Method and culture device for inducing expression of calcium channel and / or calcium pump

Abstract

To provide a method for inducing expression of a calcium channel and / or a calcium pump in a cell.SOLUTION: The present invention provides a method for inducing the expression of calcium channels and / or calcium pumps in cells, comprising a step of irradiating the cells with light in the wave length range of 315 to 325nm, wherein the calcium channels and / or calcium pumps are at least one selected from the group consisting of dihydropyridine receptors (DHPR), voltage-gated calcium channels (VGCC), ryanodine receptors (RYR), and endoplasmic reticulum calcium ATPases (SERCA).SELECTED DRAWING: Figure 6A

Description

[Technical Field]

[0001] The present invention relates to a method and culture device for inducing the expression of calcium channels and / or calcium pumps. [Background technology]

[0002] Patent Document 1 describes a compound that suppresses the expression of MMP (matrix metalloproteinase) and pro-inflammatory cytokines induced by UV exposure, and reduces intracellular Ca 2+ The paper also describes a TRPV1 inhibitory peptide that inhibits the influx of TRPV1 and increases in skin thickness, as well as a composition containing the peptide for preventing skin aging and improving skin wrinkles. The paper describes the irradiation of mouse skin with broadband UV light (275-380 nm) using a fluorescent sunlamp. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2015-515455 Summary of the Invention [Problem to be solved by the invention]

[0004] Calcium ions (Ca 2+ Calcium ions are intracellular signaling factors that regulate various biological processes, including proliferation, differentiation, gene expression, exocytosis (e.g., release of hormones, cytokines, and neurotransmitters), apoptosis, muscle contraction, and metabolism. The intracellular level of calcium ions is normally maintained at 10-100 nM and can rise to 100-200 nM upon stimulation. Calcium ions enter the cytoplasm via calcium (ion) channels and nonselective cation channels, while calcium pumps expel calcium ions from the cytoplasm. Therefore, a technique that can induce the expression of calcium channels and calcium pumps that selectively permeate calcium ions is extremely useful for controlling, regulating, or studying various biological events. [Means for solving the problem]

[0005] According to the present disclosure, there is provided a method for inducing expression of a calcium channel and / or calcium pump in a cell, the method comprising the step of irradiating the cell with light in the wavelength range of 315 to 325 nm, wherein the calcium channel and / or calcium pump is at least one calcium channel and / or calcium pump selected from the group consisting of dihydropyridine receptors (DHPRs), voltage-gated calcium channels (VGCCs), ryanodine receptors (RYRs), and endoplasmic reticulum calcium ATPases (SERCAs). The present disclosure also provides a cell culture device comprising: a first irradiation unit capable of mainly emitting light in the wavelength range of 315 to 325 nm; a first mounting unit for mounting a cell culture vessel located within the irradiation area of ​​the first irradiation unit; an irradiation control unit for controlling the first irradiation unit; and a housing for accommodating the first irradiation unit and the first mounting unit. [Effects of the Invention]

[0006] According to the methods of the present disclosure, the expression of calcium channels and / or calcium pumps can be induced in cells. According to the device of the present disclosure, the expression of calcium channels and / or calcium pumps can be induced in cells in a simple manner. [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a schematic diagram showing a first embodiment of the device of the present disclosure. [Figure 2] FIG. 1 is a schematic diagram showing a second embodiment of the device of the present disclosure. [Figure 3] FIG. 1 is a schematic diagram showing a third embodiment of the device of the present disclosure. [Figure 4]FIG. 1 shows the emission spectrum of an LED used in an experiment. [Figure 5] FIG. 10 is a Venn diagram showing the number of genes that increased under each irradiation condition. [Figure 6A] The change in the expression level of the DHPR·VGCC gene (CACNG1) due to irradiation with LED light is shown as the ratio (LED / control) of (expression level when irradiated) / (expression level when not irradiated). [Figure 6B] This shows the change in the expression level of the DHPR·VGCC gene (CACNG6) upon irradiation with LED light. [Figure 6C] This shows the change in the expression level of the SERCA gene (ATP2A1) upon irradiation with LED light. [Figure 6D] This shows the change in the expression level of the RYR gene (RYR1) upon irradiation with LED light. [Figure 6E] This shows the change in the expression level of the TRDN gene (TRDN) upon irradiation with LED light. [Figure 6F] This shows the change in the expression level of the CASQ gene (CASQ1) upon irradiation with LED light. [Figure 6G] This shows the change in the expression level of the CASQ gene (CASQ2) upon irradiation with LED light. [Figure 6H] This shows the change in the expression level of the HRC gene (HRC) upon irradiation with LED light. [Figure 6I] This shows the change in the expression level of the Cyto gene (COX6A2) due to irradiation with LED light. DETAILED DESCRIPTION OF THE INVENTION

[0008] In this specification, the numerical range "a to b" (a and b are specific numerical values) means a range including the values ​​at both ends, "a" and "b." In other words, "a to b" is synonymous with "a or more and b or less."

[0009] <Method for inducing expression of calcium channels and / or calcium pumps> In one aspect, the present disclosure provides a method for inducing expression of a calcium channel and / or calcium pump in a cell, the method comprising: irradiating the cells with light in a wavelength range of 315 to 325 nm; The method relates to a method in which the calcium channel and / or calcium pump is at least one calcium channel and / or calcium pump selected from the group consisting of dihydropyridine receptors (DHPRs), voltage-gated calcium channels (VGCCs), ryanodine receptors (RYRs), and endoplasmic reticulum calcium ATPases (SERCAs). The cells are irradiated with light in the wavelength range of 315 to 325 nm at an irradiation amount effective for inducing the expression of calcium channels and / or calcium pumps in the irradiated cells.

[0010] In the present disclosure, induction of calcium channel and / or calcium pump expression includes upregulation of calcium channel and / or calcium pump expression. In the present disclosure, "induction of expression" means that the expression level of the target calcium channel and / or calcium pump is increased, for example, by 20 times or more, more specifically, 30 times or more, more specifically, 40 times or more, more specifically, 50 times or more, more specifically, 60 times or more, more specifically, 70 times or more, and more specifically, 80 times or more, compared to cells before light irradiation according to the present disclosure (the same cells as the light-irradiated cells) or cells not subjected to light irradiation according to the present disclosure (cells different from the light-irradiated cells).

[0011] In the present disclosure, the mere mention of "calcium channels" refers to dihydropyridine receptors (DHPRs), voltage-gated calcium channels (VGCCs), and ryanodine receptors (RYRs), unless otherwise clearly indicated. Voltage-gated calcium channels include L-type channels, non-L-type (N-type, P / Q-type, R-type) channels, T-type channels, etc., and are preferably L-type channels. Ryanodine receptors (RYRs) include type 1 ryanodine receptors (RYR1) and type 2 ryanodine receptors (RYR2). Induction of calcium channel expression can promote the uptake of calcium ions from the extracellular environment or endoplasmic reticulum into the cytoplasmic compartment, thus increasing the influx of calcium ions into the cytoplasmic compartment. In this disclosure, the term "calcium pump" refers to endoplasmic reticulum calcium ATPase (SERCA), unless otherwise specified. Induction of SERCA expression can promote the export of calcium ions from the cytoplasmic compartment, thereby increasing the efflux of calcium ions from the cytoplasmic compartment to the extracellular environment or the endoplasmic reticulum.

[0012] The lower limit of the irradiation dose of light in the wavelength range of 315 to 325 nm to be irradiated onto cells is, for example, 10 mJ / cm 2 , 50 mJ / cm 2 , 100mJ / cm 2 , 200mJ / cm 2 , 300mJ / cm 2 , 400mJ / cm 2 or 500mJ / cm 2 The upper limit may be, for example, 5000 mJ / cm 2 , 4500mJ / cm 2 , 4000mJ / cm 2 , 3500mJ / cm 2 , 3000mJ / cm 2 , 2500mJ / cm 2 , 2000mJ / cm 2 or 1500mJ / cm 2 The irradiation dose of light in the wavelength range of 315 to 325 nm is 10 mJ / cm 2If the irradiation dose is less than 5000mJ / cm, it is highly likely that the induction of calcium channel and / or calcium pump expression will not be achieved. 2 If the concentration exceeds this range, it is considered that there is a high possibility that the cells will be significantly damaged and that the expression of calcium channels and / or calcium pumps will not be induced. More specifically, the dose of light in the wavelength range of 315 to 325 nm irradiated onto cells can be within a range represented by any combination of lower and upper limits selected from the above-mentioned lower and upper limits. A specific example of the range of the dose of light in the wavelength range of 315 to 325 nm is 10 to 5000 mJ / cm. 2 , more specifically 50 to 4500 mJ / cm 2 , 100~4000mJ / cm 2 , 200~3500mJ / cm 2 , 300~3000mJ / cm 2 , 400~2500mJ / cm 2 , 500~2000mJ / cm 2 , and 500 to 1500 mJ / cm 2 These include, but are not limited to:

[0013] The same cells may be irradiated once or twice or more times with light in the wavelength range of 315 to 325 nm. When irradiating twice or more times with light in the wavelength range of 315 to 325 nm, the total amount of irradiation for each irradiation may be within the range defined by the above-mentioned lower and upper limits, and the lower limit of the amount of irradiation for each irradiation may be, for example, 5 mJ / cm. 2 , 10 mJ / cm 2 , 20 mJ / cm 2 , 25 mJ / cm 2 , 50 mJ / cm 2 , 100mJ / cm 2 , 150 mJ / cm 2 , 200mJ / cm 2 or 250 mJ / cm 2 and the upper limit is 2500 mJ / cm 2 , 2000mJ / cm 2 , 1500mJ / cm 2 , 1000mJ / cm 2 , 750mJ / cm 2, 500mJ / cm 2 or 300mJ / cm 2 When irradiating two or more times, the number of irradiations may be, for example, 2 to 10 times, more specifically, 2 to 8 times, 2 to 6 times, or 2 to 4 times, and the irradiation interval may be, for example, once every 1 to 48 hours, more specifically, once every 3 hours, once every 4 hours, once every 6 hours, once every 12 hours, once every 24 hours (a day), or once every 48 hours.

[0014] On the other hand, since the absorption maximum wavelength of DNA and RNA is around 260 nm, and proteins absorb light around 200 nm and 280 nm, there is concern that light in the wavelength range of around 200 to 280 nm may have a significant adverse effect on cells. Therefore, preferably, light in the wavelength range of 200 to 300 nm (more preferably, 200 to 310 nm, more preferably, 200 to 315 nm) is not irradiated onto the target cells (i.e., the amount of irradiation is 0%), or the amount of irradiation is less than 50%, more preferably less than 40%, more preferably less than 30%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, and more preferably less than 1% of the amount of irradiation of light in the wavelength range of 315 to 325 nm.

[0015] In addition, since light in the wavelength range of 330 to 400 nm is thought to not contribute to the induction of calcium channel and / or calcium pump expression in cells and is more likely to damage cells, if the irradiance of light in the wavelength range of 330 to 400 nm is 50% or more of the irradiance of light in the wavelength range of 315 to 325 nm, the induction of expression cannot be achieved efficiently. From the viewpoint of avoiding adverse effects on the irradiated cells and from the viewpoint of energy efficiency, preferably, light in the wavelength range of 330 to 400 nm is not irradiated to the cells (i.e., the irradiance is 0%), or the irradiance is less than 50%, more preferably less than 40%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, and more preferably less than 1%.

[0016] Light in the wavelength range of 315 to 325 nm has an intensity of, for example, 0.1 to 300 mW / cm 2 The cells are irradiated with an illuminance of 0.1 mW / cm 2 If the intensity is less than 300 mW / cm, it is highly likely that the induction of calcium channel and / or calcium pump expression will not be achieved efficiently. 2 If the intensity exceeds this range, it is highly likely that cell damage will be induced. More specifically, light in the wavelength range of 315 to 325 nm has an intensity of 0.5 to 200 mW / cm. 2 , more specifically 1 to 100 mW / cm 2 , more specifically 5 to 50 mW / cm 2 , more specifically 5 to 20 mW / cm 2 In the present disclosure, the irradiance refers to the irradiance at the level of the cells to be irradiated. For biological tissues such as skin that are typically exposed to UV light, a specific dose of 300 mW / cm is required. 2 In addition, when culturing cells in mucous membranes, organs, or artificial environments, which are sensitive to strong ultraviolet light, it is possible to increase the illuminance and complete the irradiation in a short time. 2It is preferable to reduce the illuminance and irradiate for a longer period of time.

[0017] In the present disclosure, the light irradiated onto cells may include light in the wavelength range of 315 to 325 nm, and more specifically, light having a main peak wavelength in the wavelength range of 315 to 325 nm. Irradiating cells with such light can induce the expression of calcium channels and / or calcium pumps in the cells. Measurement of the wavelength range, intensity distribution in each wavelength range, main peak wavelength, and full width at half maximum can be performed by methods known in the art, for example, using a spectrometer. In this specification, the term "main peak wavelength" refers to the peak wavelength at which the intensity is maximum. For light whose spectrum exhibits a single peak, such as LED light, the term "peak wavelength" is synonymous with the term "main peak wavelength." It is preferable that light in the wavelength range of 315 to 325 nm is irradiated to two or more cells simultaneously. In other words, it is preferable that the irradiation area of ​​light in the wavelength range of 315 to 325 nm includes two or more cells. The lower limit of the irradiation area of ​​light in the wavelength range of 315 to 325 nm is, for example, 0.1 cm. 2 , 0.2cm 2 , 0.5cm 2 , 1cm 2 , 2cm 2 , 5cm 2 , 10cm 2 , 20cm 2 , 50cm 2 , 100cm 2 , 200cm 2 or 500cm 2 The upper limit may be, for example, 10000 cm 2 , 5000cm 2 , 3000cm 2 or 1000cm 2 It could be. More specifically, the irradiation range of light in the wavelength range of 315 to 325 nm can be within a range represented by any combination of lower and upper limits selected from the above-mentioned lower and upper limits. Specific examples of the range of the irradiation range of light in the wavelength range of 315 to 325 nm include 1 to 10,000 cm. 2 , more specifically 5 to 10,000 cm 2 , more specifically 10 to 10,000 cm2 , more specifically 20 to 10,000 cm 2 , more specifically 20 to 5000 cm 2 , more specifically 20 to 3000 cm 2 , more specifically 50 to 3000 cm 2 These include, but are not limited to:

[0018] Light in the wavelength range of 315 to 325 nm may be or may include artificial light. In this disclosure, "artificial light" refers to light emitted from an artificial light source and light obtained by artificially extracting a desired wavelength range component from natural light (sunlight in most cases) using, for example, an optical filter. The artificial light source is not particularly limited as long as it can irradiate light in the wavelength range of 315 to 325 nm, and examples thereof include light-emitting diodes (LEDs), laser diodes (LDs), xenon lamps, fluorescent lamps, incandescent lamps, metal halide lamps, and high-pressure mercury lamps. When the light source used emits light in the wavelength range of 200 to 300 nm (or wavelength range of 200 to 310 nm) together with light in the wavelength range of 315 to 325 nm at a radiation intensity that is 20% or more of the radiation intensity of light in the wavelength range of 315 to 325 nm, by using a filter whose transmittance for light in the wavelength range of 315 to 325 nm is greater than the transmittance for light in the wavelength range of 200 to 300 nm (or wavelength range of 200 to 310 nm), the irradiance or illuminance of light in the wavelength range of 200 to 300 nm (or wavelength range of 200 to 310 nm) irradiated to the target cells may be less than 20% of the irradiance or illuminance of light in the wavelength range of 315 to 325 nm, more specifically, less than 15%, less than 10%, less than 5%, or less than 1%. Additionally or alternatively, when the light source used emits light in the wavelength range of 315 to 325 nm as well as light in the wavelength range of 330 to 400 nm at a radiation intensity that is 50% or more of the radiation intensity of light in the wavelength range of 315 to 325 nm, by using a filter whose transmittance for light in the wavelength range of 315 to 325 nm is greater than that for light in the wavelength range of 330 to 400 nm, the irradiance or illuminance of light in the wavelength range of 330 to 400 nm irradiated onto the target cells may be less than 50% of the irradiance or illuminance of light in the wavelength range of 315 to 325 nm, more specifically, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%.

[0019] From the viewpoint of energy efficiency, the light in the wavelength range of 315 to 325 nm is irradiated as light having a main peak wavelength within, for example, 320±5 nm, more preferably 320±4 nm, more preferably 320±3 nm, or more preferably 320±2 nm. It is preferable that there is no second peak in the spectrum (i.e., the spectrum shows a single peak), or if there is a second peak, its intensity is 1 / 10 or less of the main peak. The full width at half maximum of the main peak is, for example, 1 to 20 nm, preferably 1 to 15 nm, more preferably 1 to 10 nm, and more preferably 1 to 5 nm. Having a full width at half maximum of the main peak of 20 nm or less enables selective irradiation of cells with light in a wavelength range effective for inducing calcium channel and / or calcium pump expression while avoiding irradiation with light in a wavelength range that does not contribute to the induction of calcium channel and / or calcium pump expression, thereby further improving energy efficiency. Light with a full width at half maximum of the main peak of less than 1 nm can be used by cutting out light in unnecessary wavelength ranges with a filter or the like. However, from the perspective of cost-effectiveness, it is currently preferable to use light with a full width at half maximum of the main peak of 1 nm or more. In some preferred specific embodiments, the light irradiated onto cells is light having a wavelength spectrum with a peak wavelength of 320±5 nm (more specifically, 320±3 nm) and a full width at half maximum of 1 to 20 nm (more specifically, 1 to 10 nm, more specifically, 1 to 5 nm).

[0020] As a light source for irradiating cells with light, a light-emitting diode (LED) or a laser diode (LD) having a single peak in the emission spectrum is particularly preferred. LEDs capable of emitting light in the wavelength range of 315 to 325 nm may be made of, for example, AlGaN-based materials or InAlGaN-based materials. When an LED or LD is used as a light source, it is easy to selectively irradiate cells with light in a wavelength range effective for inducing calcium channel and / or calcium pump expression while avoiding irradiation with light in a wavelength range that does not contribute to the induction of such expression. Furthermore, the use of an LED or LD is also preferable from the standpoints of energy efficiency and economy due to its energy-intensive nature, low heat generation, low power consumption, and long life. In addition, it is easy to control / manage the irradiation amount and / or illuminance. Furthermore, the use of an LED or LD allows for targeted irradiation of only cells in a required area (target cells) on a plane, thereby reducing or preventing the effects on surrounding cells other than the target cells. When light in the wavelength range of 315 to 325 nm is irradiated as laser light, the light in the wavelength range of 315 to 325 nm can be irradiated in combination with laser light of one or more other wavelengths (for example, laser light of about 640 nm and / or laser light of about 960 nm). By irradiating in combination with laser light of one or more other wavelengths, it is possible to irradiate cells inside (for example, in a deep region) of stacked cells without significantly affecting cells in the layer region by utilizing the multiphoton absorption phenomenon of two, three, or four or more photons. It can also be used inside the body by using an implantable optical device, endoscope, or catheter.

[0021] Light in the wavelength range of 315 to 325 nm may be irradiated onto cells that are irradiated with light (including natural light) from a light source other than a light source that emits light in the wavelength range of 315 to 325 nm. In this case, in the synthesized light or mixed light of the light from the light source that emits light in the wavelength range of 315 to 325 nm and the light from the other light source, the irradiance or illuminance of light in the wavelength range of 200 to 300 nm (or wavelength range of 200 to 310 nm) is preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, and more preferably less than 1% of the irradiance or illuminance of light in the wavelength range of 315 to 325 nm. In the synthetic light or mixed light, the amount of irradiation or illuminance of light in the wavelength range of 330 to 400 nm is preferably less than 50% of the amount of irradiation or illuminance of light in the wavelength range of 315 to 325 nm, more preferably less than 40%, more preferably less than 30%, more preferably less than 25%, more preferably less than 20%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, and more preferably less than 1%.

[0022] The amount of light irradiated onto a cell in the 315-325 nm wavelength range can be adjusted, for example, by controlling the on / off of a light source that emits light in the 315-325 nm wavelength range at a constant radiant intensity (i.e., controlling the on-time), or by controlling the radiant intensity of light in the 315-325 nm wavelength range from a light source that is on for a certain period of time, or by controlling the radiant intensity of light in the 315-325 nm wavelength range emitted from the light source and the on / off of the light source. The amount of light irradiated onto a cell in the 315-325 nm wavelength range may be measured by a photosensor (more specifically, an illuminance sensor) placed near the cell to be irradiated, and may be adjusted as described above based on the output signal from the photosensor.

[0023] Light in the wavelength range of 315 to 325 nm may be irradiated to cells as continuous light, intermittent light (e.g., pulsed light), or a combination thereof. Light in the wavelength range of 315 to 325 nm is preferably irradiated as intermittent light. By using intermittent light, temperature increases in the cells and / or the light source can be avoided or reduced. A specific example of intermittent light is pulsed light having a pulse width of 100 ms or less, more specifically 50 ms or less, more specifically 20 ms or less, more specifically 10 ms or less, or more specifically 5 ms or less, and a duty ratio of 50% or less, more specifically 40% or less, more specifically 30% or less, more specifically 20% or less, more specifically 10% or less, or more specifically 5% or less.

[0024] It is preferable that light in the wavelength range of 315 to 325 nm does not pass through any environment other than that required for cell survival or proliferation (e.g., air, an atmosphere commonly used for cell culture (e.g., 95% air / 5% CO2), physiological saline, or culture medium) between the irradiation surface of the irradiation unit (see below) that emits the light and the cells. When the cells to be irradiated are cultured cells, light in the wavelength range of 315 to 325 nm may be irradiated onto the cells by passing through a culture vessel containing the cells (specifically, the lid, sidewall, or bottom of the culture vessel). In this case, the culture vessel is preferably made of a material that has a transmittance for light in the wavelength range of 315 to 325 nm of 40% or more, more specifically 50% or more, more specifically 60% or more, more specifically 70% or more, more specifically 75% or more, more specifically 80% or more, more specifically 85% or more, and more specifically 90% or more. Such materials may be, for example, glass or plastic, more specifically, acrylic resin, polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, and the like.

[0025] The cells may be irradiated with light in the wavelength range of 315 to 325 nm from one direction, or from two or more directions. When irradiation is performed from two directions, the two directions are preferably, but not limited to, two opposite directions such as up-down, left-right, or front-back. When the cells to be irradiated are cultured on a substrate (e.g., an agar medium) that has low transmittance (e.g., less than 50%) to light in the wavelength range of 315 to 325 nm, it is preferable to irradiate the cells from above so that the light in the wavelength range of 315 to 325 nm does not pass through the substrate. Irradiation of cells with light in the wavelength range of 315 to 325 nm is preferably carried out in a closed space surrounded by a material that can block light in the wavelength range of 315 to 325 nm (for example, having a transmittance of less than 5%, more preferably less than 1%, and more preferably less than 0.5%).

[0026] In the present disclosure, the cell is not particularly limited and may be any type of cell at any developmental stage, and specific examples of the cell include bacterial cells, yeast cells, plant cells, and animal cells. The cells may be naturally occurring cells (including cancerous cells), artificially transformed cells (e.g., immortalized cells), genetically engineered cells, or fused cells (also called "hybridomas"). The cells are preferably eukaryotic cells. Cells derived from multicellular organisms may consist of only one type of cell or may be a mixture of two or more types of cells. In some embodiments, the cell may be a yeast cell, a plant cell, or an animal cell. The animal may be a human or non-human animal. Non-human animals include companion animals, pet animals, zoo animals, farm animals (e.g., livestock), wild animals, etc. The non-human animal may be, for example, a vertebrate, preferably selected from non-human mammals, birds, and fish, more preferably from non-human mammals and birds, more preferably from non-human mammals. The animal cell may preferably be a human cell or a non-human mammal cell.

[0027] When the cell is an animal cell, it may be a totipotent stem cell, a pluripotent stem cell, a multipotent stem cell, a progenitor cell, a differentiated cell, or any mixture thereof. Specific examples of pluripotent stem cells include embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Specific examples of multipotent stem cells include tissue stem cells and somatic stem cells. Examples of differentiated cells include skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neurons, adipocytes, epithelial cells, endothelial cells, immune cells, bone marrow cells, blood cells, and skin cells. In some specific embodiments, the cell may be a skin cell. Skin cells include keratinocytes, fibroblasts, melanocytes, and Langerhans cells. In other specific embodiments, the cell is an excitable cell. Excitable cells include neurons, glial cells, muscle cells (including cardiac muscle cells, skeletal muscle cells, and smooth muscle cells), and endocrine cells. Animal cells (especially human cells) are preferably irradiated ex vivo with light in the wavelength range of 315 to 325 nm.

[0028] The plant or animal cells may be primary cultured cells, subcultured cells, or tissue or organ cultured cells (e.g., skin tissue culture cells). The cell culture may be either adherent culture or non-adherent culture (e.g., suspension culture). In an embodiment in which the cells to be irradiated are in adhesion culture, the cells are irradiated with light in the wavelength range of 315 to 325 nm at an irradiation dose that does not cause cell detachment.In an embodiment in which the cells to be irradiated are in suspension culture, the cells are irradiated with light in the wavelength range of 315 to 325 nm at an irradiation dose that does not cause cell adhesion.

[0029] Cell culture conditions can be appropriately selected depending on the type of cell used. For example, for animal cell culture conditions, the temperature can be set to approximately 36°C to approximately 37°C, the relative humidity can be set to approximately 90% to approximately 95%, the CO2 concentration can be set to approximately 4% to approximately 10% (more specifically, approximately 5% to approximately 7%), and the pH can be set to approximately neutral to slightly alkaline (e.g., approximately 7.0 to approximately 7.7, more specifically, approximately 7.2 to approximately 7.4). The culture medium can be appropriately selected from known media depending on the type of cells used, and can be, for example, MEM, DMEM, F12, F10, M199, DMEM / F12, RPMI 1640, BME, or IMDM, or any combination or modified medium of these media. If the cells are cells with differentiation potential, the culture medium can be a differentiation-inducing medium. The differentiation-inducing medium can be a known medium (e.g., E5) depending on the type of cells used and the type of differentiated cells to be targeted. TM Medium, E6 TM Medium, E8 TM Medium, TeSR TM Medium, TeSR TM -E8 TM The medium can be appropriately selected from the following. The medium may contain additives commonly used in the art, if necessary. Additives are known in the art, and examples include buffers (e.g., HEPES, DPBS, HBSS, bicarbonate buffer), amino acids (e.g., L-glutamine), vitamins, EDTA, antibiotics, serum, and serum albumin. When the cells are differentiation-competent cells, the additives may include growth factors and / or cytokines. Specific examples of growth factors and cytokines include epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), stem cell factor (SCF), activin A, bone morphogenetic protein (BMP), interferon (INF), interleukin, tumor necrosis factor (TNF), and transforming growth factor (TGF).

[0030] Cells irradiated with light in the wavelength range of 315 to 325 nm may be subjected to measurement of physiological events involving calcium signaling. Measurement of physiological events involving calcium signaling can confirm the induction of calcium channel and / or calcium pump expression in the cells. In the present disclosure, "measurement" refers to detection, identification, or quantification (including estimation of quantity) of the object of measurement. Physiological events involving calcium signaling may be, for example, one or more selected from the group consisting of muscle contraction (particularly, extension of contraction volume and / or contraction duration), neurotransmission (particularly, an increase in transmission efficiency (e.g., the amount of neurotransmitter released)), mitochondrial activity (e.g., the degree of membrane potential change), cell death, glycogen degradation (particularly, promotion of glycogen degradation), cell differentiation (particularly, promotion of differentiation into osteoblast-like cells), amino acid biosynthesis, carbon metabolism, and antibiotic biosynthesis. Preferably, the physiological events are one or more selected from the group consisting of muscle contraction, neurotransmission, cell death, and osteoblast-like differentiation.

[0031] Cells in which calcium channel and / or calcium pump expression is induced by the above-described methods of the present disclosure can be used, for example, to study and / or control physiological events involving calcium signaling, such as the control of muscle contraction, neurotransmission, mitochondrial activity, cell death, promotion of glycogen breakdown, and promotion of differentiation into osteoblast-like cells. Alternatively, cells in which calcium channel and / or calcium pump expression is induced by the above-described methods of the present disclosure can be used to search for substances that can interact with calcium channels and / or calcium pumps (e.g., DHPR, VGCC, RYR, or SERCA). Thus, in one aspect, the present disclosure provides a method of modulating calcium signaling in a cell, the method comprising: inducing expression of calcium channels and / or calcium pumps in the cells by the above method; and measuring a physiological event involving calcium signaling in the cell. Physiological events involving calcium signaling can be measured by any known method, depending on the subject of measurement, such as one or more physiological events selected from the group consisting of muscle contraction, neurotransmission, cell death, and osteoblast-like differentiation.

[0032] From another aspect, the present disclosure provides a method for producing a cell in which expression of a calcium channel and / or a calcium pump is enhanced, the method comprising: Provided is a method comprising the steps of irradiating cells with light in the wavelength range of 315 to 325 nm and freezing the cells irradiated with the light, wherein in the irradiating step, the cells are not irradiated with light in the wavelength range of 200 to 300 nm, or the amount of irradiation is less than 30% of the amount of irradiation of light in the wavelength range of 315 to 325 nm, and the cells are not irradiated with light in the wavelength range of 330 to 400 nm, or the amount of irradiation is less than 50% of the amount of irradiation of light in the wavelength range of 315 to 325 nm.

[0033] The irradiation step is as described above for the method for inducing calcium channel and / or calcium pump expression. The method for freezing the irradiated cells is not particularly limited, and any known method that can be used for cryopreserving cells can be used. Cell freezing can be performed by, for example, slow freezing, vitrification, or ultra-rapid vitrification. For example, cells can be frozen in a suitable container in a suspension state in a suitable culture medium (such as those described above). The container can be, for example, a tube, vial, well plate, or cell freezing container (e.g., Bicell Freezing Container (Nippon Freezer), CoolCell® (Corning), or Mr. Frosty® (Thermo Fisher Scientific)). A cryoprotectant may be added to the culture medium. Any known cryoprotectant that can be used for cryopreservation of cells can be used, such as dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, propylene glycol, sucrose, glycerin, polyvinylpyrrolidone, sorbitol, dextran, trehalose, or hydroxyethyl starch. The concentration of the cryoprotectant added is usually 2 to 10% (w / v or v / v). Frozen cells can be stored, for example, in a deep freezer, for example at -50°C or below, or in liquid nitrogen, for example at -180°C or below. As described above, the expression of calcium channels and / or calcium pumps is induced in cells that have undergone the irradiation step, and thus the expression is enhanced. By freezing the cells, the enhanced state of expression of calcium channels and / or calcium pumps can be maintained even after thawing. Therefore, after thawing, the frozen cells can be easily used, for example, as an evaluation system for the effects on calcium channels and / or calcium pumps and / or calcium fluxes.

[0034] <Cell culture equipment> In another aspect, the present disclosure provides a cell culture device, A (first) irradiation unit capable of mainly emitting light in the wavelength range of 315 to 325 nm; a (first) placement unit for placing a cell culture vessel thereon, the placement unit being located within an irradiation area of ​​the (first) irradiation unit; an irradiation control unit that controls the (first) irradiation unit; a housing that houses the (first) irradiation unit and the (first) placement unit; The present invention is characterized by comprising: The cell culture device of the present disclosure is suitable for carrying out the method of inducing expression of calcium channels and / or calcium pumps of the present disclosure. As described above, the expression of calcium channels and / or calcium pumps is induced in cells irradiated with light in the wavelength range of 315 to 325 nm using the cell culture device of the present disclosure. Therefore, the cells after irradiation can be subsequently cultured for research and / or control of physiological events involving calcium signaling. Cells suitable for culturing using the cell culture device of the present disclosure are the cells as explained above in <Method for inducing expression of calcium channel and / or calcium pump>.

[0035] The cell culture device of the present disclosure includes at least one irradiation unit capable of mainly emitting light in the wavelength range of 315 to 325 nm within a housing. Preferably, the irradiation unit is capable of substantially exclusively emitting light in the wavelength range of 315 to 325 nm. As used herein, "primarily emitting" or "capable of emitting" light in a certain wavelength range means that the radiation amount of emitted light in that specific wavelength range is 50% or more, more specifically 60% or more, more specifically 70% or more, more specifically 80% or more, and even more specifically 90% or more of the radiation amount of light in all wavelength ranges emitted simultaneously, or that it is capable of being emitted in that manner. As used herein, "substantially exclusively emitting" or "capable of substantially exclusively emitting" light in a certain wavelength range means that the radiation amount of emitted light in that specific wavelength range is 95% or more, more specifically 98% or more, and even more specifically 99% or more of the radiation amount of light in all wavelength ranges emitted simultaneously, or that it is capable of being emitted in that manner.

[0036] The irradiation unit includes at least one light source capable of emitting light in the wavelength range of 315 to 325 nm. Examples of such light sources that can be used include light-emitting diodes (LEDs), laser diodes (LDs), xenon lamps (equipped with necessary filters), metal halide lamps, and high-pressure mercury lamps. When the proportion of components in this wavelength range (the proportion of radiant intensity relative to light in the entire wavelength range) in the light emitted by the light source itself to be used is relatively low (for example, below 30%, more specifically below 40%, or even below 50%), it is preferable to increase this proportion by using a filter whose transmittance for light in the wavelength range of 315 to 325 nm is higher than its transmittance for light outside of this wavelength range.

[0037] From the viewpoint of energy efficiency, the light source that emits light in the wavelength range of 315 to 325 nm is preferably a light source that mainly emits light in the wavelength range of 315 to 325 nm, and more preferably a light source that substantially exclusively emits light in the wavelength range of 315 to 325 nm. A light source that primarily or substantially exclusively emits light in the wavelength range of 315 to 325 nm may be a light source that emits light having a main peak wavelength in the wavelength range of 315 to 325 nm, preferably in the wavelength range of 316 to 324 nm, more preferably in the wavelength range of 317 to 323 nm, and more preferably in the wavelength range of 318 to 322 nm. More specifically, such a light source may be a light source (particularly one having a single peak) that emits light having a wavelength spectrum with a peak wavelength of 320±5 nm (preferably 320±3 nm) and a full width at half maximum of 1 to 20 nm (preferably 1 to 10 nm, more preferably 1 to 5 nm). Specific examples of such light sources are light-emitting diodes (LEDs) or laser diodes (LDs). LDs or LEDs may be provided as arrays or clusters. The use of LEDs or LDs is preferable from the standpoints of energy efficiency and economy due to their energy-saving, low heat generation, low power consumption, and long life. In addition, it facilitates control or management of illuminance or irradiation amount.

[0038] The illumination unit may include a light diffusing member and / or a light reflecting member on the emission path of the light source. The light diffusing member is not particularly limited and may be a known member, specific examples of which include frosted, opal, and holographic diffusing members. The light reflecting member is not particularly limited and may be a known member, specific examples of which include a mirror and a prism. In some specific embodiments, the illumination unit includes a surface light emitter including a light source (e.g., an LD or LED), a light guide member, and a light diffusing member and / or a light reflecting member. The irradiating unit is preferably capable of irradiating the cell culture vessel with light in the wavelength range of 315 to 325 nm at a substantially uniform illuminance.

[0039] The irradiation unit may be capable of emitting (e.g., mainly or substantially exclusively) light in a specific wavelength range outside the wavelength range of 315 to 325 nm. In other words, the irradiation unit may be equipped with a light source that emits (preferably mainly, more preferably substantially exclusively) light in the wavelength range of 315 to 325 nm, as well as a light source that emits (e.g., mainly or exclusively) light in another wavelength range. Alternatively, the irradiation unit may be equipped with an optical filter that transmits light in the wavelength range of 315 to 325 nm and an optical filter that transmits light in another wavelength range, and may be configured to emit (preferably mainly, more preferably substantially exclusively) light in a desired wavelength range by switching between the optical filters. The light in the other wavelength range may be, for example, light in the wavelength range of 100 to 290 nm, more specifically 190 to 290 nm, more specifically 200 to 280 nm, and even more specifically 240 to 280 nm or 210 to 220 nm. Light in this wavelength range can be used for sterilizing or disinfecting the mounting section and / or the atmosphere surrounding the mounting section (i.e., as sterilizing or disinfecting light) before and / or after cell culture. A germicidal lamp known in the art can be used as a light source emitting light in such a wavelength range, and more specifically, an LED or LD can be used. Another example of light in another wavelength range is white light. White light can be used as illumination during work and / or observation. Examples of white light sources include fluorescent lamps, incandescent lamps, white lamps, and white LEDs, with white LEDs being preferred.

[0040] The irradiating unit can be arranged so as to irradiate light from at least one of above (for example, directly above), below (for example, directly below), and side (for example, directly beside) the placing unit. In some embodiments, the irradiation unit is disposed above the mounting unit (and thus above the cell culture vessel mounted on the mounting unit). In this case, it is possible to avoid attenuation of the light in the wavelength range of 315 to 325 nm irradiated from the irradiation unit due to transmission through an ultraviolet light-absorbing substrate (such as an agar medium), and therefore it is easy to control the amount or illuminance of light in the wavelength range of 315 to 325 nm irradiated to the cells.

[0041] The cell culture device of the present disclosure includes an irradiation control unit that controls the irradiation unit. When the cell culture device of the present disclosure includes multiple irradiation units, different irradiation control units may control different irradiation units, or one irradiation control unit may control two or more irradiation units, or may control all of the irradiation units included in the device. The irradiation control unit may control the irradiation unit so that the light emitted from the irradiation unit is continuous light, intermittent light, or a combination thereof. When intermittent light is used, temperature rise of the cells and / or the irradiation unit (particularly its light source) can be avoided or reduced. The irradiation control unit may control the irradiation unit so that the light emitted from the irradiation unit is pulsed light with a pulse width of, for example, 100 ms or less, more specifically 50 ms or less, more specifically 20 ms or less, more specifically 10 ms or less, and a duty ratio of 50% or less, more specifically 40% or less, more specifically 30% or less, more specifically 20% or less, more specifically 10% or less, and more specifically 5% or less.

[0042] The irradiation control unit may also control the irradiation unit so that the illuminance (on the placement surface) of light in the wavelength range of 315 to 325 nm emitted from the irradiation unit falls within a predetermined range. The predetermined illuminance range can be appropriately set depending on the cells used and / or the degree of the desired effect (induction of calcium channel and / or calcium pump expression), but is, for example, 0.1 to 300 mW / cm2 More specifically, it can be 0.5 to 200 mW / cm 2 , more specifically 1 to 100 mW / cm 2 , more specifically 5 to 50 mW / cm 2 , more specifically 5 to 20 mW / cm 2 By controlling the illuminance of the light from the irradiation unit (hence, the radiation intensity of the light emitted from the irradiation unit) within the above range, it is possible to provide an illuminance or irradiation amount that is effective in achieving the desired effect on the cells in the cell culture vessel placed on the placement unit. 2 Less than or equal to 300mW / cm 2 If it exceeds this limit, the desired effect may not be achieved efficiently. The illumination control for these purposes can be, for example, a pulse width modulation circuit.

[0043] The irradiation control unit may also control the irradiation unit so that the irradiation time of the light in the wavelength range of 315 to 325 nm emitted from the irradiation unit is within a predetermined range. The lower limit of the irradiation time may be, for example, 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 1 minute, 2 minutes, or 5 minutes, and the upper limit may be, for example, 5 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 10 minutes. More specifically, the irradiation time may be within a range represented by any combination of lower and upper limits selected from the above-mentioned lower and upper limits. Specific examples of the range of irradiation time include, but are not limited to, 5 seconds to 5 hours, more specifically 5 seconds to 2 hours, more specifically 5 seconds to 1 hour, more specifically 5 seconds to 30 minutes, more specifically 10 seconds to 30 minutes, and more specifically 10 seconds to 15 minutes. An illumination control for this purpose can be, for example, a timer.

[0044] In a specific embodiment, the irradiation control unit controls the illuminance and irradiation time (or irradiation amount) of light in the wavelength range of 315 to 325 nm emitted from the irradiation unit on the placement unit. In this case, the irradiation control unit may be composed of, for example, a pulse width modulation circuit and a timer. In a more specific embodiment, the irradiation control unit controls the irradiation amount from the irradiation unit to be, for example, 10 to 3000 mJ / cm 2 , more specifically 50 to 2000 mJ / cm 2 , 100-1000mJ / cm 2 , 200-500mJ / cm 2 , 300-400mJ / cm 2 The irradiation unit may be controlled so that the irradiation intensity falls within the range. The irradiation control unit may control the irradiation unit based on a signal received from an optical sensor installed at the level of the placement surface within the irradiation area of ​​the irradiation unit.

[0045] In an embodiment in which the irradiation unit is also capable of emitting light in a specific wavelength range outside the wavelength range of 315 to 325 nm (for example, sterilizing light or white light), the irradiation control unit may control the irradiation unit so that light in the specific wavelength range outside the wavelength range of 315 to 325 nm can be emitted only in specific cases, or cannot be emitted in specific cases. For example, in a specific embodiment in which the irradiation unit is also capable of emitting sterilizing or disinfecting light, the irradiation unit may be controlled to emit sterilizing or disinfecting light only when a door (particularly an inner door) provided on the housing is closed. Alternatively or additionally, the irradiation control unit may control the irradiation unit not to emit sterilizing or disinfecting light simultaneously with light in the wavelength range of 315 to 325 nm.

[0046] The cell culture device of the present disclosure includes, in a housing, at least one mounting section for mounting a cell culture vessel thereon. The two or more placement sections can be provided at different positions in the horizontal direction and / or the up-down direction (preferably the vertical direction). The mounting section is located within the irradiation area of ​​the irradiation section. Therefore, when a cell culture vessel containing cells is placed on the mounting section, the cells can be irradiated with light in the wavelength range of 315 to 325 nm emitted from the irradiation section. In an embodiment including two or more mounting sections, the two or more mounting sections may be located within the irradiation area of ​​one irradiation section, or may be located within the irradiation area of ​​two or more irradiation sections, or different mounting sections may be located within the irradiation areas of different irradiation sections. The mounting portion is not particularly limited as long as it has a structure and size that allows the cell culture vessel to be mounted on its upper surface, and can be designed appropriately depending on the cell culture vessel. The cell culture vessel used with the cell culture device of the present disclosure can be any vessel known in the art for cell culture, such as a petri dish, a dish, a beaker, a flask, a bottle, or a multi-well plate. The mounting portion may be composed of, for example, a shelf, a shelf net, the upper surface of a stand or a pedestal, or at least a portion of the inner bottom surface of a housing. The mounting surface is not limited to a single continuous surface, but may be composed of multiple separate surfaces, or may be, for example, a surface such as the upper surface of a mesh or lattice-like shelf. The material constituting the mounting surface may be, for example, metal (e.g., iron, stainless steel, aluminum, brass, etc.), glass, or plastic (e.g., acrylic resin, polycarbonate, polyvinyl chloride, etc.). In some embodiments, the mounting sections are made of a material that is opaque to light in the wavelength range of 315 to 325 nm. According to this embodiment, particularly when light in the wavelength range of 315 to 325 nm is irradiated from above, the light can be prevented from passing through the mounting sections and irradiating the mounting sections that may be present below (and therefore the cells in the cell culture vessels placed on the mounting sections), thereby facilitating individual control of the irradiation amount or illuminance of light in the wavelength range of 315 to 325 nm on each mounting section.

[0047] The mounting portion may have a recess or a protrusion that defines an area for mounting the cell culture vessel. In the case of a recess, the cell culture vessel can be mounted on the bottom surface of the recess. In the case of a protrusion, the cell culture vessel can be mounted on the upper surface of one protrusion, or can be mounted within an area defined by multiple protrusions. One mounting section may be configured so that only one cell culture vessel can be mounted thereon, or may be configured so that two or more cell culture vessels can be mounted thereon.

[0048] The cell culture device of the present disclosure includes a housing that houses at least a mounting unit and an irradiation unit. The irradiation control unit may be disposed outside the housing or may be built into the housing. The housing may have an opening on one surface (preferably a side surface), and may be provided with a door for opening and closing the opening. The door may be composed of an inner door and an outer door. At least a portion of the inner door is made of a material (e.g., glass containing an inorganic ultraviolet absorber) or member (e.g., glass or a plastic plate provided with a film of an organic ultraviolet absorber) that has a transmittance of 50% or more (preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more) for visible light and a transmittance of 20% or less (preferably 10% or less, more preferably 5% or less, more preferably 1% or less) for light in the wavelength range of 315 to 325 nm, so that the cell culture compartments inside the housing can be observed. The shape of the housing may be, for example, a cube, a rectangular parallelepiped, etc. The housing is preferably made of a material that is opaque to light in the wavelength range of 315 to 325 nm, such as metal (e.g., iron, stainless steel, aluminum, brass, etc.). The internal space of the housing may be surrounded by a heat insulating material. In an embodiment in which the placing portion is a part of a shelf, a shelf net, or a stand, a support member for the shelf, the shelf net, or the stand is provided inside the housing.

[0049] The interior of the housing may be divided into two or more compartments (hereinafter also referred to as "cell culture compartments") in the up-down (or vertical) direction and / or horizontal direction. The two or more cell culture compartments may be formed in the up-down direction by dividing the interior of the housing, for example, with shelves, shelf nets, or racks, or additionally or alternatively, they may be formed in the horizontal direction by dividing the interior of the housing, for example, with side walls. The two or more cell culture compartments are preferably in fluid communication to allow circulation of atmosphere through each compartment, for example, by a shelf or stand having multiple openings (especially outside the irradiation area) or by the presence of a gap between the shelf or stand and one or two interior side walls (e.g., the front and / or rear side walls) of the housing, or by another communication mechanism (e.g., a duct).

[0050] In an embodiment in which the interior of the housing is divided into two or more cell culture compartments, the cell culture device of the present disclosure may further include a shielding member that blocks light in the wavelength range of 315 to 325 nm (e.g., having a transmittance of less than 5%, more preferably less than 1%, more preferably less than 0.5%). The shielding member is arranged in the housing so as to prevent light in the wavelength range of 315 to 325 nm from an irradiation unit arranged in one cell culture compartment (e.g., an upper compartment) from being irradiated onto a mounting unit arranged in another cell culture compartment (e.g., a lower compartment). In this embodiment, one set of an irradiation unit and a mounting unit is arranged in each cell culture compartment, and in each compartment, the mounting unit is arranged outside the irradiation area of ​​the irradiation unit located in the other compartment. According to this embodiment, it becomes possible to culture cells in multiple cell culture vessels under irradiation with light in the wavelength range of 315 to 325 nm at an illuminance controlled for each vessel or for each group of vessels.

[0051] Therefore, in some preferred embodiments, the cell culture device of the present disclosure includes, in the housing: a second irradiation unit capable of mainly emitting light in the wavelength range of 315 to 325 nm; a second mounting unit for mounting a cell culture vessel thereon, the second mounting unit being located within an irradiation area of ​​the second irradiation unit; a second irradiation control unit that controls the second irradiation unit; A shielding member that blocks light in the wavelength range of 315 to 325 nm. Further provided with the second irradiation unit is located below the first placement unit, The shielding member is positioned so as to prevent light in the wavelength range of 315 to 325 nm emitted from the first irradiation unit from being irradiated onto the second placement unit (i.e., the second placement unit is positioned outside the irradiation area of ​​the first irradiation unit). The cell culture device of this embodiment can include two or more shielding members in the housing as needed, and therefore three or more sets of irradiation units and mounting units. The first irradiation control unit may also serve as the second irradiation control unit. The mounting unit may be formed on the upper surface of the shielding member. In other words, the upper surface of the shielding member may also serve as the mounting unit. The shielding member is made of, for example, a metal (for example, iron, stainless steel, aluminum, brass, etc.).

[0052] The cell culture device of the present disclosure may further include a temperature control mechanism that heats and maintains the atmosphere in the housing and / or the cell culture vessel placed on the placement section at a predetermined temperature. According to this embodiment, the temperature of the cells being cultured in the cell culture device can be maintained at an appropriate temperature, which makes it easy to reduce the influence on cell survival caused by temperature changes before, during, and / or after irradiation with light in the wavelength range of 315 to 325 nm. The temperature adjustment mechanism may be capable of heating to a predetermined temperature or temperature range, for example, around about 36°C to about 37°C. The temperature adjustment mechanism may be composed of, for example, a heat source (for example, a heater, a Peltier element, etc.) and / or a heat medium (for example, water, oil, metal, etc.) and a temperature control unit that controls the temperature of the heat source and / or the heat medium. The temperature adjustment mechanism may be equipped with a temperature sensor that detects the atmosphere inside the housing or the temperature of the cell culture vessel placed on the placement unit.

[0053] The cell culture device of the present disclosure may further include a humidity control mechanism that humidifies and maintains the atmosphere inside the housing within a predetermined humidity level or within a humidity range. The humidity adjustment mechanism may be capable of humidifying the air to a predetermined relative humidity of, for example, about 90% to about 95% or within a relative humidity range. The humidity adjustment mechanism may be, for example, a natural evaporation type humidifier, or may be comprised of a humidity sensor that detects the humidity of the atmosphere inside the housing, a water vapor supply device, and a humidity control unit that controls the water vapor supply device in response to a signal received from the sensor.

[0054] The cell culture device of the present disclosure may further include a CO2 concentration adjustment mechanism that supplies CO2 to and maintains the CO2 concentration in the atmosphere within the housing at a predetermined concentration or within a predetermined concentration range. The CO2 concentration adjustment mechanism may be capable of supplying CO2 to a predetermined CO2 concentration or CO2 concentration range of, for example, about 5% to about 7%. The CO2 concentration adjustment mechanism may be comprised of, for example, a CO2 sensor that detects the CO2 concentration in the atmosphere inside the housing, a CO2 supply device, and a CO2 concentration control unit that controls the CO2 supply device in response to a signal received from the sensor.

[0055] The cell culture device of the present disclosure may include a display unit. The display unit can display information about light in the wavelength range of 315 to 325 nm emitted from the irradiation unit (e.g., illuminance and / or irradiation time and / or irradiation dose on the mounting unit). In this case, the display unit may display information based on a signal received from the irradiation control unit, or may display information based on a signal received from an optical sensor installed in the irradiation area of ​​the irradiation unit. Alternatively or additionally, the display unit may display the temperature and / or humidity and / or CO2 concentration inside the housing of the cell culture device of the present disclosure. In this case, the display unit may display information based on signals received from sensors provided in the temperature adjustment mechanism and / or humidity adjustment mechanism and / or CO2 concentration adjustment mechanism. The display unit may be configured with a display device and a display control unit that controls the display device.

[0056] The cell culture device of the present disclosure may be equipped with a mechanism (for example, a fan) for stirring and / or circulating the atmosphere within the housing. In the cell culture device of the present disclosure, it is preferable that no components (except for optical components constituting the irradiation unit) whose transmittance is substantially changed by irradiation with light in the wavelength range of 315 to 325 nm are disposed on the path of the light from the irradiation unit to the mounting unit (especially the cells to be irradiated). According to this preferred embodiment, it is possible to easily control the illuminance or dose of light in the wavelength range of 315 to 325 nm that is irradiated to the cells in the cell culture vessel mounted on the mounting unit.

[0057] The cell culture device of the present disclosure will be described below with reference to FIGS. 1 to 3, which are schematic diagrams showing several specific embodiments of the cell culture device of the present disclosure.

[0058] (Embodiment 1) In some embodiments of the present disclosure, as shown in FIG. 1 , the cell culture device 100 includes an irradiation unit 112 capable of irradiating mainly light in the wavelength range of 315 to 325 nm, a mounting unit 114 located within the irradiation area of ​​the irradiation unit 112 and for mounting a cell culture container 150, an irradiation control unit 110 that controls the irradiation unit 112, and a housing 118 that houses the irradiation unit 112 and the mounting unit 114. The cell culture device 100 may include an optical sensor 120 in the irradiation area of ​​the irradiation unit 112 as an optional component. In FIG. 1, the irradiation unit 112 is provided on the interior top surface of the housing 118, but this is not limited to this and may be provided on the top of one or more side walls (e.g., the rear side wall and / or the left and right side walls) of the housing or on another structure (e.g., a shelf or stand) arranged above the placing portion. In FIG. 1, the placement portion 114 is provided as a partial area of ​​the shelf 140 or the stand, but it may be defined as a partial area of ​​the bottom surface of the housing, or may be a platform structure placed on the bottom surface of the housing. In FIG. 1, the irradiation control unit 110 is provided on the top of the housing outside the housing 118, but it may also be provided on the side of the housing (either left, right, front, or back) or on the bottom of the housing, or it may be built into the housing 118. When a cell culture vessel (for example, a petri dish) 150 containing cells is placed on the placement section 114, the irradiation section 112 can irradiate the cells being cultured in the cell culture vessel with light in the wavelength range of 315 to 325 nm. When provided, the optical sensor 120 detects light in the wavelength range of 315 to 325 nm from the irradiating unit 112 at the level of the placing surface. The level of the placing surface means not only the placing surface but also a position outside the placing surface that can be regarded as equivalent. The optical sensor 120 transmits a signal according to the amount of light in the wavelength range of 315 to 325 nm detected to the irradiation control unit 110. Based on the received signal, the irradiation control unit 110 can control the irradiating unit 112 so that the irradiating unit 112 emits light in the wavelength range of 315 to 325 nm with an appropriate radiation intensity (so as to achieve the desired illuminance at the level of the placing surface).

[0059] (Embodiment 2) In some other embodiments of the present disclosure, as shown in FIG. 2, the cell culture device 200 includes a first irradiation unit 212a capable of irradiating mainly light in the wavelength range of 315 to 325 nm, a first mounting unit 214a located within the irradiation area of ​​the first irradiation unit 212a and for placing a cell culture vessel 250a thereon, a second irradiation unit 212b capable of irradiating mainly light in the wavelength range of 315 to 325 nm, a second mounting unit 214b located within the irradiation area of ​​the second irradiation unit 212b and for placing the cell culture vessel 250b thereon, an irradiation control unit 210 that controls the first and second irradiation units 212a and 212b, a first shielding member 216a that shields light in the wavelength range of 315 to 325 nm, and a housing 218 that houses the irradiation units 212a and 212b, the mounting units 214a and 214b, and the first shielding member 216a. Within the housing 218, the second irradiator 212b is located below the first mounting section 214a, and the first shielding member 216a is positioned so as to prevent the light in the wavelength range of 315 to 325 nm from the first irradiator 212a from being irradiated onto the second mounting section 214b (and therefore onto the cells in the cell culture vessel 250b mounted on the mounting section). In other words, the second mounting section 214b is located outside the irradiation area of ​​the first irradiator 212a, which is located above the second irradiator 212b. The cell culture device 200 may further include, as optional components, a third irradiation unit 212c controlled by the irradiation control unit 210, which can irradiate the interior of the housing 218 with light mainly in the wavelength range of 315 to 325 nm, a third mounting unit 214c located within the irradiation area of ​​the third irradiation unit 212c and for mounting the cell culture vessel 250c thereon, and a second shielding member 216b blocking light in the wavelength range of 315 to 325 nm. Within the housing 218, the third irradiation unit 212c is located below the second mounting unit 214b, and the second shielding member 216b is positioned so as to prevent light in the wavelength range of 315 to 325 nm from being irradiated onto the third mounting unit 214c from the first irradiation unit 212a and the second irradiation unit 212b. In FIG. 2, the interior of the housing 218 is divided into three cell culture compartments, but it may be divided into two cell culture compartments or four or more cell culture compartments as needed.

[0060] In the cell culture device 200, when cell culture vessels 250a, 250b, and 250c containing cells are placed on the placement sections 214a, 214b, and 214c, the first irradiation section 212a can irradiate light in the wavelength range of 315 to 325 nm only to the cells in the cell culture vessel 250a, the second irradiation section 212b can irradiate light in the wavelength range of 315 to 325 nm only to the cells in the cell culture vessel 250b, and the third irradiation section 212c can irradiate light in the wavelength range of 315 to 325 nm only to the cells in the cell culture vessel 250c. In FIG. 2, the three irradiation units 212a, 212b, and 212c are controlled by one irradiation control unit 210, but they may be controlled individually by three corresponding irradiation control units. In FIG. 2, the placement sections 214a, 214b, and 214c are provided as partial areas of the shelves 240a, 240b, and 240c, but as shown in FIG. 3, they may be provided as separate structures on the shelves or on the stand. 2, the entire shelves 240a, 240b also serve as the shielding members 216a, 216b, but only a portion of the shelves 240a, 240b may also serve as the shielding members 216a, 216b, as long as it can prevent light in the wavelength range of 315 to 325 nm from the irradiation unit 212a or 212a and 212b in the upper cell culture compartment from being irradiated onto the mounting unit 214b or 214c in the lower cell culture compartment. As shown in FIG. 3, the shielding member 316 may be provided as a structure separate from the shelf 340a. In Fig. 2, a humidifier and / or an airflow generating mechanism (e.g., a fan) may be disposed in the lower compartment of the third mounting portion 214c. In this embodiment, the shelves 240a, 240b, and 240c do not contact the front and / or rear side walls of the housing in Fig. 2, or have openings in parts of the shelves (outside the irradiation areas of the irradiation units 212a, 212b, and 212c). This configuration makes it possible to uniformly maintain the atmosphere in each cell culture compartment at the desired culture conditions. For other details, the explanation for the first embodiment may be applicable.

[0061] (Embodiment 3) In some other embodiments of the present disclosure, as shown in FIG. 3 , a cell culture device 300 includes a first irradiation unit 312a capable of irradiating mainly light in the wavelength range of 315 to 325 nm, a first mounting unit 314a located within the irradiation area of ​​the first irradiation unit 312a and for placing a cell culture vessel 350a thereon, a second irradiation unit 312b capable of irradiating mainly light in the wavelength range of 315 to 325 nm, a second mounting unit 314b located within the irradiation area of ​​the second irradiation unit 312b and for placing the cell culture vessel 350b thereon, an irradiation control unit 310 that controls the first and second irradiation units 312a and 312b, a shielding member 316 that shields light in the wavelength range of 315 to 325 nm, and a housing 318 that houses the irradiation units 312a and 312b, the mounting units 314a and 314b, and the shielding member 316. In this embodiment, the placement portions 314a and 314b are plates provided on the shelf nets 340a and 340b, and the placement portion 314a also serves as the shielding member 316. Within the housing 318, the second irradiation unit 312b is located below the first mounting unit 314a, and the shielding member 316 is positioned so as to prevent the light in the wavelength range of 315 to 325 nm from the first irradiation unit 312a from being irradiated onto the second mounting unit 314b (and therefore onto the cells in the cell culture vessel 350b mounted on the second mounting unit). In other words, the second mounting unit 314b is located outside the irradiation region of the first irradiation unit 312a, which is located above the second irradiation unit 312b. The irradiating units 312a and 312b may also be capable of primarily irradiating light in a wavelength range outside the wavelength range of 315 to 325 nm (for example, sterilizing ultraviolet light or white light), and may be switchable between an off state, a state in which primarily irradiates light in the wavelength range of 315 to 325 nm, and a state in which primarily irradiates light in a wavelength range outside the wavelength range of 315 to 325 nm.

[0062] As shown in FIG. 3A, in the cell culture device 300, when cell culture vessels 350a and 350b containing cells are placed on the placement sections 314a and 314b, the first irradiation section 312a can irradiate the cells being cultured in the cell culture vessel 350a with light in the wavelength range of 315 to 325 nm, and the second irradiation section 312b can irradiate the cells being cultured in the cell culture vessel 350b with light in the wavelength range of 315 to 325 nm. 3B shows an embodiment in which the light in a wavelength range outside the 315-325 nm wavelength range is sterilizing ultraviolet light. The irradiators 312a and 312b can sterilize the cell culture compartments by irradiating them with sterilizing ultraviolet light (e.g., light in a wavelength range of 200-280 nm, more specifically, light in a wavelength range of 240-280 nm or 210-220 nm) before and / or after cell culture in the cell culture device 300. The irradiation controller 310 can control the irradiators 312a and 312b not to simultaneously emit light in the wavelength range of 315-325 nm and sterilizing ultraviolet light. Additionally or alternatively, the irradiation controller 310 may control the irradiators 312a and 312b to emit sterilizing ultraviolet light only when a door (particularly an inner door) provided to the housing 318 is closed. The irradiators 312a and 312b may be capable of emitting white light as another light in a wavelength range outside the wavelength range of 315 to 325 nm. In this case, the white light can be used to illuminate the cell culture compartment of the cell culture device 300, for example, to facilitate work and / or cell observation. In an embodiment in which the irradiators 312a and 312b are capable of emitting sterilizing ultraviolet light and white light, the irradiation controller 310 can control the irradiators 312a and 312b so as not to simultaneously emit sterilizing ultraviolet light and white light. For the rest, the explanations for the first and / or second embodiments may apply.

[0063] experiment The shaved back skin of three beagle dogs (2 to 11 years old) was irradiated with LED light with peak wavelengths of 310, 320, and 330 nm. Figure 4 shows the LED emission spectrum. Specifically, LED light with a peak wavelength of 310 nm was irradiated at an irradiation dose of 300 mJ / cm. 2 Once a day for a total of 4 days, or at an irradiation dose of 1500 mJ / cm2 The LED light with a peak wavelength of 320 nm or 330 nm was irradiated once at a dose of 300 mJ / cm 2 The irradiation was carried out once a day for a total of 4 days. In this experimental example, wavelength measurement was performed by measuring the emission spectrum using a multi-channel detector (model: PMA-11 C7473; Hamamatsu Photonics). Illuminance [mW / cm 2 The irradiance [mJ / cm ] was measured using a photodiode sensor (model: PD300-UV; Ophir) whose sensitivity was calibrated to the peak wavelength of the LED used. 2 ] is the illuminance [mW / cm 2 ]×irradiation time [seconds]. A biopsy trephine (BP-80F, Kai Medical) was used to examine the skin in the center of the light irradiation field and the skin in the non-irradiated field. The obtained skin tissue samples were incubated with RNAlater (registered trademark) (Thermo Fisher) overnight at 4°C and then stored at -80°C until use. Total RNA was crudely extracted from the thawed skin tissue samples using RNAisoPlus (MACHEREY-NAGEL GmbH & Co.) and then purified using NucleoSpin® RNA Clean-up XS (MACHEREY-NAGEL GmbH & Co.). RNA-seq analysis was performed on purified total RNA (Takara Bio Inc.). Briefly, PolyA was extracted from total RNA. +RNA was isolated and fragmented, and single-stranded cDNA was synthesized by reverse transcription using the resulting RNA fragments as templates. Next, double-stranded cDNA was synthesized using the single-stranded cDNA as a template in the presence of dUTP. Both ends of the resulting double-stranded cDNA (containing dUTP) were blunted and phosphorylated, after which 3'-dA overhangs and indexed adapters were ligated. PCR amplification was performed using the resulting adapter-ligated double-stranded cDNA as a template with a polymerase that selectively inhibits the amplification of DNA strands containing dUTP. The amplified cDNA fragments were used as a sequencing library. Sequence analysis was performed using a NovaSeq system (Illumina, Inc.), and information analysis was performed on known genes only.

[0064] Excluding genes for which the FPKM (Fragments Per Kilobase of transcript per Million fragments sequenced) value or count value was 0 for all skin tissue samples, genes whose FPKM and / or count values ​​were significantly different in the light-irradiated field compared to the non-irradiated field (p≦0.01; t-test) were extracted for each irradiation condition. Figure 5 shows a Venn diagram of the number of genes that changed under each irradiation condition. Venny (BioinfoGP) was used to create the Venn diagram. From Figure 5, it can be seen that irradiation with light with a peak wavelength of approximately 320 nm specifically changed the expression of 248 genes. Therefore, it is inferred that irradiation with light with a peak wavelength of approximately 320 nm can control the expression of specific genes. Next, the extracted genes were subjected to pathway analysis and Gene Ontology (GO) analysis using the analysis software DAVID 6.8 (NIAID). Pathway analysis was performed based on the Pathway database. GO analysis was performed based on biological processes (BPs).

[0065] In the pathway analysis, pathways that were significantly (p≦0.05) changed under each irradiation condition were extracted. The results are shown in Table 1. [Table 1]

[0066] The increase or decrease in gene expression levels was examined, and the direction of change for the extracted pathways is summarized below. (a) LED light with a peak wavelength of 310 nm is used at an irradiation dose of 300 mJ / cm 2 Once a day for a total of 4 days Purine metabolism: Promotes the movement of ATP-related compounds and the synthesis of dissociated uric acid. Aminoacyl-tRNA biosynthesis: Promotion of glycine synthesis, inhibition of histidine and phenylalanine synthesis. (b) LED light with a peak wavelength of 320 nm is used at an irradiation dose of 300 mJ / cm 2 Once a day for a total of 4 days Calcium signaling pathway: Ca 2+ Promotes uptake, muscle contraction and glycogenolysis. Cardiac muscle contraction: Ca 2+ Facilitates uptake and activates cytochrome C oxidase and myosin. Glycolysis / Gluconeogenesis: Activation of glycolysis / gluconeogenesis. Biosynthesis of amino acids: Activation of amino acid biosynthesis. Carbon metabolism: Activation of carbon metabolism. Biosynthesis of antibiotics: Activation of antibiotic biosynthesis. Insulin signaling pathway: Suppression of glycogen synthesis. Promotion of glycolysis Adrenergic signaling in cardiomyocytes: Ca2+ Facilitates uptake. Activates myosin. Glucagon signaling pathway: Promotion of glycogenolysis Oxytocin signaling pathway: Ca into the cell 2+ Ca uptake and Ca from the endoplasmic reticulum 2+ Promote discharge. (c) LED light with a peak wavelength of 330 nm is used at an irradiation dose of 300 mJ / cm 2 Once a day for a total of 4 days · Metabolic pathways: metabolic activation. ·RNA transport (metabolic pathways): Inhibition of transport from the nucleus to the cytoplasm (d) LED light with a peak wavelength of 310 nm is used at an irradiation dose of 1500 mJ / cm 2 Irradiated once at β-Alanine metabolism: Inhibition of β-alanine metabolism

[0067] In the pathway analysis of (b), the hit genes and expression changes (fold change) for each pathway are shown in Tables 2 to 11. Here, the expression changes are values ​​obtained by dividing the FPKM value for the irradiated field by the FPKM value for the non-irradiated field and then logarithmically transforming the result.

[0068] [Table 2] [Table 3]

[0069] [Table 4] [Table 5]

[0070] [Table 6] [Table 7]

[0071] [Table 8] [Table 9]

[0072] [Table 10] [Table 11]

[0073] Table 2 shows that there was a significant increase in gene expression for TnC (cardiac troponin C), RYR (ryanodine receptor), SERCA (sarcoplasmic reticulum calcium ATPase), GPCR (G protein-coupled receptor), PHK (phosphorylase kinase), VDAC (voltage-dependent anion channel), and ROC (receptor-operated calcium channel). Therefore, irradiation with light with a peak wavelength of approximately 320 nm induces the transfer of Ca to the endoplasmic reticulum in the calcium signaling pathway. 2+ It can be seen that this can promote uptake and stimulate muscle contraction and glycogenolysis. Table 3 shows that there was a significant increase in gene expression related to DHPR (dihydropyridine receptor), ATP (ATP synthase), Cyto (cytochrome C oxidase), and Myosin. Therefore, irradiation with light with a peak wavelength of approximately 320 nm induces Ca ions in the myocardial contraction pathway. 2+ It is understood that it can promote uptake and activate cytochrome C oxidase and myosin.

[0074] Table 4 shows that there was a significant increase in the expression of genes related to EC 5.4.2.11 (phosphoglycerate mutase), EC 4.2.1.11 (phosphopyruvate hydratase), EC 3.1.3.11 (fructose-bisphosphatase), and EC 2.7.1.11 (6-phosphofructokinase). Therefore, it can be understood that irradiation with light having a peak wavelength of approximately 320 nm can activate glycolysis / gluconeogenesis in the glycolysis / gluconeogenesis pathway. Table 5 shows that there was a significant increase in the gene expression of phosphoglycerate mutase 2 (PGAM2), enolase 3 (ENO3), phosphofructokinase, muscle (PFKM), and glutamic-oxaloacetic transaminase 2 (GOT2). Therefore, it can be understood that irradiation with light having a peak wavelength of approximately 320 nm can activate amino acid biosynthesis in the amino acid biosynthetic pathway.

[0075] Table 6 shows that there was a significant increase in the gene expression of phosphoglycerate mutase 2 (PGAM2), enolase 3 (ENO3), fructose-1,6-bisphosphatase isozyme 2 (LOC476300), phosphofructokinase, muscle (PFKM), and glutamic-oxaloacetic transaminase 2 (GOT2). Therefore, it can be understood that irradiation with light having a peak wavelength of approximately 320 nm can activate carbon metabolism in the carbon metabolic pathway. From Table 7, phosphoglycerate mutase 2 (PGAM2), adenosine monophosphate deaminase 1 (AMPD1) enolase 3(ENO3), fructose-1,6-bisphosphatase isozyme 2(LOC476300), phosphofructokinase, muscle(PFKM), carboxymethylenebutenolidase homolog(CMBL), glutamic-oxalo A significant increase in the gene expression of acetic transaminase 2 (GOT2) was observed. Therefore, it can be seen that irradiation with light having a peak wavelength of approximately 320 nm can activate the biosynthesis of antibiotics in the antibiotic biosynthetic pathway.

[0076] Table 8 shows that there was a significant increase in the gene expression of PP1 (protein phosphatase 1), PYG (glycogen phosphorylase), FBP (fructose-1,6-bisphosphatase), and PHK (phosphorylase kinase). Therefore, it can be understood that irradiation with light having a peak wavelength of approximately 320 nm can suppress glycogen synthesis and promote glycolysis in the insulin signaling pathway. Table 9 shows that there was a significant increase in gene expression related to DHPR (dihydropyridine receptor), InaK (Na+ / K+-ATPase), and Myosin. Therefore, irradiation with light with a peak wavelength of approximately 320 nm induces intracellular Ca in the adrenergic signaling pathway in cardiomyocytes. 2+ It can be seen that this can promote uptake and activate myosin.

[0077] Table 10 shows that there was a significant increase in the gene expression of PGM (phosphoglycerate mutase), PYGL (glycogen phosphorylase), and PHK (phosphorylase kinase). Therefore, it can be seen that irradiation with light having a peak wavelength of approximately 320 nm can promote glycogenolysis in the glucagon signaling pathway. Table 11 shows that there was a significant increase in gene expression related to VGCC (voltage-gated calcium channel), RYR (ryanodine receptor), and GIRK (G protein-activated inwardly rectifying potassium channel). Therefore, irradiation with light with a peak wavelength of approximately 320 nm induces Ca2+ into the cell in the oxytocin signaling pathway. 2+ Facilitates Ca uptake from the endoplasmic reticulum 2+ It can be seen that this can promote excretion.

[0078] In the GO analysis, significant changes were observed in multiple biological processes upon irradiation with light with a peak wavelength of approximately 320 nm (Table 12). The biological processes in which changes were observed were related to muscle tissue. This was the effect of inducing the expression of calcium channels and / or calcium pumps in the cells. [Table 12]

[0079] Genes related to the calcium signaling pathway whose expression levels were specifically increased by irradiation with light with a peak wavelength of approximately 320 nm included DHPR, VGCC, SERCA, RYR, TRDN, CASQ, HRC, and Cyto. The changes in expression levels due to irradiation with light of each wavelength are shown in Figures 6(A) to 6(I). From the data shown in the figures, it can be seen that irradiation with light with a peak wavelength of approximately 320 nm specifically upregulated (at least approximately 30-fold) the expression of DHPR, VGCC, SERCA, RYR, TRDN, CASQ, HRC, and Cyto. In summary, irradiation of cells with light having a peak wavelength of approximately 320 nm can modulate the calcium signaling pathway, insulin signaling pathway, adrenergic signaling pathway in cardiomyocytes, glucagon signaling pathway, or oxytocin signaling pathway; control muscle contraction, neurotransmission, mitochondrial activity, or cell death (or apoptosis); activate glycolysis / gluconeogenesis, amino acid biosynthesis, carbon metabolism, or antibiotic biosynthesis; promote glycogenolysis; and promote osteoblast-like differentiation in the cells.

[0080] The methods and apparatus of the present disclosure are expected to be applicable to the following applications: (1) Regenerative medicine for bones and teeth It is known that progenitor cells such as mesenchymal stem cells and osteoblasts take up calcium ions to produce bone cells. Therefore, the method and apparatus disclosed herein can be applied to a method for efficiently inducing differentiation of progenitor cells into bone cells in vitro, and can be expected to be applicable to a method for producing a transplant (bone) material containing cultured bone cells. Furthermore, the method disclosed herein can be expected to be applied to a method for efficiently inducing differentiation of progenitor cells transplanted into a patient into bone cells.

[0081] (2) Improves the barrier function of the skin and mucous membranes The method and device disclosed herein are expected to promote calcium ion uptake by epidermal cells. It is known that promoting calcium ion uptake by epidermal cells activates epidermal transglutaminase (Tgase), crosslinking proteins and leading to skin formation. Therefore, the method disclosed herein is expected to promote skin formation, increase the physical strength of the skin and mucous membranes, and enhance their moisturizing function. By irradiating with light in the wavelength range of 315 to 325 nm, gene expression of FGF6 (fibroblast growth factor 6), MYOD1 (myoblast determination protein 1), MYOG (myogenic factor 4), etc. was also confirmed. Therefore, the light irradiation method disclosed herein is expected to have a skin-beautifying effect by promoting collagen synthesis in fibroblasts.

[0082] (3) Neuromuscular disease research and treatment Calcium ion signals have been the subject of much research in relation to neurotransmission and muscle control. Currently, calcium ionophores (drugs that increase the permeability of cell membranes to calcium ions) and optogenetics (a method of expressing light-activated ion channels such as channelrhodopsin in target cells by genetic engineering techniques, and then irradiating these cells with light of a specific wavelength to control calcium ion signals in the target cells) are used to modulate calcium ion signals. According to the method and device disclosed herein, calcium ion signals can be modulated by light irradiation alone. Since it is possible to modulate the ion signal, it is simple and can be used more clinically. It is known that muscle contraction is regulated by changes in intracellular calcium ion concentration. The method and device disclosed herein are expected to be applicable to cardiomyocyte culture in regenerative medicine. Furthermore, it has been reported that changes in intracellular calcium ion concentration differ from normal in some neurological diseases, such as Alzheimer's disease and Parkinson's disease. The method and device disclosed herein are expected to be useful in elucidating the mechanisms and proposing treatments such as nerve regeneration.

[0083] (4) Infertility The influence of calcium ions is also known to affect infertility. For example, it is known that sperm undergo changes in the motility of flagella and cilia induced by a transient increase in intracellular calcium ion concentration due to the influx of calcium ions from outside the cell, followed by a decrease in intracellular calcium ion concentration due to the subsequent excretion of calcium ions from the cell. It is also known that increasing intracellular calcium ion concentration leads to capacitation (acquisition of fertilization capacity) and the acrosome reaction, leading to penetration into the egg. Therefore, the method and device disclosed herein can be used to control sperm motility and the acrosome reaction, which are important parameters of sperm function. In other words, it is expected that the method and device disclosed herein will improve the motility of sperm that are unable to penetrate an egg due to their weak ability to control calcium ions. Furthermore, the method and apparatus of the present disclosure are expected to activate eggs and improve fertilization rates. For example, one of the reasons why fertilization does not occur even after intracytoplasmic sperm injection (ICSI) is due to impaired egg activation, which is thought to be an insufficient increase in calcium ions caused by the fusion of sperm and eggs. While prior art methods have been used to activate eggs by increasing the calcium ion permeability of the egg cell membrane using calcium ionophores, the method and apparatus of the present disclosure are simpler. Fertilized eggs periodically change their intracellular calcium ion concentration over a long period of time. This is called calcium ion oscillation and affects embryonic development. Specifically, disruption of the oscillation is known to cause abnormalities in gene expression and a decrease in normal birth rates. It is expected that normal birth rates can be increased by controlling the intracellular calcium ion concentration using the method and device disclosed herein.

[0084] (5) Induction of cell apoptosis Prior art techniques for removing unwanted cells from living tissues and cultured cells include ablation by laser irradiation and apoptosis induction by calcium ion overload (uptake of excess calcium ions) using calcium ionophores such as ionomycin. However, this technology can induce apoptosis by irradiating unwanted cells with light locally at low light intensity while suppressing heat generation, so it is expected to remove only the unwanted cells without causing heat or light damage to normal cells. [Explanation of symbols]

[0085] 100, 200, 300 cell culture equipment 110, 210, 310 Irradiation control unit 112, 212a, 212b, 212c, 312a, 312b Irradiation part 114, 214a, 214b, 214c, 314a, 314b Placement section 216a, 216b, 316 Shielding member 118, 218, 318 housing 120 Optical Sensor 140, 240a, 240b, 240c, 340a, 340b Shelf board (or stand or shelf net) 150, 250a, 250b, 250c, 350a, 350b Cell culture vessel

Claims

1. 1. A method for inducing expression of a calcium channel and / or calcium pump in a cell, comprising: The cells were exposed to 10 mJ / cm of light in the wavelength range of 315 to 325 nm. 2 More than 5000mJ / cm 2 The irradiation step includes irradiating with the following irradiation amount: the calcium channel and / or calcium pump is at least one calcium channel and / or calcium pump selected from the group consisting of CACNG1, CACNG6, ATP2A1, RYR1, TRDN, CASQ1, CASQ2, HRC, and COX6A2; the cells are human cells, In the irradiating step, the cells are not irradiated with light in the wavelength range of 330 to 400 nm, or the amount of irradiation is less than 50% of the amount of irradiation of light in the wavelength range of 315 to 325 nm.

2. 2. The method according to claim 1, wherein in the irradiating step, the cells are not irradiated with light in the wavelength range of 200 to 300 nm, or the amount of light irradiated is less than 30% of the amount of light irradiated with light in the wavelength range of 315 to 325 nm.

3. 3. The method according to claim 1, wherein the light in the wavelength range of 315 to 325 nm has a wavelength spectrum with a peak wavelength of 320±5 nm and a full width at half maximum of 1 to 20 nm.

4. The method according to any one of claims 1 to 3, wherein the light in the wavelength range of 315 to 325 nm is emitted from an LED as a light source.

5. The method according to any one of claims 1 to 4, wherein the cell is an excitable cell.

6. 1. A method of modulating calcium signaling in a cell, comprising: Inducing expression of calcium channels and / or calcium pumps in the cells by the method of any one of claims 1 to 5; and measuring a physiological event involving calcium signaling in said cell.

7. The method of claim 6, wherein the physiological event involving calcium signaling is one or more selected from the group consisting of muscle contraction, neurotransmission, cell death, and osteoblast-like differentiation.

8. A method for producing a cell in which expression of a calcium channel and / or a calcium pump is enhanced, comprising: The cells were exposed to light in the wavelength range of 315 to 325 nm at 10 mJ / cm 2 More than 5000mJ / cm 2 an irradiation step of irradiating with the following irradiation dose; freezing the cells irradiated with light; the cells are human cells, the calcium channel and / or calcium pump is at least one calcium channel and / or calcium pump selected from the group consisting of CACNG1, CACNG6, ATP2A1, RYR1, TRDN, CASQ1, CASQ2, HRC, and COX6A2; wherein, in the irradiating step, the cells are not irradiated with light in the wavelength range of 200 to 300 nm, or the amount of irradiation is less than 30% of the amount of irradiation of light in the wavelength range of 315 to 325 nm, and the cells are not irradiated with light in the wavelength range of 330 to 400 nm, or the amount of irradiation is less than 50% of the amount of irradiation of light in the wavelength range of 315 to 325 nm.

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