuli such as ultrasonic stimulation or vibration are applied to these cells in this process, the SHH signal is enhanced, and in NIH3T3 cells, the expression levels of Gli1 and Ptc1 increase with the duration of Sonic Hedgehog signaling.

[0059] (Preculture step) In the cell stimulation method of the present invention, the "pre-culture step" is a step of pre-culturing cells before the stimulation step described above. This step is optional and may be performed as needed. The culture conditions for pre-culturing in this step can be the same as those for the culture step described later. For example, cells may be cultured statically at 37°C and 5% CO2, with half the culture medium replaced every two days, and cultured for 2 to 40 days depending on the colony formation state. In this step, for example, cells can be cultured with adhesion to form colonies or cell sheets, and these can be subjected to the stimulation step described above to efficiently deliver physical stimulation to the cells.

[0060] (Culture process) In the cell stimulation method of the present invention, the "culture step" refers to the step of culturing the cells after the stimulation step. This step is an optional step and may be performed as needed.

[0061] The culture period in this process is not limited as long as the cells that have received physical stimulation after the stimulation step can proliferate, differentiate, survive, adhere, and / or migrate. The culture period is, for example, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 12 hours or more, 1 day or more, or 2 days or more, and / or 30 days or less, 25 days or less, 20 days or less, or 15 days or less, preferably 1 to 7 days or 2 to 5 days.

[0062] In this process, the culture conditions, such as CO2 concentration and the frequency of medium changes, are not limited. For example, the culture temperature may be approximately 30°C to 40°C (e.g., 37°C), the CO2 concentration approximately 2% to 10% (e.g., 5% CO2), and the medium may be changed every two days. Furthermore, the culture method is not limited; for example, adherent culture, suspension culture, or suspension culture may be used, and culture may also be performed in a three-dimensional matrix. In addition, the seeding density of the cells used in this process is not particularly limited; for example, 1.0 × 10⁶ 2 cells / cm 2 ~1.0×10 5 cells / cm 2 or 1.0 × 10 3 cells / cm 2 ~1.0×10 4 cells / cm 2 That's fine.

[0063] The culture medium used in this process is not particularly limited and should be selected appropriately depending on the type of cells being cultured. For example, serum or plasma-containing media, basic media, or serum-free media can be used. Examples of basic media include EMEM medium (also referred to as αMEM medium), DMEM medium, IMDM medium, GMEM medium, Ham F10 medium, Ham F12 medium, RPMI1640 medium, and combinations thereof. Serum-free media are media that contain components necessary for cell survival and proliferation but do not contain unprocessed or unpurified serum, and the specific media are not particularly limited.

[0064] This step is performed after the stimulation step described above, but the stimulation step can also be repeated after this step. For example, the stimulation step may be performed during the culture in this step, and / or this step and the stimulation step can be performed alternately. The cells cultured in this step may be maintained or proliferated by performing expansion culture or subculture as appropriate after this step.

[0065] (Recovery process) In the cell stimulation method of the present invention, the "recovery step" is a step of recovering the stimulated cells from the culture substrate or three-dimensional matrix, etc., after the stimulation step or culture step described above. This step is an optional step and may be performed as needed.

[0066] In this process, the method for recovering cells from the culture substrate or three-dimensional matrix is ​​not limited and can be appropriately selected depending on the type of cell. For example, cells may be separated from the culture substrate or three-dimensional matrix by enzymatic treatment, chemical treatment, temperature change, and / or physical recovery, and then the cell suspension containing the cells may be recovered by aspiration or decantation. For example, differentiated cells can also be selectively recovered by separating differentiated cells from undifferentiated cells by physical stimulation.

[0067] 1-4. Effects In the cell stimulation method of the present invention, by applying physical stimuli to cells and / or ligand molecules during the stimulation step, extracellular signals based on ligand molecules are enhanced, and as a result, cell proliferation, differentiation, survival, adhesion, movement, etc., are controlled based on the enhanced extracellular signals. For example, when BMP-2 immobilized on a culture substrate is bound to osteomorphic protein receptor 1A (BMPR1A) expressed on osteoblast cells, mechanical stimulation is applied to the culture substrate by irradiating it with ultrasound, thereby enhancing the BMP signal input to the osteoblasts and promoting osteoblast differentiation.

[0068] 2.Cell manufacturing method 2-1. Overview A second aspect of the present invention is a method for producing differentiated cells from undifferentiated cells (hereinafter sometimes abbreviated as "cell production method"). The cell production method of the present invention can enhance extracellular signals that induce differentiation of undifferentiated cells into differentiated cells based on the application of physical stimuli to cells and / or ligand molecules.

[0069] 2-2. Method The cell production method of the present invention includes a stimulation step and a culture step as essential steps, and a pre-culture step and a harvesting step as optional steps. Each step will be described below.

[0070] (stimulation process) In the cell production method of the present invention, the "stimulation step" is a step of applying physical stimulation to undifferentiated cells and / or ligand molecules. In this step, the physical stimulation may be applied to undifferentiated cells only, to ligand molecules only, or to both undifferentiated cells and ligand molecules.

[0071] In this specification, "stem cells" refer to cells that possess the ability to self-replicate by dividing to produce cells identical to themselves, the ability to differentiate into various cell lineages, and the ability to proliferate without limit. Examples of stem cells include adult stem cells and pluripotent stem cells. Specific examples of adult stem cells include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, and pigment stem cells. Specific examples of pluripotent stem cells include induced pluripotent stem cells and embryonic stem cells.

[0072] In this specification, "progenitor cell" refers to a cell that is in the process of differentiating from a stem cell to a final differentiated cell. Examples include cells in the process of differentiating from mesenchymal stem cells into osteoblasts, as well as vascular endothelial progenitor cells, satellite cells, blast cells, etc.

[0073] In this specification, "differentiated cells" refers to cells that have been differentiated from undifferentiated cells such as stem cells or progenitor cells, and more specifically, differentiated cells whose fate has been determined to become a specific type of cell. Examples include differentiated osteoblasts, as well as cardiomyocytes, muscle cells, fibroblasts, nerve cells, immune cells such as lymphocytes, vascular cells, ophthalmic cells such as retinal pigment epithelial cells, and blood cells such as megakaryocytes and erythrocytes. The aforementioned progenitor cells are also considered differentiated cells from stem cells.

[0074] In this specification, "undifferentiated cells" refers to any cells that exist before the differentiation of the differentiated cells described above. Typical examples of undifferentiated cells are the stem cells or progenitor cells described above.

[0075] In this process, the ligand molecule is bound to a receptor protein expressed on undifferentiated cells such as stem cells or progenitor cells. This receptor protein can mediate the transmission of extracellular signals to undifferentiated cells based on its binding to the ligand molecule. In this process, the extracellular signals that induce the differentiation of undifferentiated cells into differentiated cells are enhanced based on the application of physical stimuli to the undifferentiated cells and / or the ligand molecule.

[0076] Furthermore, in this process, the ligand molecules are immobilized on the culture substrate and / or matrix molecules used for culturing undifferentiated cells.

[0077] The further configuration of this process is the same as the stimulation process in the first embodiment, so a detailed explanation is omitted here.

[0078] For example, the undifferentiated cells in this process may be stem cells or progenitor cells, and furthermore, the stem cells or progenitor cells may be mesenchymal stem cells, stromal cells, or osteoblasts. When extracellular signals mediated by BMP-2 and BMPR1A (BMP signals) are input to these cells, mesenchymal stem cells can differentiate into stromal cells, and stromal cells can differentiate into osteoblasts. If physical stimuli such as ultrasonic stimulation or vibration are applied to these cells in this process, the BMP signal can be enhanced, and the differentiation of mesenchymal stem cells into stromal cells and the differentiation of stromal cells into osteoblasts can be promoted.

[0079] (Preculture step) In the cell manufacturing method of the present invention, the "pre-culture step" refers to the step of culturing undifferentiated cells before the stimulation step. The method for culturing undifferentiated cells in this step is the same as the method for culturing cells in the pre-culture step of the first embodiment, so a detailed explanation is omitted here.

[0080] (Culture process) In the cell manufacturing method of the present invention, the "culture step" refers to the step of culturing undifferentiated cells after the stimulation step. The method for culturing undifferentiated cells in this step is the same as the method for culturing cells in the culture step of the first embodiment, so a detailed explanation is omitted here.

[0081] This step is performed after the stimulation step described above, but the stimulation step can also be repeated after this step. For example, the stimulation step may be performed during the culture in this step, and / or this step and the stimulation step can be performed alternately. The cells cultured in this step may be maintained or proliferated by performing expansion culture or subculture as appropriate after this step.

[0082] In this process, when undifferentiated cells after the stimulation step are cultured, differentiated cells are obtained. For example, when BMP-2 is used as the ligand molecule, stromal cells differentiated from mesenchymal stem cells, osteoblasts differentiated from stromal cells, and / or osteoblasts with advanced differentiation are obtained.

[0083] (Recovery process) In the cell production method of the present invention, the "recovery step" is a step of recovering differentiated cells from a culture substrate or three-dimensional matrix after the stimulation step and the culture step. The method for recovering differentiated cells in this step is the same as the method for recovering cells in the recovery step of the first embodiment, so a detailed explanation is omitted here. For example, when BMP-2 is used as the ligand molecule in the stimulation step, stromal cells and / or osteoblasts are recovered in this step.

[0084] 2-3. Effects In the cell production method of the present invention, physical stimulation is applied to undifferentiated cells and / or ligand molecules during the stimulation step, thereby enhancing extracellular signals based on ligand molecules. Based on these enhanced extracellular signals, undifferentiated cells are efficiently induced to differentiate, and differentiated cells are produced during the culture step. For example, when BMP-2 immobilized on a culture substrate is bound to osteomorphic protein receptor 1A (BMPR1A) expressed on osteoblast cells, mechanical stimulation is applied to the culture substrate by irradiating it with ultrasound, thereby enhancing the BMP signal input to the osteoblasts, promoting osteoblast differentiation, and enabling the efficient production of differentiated osteoblasts.

[0085] 3. Cell stimulator 3-1. Overview A third aspect of the present invention is a device for stimulating cells with ligand molecules (hereinafter sometimes abbreviated as "cell stimulation device"). The cell stimulation device of this aspect includes a mounting section and a physical stimulation delivery section, and can be used in the cell stimulation method of the first aspect or the cell production method of the second aspect.

[0086] 3-2. Composition The cell stimulation device of this embodiment includes a mounting section and a physical stimulation delivery section as essential components, and includes a cell retrieval section as an optional component. Each component will be described below.

[0087] (Mounting section) In the cell stimulation device of this embodiment, the mounting section can accommodate a culture substrate, a cell culture substrate, or a culture vessel containing a three-dimensional matrix.

[0088] The mounting section may include temperature control means capable of controlling the temperature of a culture vessel containing a culture substrate, cell culture substrate, or three-dimensional matrix. The temperature control means can maintain the temperature of the culture substrate, cell culture substrate, or culture vessel at approximately 30°C to approximately 40°C (e.g., 37°C).

[0089] Furthermore, the mounting section may include CO2 control means capable of controlling the CO2 concentration of a culture vessel containing a culture substrate, cell culture substrate, or three-dimensional matrix. The CO2 control means can maintain the CO2 concentration of the culture substrate, cell culture substrate, or culture vessel at approximately 2% to approximately 10% (e.g., 5% CO2).

[0090] (Physical stimulation delivery unit) In the cell stimulation device of this embodiment, the physical stimulation unit can deliver physical stimulation to ligand molecules and / or cells placed on the mounting unit, on the culture substrate, on the cell culture substrate, or in the culture vessel.

[0091] The physical stimulus delivery unit can be configured according to the type of physical stimulus to be applied to the cells. If the physical stimulus is ultrasound, an ultrasonic oscillator can be provided as an ultrasonic irradiation means capable of applying ultrasonic-based mechanical stimulation to ligand molecules and / or cells. The ultrasonic oscillator can be equipped with a probe for irradiating ultrasound. If the physical stimulus is vibration, a vibration motor or vibration generator can be provided as a vibration generating means. If the physical stimulus is a change in gravity, a centrifuge can be provided as a gravity control means. If the physical stimulus is pressurization or depressurization, a pressurization pump or depressurization pump can be provided as a pressure control means.

[0092] (Cell retrieval section) In the cell stimulation device of this embodiment, the cell recovery unit can recover cells from a culture vessel containing a culture substrate, cell culture substrate, or three-dimensional matrix placed on the mounting unit.

[0093] The cell recovery unit can recover cells by mechanically removing cells from the culture substrate or cell culture substrate surface using cell removal means such as a cell scraper; by splashing a discharge solution onto the culture substrate or cell culture substrate surface; by enzymatic or chemical removal by applying enzymes such as trypsin or compounds such as chelating agents to the culture substrate or cell culture substrate to remove cells; by enzymatic or chemical removal by applying enzymes such as trypsin or compounds such as chelating agents to the three-dimensional matrix to separate cells from the three-dimensional matrix; or by shaking, vibrating, or irradiating the culture substrate, cell culture substrate, or culture vessel with ultrasound.

[0094] 4. Cell stimulation kit 4-1. Overview A fourth aspect of the present invention is a kit for stimulating cells with ligand molecules (hereinafter sometimes abbreviated as "cell stimulation kit"). The cell stimulation kit of the present invention includes a culture substrate on which ligand molecules are immobilized, and can be used in the cell stimulation method of the first aspect or the cell production method of the second aspect. According to the cell stimulation kit of the present invention, extracellular signals based on ligand molecules can be enhanced. For example, it becomes possible to efficiently induce cell differentiation based on growth factors such as BMP-2.

[0095] 4-2. Composition The cell stimulation kit of this embodiment includes, as essential components, at least a ligand molecule for immobilization on a culture substrate and / or matrix molecule, or a culture substrate and / or matrix molecule on which the ligand molecule is immobilized. The culture substrate, matrix molecule, ligand molecule, etc., are described in the first embodiment, so a detailed explanation is omitted here.

[0096] The cell stimulation kit of this embodiment may include, as selective components, instruments necessary for physical stimulation such as ultrasound irradiation, culture media and antibiotics used for cell culture, and instructions describing methods for physical stimulation such as ultrasound irradiation.

[0097] According to the cell stimulation kit of this embodiment, by applying physical stimulation to cells bound to ligand molecules and / or ligand molecules bound to receptor proteins, it is possible to enhance extracellular signals transmitted to cells based on the binding of receptor proteins to ligand molecules. [Examples]

[0098] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0099] <Example 1: Induction of osteogenic differentiation based on immobilized BMP-2> (the purpose) BMP-2 immobilized on the surface of the culture substrate is used to induce osteoblast differentiation.

[0100] (Methods and Results) (1) Immobilization of BMP-2 onto the substrate surface DOPA is known to have high binding affinity to various surfaces through interactions via catechol groups. Therefore, a YKYKY-BMP-2 fusion protein (SEQ ID NO: 3; using the sequence described in Xueli R., et al., J. Mater. Chem. B., 12, 3006-3014 (2024)) was prepared by expressing it in E. coli, in which the Tyr-Lys-Tyr-Lys-Tyr pentapeptide (SEQ ID NO: 1) was linked to the N-terminus of the BMP-2 protein along with a histidine tag. By converting the Tyr residue to DOPA through tyrosinase-based enzymatic modification, an XKXKX-BMP-2 fusion protein (SEQ ID NO: 4; using the sequence described in Xueli R., et al., J. Mater. Chem. B., 12, 3006-3014) was prepared, in which DOPA-Lys-DOPA-Lys-DOPA (SEQ ID NO: 2) was linked to the N-terminus of the BMP-2 protein along with a histidine tag. Using the sequence described in (2024); "X" represents DOPA, and the resulting fusion protein will be referred to as "DOPA-BMP-2" below. DOPA-BMP-2 was immobilized on the surface of a TCPS (Tissue Culture Polystyrene) substrate (Iwaki, AGC Techno Glass Co., Ltd.) by incubating a solution of these DOPA-BMP-2s at room temperature for 30 minutes (Figure 1A).

[0101] Figure 1B shows the relationship between the amount of DOPA-BMP-2 applied and the amount of DOPA-BMP-2 bound to the substrate surface.

[0102] (2) Evaluation of the activity of BMP-2 immobilized on the substrate surface The activity of DOPA-BMP-2 in inducing osteogenic differentiation was evaluated by culturing cells on a substrate immobilized with DOPA-BMP-2.

[0103] Mouse-derived osteoblasts (MC3T3-E1) were purchased from the Japanese Collection of Research Bioresources Cell Bank and maintained at 37°C and 5% CO2 using α-MEM (Fujifilm Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin antibiotic (Nacalai Tesque Corporation). Cell passage was performed after the cells reached 90% confluence.

[0104] DOPA-BMP-2 was immobilized on each well of a 12-well plate (hereinafter, DOPA-BMP-2 immobilized on the well surface will simply be referred to as "immobilized BMP-2"). MC3T3-E1 cells were seeded in each well at a seeding density of 50,000 cells / mL and cultured for 3 days (Figure 2A, right side). As a control, MC3T3-E1 cells were cultured in wells to which an equivalent amount of soluble BMP-2 was added (Figure 2A, left side).

[0105] BMP-2 activity was evaluated by quantifying the activity of alkaline phosphatase (ALP), a marker of osteogenic differentiation. ALP activity was measured using the LabAssay® ALP kit (Fujifilm Wako Pure Chemical Corporation) according to the attached protocol.

[0106] The results are shown in Figure 2B. ALP activity was significantly higher when cells were stimulated with immobilized BMP-2 compared to when stimulated with soluble BMP-2. When cells were stimulated with immobilized DOPA-BMP-2, ALP activity showed a concentration-dependent increase, gradually reaching a plateau at concentrations of 6 μg / well or higher. In contrast, when cells were stimulated with soluble BMP-2, ALP activity was significantly lower at all concentrations.

[0107] (3) Evaluation of calcium deposition The effect of immobilized BMP-2 was evaluated by calcium deposition, an indicator of late osteogenic differentiation. Calcium deposition was evaluated in MC3T3-E1 cells cultured for 7 days using the same culture method as described in (2) above.

[0108] To evaluate calcium deposition, calcification of differentiated cells was assessed using Alizarin Red S staining. After 7 days of culture, cells were washed three times with D-PBS and fixed with 4% paraformaldehyde phosphate. Subsequently, cells were washed twice with MilliQ, and 2% Alizarin Red S solution (pH 4.2) was added to each well, and incubated at room temperature for 20 minutes. After that, cells were washed three times with MilliQ to remove unchelated staining. The percentage of stained area was calculated by image analysis.

[0109] The results of the calcium deposition evaluation are shown in Figure 3. No calcium deposition was observed in MC3T3-E1 cells cultured for 7 days in the presence of soluble BMP-2 (Figure 3A). In contrast, calcium deposition appeared in MC3T3-E1 cells cultured for 7 days in the presence of immobilized BMP-2. DOPA-BMP-2 at 4 μg / well was 1051 ng / cm³. 2 This corresponds to the amount of binding, and 55.1% of the area was calcified (Figure 3B).

[0110] <Example 2: Verification of synergistic effects based on the combination of immobilized BMP-2 and ultrasonic irradiation> (the purpose) We will investigate whether the efficiency of osteoblast differentiation induction increases when irradiating with ultrasound while inducing osteoblast differentiation of osteoblasts using BMP-2 immobilized on the surface of a culture substrate (Figure 4).

[0111] (Methods and Results) (1) Evaluation of ossification The effect of ultrasound on BMP-2-dependent osteogenic differentiation was investigated by applying ultrasound stimulation (low-intensity pulsed ultrasound; LIPUS) to MC3T3-E1 cells during a 4-7 day culture period using the same culture method as in Example 1(2) above.

[0112] Ultrasound was applied using a functional ultrasound device (Osteotron 5, ITO Corporation) under the conditions of a pulse frequency of 100 Hz, an ultrasound frequency of 1 MHz, and a 20% duty cycle. An ultrasound gel was placed between the probe and the TCPS (Tissue Cell Structure). The distance between the probe and the bottom of the cell culture plate was set to approximately 3 mm to allow the ultrasound to reach the cell layer to its maximum extent. The cell culture plate was placed horizontally to avoid uneven ultrasound irradiation. One day after cell seeding, the cells were incubated at 37°C with 5% CO2 at a rate of 30 mW / cm². 2 or 60 mW / cm² 2 Ultrasound of a certain intensity was applied for 20 or 60 minutes per day. Note that these ultrasound irradiation conditions have little effect on the cell proliferation rate.

[0113] Figure 5A shows the results of evaluating ALP activity to assess early osteogenic differentiation after culturing cells under the above conditions for 4 days. In the control group (without BMP-2) and the soluble BMP-2 group, ultrasound irradiation had little effect on ALP expression. In contrast, when ultrasound was applied in the presence of immobilized BMP-2, ALP activity increased significantly compared to the control group (without ultrasound).

[0114] Next, Figure 5B shows the results of evaluating calcium deposition to assess late-stage osteogenic differentiation after culturing cells under the above conditions for 7 days. Similar to ALP activity, little calcium deposition was observed in the control group and the soluble BMP-2 group even after ultrasound irradiation. In contrast, cell calcification was significantly and substantially enhanced when ultrasound was irradiated in the presence of immobilized BMP-2.

[0115] These results demonstrate that osteogenic differentiation is promoted only when BMP-2 is immobilized, and that a synergistic effect is obtained between BMP-2 immobilization and ultrasound.

[0116] (2) Evaluation of focal adhesion Focal adhesion is a molecular complex bound to integrins and the cytoskeleton, promoting interactions between the extracellular matrix, integrins, and the cytoskeleton. Focal adhesion plays a crucial role in receiving extracellular physical forces and transmitting those forces to the cytoplasm and nucleus to regulate downstream transcription. Therefore, we analyzed how ultrasound irradiation affects intracellular structures by immunostaining vinculin and actin filaments, which are components of focal adhesion.

[0117] Specifically, 4 x 10 4 MC3T3-E1 cells were seeded at a concentration of cells / mL onto untreated TCPS or TCPS immobilized with DOPA-BMP-2, and cultured for 24 hours in a humid incubator at 37°C and 5% CO2. After culture, ultrasonic stimulation (intensity 30 mW / cm²) was performed. 2 or 60 mW / cm² 2 The cells were irradiated for 20 minutes or 60 minutes per day, and then incubated for another hour before immunostaining for vinculin and actin filaments.

[0118] The immunohistochemical staining procedure was as follows: Cells were washed twice with D-PBS and then fixed at room temperature for 15 minutes using 4% paraformaldehyde phosphate buffer. After washing the cells, they were permeabilized with 0.1% TritonX-100 solution for 5 minutes, and then blocked with 1% bovine serum albumin at room temperature for 1 hour. After three washes, the cells were incubated with Alexa Fluo488-labeled goat anti-mouse IgG (1:500) and TRITC-labeled phalloidin (1:1000) for 1 hour. Cell adhesion was shown as green clusters around the cell periphery. The number of focal adhesions per cell was analyzed by Fiji in vinculin-stained images.

[0119] Figure 6 shows the results of the analysis of focal adhesions. Under conditions without ultrasound irradiation, cells cultured in the presence of immobilized BMP-2 showed an increased number of focal adhesions compared to the soluble BMP-2 group and the control group. Under conditions combined with ultrasound, cells cultured in the presence of immobilized BMP-2 showed larger focal adhesion sizes and a greater number of adhesions. In contrast, ultrasound irradiation did not affect the formation of focal adhesions in the soluble BMP-2 group and the control group.

Claims

1. A method of stimulating cells with ligand molecules, Stimulation process: Applying physical stimuli to cells and / or ligand molecules. Includes, The ligand molecule is bound to or interacts with a receptor protein expressed on the cell, and is immobilized on a culture substrate and / or matrix molecule used for culturing the cell. The method wherein the receptor protein mediates the transmission of an extracellular signal to the cell based on its binding to the ligand molecule, and the extracellular signal is enhanced by the physical stimulus.

2. The method according to claim 1, wherein the cells are arranged on the culture substrate or on a cell culture substrate coated with the matrix molecules.

3. The method according to claim 1, wherein the cells are contained in a three-dimensional matrix containing the matrix molecules.

4. The method according to claim 1, wherein the receptor protein is an enzyme-coupled receptor and / or a G protein-coupled receptor.

5. The method according to claim 1, wherein the ligand molecule is a secretory factor, an extracellular domain of a membrane protein, an artificial peptide, and / or a small molecule compound.

6. The method according to claim 5, wherein the secreted factor is selected from the group consisting of the TGF-β superfamily, epidermal growth factor (EGF) family, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) family, tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Wnt protein, SHH protein, interleukin (IL), interferon (IFN), and colony-stimulating factor (CSF).

7. The method according to claim 6, wherein the TGF-β superfamily is bone morphogenetic protein 2 (BMP-2).

8. The method according to claim 1, wherein the physical stimulus is a mechanical stimulus and / or an electrical stimulus.

9. The method according to claim 8, wherein the mechanical stimulus is ultrasound, compression, extension, pressurization, depressurization, vibration, or change in gravity.

10. The intensity of the ultrasound is 1 mW / cm 2 ~500 mW / cm 2 The method according to claim 9, wherein the frequency of the ultrasonic wave is 1 MHz to 3 MHz.

11. The method according to claim 10, wherein the ultrasonic waves are applied in pulse form.

12. The method according to any one of claims 9 to 11, wherein the ultrasound is applied for 10 seconds to 60 minutes per day.

13. The method according to claim 1, further comprising a culture step of culturing the cells after the stimulation step.

14. A method for producing differentiated cells from undifferentiated cells, A stimulation step, which involves applying physical stimuli to undifferentiated cells and / or ligand molecules, A culture step in which the undifferentiated cells after the stimulation step are cultured. Includes, The ligand molecule binds to or interacts with a receptor protein expressed on the undifferentiated cells and is immobilized on a culture substrate and / or matrix molecule used for culturing the undifferentiated cells. The method wherein the receptor protein mediates the transmission of an extracellular signal that induces the differentiation of the undifferentiated cells into differentiated cells based on its binding to the ligand molecule, and the extracellular signal is enhanced by the physical stimulus.

15. The method according to claim 14, wherein the undifferentiated cell is an osteoblast and the ligand molecule is bone morphogenetic protein 2 (BMP-2).

16. An apparatus for use in the method according to claim 2 or 3, A mounting section for placing a culture vessel containing a culture substrate, cell culture substrate, or three-dimensional matrix, and A physical stimulus delivery unit that can deliver the aforementioned physical stimulus to the ligand molecules and / or cells on the culture substrate, the cell culture substrate, or the culture vessel, which is placed on the aforementioned mounting unit. The apparatus, including the above.

17. The apparatus according to claim 16, wherein the mounting section includes temperature control means capable of controlling the temperature of the culture substrate, the cell culture substrate, or the culture vessel.

18. The apparatus according to claim 16, wherein the physical stimulation unit includes an ultrasonic irradiation means capable of providing ultrasonic-based mechanical stimulation to the ligand molecules and / or the cells.

19. A kit for stimulating cells with ligand molecules, Ligand molecules for immobilization on culture substrates and / or matrix molecules, or Culture substrates and / or matrix molecules immobilized with ligand molecules Includes, The ligand molecule is immobilized on the culture substrate and / or matrix molecule used for culturing the cells, and is intended to bind to or interact with the receptor protein expressed on the cells. A kit for enhancing extracellular signals transmitted to a cell based on the binding of a receptor protein to a ligand molecule, by applying a physical stimulus to the cell bound to the ligand molecule and / or to the ligand molecule bound to the receptor protein.

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