A method for determining the wavelength of light and irradiance used by subjects to perceive information from the external world.

Abstract

Provided is a method for determining the properties of light, the method having: a relationship acquisition step for obtaining, in advance, a relationship 1 between the wavelength and irradiance of light showing whether or not a morphological change is caused in ocular cells and a relationship 2 between the wavelength and irradiance of light showing whether or not migration of ocular cells is changed in a subject; a relationship selection step for selecting at least one of relationship 1 and relationship 2 in accordance with the degree 1 required for a morphological change and the degree 2 required for migration; and a determination step for determining the wavelength and irradiance of the light that should enter the eye of the subject on the basis of the selected relationship.

Description

[Technical Field]

[0001] This invention relates to a method for determining the properties of light. [Background technology]

[0002] Patent Document 1 describes the effects of light on retinal pigment epithelial cells (RPE). It states that RPE apoptosis was strongly induced in a 10 nm bandwidth centered on 420, 430, 440, and 450 nm (415-455 nm). It then proposes blocking the transmission of light with wavelengths in the harmful bandwidth using selective filtering means. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent No. 6773391 Specification [Overview of the project] [Problems that the invention aims to solve]

[0004] The inventors of this invention have studied symptoms and diseases caused by abnormalities in the lens epithelial cells, such as posterior capsule opacification. Posterior capsule opacification is defined as follows:

[0005] Cataract surgery is a treatment option for patients who have developed cataracts. Generally, the cloudy lens is removed from the lens capsule through an incision (phacoemulsification), and an intraocular lens is inserted into the lens capsule in its place. The intraocular lens is covered by a portion of the incised lens capsule.

[0006] Postoperative complications can occur. One such complication is that, after surgery, remaining lens epithelial cells and other tissues in the lens capsule proliferate, particularly in the space between the posterior surface of the intraocular lens (the surface facing the eye, opposite to the side facing the object in front of the eye) and the lens capsule. This can cause clouding of the lens capsule (posterior capsule opacity), which can progress further, reducing light transmittance into the eye and impairing visual function. This condition is called posterior capsule opacification. In such cases, the typical treatment is to remove the opacity using a YAG laser.

[0007] In Japan, around 2020, approximately 30% of cataract surgeries (lens reconstruction surgery) were performed to treat posterior capsule opacification, which is considered a problem. Furthermore, abnormalities in the lens epithelial cells are thought to be related to other symptoms and diseases affecting the lens.

[0008] The inventors have found that it is extremely important to know, in relation to lens epithelial cells, which are a contributing factor to posterior capsule opacification, what characteristics of light incident on the eye of each individual (hereinafter also referred to as the Subject) cause changes in lens epithelial cells, or how those changes occur, within the living environment of each individual.

[0009] Based on this finding, the inventors investigated the effects of the characteristics of light entering the eye (e.g., wavelength and irradiance) on lens epithelial cells. As a result, they found that depending on the characteristics of light entering the eye, morphological changes and morphological abnormalities (irregular morphology) can occur in lens epithelial cells, and that the migration of lens epithelial cells can be altered (e.g., reduced). Furthermore, they found that these findings are also applicable to other cells related to the eye (hereinafter referred to as ocular cells; specific examples of ocular cells will be described later).

[0010] One embodiment of the present invention aims to determine the characteristics of light incident on the eye based on the relationship between changes in the morphology and migration of eye cells and the properties of light. [Means for solving the problem]

[0011] It is significant for the subject to know in advance what characteristics of light will cause morphological changes in eye cells and what characteristics of light will change the migratory ability of eye cells when entering the eye.

[0012] As a result of intensive studies based on the above findings, a method for determining what characteristics of light should be incident on the subject's eyes was conceived. The following aspects were created based on this finding.

[0013] The first aspect of the present invention is a relationship acquisition step of obtaining in advance a relationship 1 between the wavelength and irradiance of light that divides whether it causes morphological changes in eye cells for the subject, and a relationship 2 between the wavelength and irradiance of light that divides whether it changes the migratory ability of eye cells; a relationship selection step of selecting at least one of the relationship 1 and the relationship 2 according to a degree 1 required for the morphological change and a degree 2 required for the migratory ability; a determination step of determining the wavelength and irradiance of light to be incident on the subject's eyes based on the selected relationship; and it is a method for determining the characteristics of light.

[0014] The second aspect of the present invention is wherein the relationship 1 is a plot 1 that divides whether it causes morphological changes in eye cells in the relationship between the wavelength and irradiance of light, and the relationship 2 is a plot 2 that divides whether it changes the migratory ability of eye cells in the relationship between the wavelength and irradiance of light, and it is the method for determining the characteristics of light according to the first aspect.

[0015] The third aspect of the present invention is in the relationship between the wavelength and irradiance of light, the plot 1 is on the short wavelength side of the plot 2, in the relationship between the wavelength and irradiance of light, the plot 1 divides into a plurality of regions 1 composed of a region that causes the morphological change and a region on the side that does not cause it, In the relationship between the wavelength of light and irradiance, the plot 2 divides the area into multiple regions 2, consisting of regions that cause the change in migratory behavior and regions that do not. In the relationship selection step, plot 1 and plot 2 are selected. The process further includes a region selection step in which one region is selected from region 1 and one region is selected from region 2, after the relationship selection step. In the region selection step, at least (1) of the following (1) and (2) is performed, and (1) the region that does not cause the morphological change is selected from region 1. (2) Select the region that does not cause the change in migratory behavior from region 2. The method for determining the properties of light, as described in the second embodiment, is to obtain the wavelength and irradiance of light that belong to each of the selected regions in the determination step as the wavelength and irradiance of light that should be incident on the eyes of the subject.

[0016] A fourth aspect of the present invention is: In addition to the plot 2, the third embodiment of the method for determining the properties of light is further obtained in the relationship acquisition step, in which a plot 3 is obtained in which the region that causes the change in migratory properties in region 2 is further subdivided according to the degree of the change in migratory properties.

[0017] A fifth aspect of the present invention is: Plots 1 and 2 are methods for determining the characteristics of light, as described in any one of the second to fourth embodiments, and are created based on the geographical information of the subject.

[0018] A sixth aspect of the present invention is: The method for determining the properties of light according to any one of the first to fifth embodiments, wherein the ophthalmic cells are lens epithelial cells, retinal pigment epithelial cells, corneal endothelial cells, corneal epithelial stem cells, or subconjunctival fibroblasts.

[0019] A seventh aspect of the present invention is: For the target individuals, • The relationship between light wavelength and irradiance that determines whether or not morphological changes in eye cells occur. • The relationship between light wavelength and irradiance that determines whether or not the migratory behavior of eye cells is altered. • The relationship between light wavelength and irradiance that determines whether or not it enhances the infiltration of eye cells (Part 3) A relationship acquisition process to obtain at least one of the following in advance, A relationship selection step in which at least one of the relationship 1, relationship 2, and relationship 3 is selected according to at least one of the degree required for the morphological change 1, the degree required for the migratory properties 2, and the degree required for the infiltration properties 3, A determination step is to determine the wavelength and irradiance of light to be incident on the subject's eye based on the selected relationship, This is a method for determining the properties of light, which has the following characteristics.

[0020] An eighth aspect of the present invention is: The aforementioned relationship 1 is a plot 1 that distinguishes whether or not a change in the morphology of eye cells occurs in relation to the wavelength of light and irradiance. The aforementioned relationship 2 is plot 2 which distinguishes whether or not the relationship between the wavelength of light and irradiance alters the migratory behavior of eye cells. The aforementioned relationship 3 is plot 4, which determines whether or not the infiltration of eye cells is enhanced in relation to the wavelength of light and irradiance, and is a method for determining the properties of light as described in the seventh embodiment.

[0021] A ninth aspect of the present invention is: The method for determining the properties of light according to the seventh or eighth embodiment is characterized in that, in the relationship acquisition step, at least the relationship 3 is obtained in advance, and in the relationship selection step, at least the relationship 3 is selected.

[0022] A tenth aspect of the present invention is: The method for determining the properties of light according to the seventh or eighth embodiment is characterized in that the relationship acquisition step obtains the relationship 1 to 3 in advance, and the relationship selection step selects the relationship 1 to 3.

[0023] An eleventh aspect of the present invention is: In the relationship between the wavelength of light and irradiance, plot 1 is on the shorter wavelength side of plot 2. In the relationship between the wavelength of light and irradiance, plot 1 divides the area into multiple regions 1 consisting of regions that cause the morphological change and regions that do not. In the relationship between the wavelength of light and irradiance, the plot 2 divides the area into multiple regions 2, consisting of regions that cause the change in migratory behavior and regions that do not. In the relationship between the wavelength of light and irradiance, the plot 4 divides the area into multiple regions 4, consisting of regions that cause increased penetration and regions that do not. In the relationship selection step, plot 1, plot 2, and plot 4 are selected. The process further includes a region selection step in which, after the relationship selection step, one region is selected from region 1, one region is selected from region 2, and one region is selected from region 4. In the region selection step, at least (1) of the following (1) to (3) is performed: (1) Select the region that does not undergo the aforementioned morphological change from region 1. (2) Select the region that does not cause the change in migratory behavior from region 2. (3) Select the region on the side that does not result in increased invasiveness from region 4. The method for determining the properties of light according to the tenth embodiment is to obtain the wavelength and irradiance of light that belong to each of the selected regions in the determination step as the wavelength and irradiance of light that should be incident on the eyes of the subject.

[0024] A twelfth aspect of the present invention is: In addition to the plot 2, the eleventh embodiment of the method for determining the properties of light is further obtained in the relationship acquisition step, in which a plot 3 is obtained in which the region that causes the change in migratory properties in region 2 is further subdivided according to the degree of the change in migratory properties.

[0025] A thirteenth aspect of the present invention is: In the relationship between the wavelength of light and irradiance, plot 4 is located on the shorter wavelength side of plot 3 and on the longer wavelength side of plot 2, according to the method for determining the properties of light as described in the twelfth embodiment.

[0026] A fourteenth aspect of the present invention is: At least plots 1, 2, and 4 are a method for determining the properties of light according to the tenth embodiment, which is created based on the geographical information of the subject.

[0027] A fifteenth aspect of the present invention is: The method for determining the properties of light according to any one of the seventh to fourteenth embodiments, wherein the ophthalmic cells are lens epithelial cells, retinal pigment epithelial cells, corneal endothelial cells, corneal epithelial stem cells, or subconjunctival fibroblasts. [Effects of the Invention]

[0028] According to one embodiment of the present invention, the properties of light can be determined based on the relationship between changes in the morphology and migration of eye cells and their properties of light. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 is a plot showing the spectral irradiance (vertical axis: unit is W / m2 / nm) and wavelength (horizontal axis: unit is nm) of sunlight (AM1.5 and after corneal transmission) on the anterior surface of the lens. [Figure 2] Figure 2 is a plot showing the irradiance (vertical axis: in W / m2) and wavelength (horizontal axis: in nm) of each LED light (half-width of approximately 20 nm) used in the experiment. For reference, the sunlight in front of the crystalline lens for each frequency band under the same conditions is also included in the plot. [Figure 3] Figure 3 shows photographs illustrating the observation results in the qualitative evaluation. (a) The light irradiation conditions were a wavelength of 430 nm and an irradiance of 9.0 W / m2, (b) the light irradiation conditions were a wavelength of 430 nm and an irradiance of 5.1 W / m2, and (c) the light irradiation conditions were a wavelength of 450 nm and an irradiance of 4.6 W / m2. [Figure 4]Figure 4 is a plot summarizing the relationship between irradiance (vertical axis: in W / m2) and wavelength (horizontal axis: in nm) for each LED light (half-width of approximately 20 nm) used in the experiment, categorized by the type of morphological change in lens epithelial cells. For reference, sunlight in front of the lens for each frequency band under the same conditions is also included in this plot. [Figure 5] Figure 5 illustrates the circularity and aspect ratio of lens epithelial cells. When the area is S and the circumference is L, the circularity is defined as 2πS / L² in this specification. When the lens epithelial cells are approximated as an ellipse using ImageJ, the major axis is a and the minor axis is b, and the aspect ratio is defined as a / b in this specification. [Figure 6] Figure 6 is a bar graph showing the results of the quantitative evaluation, where (a) is the area S, (b) is the circularity, and (c) is the aspect ratio. [Figure 7] Figure 7 is a schematic plan view of a well used to create a gap (wound) of a predetermined width on a surface containing numerous cells. [Figure 8] Figure 8 shows the gap closing process at a wavelength of 430 nm and an irradiance of 9.0 W / m2. (a) is a photograph showing the gap at 0h (immediately after the start of light irradiation), (b) is a photograph showing the gap 24h after the start of light irradiation, and (c) is a photograph showing the gap 48h after the start of light irradiation. [Figure 9] Figure 9 shows the gap closing process at a wavelength of 430 nm and an irradiance of 5.1 W / m2. (a) is a photograph showing the gap at 0h (immediately after the start of light irradiation), (b) is a photograph showing the gap 24h after the start of light irradiation, and (c) is a photograph showing the gap 48h after the start of light irradiation. [Figure 10] Figure 10 shows the gap closing process at a wavelength of 430 nm and an irradiance of 2.7 W / m2. (a) is a photograph showing the gap at 0h (just before the start of light irradiation), (b) is a photograph showing the gap 24h after the start of light irradiation, and (c) is a photograph showing the gap 48h after the start of light irradiation. [Figure 11] Figure 11 shows the gap closing process at a wavelength of 470 nm and an irradiance of 2.5 W / m2. (a) is a photograph showing the gap at 0h (immediately after the start of light irradiation), (b) is a photograph showing the gap 24h after the start of light irradiation, and (c) is a photograph showing the gap 48h after the start of light irradiation. [Figure 12] Figure 12 shows plots grouped by wavelength, illustrating the relationship between the distance between gap edges (in μm) and the time since the start of light irradiation (horizontal axis: in h). The slope of the plot is distance / time, representing the migration velocity of lens epithelial cells. [Figure 13] Figure 13 is a histogram showing the distribution of the rate of decrease in migratory ability during gap closure. [Figure 14] Figure 14 is a plot showing the relationship between irradiance (vertical axis: in W / m2) and wavelength (horizontal axis: in nm) for each light source used in the experiment (half-width 10 nm), and the patterns of morphological and migratory changes in lens epithelial cells. Sunlight is also included in the plot for reference. [Figure 15] Figure 15 is a plan view showing how eye cells pass through the opening during NC (no light irradiation), with (a) being 0h (immediately after the start of light irradiation), (b) 10 minutes after the start of light irradiation, (c) 20 minutes after the start of light irradiation, (d) 30 minutes after the start of light irradiation, (e) 40 minutes after the start of light irradiation, and (f) 50 minutes after the start of light irradiation. [Figure 16] Figure 16 is a plan view showing the infiltration (into the grooves) of eye cells during NC without light irradiation, with (a) being 0h (immediately after the start of light irradiation), (b) 10 minutes after the start of light irradiation, (c) 20 minutes after the start of light irradiation, (d) 30 minutes after the start of light irradiation, (e) 40 minutes after the start of light irradiation, and (f) 50 minutes after the start of light irradiation. [Figure 17] Figure 17 is a plan view showing the incomplete invasion behavior (invasion failure) of ophthalmic cells when NC (no light irradiation) is performed, and (a) is a photograph showing the state at 0h (immediately after the start of light irradiation), (b) is 10 minutes after the start of light irradiation, (c) is 20 minutes after the start of light irradiation, (d) is 30 minutes after the start of light irradiation, (e) is 40 minutes after the start of light irradiation, (f) is 50 minutes after the start of light irradiation, (g) is 60 minutes after the start of light irradiation, and (h) is a photograph showing the state at 70 minutes after the start of light irradiation. [Figure 18]Figure 18 is a plan view showing how eye cells infiltrate (climb up from the grooves) during NC (no light irradiation), with (a) being 0h (immediately after the start of light irradiation), (b) 10 minutes after the start of light irradiation, (c) 20 minutes after the start of light irradiation, (d) 30 minutes after the start of light irradiation, (e) 40 minutes after the start of light irradiation, (f) 50 minutes after the start of light irradiation, and (g) 60 minutes after the start of light irradiation. [Figure 19] Figure 19 shows plots NC-1, NC-2, NC-3, and NC-4, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis. [Figure 20] Figure 20 shows plots NC-1, NC-2, NC-3, and NC-4, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis, along with the results at wavelengths of 430-460 nm. [Figure 21] Figure 21 shows plots NC-1, NC-2, NC-3, and NC-4, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis, along with the results at a wavelength of 480 nm. [Figure 22] Figure 22 shows plots NC-1, NC-2, NC-3, and NC-4, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis, along with the results at a wavelength of 500 nm. [Figure 23] Figure 23 shows plots NC-1, NC-2, NC-3, and NC-4, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis, along with the results at a wavelength of 520 nm. [Figure 24] Figure 24 shows plots NC-1, NC-2, NC-3, and NC-4, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis, along with the results at a wavelength of 540 nm. [Figure 25] Figure 25 shows the results for low irradiance of 1.4 to 1.5 W / m2, with infiltration (inside the groove) on the vertical axis and groove width w (μm) on the horizontal axis. [Figure 26] Figure 26 shows the results for a medium irradiance of 3.2 to 5.5 W / m2, with infiltration (inside the groove) on the vertical axis and the groove width w (μm) on the horizontal axis. [Figure 27] Figure 27 shows the results when the infiltration (inside the groove) is on the vertical axis and the groove width w (μm) is on the horizontal axis, for high irradiance of 7.5 W / m2 or more. [Figure 28] Figure 28 shows the results when the number of passages is on the vertical axis and the groove width w (μm) is on the horizontal axis. [Figure 29] Figure 29 shows the results when the number of incomplete invasion behaviors (invasion failures) is plotted on the vertical axis and the groove width w (μm) is plotted on the horizontal axis. [Figure 30] Figure 30 shows the results when the number of infiltrations (climbing out of the groove) is on the vertical axis and the groove width w (μm) is on the horizontal axis. [Figure 31A] Figure 31A plots the relationship between irradiance (vertical axis: in W / m2) and wavelength (horizontal axis: in nm) for each light source used in the experiment (full width at half maximum: 10 nm), showing the changes in the invasiveness of lens epithelial cells. Sunlight is also included in the plot for reference. Figure 31A is shown when the groove width w is 8.9 μm. [Figure 31B] Figure 31B is a plot showing the pattern of invasive changes in lens epithelial cells in relation to the relationship between irradiance (vertical axis: unit is W / m2) and wavelength (horizontal axis: unit is nm) for each light source used in the experiment (half-width 10 nm). Sunlight is also included in the plot for reference. Figure 31B is a diagram when the groove width w is 9.6 μm. [Modes for carrying out the invention]

[0030] {Embodiment 1} [Detailed insights] The insights that led to the creation of one embodiment of the present invention were obtained through the following tests.

[0031] <Cells used> The cells used in one embodiment of the present invention were selected from the following perspectives.

[0032] First, in order to investigate the relationship between light wavelength and irradiance that determines whether or not lens epithelial cells undergo changes in the entire process described above, the type of lens epithelial cells to be used in the test is determined. Hereafter, the combination of light wavelength and irradiance will be referred to as the "irradiation condition." To solve problems related to symptoms and diseases concerning human lens epithelial cells, it is preferable to obtain the relationship using primary human lens epithelial cells.

[0033] However, primary human cells have several drawbacks: their properties vary from donor to donor, they have a limited number of cell divisions, and their properties change with each division, making it difficult to guarantee the reproducibility of evaluation results; they are expensive overall, including the cells themselves and the reagents required for cell culture, making them economically unviable; and they are not easily available.

[0034] Therefore, it is preferable to obtain the relationship using human cell lines. Since there are different types of cell lines, it is even more preferable to validate the results using multiple types after obtaining the initial data. Furthermore, it is preferable to finally confirm the validity of the relationship using primary human cells. In this case, it is even more preferable to use primary human cells derived from multiple donors. Even more preferable is to simultaneously obtain lens epithelial cells from the subject during cataract surgery and confirm their properties (resistance) to light.

[0035] If ease of availability, handling, and cost-effectiveness are considered, animal cells can also be used. In this case, it is preferable to examine the validity using human cells after obtaining the relationship. When selecting the type of animal cell, it is preferable to choose a species that has properties similar to those of humans.

[0036] Based on the above considerations, the details of the cell culture selected as one embodiment of the present invention are as follows.

[0037] The cells used were human lens epithelial cells SRA01 / 04 (RCB1591, RIKEN BRC).

[0038] The reagents are as follows: D-MEM (Low Glucose, 041-29775, Wako) + 20% Fetal Bovine Serum (FBS, Gibco, 10437028) + 100 units / mL Penicillin and 100 μg / mL Streptomycin (P / S, Gibco, 15140-122) (hereinafter also simply referred to as "culture medium"). ·Phosphate buffer (PBS(-) solution, Nacalai Tesque, 07269-84) Trypsin / EDTA solution 0.25% solution (Sigma, T4049)

[0039] Cell subculturing was performed when the cells reached 80-100% confluence. The culture medium in the 60 mm dish was removed with an aspirator and washed with PBS(-) solution. Then, 1 mL of Trypsin / EDTA solution was added, and the mixture was allowed to stand for 3 minutes at 37°C under 5% CO2 to detach the cells from the dish.

[0040] The reaction of the Trypsin / EDTA solution was stopped by adding culture medium, and the mixture was centrifuged for 5 minutes (using a Kubota LY3690-A000). The supernatant was removed using an aspirator, and culture medium was added to prepare a cell suspension. After cell counting, the cells were placed in 35 mm or 60 mm dishes in a 5.0 × 10⁶ dish. 4 Seeds were seeded at Cells / ml. The culture medium was changed every 2-3 days.

[0041] <Light source and light irradiation conditions used> The details regarding light irradiation are as follows:

[0042] The equipment used for light irradiation is listed below. • LED controller (CL-1501, ASAHI SPECTRA) • LED head 430-900B (CL-H1-430-9-1-B, ASAHI SPECTRA) • LED head 450-900B (CL-H1-450-9-1-B, ASAHI SPECTRA) • LED head 470-900B (CL-H1-470-9-1-B, ASAHI SPECTRA) • LED head 505-900B (CL-H1-505-9-1-B, ASAHI SPECTRA) • LED head 590-900B (CL-H1-590-9-1-B, ASAHI SPECTRA) • LED head cover glass (CL-H1GCQ01, ASAHI SPECTRA)

[0043] The irradiation conditions are as follows:

[0044] Light irradiation was performed from 15 cm above the cell culture surface under cell culture conditions (at a temperature of 37°C and an atmospheric environment with a CO2 concentration of 5%). It was anticipated that the effect on cells would differ depending on the intensity of the light, even at the same wavelength. Therefore, multiple LED heads were used, and the intensity was varied for each experiment using a controller. Table 1 shows the relationship between the LED controller intensity and irradiance at each wavelength. [Table 1]

[0045] Figure 1 shows the spectral irradiance of sunlight (AM1.5 and after corneal transmission) on the anterior surface of the lens (vertical axis: unit is W / m²). 2 This is a plot showing wavelength (x axis: unit is nm) and (x axis: unit is nm). Figure 2 shows the irradiance of each LED light (half-width of approximately 20 nm) used in the experiment (vertical axis: unit is W / m²). 2 This plot shows the solar cell's front-facing sunlight in each frequency band under the same conditions, and wavelength (horizontal axis: unit is nm). AM stands for Air Mass, and it is the ratio of the path of sunlight to the reference path (AM1) when sunlight is incident perpendicularly on the Earth's surface. For Japan's latitude, AM1.5 is usually used.

[0046] In some of the multiple tests, the wavelength was standardized to 430nm, and only the irradiance was varied. The irradiance used was 100% LED controller intensity (approximately 9W / m²). 2 ), 75% (approximately 7.2W / m 2 ), 50% (approximately 5.1W / m 2 ), 25% (approximately 2.7W / m 2 The light irradiation time was set to 24 hours.

[0047] In some of the multiple tests, the irradiance was set to 2.4-2.7 W / m². 2 The only change was the wavelength. Five wavelengths were used: 430nm, 450nm, 470nm, 505nm, and 590nm. The light irradiation time was 24 hours for wavelengths of 430nm and 450nm, and 48 hours for wavelengths of 470nm, 505nm, and 590nm.

[0048] <Evaluation of cell peripheral shape> First, we investigated the relationship between light wavelength and irradiance that determines whether or not a change in the morphology of eye cells (hereinafter, lens epithelial cells are used as an example) occurs in the subjects. Relationship 1 may also be plot 1, which determines whether or not a change in the morphology of eye cells occurs in relation to the wavelength of light and irradiance. Relationship 1 is not limited to being concretely expressed in the form of a plot, and may also be obtained as data.

[0049] To obtain relationship 1, qualitative and quantitative evaluations were performed on the morphological changes of lens epithelial cells. In the qualitative and quantitative evaluation tests, cells that reached 90-100% confluence were irradiated with light.

[0050] (Qualitative evaluation) Cell membrane staining was performed after light irradiation at a predetermined wavelength and a predetermined irradiance. The supernatant was removed using an aspirator, 0.5 ml of 0.1% CellMask Orange Plasma Membrane Stains (C10045, Life Technologies) was added to the culture medium, and the mixture was allowed to stand for 10 minutes in an atmosphere at 37 °C with a CO2 concentration of 5%. Then, the supernatant was removed with an aspirator and 2 ml of the medium was added, and the same operation of allowing it to stand for 10 minutes in an atmosphere at 37 °C with a CO2 concentration of 5% was repeated three times. Thereafter, the cells were observed.

[0051] Fluorescent microscopy was employed for the observation of the cells. Specifically, the stained cells were photographed at a magnification of 10 times (NA = 0.55, OLYMPUS) using an inverted microscope (FSX100, OLYMPUS).

[0052] Figure 3 is a photograph showing the observation results in the qualitative evaluation. The light irradiation conditions in (a) are a wavelength of 430 nm and an irradiance of 9.0 W / m 2 , the light irradiation conditions in (b) are a wavelength of 430 nm and an irradiance of 5.1 W / m 2 , and the light irradiation conditions in (c) are a wavelength of 450 nm and an irradiance of 4.6 W / m 2 .

[0053] The lens epithelial cells in Figure 3(a) are fragmented and shrunk. The lens epithelial cells in Figure 3(b) are shrunk. That is, apoptosis is progressing in the lens epithelial cells in Figure 3(a) and Figure 3(b). The morphology of the lens epithelial cells in Figure 3(c) has not changed.

[0054] Figure 4 is a plot summarizing the modes of morphological changes of lens epithelial cells in the relationship between the irradiance (vertical axis: unit is W / m 2 ) and the wavelength (horizontal axis: unit is nm) of each LED light (half-value width around 20 nm) used in the experiment. The solar light on the front surface of the lens for each band under the same conditions is also shown in the plot for reference.

[0055] (Quantitative evaluation) Lens epithelial cells prepared separately from those used for qualitative evaluation were cultured under the same conditions as those used to obtain the observational lenses for qualitative evaluation, thereby obtaining lens epithelial cells for quantitative evaluation. Quantitative analysis of the extracellular shape (cell peripheral shape) was then performed on these lens epithelial cells.

[0056] Cell staining images captured using the FSX100 were analyzed using image analysis software (ImageJ) to measure their shape. Thirty cells were randomly selected, and the contour of each cell membrane was manually extracted and its shape measured.

[0057] The parameters of the shape measured were the cell membrane area, circularity, and aspect ratio.

[0058] Figure 5 illustrates the roundness and aspect ratio of lens epithelial cells. When the area is S and the circumference is L, the roundness is defined as 2πS / L in this specification. 2 In this specification, when the lens epithelial cells are approximated as an ellipse in ImageJ, the major axis is a and the minor axis is b, and the aspect ratio is defined as a / b.

[0059] In Figure 3(c), which shows a normal cell, the circularity is 0.63, the aspect ratio is 1.8, and the area is 4.0 × 10⁻⁶. -9 m 2 That was it. On the other hand, Figure 3(a), which shows cells that have undergone morphological changes, has a circularity of 0.21, an aspect ratio of 1.8, and an area of ​​2.1 × 10⁻⁶. -9 m 2 That was it. Figure 3(b), which shows cells that have undergone morphological changes, has a circularity of 0.51, an aspect ratio of 1.1, and an area of ​​2.4 × 10⁻⁶. -9 m 2 That was it.

[0060] In conjunction with quantitative evaluation, to determine the relationship between light wavelength and irradiance that determines whether or not morphological changes occur in lens epithelial cells in subjects, evaluation metrics for assessing whether or not morphological changes occur will be established. Incidentally, since the morphology of cells used in the test differs depending on the type, it is best to evaluate and determine the morphological change metrics according to the type of cell.

[0061] One concrete example of determining evaluation indicators is the following list of possible measurement items, and it is also possible to combine multiple items. <1> Microscope image tonal scaling <2> Fluorescent staining of cell membranes and image interpretation of fluorescence microscopy images using computer software. a. Determination of circularity by automatic measurement of cell membrane circumference and cell extension area. b. Determination of the aspect ratio of cell shape

[0062] For scaling the tonal range of a microscope image, it is practical to scale the image by approximately 2-5 steps. <2> The software-based image recognition system recognizes the difference between the cell area and the background, and measures the cell's area, perimeter, and the distance between any two points. At this time, considering the variability in cell morphology, measurements are performed on multiple fields of view of the microscope image.

[0063] Based on the results obtained from the initial light irradiation condition experiment (preliminary feasibility study) (results from the initial experimental state), the measurement items to be used are selected. In this process, measurement items that can sensitively detect changes are selected based on the results obtained from the initial test light irradiation condition experiment under generally known abnormal conditions. Furthermore, an abnormal condition is defined for the selected measurement item and used as an evaluation index. In addition, the irradiation time, rest time, and timing of observations for observing morphological changes are also determined based on the results of the initial light irradiation condition experiment.

[0064] An example of determining evaluation indicators is as follows. Table 2 below shows the percentage of morphologically abnormal cells (negative control) in the dark. [Table 2] Table 2 shows that apoptosis or apoptosis-like morphological abnormalities occur even when light is not irradiated. The proportion was approximately 4.37% on average, with an unbiased variance of 4.26(S2).

[0065] Therefore, in this embodiment, when 20% of cells exhibit morphological abnormalities, it is considered that the conditions are different from those under dark conditions, and that phototoxicity is occurring. This value is sufficiently larger than the 5% significance level (t-distribution (t=1.833 when degrees of freedom (n-1):9)) (t-test).

[0066] Next, using this evaluation index, we will clarify the relationship between the wavelength of light and irradiance that determines whether or not morphological changes occur in lens epithelial cells.1 To do this, we will observe morphological changes at predetermined timings under different light wavelength and irradiance conditions, and plot whether or not morphological abnormalities occurred at each point. Such a plot is called an action spectrum.

[0067] From that plot, we roughly determine the areas that cause morphological abnormalities and those that do not. Next, we tentatively determine the boundaries between these areas, and then determine the boundaries from the group of points located at those boundaries. Methods for determining these boundaries include the least squares method. This boundary is the plot that represents relationship 1.

[0068] The reason why it is possible to determine the boundary in this way is based on the principle that shorter wavelengths of light (where each photon has greater energy and can cause chemical changes in matter even at low irradiance) and higher irradiances generally cause stronger anomalies.

[0069] Here, the evaluation was explained using the no-light irradiation state as a comparison (reference) and focusing on whether or not it causes morphological abnormalities. However, there may be other light irradiation conditions that result in a state where the morphology is presumed to be better than the no-light state (e.g., a state with good cell roundness), and there is also a relationship 1 where a more desirable state is obtained without causing abnormalities.

[0070] A quantitative evaluation was performed using the above action spectrum. Figure 6 is a bar graph showing the results of the quantitative evaluation, where (a) is the area S, (b) is the circularity, and (c) is the aspect ratio.

[0071] As shown in Figure 6, quantitative evaluation revealed that as irradiance increased, the area of ​​lens epithelial cells increased in some samples, while decreasing in others. Circularity also increased with increasing irradiance.

[0072] Generally, apoptosis reduces both cell area and circularity. The aspect ratio decreases or remains constant. Therefore, the changes in area and circularity in the quantitative evaluation described above are distinct from the behavior generally understood to result from apoptosis.

[0073] The inventors found that increasing irradiance reduces the active stretching and contracting function of lens epithelial cells. The inventors further investigated the relationship between irradiance and changes in the migration of lens epithelial cells.

[0074] <Evaluation of cell migration> The relationship between light wavelength and irradiance, which determines whether or not the migration of lens epithelial cells is altered, is determined using the same steps as for relationship 1. In other words, the cell type decided to be used throughout the entire process is used first, the evaluation index is determined first, and then the relationship is studied.

[0075] First, we determine metrics to evaluate whether or not a change in migration occurs. Here, migration refers to the distance a cell travels per unit time within a certain period of time on a polystyrene culture surface for tissue culture (hereinafter also referred to as "movement within the culture medium"). To determine migration, we determine the cell culture conditions that allow us to observe the amount of cell movement and the conditions for measuring the distance traveled. Then, we observe the cells over time to determine the migration rate per unit time. An example of culture conditions that allow us to observe the amount of movement is to divide the culture medium into blocks with partitions, etc., creating areas where cells are seeded and areas where they are not (gaps). This can involve the formation of linear or circular non-seeding areas of a specific width. In this state, the partitions are removed, marking the start of measurement, and then the movement of the cells is observed. If the migration rate is appropriate, the cells will move, the non-seeding areas (gaps) will decrease, and then become occluded. The distance traveled per unit time until the non-seeding area decreases or becomes occluded is called the migration rate under those culture conditions.

[0076] Furthermore, initial experiments studying migration and light irradiation conditions revealed the principle that shorter wavelengths of light and higher intensity irradiation induced more pronounced abnormalities in migration. Therefore, possible evaluation indicators include plots of migration (cell movement distance per unit of time) for specific cell types under each light irradiation condition, and plots of the change in migration (difference or ratio) between no light irradiation and specific light irradiation conditions. Relationship 2 is determined by obtaining such plots. A specific example of the above content is as follows.

[0077] To evaluate the migration of lens epithelial cells, a cell-free region (gap) of a predetermined width was created on a surface containing a large number of cells. Then, the effect of light irradiation on cell migration was investigated based on the closure process of this gap.

[0078] The cells used in this experiment were prepared in the same manner as in the previous experiment (<Cells Used>) up to centrifugation. Then, a cell suspension of 10,000 cells / ml was prepared. Subsequently, 700 cells were seeded into each well of two culture inserts (ib81176, ibidi) attached to a 60 mm dish to form a gap of approximately 500 μm in width.

[0079] Figure 7 is a schematic plan view of a well used to create a gap (wound) of a predetermined width on a surface containing numerous cells.

[0080] One day after seeding the cells, the two culture insert wells were removed, and light irradiation was started after confirming gap formation. Immediately after the start of light irradiation, and at 6, 12, 24, and 48 hours later, images were taken at 4x magnification using the above-mentioned inverted microscope to observe the gap. The distance between the edges of the captured gaps was measured using image analysis software (ImageJ). Measurements were taken at 10 arbitrary locations at each time point after light irradiation, and the average value was taken as the distance between the edges of the gap.

[0081] Figure 8 shows the results for a wavelength of 430 nm and an irradiance of 9.0 W / m². 2 The diagram shows the state of gap closure at the following times: (a) is a photograph showing the state of the gap at 0h (immediately after the start of light irradiation), (b) is a photograph showing the state of the gap 24h after the start of light irradiation, and (c) is a photograph showing the state of the gap 48h after the start of light irradiation. Figure 9 shows a wavelength of 430 nm and an irradiance of 5.1 W / m². 2 The diagram shows the state of gap closure at the following times: (a) is a photograph showing the state of the gap at 0h (immediately after the start of light irradiation), (b) is a photograph showing the state of the gap 24h after the start of light irradiation, and (c) is a photograph showing the state of the gap 48h after the start of light irradiation. Figure 10 shows a wavelength of 430 nm and an irradiance of 2.7 W / m². 2 The diagram shows the state of gap closure at the following times: (a) is a photograph showing the state of the gap at 0h (just before the start of light irradiation), (b) is a photograph showing the state of the gap 24h after the start of light irradiation, and (c) is a photograph showing the state of the gap 48h after the start of light irradiation. Figure 11 shows the wavelength at 470 nm and irradiance at 2.5 W / m². 2The diagram shows the state of gap closure at the following times: (a) is a photograph showing the state of the gap at 0h (immediately after the start of light irradiation), (b) is a photograph showing the state of the gap 24h after the start of light irradiation, and (c) is a photograph showing the state of the gap 48h after the start of light irradiation. The scale (horizontal lines) in the photographs in Figures 8 to 11 represents 500 nm. Figure 12 shows plots grouped by wavelength, illustrating the relationship between the gap edge distance (in μm) and the time since the start of light irradiation (horizontal axis: in h). The slope of the plot is distance / time, representing the migration velocity of lens epithelial cells. As shown in Figure 12, a correlation was found between the wavelength of the irradiated light and the migration speed.

[0082] Furthermore, the inventors discovered that by observing the gap closure in each figure, the rate of decrease in migratory ability can be divided into several groups.

[0083] Figure 13 is a histogram showing the distribution of the rate of decrease in migratory ability during gap closure.

[0084] As shown in Figure 13, two distinct stages of change in the rate of decrease were observed. Therefore, we decided to distinguish between those with a large rate of decrease (80% or more) as the "Large effect" and those with a moderate rate of decrease (40-70%) as the "Middle effect". From this point forward, the classification of migratory behavior is based on this two-stage change as the boundary.

[0085] <Insights gained from the evaluation of morphological and migratory changes in lens epithelial cells> Figure 14 shows the irradiance of each light source (full width 10 nm) used in the experiment (vertical axis: unit is W / m²). 2 This plot shows the relationship between light and wavelength (horizontal axis: unit is nm), combining the morphological changes and migratory changes of lens epithelial cells. Sunlight is also included in the plot for reference.

[0086] The leftmost dotted line plot in Figure 14 represents the relationship between light wavelength and irradiance that determines whether or not a change in the morphology of eye cells occurs in the subjects. Specifically, it is a plot that determines whether or not a change in the morphology of eye cells occurs in relation to the wavelength of light and irradiance. The rightmost dotted line plot in Figure 14 represents the relationship between light wavelength and irradiance that determines whether or not the migration behavior of eye cells is altered in the subjects. Specifically, it is a plot that determines whether or not the migration behavior of eye cells is altered in relation to the wavelength of light and irradiance.

[0087] In the relationship between the wavelength of light and irradiance, plot 1 is on the shorter wavelength side of plot 2. In the relationship between the wavelength of light and irradiance, plot 1 divides the area into multiple regions 1 consisting of regions that cause the morphological change and regions that do not. In the relationship between the wavelength of light and irradiance, the plot 2 divides the data into multiple regions 2, consisting of regions that cause a change in migratory properties (for example, a decrease) and regions that do not.

[0088] The dotted line plot in the middle of Figure 14 is plot 3, which further subdivides the region in region 2 that causes the change in migratory properties, according to the degree of the change in migratory properties. Plots 1 to 3 are approximation curves that divide each region. Alternatively, two points closest to the boundary in each adjacent region may be selected from each region, and an approximation curve representing the boundary may be created from a total of four points.

[0089] It is preferable to obtain the plots in Figure 14 before carrying out one embodiment of the present invention. In other words, one embodiment of the present invention does not need to include the step of creating the plots in Figure 14 from scratch. Also, each plot in Figure 14 becomes a straight line (approximate straight line) if the vertical axis is displayed on a logarithmic scale.

[0090] Figure 14 shows how, for a given person (subject), the wavelength and irradiance of light entering the eye affect the eye cells in a given way. As Figure 14 shows, if no short-wavelength light enters the eye at all, the effect on eye cells is extremely small. On the other hand, if no short-wavelength light enters the eye at all, it is also possible that no short-wavelength visible light enters the eye at all.

[0091] <An embodiment of the present invention derived from findings> Based on the above findings, in one embodiment of the present invention, a relationship acquisition step is preferably performed in advance to obtain a relationship 1 between the wavelength of light and irradiance that determines whether or not a change in the morphology of eye cells in a subject, and a relationship 2 between the wavelength of light and irradiance that determines whether or not a change in the migratory properties of eye cells is caused. Then, a relationship selection step is performed to select at least one of the relationship 1 and relationship 2 according to the degree required for the morphological change 1 and the degree required for the migratory properties 2. Then, a determination step is performed to determine the wavelength of light and irradiance that should be incident on the subject's eye based on the selected relationship. This determination step may also be called a spectral irradiance range determination step. The characteristics of the light incident on the eye are determined by each of the above steps.

[0092] The aforementioned relationship selection process will be explained below.

[0093] Through the aforementioned steps, the light irradiation conditions that cause cells to enter an abnormal state were plotted, and their relationships were clarified. Next, in order to determine the characteristics of the light incident on the eye, it was decided which relationship to base the determination on.

[0094] The underlying principle of the decision is whether the abnormal condition induced in the cells is harmful to the subject. At first glance, it seems most desirable to have no harmful effects at all and to remove all light with harmful irradiation conditions. However, in reality, a diverse range of wavelengths of light are necessary, and there is a fundamental principle that it is preferable not to alter the ambient light to avoid taking on other undiscovered risks unless it is clearly harmful.

[0095] Since the human body uses light to perceive information from the outside world, it is thought that light exposure across a diverse range of wavelengths is necessary. Furthermore, the harmful effects vary depending on the exposure time and the length of the rest period.

[0096] When focusing on individual subjects, the results are influenced by individual differences in the ocular optics related to age, the strength of cell resistance depending on the presence or absence of disease, and the condition at that particular time (e.g., immediately after surgery, immediately after injury, etc.). Therefore, if necessary, additional research may be required to determine how much the relationship changes in response to changes in the above conditions.

[0097] Taking the above conditions into consideration, and considering the degree of morphological change 1 and the degree required for migratory ability 2, it is determined which relationship and degree is preferable to base the determination on. Then, based on the selected relationship, the wavelength and irradiance of the light to be incident on the subject's eye are determined. In this determination, for example, the region in Figure 14 may be used as a criterion.

[0098] Generally, there is a principle that shorter wavelengths of light and higher intensity irradiation cause stronger anomalies. Therefore, the irradiance for each wavelength is plotted, and the amount of incident light is determined by keeping it below that irradiance. The specific example described in this embodiment so far is based on this principle.

[0099] If the degree required for morphological change (1) is high and the degree required for migratory behavior (2) is low, then it is sufficient to simply select the region that does not cause morphological change from region 1. If there is a plot 3, the wavelength and irradiance in the region where the region that causes a change in migratory behavior more than plot 3 overlaps with the region selected from region 1 should be determined as the characteristics of the light incident on the eye.

[0100] In the relationship selection step, it is preferable to select plot 1 and plot 2. Furthermore, it is preferable to have a region selection step after the relationship selection step in which one region is selected from region 1 and one region is selected from region 2.

[0101] In the region selection step, it is preferable to perform at least (1) of the following (1) and (2). (1) Select the region that does not undergo the aforementioned morphological change from region 1. (2) Select the region that does not cause the change in migratory behavior from region 2. Then, in the determination step, the wavelength and irradiance of light that belong to each of the selected regions in common are obtained as the wavelength and irradiance of light that should be incident on the eyes of the subject.

[0102] Plots 1 and 2 are preferably created based on regional information of the location of the subject. "Regional information" may be, for example, latitude information such as AM1.5 above, or climate information. "Climate information" may be, for example, the average annual sunshine hours per day, or the average annual total solar radiation, etc.

[0103] The aforementioned ophthalmic cells may be lens epithelial cells, retinal pigment epithelial cells, corneal endothelial cells, corneal epithelial stem cells, or subconjunctival fibroblasts. Among these, lens epithelial cells have high phototoxicity sensitivity, and as a result, the effects of the present invention are particularly pronounced.

[0104] The technical scope of the present invention is not limited to the embodiments described above, and includes various modified and improved forms to the extent that specific effects can be obtained by the constituent elements of the invention or combinations thereof.

[0105] There are no restrictions on who can participate; participants may or may not have cataracts.

[0106] In addition to plot 2, a plot 3 may be obtained in the relationship acquisition step, which further subdivides the region that does not cause the change in migratory behavior in region 2, in order to determine the degree of the change in migratory behavior.

[0107] {Embodiment 2} For details not described in Embodiment 2 below, refer to the content of Embodiment 1.

[0108] [Detailed insights] In Embodiment 2, the following two relationships with respect to the target person, as described in Embodiment 1, • The relationship between light wavelength and irradiance that determines whether or not morphological changes in eye cells occur. • The relationship between light wavelength and irradiance that determines whether or not the migratory behavior of eye cells is altered. In addition, • The relationship between light wavelength and irradiance that determines whether or not it alters the invasiveness of eye cells. Add this to the list of possible relationships to acquire. In Embodiment 2, a relationship acquisition step is performed (preferably in advance) to obtain at least one of the relationships 1 to 3. Obtaining at least two of the relationships 1 to 3 is preferable because it allows for the determination of more suitable optical characteristics.

[0109] Then, a relationship selection step is performed in which at least one of the relationships 1, 2, and 3 is selected according to at least one of the degree required for morphological change 1, the degree required for migratory ability 2, and the degree required for invasiveness 3. Then, a determination step is performed to determine the wavelength and irradiance of light to be incident on the subject's eye based on the selected relationship. This determination step may also be called a spectral irradiance range determination step.

[0110] The aforementioned relationship 3 may also be plot 4, which distinguishes whether or not the relationship between the wavelength of light and irradiance enhances the infiltration of eye cells.

[0111] In the relationship acquisition step, at least relationship 3 may be obtained in advance, and in the relationship selection step, at least relationship 3 may be selected. In other words, relationship 3 and relationship 1 may be adopted, or relationship 3 and relationship 2 may be adopted.

[0112] In the relationship acquisition step, relationships 1 to 3 may be obtained in advance, and in the relationship selection step, relationships 1 to 3 may be selected.

[0113] The following embodiments are also preferred. "In the relationship between the wavelength of light and irradiance, the plot 4 divides the area into multiple regions 4 consisting of regions that cause increased penetration and regions that do not, In the relationship selection step, plot 1, plot 2, and plot 4 are selected. The process further includes a region selection step in which one region is selected from region 1, one region is selected from region 2, and one region is selected from region 4, after the relationship selection step. In the region selection step, at least (1) of the following (1) to (3) is performed: (1) Select the region that does not undergo the aforementioned morphological change from region 1. (2) Select the region that does not cause the change in migratory behavior from region 2. (3) Select the region from region 4 that does not result in the increased invasiveness.

[0114] In the relationship between the wavelength of light and irradiance, plot 4 may be on the shorter wavelength side of plot 3 and on the longer wavelength side of plot 2.

[0115] The specific invasiveness of Embodiment 2 will be described in detail below. The morphological changes and migratory properties described in Embodiment 1 may be understood in relation to the invasiveness described below, and area selection may be made accordingly, or is preferable.

[0116] <Cells Used> Human lens epithelial cells SRA01 / 04 (RCB1591, RIKEN BRC) were used. The reagents were the culture medium used in Embodiment 1. The cell density was 2 × 10⁻⁶. 4 I named it cells / dish.

[0117] <Light source and light irradiation conditions used> The equipment used for light irradiation was a xenon light source (MAX-350, ASAHI SPECTRA). The light irradiation conditions were as follows:

[0118] Light irradiation was performed from 15 cm above the cell culture surface under cell culture conditions (at a temperature of 37°C and an atmospheric environment with a CO2 concentration of 5%). It was anticipated that the effect on cells would differ depending on the intensity of the light, even at the same wavelength. Therefore, multiple bandpass filters were used with the xenon light source, and the intensity was varied for each experiment using the light source controller. Table 3 shows the irradiance conditions for each wavelength. The light irradiation time was 24 hours. [Table 3]

[0119] Furthermore, the tests under conditions without light irradiation (Negative Control: NC) were conducted four times under the same conditions for each test. The plots for each test are referred to as NC-1, NC-2, NC-3, and NC-4.

[0120] <Evaluation of invasion> The relationship between light wavelength and irradiance, which determines whether or not the invasiveness of lens epithelial cells is enhanced, will be determined using the same steps as for relationship 1. In other words, the cell type to be used throughout the entire process will be used first, the evaluation index will be determined, and then the relationship will be studied.

[0121] First, we determine the metrics used to assess whether or not there will be a change in invasiveness. The term "invasiveness" as used in this specification is explained below.

[0122] First, a mold is prepared with a pattern of straight convex ridges in plan view. Polydimethylsiloxane (PDMS) is injected into this mold to form grooves in the PDMS corresponding to the convex ridges of the mold. Then, the PDMS is released from the mold to obtain a PDMS substrate with grooves formed therein. In this specification, infiltration is defined as the entry of eye cells into the grooves from the entrance of the grooves (hereinafter referred to as the "opening") when eye cells are placed in front of the grooves on the PDMS substrate. As shown in Figures 15 to 18 below, the PDMS substrate is provided with a plurality of grooves of equal width.

[0123] For each PDMS substrate, the following groove widths were prepared. The groove depth and groove spacing were as follows. Groove widths: 6.2μm, 7.2μm, 7.9μm, 8.9μm, 9.6μm, 10.6μm, 11.6μm Groove depth: 10.6 μm Groove spacing: 200μm

[0124] Cell behavior at the groove openings was captured at 10-minute intervals, and the invasiveness of ophthalmic cells was analyzed using image analysis software (ImageJ).

[0125] Regarding invasiveness, eye cells were classified into the following four categories. • Eye cells that passed through the opening (demonstrated passage behavior) (Figure 15) • Infiltrated (into the groove) ophthalmic cells, where the entire cell has entered the groove (Figure 16) • Ophthalmic cells exhibiting incomplete invasion behavior (invasion failure), where a portion of the cell entered the groove, but the entire cell crawled out before it could fully penetrate (Figure 17). • Ophthalmic cells that, in whole or in part, invaded the groove, but then climbed the side walls of the groove and crawled out through the upper opening of the groove, showing infiltration (crawling out of the groove) (Figure 18). Figure 15 is a plan view showing how eye cells pass through the opening during NC (no light irradiation), with (a) being 0h (immediately after the start of light irradiation), (b) 10 minutes after the start of light irradiation, (c) 20 minutes after the start of light irradiation, (d) 30 minutes after the start of light irradiation, (e) 40 minutes after the start of light irradiation, and (f) 50 minutes after the start of light irradiation. Figure 16 is a plan view showing the infiltration (into the grooves) of eye cells during NC without light irradiation, with (a) being 0h (immediately after the start of light irradiation), (b) 10 minutes after the start of light irradiation, (c) 20 minutes after the start of light irradiation, (d) 30 minutes after the start of light irradiation, (e) 40 minutes after the start of light irradiation, and (f) 50 minutes after the start of light irradiation. Figure 17 is a plan view showing the incomplete invasion behavior (invasion failure) of ophthalmic cells when NC (no light irradiation) is performed, and (a) is a photograph showing the state at 0h (immediately after the start of light irradiation), (b) is 10 minutes after the start of light irradiation, (c) is 20 minutes after the start of light irradiation, (d) is 30 minutes after the start of light irradiation, (e) is 40 minutes after the start of light irradiation, (f) is 50 minutes after the start of light irradiation, (g) is 60 minutes after the start of light irradiation, and (h) is a photograph showing the state at 70 minutes after the start of light irradiation. Figure 18 is a plan view showing how eye cells infiltrate (climb up from the grooves) during NC (no light irradiation), with (a) being 0h (immediately after the start of light irradiation), (b) 10 minutes after the start of light irradiation, (c) 20 minutes after the start of light irradiation, (d) 30 minutes after the start of light irradiation, (e) 40 minutes after the start of light irradiation, (f) 50 minutes after the start of light irradiation, and (g) 60 minutes after the start of light irradiation.

[0126] The parameters used for evaluation are as follows: • Number of cells passing through = Number of cells showing passage behavior / Number of openings • Number of invading cells (in the groove) = Number of cells that have completely entered the groove, indicating invasion / Number of openings • Number of incomplete invasions (invasion failures) = Number of cells showing incomplete invasion behavior (invasion failures) / Number of openings • Number of invading cells (crawling out of grooves) = Number of cells exhibiting invading behavior (crawling out of grooves) / Number of openings

[0127] Figure 19 shows plots NC-1, NC-2, NC-3, and NC-4, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis.

[0128] In Embodiment 2, the invasiveness was evaluated as follows. For each groove width w, a number of infiltrations (within the groove) within the upper and lower limits of NC was considered to be "less affected by light irradiation." We considered an increase in infiltration to occur when the sum of the number of infiltrations (within the groove) and the number of infiltrations (climbing out of the groove) was higher than the maximum value among each NC plot. "Invasion suppression" was defined as the sum of the number of invading lesions (within the groove) and the number of invading lesions (climbing out of the groove) being lower than the minimum value among each NC plot. All other NC plots were considered "low effect".

[0129] Figure 20 shows plots NC-1, NC-2, NC-3, and NC-4, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis, along with the results at wavelengths of 430-460 nm. Figure 21 shows the plots NC-1, NC-2, NC-3, and NC-4, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis, along with the results at a wavelength of 480 nm. Figure 22 shows the plots NC-1, NC-2, NC-3, and NC-4, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis, along with the results at a wavelength of 500 nm. Figure 23 shows the plots NC-1, NC-2, NC-3, and NC-4, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis, along with the results at a wavelength of 520 nm. Figure 24 shows the plots NC-1, NC-2, NC-3, and NC-4, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis, along with the results at a wavelength of 540 nm.

[0130] At wavelengths of 430-460 nm (Figure 20) and 480 nm (Figure 21), a tendency was observed for a higher number of infiltrations (in the grooves) compared to NC. On the other hand, at wavelengths of 500 nm (Figure 22), 520 nm (Figure 23), and 540 nm (Figure 24), there was a large variation in the number of infiltrations (in the grooves), and a tendency for a lower number of infiltrations (in the grooves) was observed.

[0131] The following diagram shows the number of infiltrations (inside the trench) organized according to irradiance.

[0132] Figure 25 shows 1.4~1.5 W / m². 2 This figure shows the results for low irradiance, with the number of infiltrations (in the groove) on the vertical axis and the groove width w (μm) on the horizontal axis. Figure 26 shows 3.2–5.5 W / m². 2 This figure shows the results when the number of penetrations (in the groove) is plotted on the vertical axis and the groove width w (μm) is plotted on the horizontal axis, for moderate irradiance. Figure 27 shows 7.5 W / m 2 This figure shows the results when the number of infiltrations (inside the groove) is on the vertical axis and the groove width w (μm) is on the horizontal axis, under the above high irradiance conditions.

[0133] The number of infiltrations (within the trenches) tended to be lower at lower irradiance levels and higher at higher irradiance levels.

[0134] The following figure summarizes the effects of light irradiation, focusing on the number of particles passing through and the number of incomplete invasions.

[0135] Figure 28 shows the results when the number of passages is on the vertical axis and the groove width w (μm) is on the horizontal axis. Figure 29 shows the results when the number of incomplete invasion behaviors (invasion failures) is plotted on the vertical axis and the groove width w (μm) is plotted on the horizontal axis. Figure 30 shows the results when the number of infiltrations (climbing out of the groove) is on the vertical axis and the groove width w (μm) is on the horizontal axis.

[0136] In terms of the number of passes (Figure 28), 480 nm - 7.6 W / m 2 This plot showed a higher value compared to the other plots. In cases of "invasion failure" (Figure 29) and "climbing out of the groove" (Figure 30), the values ​​were extremely low compared to the number of successful invasions (within the groove), regardless of the light irradiation conditions or groove width.

[0137] Considering the above, light with wavelengths below 480 nm may enhance invasiveness. Therefore, to inhibit enhanced invasiveness, it is preferable to determine the characteristics of the light so that the irradiance of light with wavelengths below 480 nm that enters the subject's eye is low. In particular, 1.49 W / m at a wavelength of 460 nm. 2 Even at such low irradiance levels, it showed higher penetration than in the dark, so it is preferable to set the irradiance at a wavelength of 460 nm in the above characteristics to be sufficiently low. On the other hand, even with light exceeding 480 nm, at a wavelength of 540 nm, it was 20.1 W / m 2 Since invasiveness can be enhanced at relatively low irradiance, it is preferable to set the irradiance at a wavelength of 540 nm in the above characteristics to a low value. The width w (μm) of the groove in which cells can easily invade varies depending on the size and stiffness (ease of deformation) of the cell. Therefore, the relationship between the wavelength of light and irradiance that determines whether or not invasiveness is enhanced is preferably evaluated for grooves of multiple widths less than the diameter of the cell being evaluated, in an undeformed state, for example, in a suspended state.

[0138] {Embodiment 3} For details not described in Embodiment 3 below, refer to the contents of Embodiments 1 and 2. Embodiment 3 describes the test results for human cell lines (LEC, Cell Line) in Embodiments 1 and 2, as well as for human primary lens epithelial cells (LEC, Primary) and retinal pigment epithelial cells (RPE).

[0139] Figure 31 shows the irradiance of each light source (full width 10 nm) used in the experiment (vertical axis: unit is W / m²). 2 This plot shows the pattern of invasive changes in lens epithelial cells in relation to the relationship between (x) and wavelength (horizontal axis: unit is nm). Sunlight is also included in the plot for reference. Figure 31A shows the case when the groove width w is 8.9 μm, and Figure 31B shows the case when the groove width w is 9.6 μm. As shown in the above figures, even if relationship 3 is obtained in advance in the relationship acquisition step and relationship 3 is selected in the relationship selection step, the present invention is valid and the problems of the present invention can be solved.

[0140] In Figure 31, the upper part of the V-shaped boundary line indicates increased invasion, while the lower part indicates no change in invasion or inhibition of invasion. The boundary line is the boundary between increased invasion and low effect in each NC plot, and it is a line obtained using the least squares method.

Claims

1. The ophthalmic cells are lens epithelial cells, retinal pigment epithelial cells, corneal endothelial cells, corneal epithelial stem cells, or subconjunctival fibroblasts, Light is the light that an object uses to perceive information from the outside world. A relationship acquisition step to obtain in advance a relationship between the wavelength of light and irradiance 1 that determines whether or not a morphological change, which is contraction of the eye cells, occurs in the subject, and a relationship between the wavelength of light and irradiance 2 that determines whether or not the migration distance, which is the migration distance of the eye cells, decreases. A relationship selection step in which at least one of the relationship 1 and the relationship 2 is selected according to the degree 1 required for the morphological change and the degree 2 required for the migratory ability, A determination step of determining the wavelength and irradiance of the light to be incident on the eyes of the subject based on the selected relationship, It has, The aforementioned relationship 1 is a plot 1 that determines whether or not the aforementioned morphological change occurs in the relationship between the wavelength of light and the irradiance. The aforementioned relationship 2 is a plot 2 that determines whether or not the migratory properties are reduced in relation to the wavelength of light and the irradiance. In the relationship between the wavelength of light and irradiance, plot 1 is on the shorter wavelength side of plot 2. In the relationship between the wavelength of light and irradiance, the plot 1 divides the area into multiple regions 1 consisting of regions that cause the morphological change and regions that do not. In the relationship between the wavelength of light and irradiance, the plot 2 divides the relationship into multiple regions 2 consisting of regions that cause a decrease in migratory behavior and regions that do not. In the relationship selection step, plot 1 and plot 2 are selected. The process further includes a region selection step in which one region is selected from region 1 and one region is selected from region 2, after the relationship selection step. In the region selection step, at least (1) of the following (1) and (2) is performed: (1) Select the region that does not undergo the morphological change from region 1. (2) Select the region from region 2 that does not result in the reduction of motility. A method for determining the wavelength and irradiance of light used by a subject to perceive information from the outside world, wherein the wavelength and irradiance of light that belong to each of the selected regions in common are obtained as the wavelength and irradiance of light to be incident on the subject's eyes.

2. A method for determining the wavelength of light and irradiance used by a subject to perceive information from the outside world, according to claim 1, further comprising obtaining in the relationship acquisition step a plot 3 in which, in addition to the plot 2, the region in region 2 that causes the decrease in migratory ability is further subdivided according to the degree of the decrease in migratory ability.

3. The plots 1 and 2 are created based on the geographical information of the subject, and the method for determining the wavelength of light and irradiance used by a subject to perceive information about the outside world, according to claim 1 or 2.

4. The ophthalmic cells are lens epithelial cells, retinal pigment epithelial cells, corneal endothelial cells, corneal epithelial stem cells, or subconjunctival fibroblasts, Light is the light that an object uses to perceive information from the outside world. For the target individuals, - The relationship between the wavelength of light and irradiance that determines whether or not a morphological change, namely contraction of the eye cells, occurs. - Relationship between the wavelength of light and irradiance that determines whether or not the migratory ability, which is the distance traveled by the eye cells over a certain period of time, is reduced. - The relationship between the wavelength of light and irradiance that determines whether or not the infiltration, which is the number of eye cells that pass through the opening of the groove into the interior, is enhanced. A relationship acquisition process to obtain at least one of the following in advance, A relationship selection step in which at least one of the relationship 1, relationship 2, and relationship 3 is selected according to at least one of the degree required for the morphological change 1, the degree required for the migratory properties 2, and the degree required for the infiltration properties 3, A determination step of determining the wavelength and irradiance of the light to be incident on the eyes of the subject based on the selected relationship, It has, The aforementioned relationship 1 is a plot 1 that determines whether or not the aforementioned morphological change occurs in the relationship between the wavelength of light and the irradiance. The aforementioned relationship 2 is a plot 2 that determines whether or not the migratory properties are reduced in relation to the wavelength of light and the irradiance. The aforementioned relationship 3 is plot 4 which determines whether or not the penetration is enhanced in relation to the wavelength of light and the irradiance. In the relationship acquisition step, relationships 1 to 3 are obtained in advance, and in the relationship selection step, relationships 1 to 3 are selected. In the relationship between the wavelength of light and irradiance, plot 1 is on the shorter wavelength side of plot 2. In the relationship between the wavelength of light and irradiance, the plot 1 divides the area into multiple regions 1 consisting of regions that cause the morphological change and regions that do not. In the relationship between the wavelength of light and irradiance, the plot 2 divides the relationship into multiple regions 2 consisting of regions that cause a decrease in migratory behavior and regions that do not. In the relationship between the wavelength of light and irradiance, the plot 4 divides the area into multiple regions 4, consisting of regions that cause increased penetration and regions that do not. In the relationship selection step, plot 1, plot 2, and plot 4 are selected. The process further includes a region selection step in which, after the relationship selection step, one region is selected from region 1, one region is selected from region 2, and one region is selected from region 4. In the region selection step, at least (1) of the following (1) to (3) is performed: (1) Select the region that does not undergo the morphological change from region 1. (2) Select the region from region 2 that does not result in the reduction of motility. (3) Select the region on the side that does not result in increased invasiveness from region 4. A method for determining the wavelength and irradiance of light used by a subject to perceive information from the outside world, wherein the wavelength and irradiance of light that belong to each of the selected regions in common are obtained as the wavelength and irradiance of light to be incident on the subject's eyes.

5. A method for determining the wavelength of light and irradiance used by a subject to perceive information from the outside world, according to claim 4, further obtained in the relationship acquisition step, in addition to the plot 2, a plot 3 is obtained in which the region on the side causing the decrease in migratory ability in region 2 is further subdivided according to the degree of the decrease in migratory ability.

6. The method for determining the wavelength of light and irradiance used by a subject to perceive information from the outside world, according to claim 4, wherein, in the relationship between the wavelength of light and irradiance, plot 4 is on the shorter wavelength side of plot 3 and on the longer wavelength side of plot 2.

7. A method for determining the wavelength of light and irradiance used by a subject to perceive information about the outside world, according to any one of claims 4 to 6, wherein at least plots 1, 2, and 4 are created based on regional information of the subject's location.

8. The method for determining the wavelength of light and irradiance used by a subject to perceive information from the external world, according to claim 1 or 4, wherein the eye cells are lens epithelial cells.

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