JP5869810B2 - Method for evaluating the load on a living body - Google Patents

Description

  The present invention relates to a method for evaluating various loads on a living body, a method for detecting an influence due to a chemical load, for example, a drug effect or toxicity of a test substance, or an effect due to a physical load.

In vitro test methods using organ culture and cell culture are used to detect various loads on living organisms, for example, the efficacy and toxicity of substances on living organisms, in order to replace conventional animal testing systems. Has been.
For example, in order to detect the eye toxicity of eye drops that are administered directly to the eye or cosmetics such as facial cleansers, hair wash agents, and cosmetics that accidentally enter the eye, a drayed eye that puts the test substance into the conjunctival sac of a rabbit Although an irritation test has been conducted, the present inventor has developed a test method using rabbit cultured corneal epithelial cells reconstructed three-dimensionally in vitro, and has succeeded in detecting eye irritation ( Non-patent documents 1 to 4). A test method for exposing a test substance to cells cultured in a monolayer is also well known.
Furthermore, it is expected that cultured cells reconstructed in the same manner as in vivo can be detected with higher sensitivity than cells cultured in a monolayer.

Biol Pharm Bull 2009; 32: 807-12. Toxicol In Vitro 2009; 23: 555-60. J Oleo Sci 2009; 58: 437-42. Yakugaku Zasshi 2009; 129: 1113-20.

However, in the in vitro test method using cultured cells, there is a problem that the quality of the cells subjected to the test differs between wells (culture units). In addition, since one culture unit becomes one test unit, there is a problem that in one test, it is not possible to increase the test unit and improve accuracy, or to test many kinds of test substances at one time. In order to conduct a test closer to a living body with cultured cells, a tissue that mimics the living body is reconstructed in vitro using tissue-derived cultured cells. This method is time consuming and laborious, cannot produce a large number of culture units at a time, and cannot naturally provide a large number of test units.
Accordingly, an object of the present invention is to provide a plurality of test units from one unit region such as one culture unit, and to provide an evaluation method for various loads using the test units.

The present invention
[1] (1) A process of processing a cell mass in one unit region so as to provide two or more test units (hereinafter referred to as a processing process),
(2) A step of providing two or more test units with a cell mass moved from the original one unit region to a new culture region and / or a cell mass left in the original one unit region (hereinafter referred to as a test unit providing step) ),
(3) A step of exposing the cell mass to a chemical or physical load in each test unit provided in the step (2) and detecting the influence (hereinafter referred to as a test step).
Including the load evaluation method,
[2] As the processing and movement, an incision is made in the cell mass of one unit area, and all or part of the cell mass moved to a new culture area and / or all of the cell mass left in the original area Or, in part, providing two or more test units, the method of [1],
[3] As the processing and movement, cells are cultured in a state in which one or more movable substrates are arranged in advance on the entire surface or a part of one unit region, and all or a part of the substrate is moved. Alternatively, two or more test units are provided with all or part of the cell mass moved to the new culture area and / or with all or part of the cell mass left in the original area without being moved. , The method of [1],
[4] The method according to [3], wherein a movable substrate is disposed on the entire surface of one unit region.
[5] The method according to [3], wherein a movable substrate is disposed in a part of one unit region.
[6] The method according to any one of [3] to [5], wherein only a cell cultivatable substrate is used as the movable substrate.
[7] The method according to any one of [3] to [5], wherein only a substrate that is difficult to culture is used as the movable substrate.
[8] The method according to any one of [3] to [5], wherein a cell cultivatable substrate and a cell cultivated substrate are used in combination as the movable substrate.
[9] The method according to any one of [3] to [8], wherein the entire movable substrate is moved.
[10] The method according to any one of [3] to [8], wherein a part of the movable substrate is moved.
[11] The method according to any one of [3] to [8], wherein the movable substrate is not moved.
[12] The method according to any one of [3] to [11], wherein the entire surface of one unit region can be cultured.
[13] The method according to any one of [3] to [11], wherein the entire surface of one unit region is difficult to culture.
[14] The method according to any one of [3] to [11], wherein a part of one unit region is capable of cell culture and the rest is difficult to culture.
[15] The method according to any one of [1] to [14], wherein the test unit is provided only from a cell mass that has moved to a new culture region.
[16] The method according to [1] to [14], wherein the test unit is provided only from the cell mass left in one unit region.
[17] The method according to any one of [1] to [14], wherein the test unit is provided from a cell mass moved to a new culture region and a cell mass left in one unit region.
[18] The method according to any one of [1] to [17], wherein the cell mass is a non-immortalized cell derived from a living tissue,
[19] The method according to any one of [1] to [17], wherein the cell mass is an immortalized cultured cell,
[20] The method according to any one of [1] to [19], wherein the cell mass is derived from the cornea.
[21] The method according to any one of [1] to [19], wherein the cell mass is derived from a corneal epithelium.
[22] The method according to any one of [1] to [21], wherein the evaluation after exposure to a chemical or physical load is analyzed by cell extension from a partial cell mass,
About.
In the present invention, the processing step and the test unit providing step can be performed in this order, or the processing step and the test unit providing step can be performed simultaneously in parallel. That is, after completing the processing of the cell mass in one unit area, the next movement can be performed, or both processes can be performed simultaneously by moving simultaneously with the processing of the cell mass in one unit area. Can also be implemented.

  According to the present invention, a plurality of test units can be produced from one unit region.

It is explanatory drawing which shows typically the state which made the cell mass of one unit area | region divided radially so that two or more test units could be provided. It is explanatory drawing which shows typically the state which enabled the division | segmentation of the cell cluster of one unit area | region so that two or more test units could be provided. It is explanatory drawing which shows typically the state which enabled the division | segmentation of the cell mass of one unit area | region so that two or more test units could be provided. Explanatory drawing schematically showing a state where a cell mass in one unit area can be divided into sea-island types (island portions are concentrically arranged) so that two or more test units can be provided. It is. Explanatory drawing schematically showing a state in which a cell mass in one unit region can be divided into sea-island types (island portions are arranged in a grid) so that two or more test units can be provided. It is. Photomicrograph showing the state of cell extension from the edge of the disk-shaped cell mass on the third day from the start of the BAK exposure test in the test process in which the disc-shaped island-side cell mass that has been divided and moved is provided as a test unit It is. It is a graph which shows the time-dependent change of the cell extension distance from the edge of the cell mass in the test process by which the cell mass of the disk-shaped island side which has been divided and moved in the BAK exposure test was provided as a test unit. Time-dependent change in cell extension distance from the edge of the circular hole of the cell mass in the test process in which the sea side cell mass left after the movement of the divided and disk-shaped island side cell mass in the BAK exposure test was provided as a test unit It is a graph which shows.

  A feature of the present invention is that a cell mass in one unit region is processed so that two or more test units can be provided, and (a) all or part of these cell masses are moved to a new culture region. By using the moved cell mass and / or (b) after moving all or part of these cell masses or without moving, the original one unit region (hereinafter simply referred to as the original It is to provide two or more test units by utilizing the cell mass left in the region. In the method of the present invention, the load can be evaluated by exposing a desired load to these test units. In the method of the present invention, the test step can be performed using only the cell mass moved to the new culture region, or using only the cell mass left in the original region, or Both of these can be used to carry out the test process.

  The cell mass to which the method of the present invention can be applied is not particularly limited as long as it is a cell group capable of obtaining a cell mass that can be cultured even if processed so as to provide two or more test units, For example, a living organism organ of an animal, a plant, or an insect, or a living tissue, or a cell group composed of cultured cells such as a non-immortal cell or an immortalized cell derived from a living tissue can be mentioned. Examples of the origin of the living organ or living tissue or the cultured cell include a visual organ eye. In the case of the eye, examples of the biological tissue constituting the organ include the cornea, sclera, conjunctiva, iris, ciliary body, choroid, retina, lens, and vitreous body. Examples include corneal epithelium, Bowman's layer, corneal stroma, Descemet's membrane, and corneal endothelium.

  When a biological organ or biological tissue is used as the cell mass, a cell mass obtained by processing from the biological organ or biological tissue can be transferred to a new culture region to obtain a test unit. In this case, an organ or tissue corresponds to a cell mass of one unit region. In addition, the cell mass obtained from the organ or tissue can be transferred to a processing container and the following processing can be performed. In this case, the cell mass in one processing container corresponds to the cell mass of one unit region. In addition, a cell mass obtained from an organ or tissue can be once transferred to a culture vessel and cultured, so that it can also be used as a non-immortal cell derived from a living body.

  When a cell group composed of non-immortalized cells or immortalized cells is used as the cell mass, it is a monolayer or a layered layer, or is reconstructed so as to mimic the living body. Any cultured cell can be used. These cultured cells can be subjected to the following processing after being maintained or grown to a state where they can be processed by appropriately selecting culture conditions suitable for each cell. In this case, the cultured cells in one culture container correspond to a cell mass in one unit region.

The processing step and the test unit providing step in the method of the present invention are not particularly limited as long as two or more test units can be provided from a cell mass in one unit region to a new culture region or an original region. However, it can be carried out, for example, by an operation using a movable substrate or by directly incising a cell mass.
Hereinafter, the aspect using a movable base material is demonstrated, and the aspect by incision is demonstrated subsequently.

In an embodiment using a movable base material, one unit region in which the base material is arranged is roughly divided into two modes depending on whether (A) it is arranged on the entire surface or (B) part of the unit region. can do.
In addition, regarding the base material arranged in one unit region, (a) only a base material capable of cell culture is used, (b) only a base material difficult to culture cells is used, or (c) cell culture Depending on whether a possible substrate and a substrate that is difficult to culture cells are used in combination, it can be roughly divided into three modes.
In addition, the state of the culture surface of one unit region (particularly, the region exposed without being covered by the substrate when the substrate is arranged, or the region immediately under the substrate covered by the substrate when the substrate is arranged) ) (1) Is it composed only of a region where cell culture is possible, (2) Is it composed of a region where cell culture is difficult, or (3) Is it composed of a partial region where cells can be cultured and a partial region where cell culture is difficult Can be roughly divided into three modes.
Moreover, regarding the movement of the substrate after cell culture, it can be roughly divided into two modes depending on whether (i) the substrate is moved or (ii) the substrate is not moved.

As an aspect (A) of disposing a movable substrate on the entire surface of one unit region, for example,
(A-ai) The cells are cultured in a state where one or more movable base materials capable of cell culture and movable are arranged in advance on the entire surface of one unit region, and all or a part of the base materials are moved, A mode in which two or more test units are provided in all or part of the cell mass that has migrated to the culture area and / or in whole or part of the cell mass that has been left in the original area;
(A-c-i) Cells are cultured in a state in which one or more base materials that can be cultured and movable and one or more base materials that are difficult to culture can be placed in advance on one unit area. 2 or all of a part of the cell mass moved to a new culture region and / or all or a part of the cell mass left in the original region. An embodiment providing the above test units;
(A-c-ii) Cells are cultured in a state where one or more base materials that can be cultured and movable and one or more base materials that are difficult to cultivate are arranged in advance on the entire surface of one unit region. And providing two or more test units with all or part of the cell mass left in the original region without moving the substrate;
Can be mentioned.

As an aspect (B) in which a movable base material is arranged in a part of one unit region and only a base material capable of cell culture is used as the base material,
(Ba-1-i) Cells are cultured in a state in which one or more substrates that can be cultured in a cell and are movable are preliminarily disposed in a part of one unit region in which cells can be cultured. A mode in which two or more test units are provided by all or part of the cell mass that has moved part and moved to a new culture region and / or all or part of the cell mass left in the original region ;
(B-a-2-i) The cells are cultured in a state in which one or more substrates that can be cultured in a cell and are movable are arranged in advance in a part of one unit region where cell culture is difficult, A mode in which two or more test units are provided by all or part of the cell mass that has moved part and moved to a new culture region and / or all or part of the cell mass left in the original region ;
(B-a-2-ii) Cells are cultured in a state in which one or more base materials capable of cell culture and movable are placed in advance in a part of one unit region where cell culture is difficult, and the base materials are moved. Without providing two or more test units in all or part of the cell mass left in the original region without;
(B-a-3-i) A state in which one or more movable substrates capable of cell culture are arranged in advance in a part of one unit region consisting of a partial region where cell culture is possible and a partial region where cell culture is difficult Cultivate the cells, move all or part of the substrate, move all or part of the cell mass moved to the new culture area, and / or all or one of the cell mass left in the original area. Embodiment in which two or more test units are provided;
(B-a-3-ii) A state in which one or more base materials capable of cell culture and movable are arranged in advance in a part of one unit region composed of a partial region where cell culture is possible and a partial region where cell culture is difficult A mode in which cells are cultured in the above, and two or more test units are provided in all or part of the cell mass left in the original region without moving the substrate;
Can be mentioned.

As an aspect (B) in which a movable base material is arranged in a part of one unit region and only a base material in which cell culture is difficult is used as the base material,
(Bb-1-i) culturing cells in a state in which one or more substrates that are difficult to cultivate cells are arranged in advance in a part of one unit region where cells can be cultured, An embodiment in which two or more test units are provided for all or part of the cell mass that has moved part and remained in the original region;
(Bb-1-ii) Cells are cultured in a state in which one or more base materials that can be moved due to difficulty in cell culture are arranged in a part of one unit region where cells can be cultured, and the base materials are moved. Without providing two or more test units in all or part of the cell mass left in the original region without;
(Bb-3-i) One or more substrates that are difficult to cultivate cells and at least part of one unit region consisting of a partial region that can be cultured and a partial region that is difficult to culture. Culturing the cells in a pre-arranged state so that a part of the possible partial region is exposed, moving all or a part of the base material, all or a part of the cell mass left in the original region, An embodiment providing two or more test units;
(Bb-3-ii) One or more substrates that are difficult to cultivate cells and at least part of one unit region consisting of a partial region that can be cultured and a partial region that is difficult to cultivate the cell The cells are cultured in a pre-arranged state so that a part of the possible partial region is exposed, and all or part of the cell mass left in the original region without moving the substrate, two or more An aspect of providing test units;
Can be mentioned.

A mode (B) in which a movable base material is arranged in a part of one unit region, and a base material capable of cell culture and a base material difficult to culture are used as the base material (c) For example,
(Bc-1-i) One or more base materials capable of cell culture and movable, and one or more base materials capable of movement due to difficulty in cell culture, in advance as part of one unit region capable of cell culture. Cells are cultured in the arranged state, all or part of the substrate is moved, all or part of the cell mass moved to a new culture area, and / or cell mass left in the original area Embodiments that provide more than one test unit in whole or in part;
(Bc-1-ii) One or more base materials capable of cell culture and movable, and one or more base materials capable of movement due to difficulty in cell culture, are preliminarily formed in a part of one unit region capable of cell culture. A mode in which cells are cultured in an arranged state, and two or more test units are provided for all or part of the cell mass left in the original region without moving the substrate;
(Bc-2-i) One or more base materials capable of cell culture and movable, and one or more base materials capable of movement due to difficulty in cell culture, are preliminarily formed in a part of one unit region difficult to culture cells. Cells are cultured in the arranged state, all or part of the substrate is moved, all or part of the cell mass moved to a new culture area, and / or cell mass left in the original area Embodiments that provide more than one test unit in whole or in part;
(Bc-2-ii) One or more base materials capable of cell culture and movable, and one or more base materials capable of movement due to difficulty in cell culture are preliminarily formed in a part of one unit region difficult to culture cells. A mode in which cells are cultured in an arranged state, and two or more test units are provided for all or part of the cell mass left in the original region without moving the substrate;
(Bc-3-i) One or more base materials that can be cultured and movable, and one or more base materials that are difficult to cultivate cells, and a partial region in which cell culture is difficult and a portion that is difficult to cultivate cells Culturing the cells in a state of being arranged in advance in a part of one unit region consisting of the region, moving all or part of the base material, all or part of the cell mass moved to a new culture region, and Or / or an aspect of providing two or more test units in all or part of the cell mass left in the original region;
(Bc-3-ii) One or more base materials capable of cell culture and movable, and one or more base materials capable of movement due to difficulty in cell culture. Cells are cultured in a state of being arranged in advance in a part of one unit area consisting of the area, and two or more test units in the whole or part of the cell mass left in the original area without moving the base material An aspect providing
Can be mentioned.

Hereinafter, these aspects using the movable substrate will be further described with reference to FIGS.
1 to 5 are explanatory diagrams schematically showing a state in which a cell cluster in one unit region can be divided into various patterns so that two or more test units can be provided. In addition, these patterns are not limited to the aspect using a movable base material, For example, it is applicable to the method of this invention in general including the aspect by incision.

  When cells are cultured in the part 1 shown in white and the part 2 shown in black shown in FIGS. 1 to 3, move any number of cell masses at any position regardless of white or black, and others It is possible to leave a cell mass. For example, the cell mass of the portion 1 shown in white can be moved and the cell mass of the portion 2 shown in black can be left in the original region, and conversely, the cell mass of the portion 2 shown in black can be moved and white It is also possible to leave the cell mass of the portion 1 indicated by in the original area. Either the migrated cell mass or the remaining cell mass can provide a test unit.

  In FIG. 1 to FIG. 3, a movable base material is arranged on the entire surface of one unit region, and when the base material is a base material capable of cell culture [the embodiment (Aai)], it is shown in white. After culturing the cells in a state where the base material is arranged in both the part 1 and the part 2 shown in black, the whole or a part of the base material is moved to leave the moved cell mass and / or the original part. Test units can be provided in the cell mass. For example, the movable substrate of the part shown in white and black can be moved, or the movable substrate of the part shown in white is moved, leaving the movable substrate of the part shown in black Alternatively, the portion of the movable substrate shown in black can be moved, leaving the portion of the movable substrate shown in white. The transferred cell mass can provide test units, and the remaining cell mass can provide test units. If the portion of the trace of movement of the movable substrate can be cultured, a test unit for evaluating cell extension with the remaining cell mass can be provided.

  1 to 3, when a cell cultureable and movable substrate and a cell culture difficult and movable substrate are used in combination and arranged on the entire surface of one unit region [said embodiment (A-c-i ) Or (A-c-ii)], after culturing cells at an arbitrary position, all or part of the substrate is moved, and the transferred cell mass and / or the remaining cell mass is tested. Units can be provided, or the test units can be provided in the original cell mass without moving the substrate. For example, when a cell cultivatable substrate and a cell cultivated substrate are arranged in the white portion 1 and the black portion 2 respectively, cells are cultured in the white portion, and the state is kept as it is. The test unit can be provided on the unit area, or all or part of the movable substrate of the part shown in white can be moved, and the test unit can be provided by the moved cell mass, or black It is possible to provide a test unit by moving the whole or a part of the movable substrate that is difficult to cultivate in the portion indicated by, and the remaining cell mass indicated by white. If the part of the trace that the movable substrate has moved can be cultured, move the movable substrate due to the difficulty of cell culture in the part shown in black, and evaluate the cell extension with the remaining cell mass in white Can provide test units.

In FIG. 1 to FIG. 3, the movable base material is disposed in a part of one unit region, and the base material is a base material capable of cell culture [the embodiment (Ba)], for example, In the case where a cell cultureable and movable substrate is disposed in the portion 1 shown in white, and the portion 2 other than the movable substrate shown in black is difficult to culture [the embodiment (B-a-2-i ) Or (B-a-2-ii)], cells are cultured in the portion shown in white, and the test unit can be provided as it is, or all or part of the movable substrate is moved and moved cells Test units can be provided by the clumps and / or the remaining cell clumps.
On the other hand, when a cell-cultivatable and movable substrate is arranged in the portion shown in white, and a portion other than the movable substrate shown in black can be cultured [said embodiment (B-a-1-i)] In addition to the part shown in white, cells are cultured in the part shown in black, and all or part of the movable substrate is moved, and the transferred cell mass and / or the remaining cell mass is tested. Can provide units. If the portion other than the movable substrate shown in black and the portion of the trace where the movable substrate has moved can be cultured, it is possible to provide a test unit for evaluating cell extension using the remaining cell mass.

  In FIG. 1 to FIG. 3, a movable base material is disposed in a part of one unit region, and the base material is a base material that is difficult to cultivate cells (the mode (Bb)). In the case where a mobile substrate that is difficult to cultivate cells is disposed in the portion 1 shown in white, and the portion 2 other than the mobile substrate that is shown in black is capable of cell culture [the above embodiment (B-b-1-i ) Or (B-b-1-ii)], cells are cultured in the part shown in black, can provide a test unit, move all or part of the movable substrate, and test with the remaining cell mass Can provide units. If the part other than the movable substrate shown in black and the trace of the moved movable substrate can be cultured, all or part of the movable substrate is moved, and the remaining cell mass A test unit for assessing cell spreading can be provided.

  1 to 3, when a cell cultureable and movable substrate and a cell culture difficult and movable substrate are used in combination and arranged in a part of one unit region [said embodiment (Bc) In the upper half of the figure, for example, a cell cultivatable and movable base material is disposed in the upper half portion shown in white, and in the upper half portion of the black portion, a movable base material that is difficult to cultivate and is movable. When arranged, the upper half portion can be considered in the same manner as the case where the movable base material capable of cell culture and difficult to be arranged is arranged on the entire surface of the one unit region. Cells can be cultured in the part shown in white and the test unit can be provided as it is, or all or part of the movable substrate in the part shown in white can be moved, and the test unit can be placed in the transferred cell mass. Alternatively, the whole or part of the movable substrate which is difficult to cultivate the part shown in black can be moved, and the test unit can be provided by the cell mass in the part shown in white. If the part of the trace that the movable substrate has moved can be cultured, move the movable substrate due to the difficulty of cell culture in the part shown in black, and evaluate the cell extension with the remaining cell mass in white Can provide test units. If the lower half, which is a part other than the movable base material, and the part where the base material has moved can be cultured, the cells are also cultured in the lower half part, and the lower half part moves in contact with the lower half part. A test unit can be provided that moves all or part of the possible substrate and evaluates cell spreading with the remaining cell mass.

4 and 5 are explanatory diagrams schematically showing a state in which a cell mass in one unit region can be divided into a sea-island type so that two or more test units can be provided. .
When cells are cultured in the portion 1 shown in white and the portion 2 shown in black shown in FIG. 4 or 5, move any number of cell masses at any position regardless of white or black, and others It is possible to leave a cell mass. For example, the cell mass of the portion 1 shown in white can be moved and the cell mass of the portion 2 shown in black can be left in the original region, and conversely, the cell mass of the portion 2 shown in black can be moved and white It is also possible to leave the cell mass of the portion 1 indicated by in the original area. Either the migrated cell mass or the remaining cell mass can provide a test unit.

  In FIG. 4 or FIG. 5, when a movable base material is disposed on the entire surface of one unit region and the base material is a base material capable of cell culture [the embodiment (Aai)], it is shown in white. After culturing the cells in a state where the base material is arranged in both the part 1 and the part 2 shown in black, the whole or a part of the base material is moved to leave the moved cell mass and / or the original part. Test units can be provided in the cell mass. For example, it is possible to move the movable substrate of the portion shown in white and black, or move the movable substrate on the sea side shown in white and move the movable substrate on the island side shown in black Or the island-side movable substrate shown in black can be moved to leave the sea-side movable substrate shown in white. The transferred cell mass can provide test units, and the remaining cell mass can provide test units. If the portion of the trace of movement of the movable substrate can be cultured, a test unit for evaluating cell extension with the remaining cell mass can be provided.

In FIG. 4 or FIG. 5, when a cell cultureable and movable substrate and a cell culture difficult and movable substrate are used in combination and arranged on the entire surface of one unit region [said embodiment (A-c-i ) Or (A-c-ii)], after culturing cells at an arbitrary position, all or part of the substrate is moved, and the transferred cell mass and / or the remaining cell mass is tested. Units can be provided, or test units can be provided in the original cell mass without moving the substrate.
For example, when a base material capable of cell culture and a base material that is difficult to culture are arranged in the sea side part 1 shown in white and the island side part 2 shown in black, respectively, the cells are cultured on the sea side shown in white. Can move the movable cell base that can be cultured on the sea side shown in white, and can provide the test unit with the moved cell mass, or the mobile side that is difficult to move on the island side shown in black The test unit can be provided by the seaside cell mass, which is moved in whole or in part and shown in white. If the part of the trace that the movable substrate has moved can be cultured, the part of the movable substrate that is difficult to cultivate in black is moved all or part of the movable substrate, and the remaining white cells on the sea A test unit can be provided for assessing cell spreading in a mass.
On the other hand, when a substrate that is difficult to culture and a cell cultureable and movable substrate is disposed on the sea side shown in white and the island side shown in black, respectively, cells are cultured on the island side shown in black, The test unit can be provided as it is, or all or part of the movable substrate capable of cell culture on the island side shown in black is moved, and / or the transferred cell mass. A test unit can be provided by the remaining cell mass, or a test substrate can be provided by an island-side cell mass indicated by the black color by moving a movable substrate that is difficult to cultivate the sea side cell indicated by white. If the part of the trace where the movable substrate has moved can be cultured, the movable substrate is moved due to the difficulty of cell culture on the sea side shown in white, and the cell extension is performed with the remaining island-side cell clusters shown in black A test unit can be provided.

In FIG. 4 or FIG. 5, the movable substrate is arranged in a part of one unit region, and the substrate is a substrate capable of cell culture [the embodiment (Ba)], for example, In the case where a cell-cultivatable and movable base material is disposed in the sea-side portion 1 shown in white, and the portion 2 other than the island-side movable base material shown in black is difficult to culture cells [said embodiment (B -A-2-i) or (B-a-2-ii)], cells are cultured on the sea side shown in white, the movable substrate on the sea side shown in white is moved, and the transferred cell mass Can provide test units.
On the other hand, when the portion other than the movable substrate shown in black is capable of cell culture [the embodiment (B-a-1-i)], cells are also cultured in the portion shown in black, and the sea side shown in white The mobile unit can be moved and the test unit can be provided by the sea-side migrated cell mass shown in white and / or the island-side cell mass shown in black. If the part other than the movable substrate shown in black and the part of the trace that the movable substrate has moved can be cultured, the movable substrate on the sea side shown in white is moved and shown in the remaining black It is possible to provide a test unit for evaluating cell extension in the cell mass on the island side.

On the other hand, when a cell-cultivatable and movable base material is arranged on the island side shown in black, and a portion other than the sea-side movable base material shown in white is difficult to cell culture [said embodiment (B-a -2-i) or (B-a-2-ii)], cells are cultured on the island side shown in black, and the test unit can be provided as it is, or the island side shown in black is movable All or part of the substrate can be moved to provide test units with the transferred cell mass and / or the remaining cell mass.
On the other hand, when a portion other than the sea-side movable substrate shown in white is culturable [said embodiment (B-a-1-i)], cells are also cultured in the portion shown in white and shown in black All or part of the island-side movable substrate can be moved to provide test units with island-side migrated cell mass shown in black and / or sea-side cell mass shown in white. If the part other than the seaside movable substrate shown in white and the part of the trace where the movable substrate moved can be cultured, all or part of the movable substrate on the island side shown in black It is possible to provide a test unit for evaluating cell spreading with the seaside cell mass that has migrated and remained in white.

  In FIG. 4 or FIG. 5, in the case where a movable base material is arranged in a part of one unit region, and the base material is a base material that is difficult to cultivate cells (the mode (Bb)), In the case where a mobile substrate that is difficult to cultivate cells is disposed in the sea-side portion 1 shown in white, and the portion 2 other than the island-side mobile substrate that is shown in black is capable of cell culture [said embodiment (B -B-1-i) or (B-b-1-ii)], cells are cultured on the island side shown in black, and the test unit can be provided as it is, or cells on the sea side shown in white It is possible to provide a test unit with the remaining cell mass by moving a movable substrate that is difficult to culture. If the part other than the movable substrate on the island side shown in black and the part of the trace where the movable substrate has moved can be cultured, move the movable substrate with difficulty in cell culture on the sea side, It is possible to provide a test unit for evaluating cell extension with the remaining cell mass.

  On the contrary, when a movable base material that is difficult to cultivate cells is arranged on the island side shown in black, and a portion other than the movable base material on the sea side shown in white is cell cultivated [said embodiment (BB) -1-i) or (Bb-1-ii)], cells are cultured on the sea side shown in white, and the parts other than the sea-side movable substrate shown in white and the movable substrate move If the traced portion can be cultured, the whole or part of the movable substrate that is difficult to cultivate on the island side shown in black can be moved, and the test unit can be provided with the remaining cell mass. A test unit for assessing cell spreading can also be provided.

  In the case where a cell cultureable and movable substrate and a cell culture difficult and movable substrate are arranged in a single unit region [the above-described embodiment (Bc-1-i) or (Bcc-1-ii) ) Or (Bc-2-i) or (Bcc-ii)]], for example, when placing a movable base material on the nine island-side parts shown in black in the figure, When a substrate capable of culturing cells is arranged in the part and a substrate difficult to cultivate cells is arranged in the other four parts, the part in which the cell cultivatable and movable substrate is arranged is placed in the one unit region. It can be considered in the same way as the case where a cell cultivatable and movable substrate is placed on the part, and the part on which the cell cultivated substrate is placed can be moved to the above-mentioned one unit area due to the difficulty of cell culture. It can be considered in the same manner as when a simple base material is arranged. Test units can be provided by migrated cell mass and / or left cell mass. Move all or part of the movable substrate by properly using the state where the part other than the movable substrate and / or the part where the movable substrate has moved can be cultured or difficult. The test unit can be provided by the cell mass that has migrated and / or remained.

  In the method of the present invention, any cell cultureable and movable substrate that can be cultured can be used as long as cell culture is possible. For example, non-biological constituents, biological constituents of animals, plants, and insects, and biological tissues can be used as those capable of cell culture. As the non-biological constituent material, for example, glass, plastic, polymer, and metal can be used. Examples of biological constituents that can be used include carbohydrates, lipids, amino acids, peptides and proteins, glycolipids, and glycoproteins. As biological constituents, collagen, proteoglycan, glycosaminoglycan, fibronectin, laminin, tenascin, entactin, elastin, fibrillin, cellulose, chitin and chitosan, which are extracellular matrix components, can be used. Examples of the biological tissue include animals such as amniotic membrane, Descemet's membrane, lens capsule, and skin. Even if it is difficult to cultivate cells, it is possible to use those whose surfaces are coated with the above-mentioned non-biological constituents, biological constituents, and biological tissues that can be cultured in cells. The above-mentioned substances that do not have the performance as a movable substrate can be used after being processed to have the performance as a movable substrate.

  In the method of the present invention, any movable or difficult-to-be-cultured substrate can be used as long as cell culture is difficult. Even those that can be cultured in cells can be used whose surfaces are coated with materials that are difficult to culture. As a method for making cell culture difficult, for example, a method in which cells cannot physically expand by changing the height and temperature, for example, can be used.

In the method of the present invention, as the culture surface providing one unit region, the whole surface can be cultured, the whole surface is difficult to culture, or a part can be cultured and the remaining part is difficult to culture. Can be used.
Any culture surface capable of cell culture can be used as long as cell culture is possible or processed. For example, non-biological constituents, biological constituents of animals, plants, and insects, and biological tissues can be used as those capable of cell culture. As the non-biological constituent material, for example, glass, plastic, polymer, and metal can be used. Examples of biological constituents that can be used include carbohydrates, lipids, amino acids, peptides and proteins, glycolipids, and glycoproteins. As biological constituents, collagen, proteoglycan, glycosaminoglycan, fibronectin, laminin, tenascin, entactin, elastin, fibrillin, cellulose, chitin and chitosan, which are extracellular matrix components, can be used. Examples of the biological tissue include, in animals, amniotic membrane, Descemet's membrane, lens capsule, or skin. Even if it is difficult to cultivate cells, it is possible to use those whose surfaces are coated with the above-mentioned non-biological constituents, biological constituents, and biological tissues that can be cultured in cells. Those that can be processed as a culture vessel in the above can be used as a culture vessel, or those that are difficult to process as a culture vessel can be used after being processed for use on the culture surface of the culture vessel.

  In the method of the present invention, any culture surface that is difficult to cultivate cells providing one unit region can be used as long as cell culturing is difficult, or any processed surface can be used. Even those that can be cultured in cells can be used whose surfaces are coated with materials that are difficult to culture. Those that can be processed as a culture vessel in the above can be used as a culture vessel, or those that are difficult to process as a culture vessel can be used after being processed for use on the culture surface of the culture vessel.

  In the method of the present invention, as a culture surface in which a part providing one unit region can be cultured in a cell and the remaining part is difficult to culture, any part can be used as long as a part can be cultured and the remaining part is difficult to culture. Alternatively, processed products can also be used. A cell cultivated one that has a part of its surface coated with a cell culture difficult or a cell cultivated one that has a part of its surface coated with a cell cultivatable one can be used. Can be used. Those that can be processed as a culture vessel in the above can be used as a culture vessel, or those that are difficult to process as a culture vessel can be used after being processed for use on the culture surface of the culture vessel.

  When the culture surface providing one unit region has the performance as a movable substrate, the culture surface can be used as a movable substrate. For example, when co-culture using a transwell is performed, the membrane of the transwell, which is the culture surface, can be moved along with the cell mass cultured thereon as a movable substrate. As a process to provide two or more test units, an incision can be made in the cell mass with the culture surface before movement, or an incision can be made in the cell mass with the culture surface at the same time as movement. Alternatively, the cell mass can be incised with the culture surface after movement.

  In the method of the present invention, when cell culture is performed, cell culture for preparing a cell mass used in a processing step, cell culture in a test unit providing step, or cells in a test step Regardless of whether it is a culture or not, the use of a culture substrate is optional, but when a culture substrate is used, for example, living tissues derived from animals, plants, and insects can be used as they are, or extracted. A refined product or a synthetic product can also be used. Examples of the biological tissue include, in animals, amniotic membrane, Descemet's membrane, lens capsule, or skin. Examples of extracted purified products or synthetic products include collagen, proteoglycan, glycosaminoglycan, fibronectin, laminin, tenascin, entactin, elastin, fibrillin, cellulose, chitin, and chitosan, which are extracellular matrix components. It can be used by coating the substrate or in a processed state, for example, a gel or a sheet, but is not limited thereto. In the case of using the living body-derived tissue, the tissue itself having performance as a base material can also be used as the base material. In the case of using an extracted refined product or a synthetic product, those processed so as to have the performance as a base material can also be used as the base material.

  Regardless of whether or not cell culture is possible, the shape of the movable substrate can be arbitrarily selected. If the substrate is flat, for example, a circular shape, a rectangular shape, a fan shape, a donut shape, a star shape, and the like can be selected. A square shape and a long shape can be mentioned. As long as it is a solid, for example, a cylinder, a rectangular parallelepiped, a triangular prism, a polygonal cylinder whose cross section includes a star shape, and a shape in which a sphere is divided in half can be given.

  The movable base material can be arranged as a plurality of continuous base materials that have been separated in advance or processed so as to be easily separated before culturing, or those that are scheduled to be separated are separated. It is possible to dispose the base material in a state of not being separated and separate in one unit area before the movement, or simultaneously with the movement, or after the movement.

  When a cell cultivatable substrate is used as the movable substrate, the transferred cell mass can provide a test unit together with the substrate, or the substrate can be removed from the cell mass to provide the test unit. You can also. This separation of the cell mass and the substrate can be performed at any time, for example, it can be performed in one unit area before the movement, or can be performed simultaneously with the movement, or after the movement. You can also.

  The movable substrate, the culture surface that provides a unit area that can be used as a movable substrate, and the culture substrate that can be used as a movable substrate can be used either alone or in any combination. Can be used. For example, when a culture surface container that provides one unit region that can be used as a movable substrate and a culture substrate that can be used as a movable substrate, the cultured cell mass is transferred to the movable substrate. It can move with a culture surface that provides a unit area that can be used as a culture substrate that can be used as a movable substrate, or it can move with a culture substrate that can be used as a movable substrate .

In the method of the present invention, when the cell mass is divided using an incision, for example, a method of moving the cell mass after incising the cell mass using a biopsy trepan or knife can be mentioned.
When moving after making a circular incision using a biopsy trepan, one or more excised cell clusters and one or more remaining cell clusters with one or more holes are obtained from one cell cluster. For example, when using a single layer or multiple layers, or a cell mass reconstructed so as to imitate a living body and making a circular incision with a biopsy trepan, for example, as shown in FIG. 4 or FIG. A plurality of disc-shaped cell masses shown in black (2 in FIG. 4 or 5) moved from a cell mass of one culture unit, which is one unit region, to a new culture region, and a honeycomb shape left in the original container A cell cluster (1 in FIG. 4 or 5) shown in white with a plurality of circular holes is obtained. The embodiment using the biopsy trepan is an embodiment in which a disk-shaped cell mass is moved and a holed cell mass is left, but in the method of the present invention, on the contrary, a cell cell with a hole is moved and the disk is moved. It is also possible to leave a cellular cell mass.

  The shape of the division using the incision is not particularly limited, and examples thereof include a circular shape, a rectangular shape, a fan shape, a long shape, a donut shape, and a polygon including a star shape. Further, the size of the divided and moved can be appropriately set with a size smaller than the area of one unit region, but when the shape is circular, for example, the diameter of a commercially available trepan is 0.5 mm to 8 mm. However, the present invention is not limited to this.

  In the method of the present invention, regardless of whether a movable substrate is used for processing or an incision is used, when a cell mass is moved from the original unit region to a new culture region and used, a new culture is used. The test can be carried out immediately after moving to the region, or the next test can be carried out after a suitable stabilization period or culture period has passed.

Moving to a new culture area can move one cell mass per culture area (for example, 1 container, 1 well), or a plurality of cells in 1 culture area (for example, 1 container, 1 well). You can also move the mass.
In the method of the present invention, by using uniform cells cultured in the same unit region, that is, in the same culture unit (for example, well), or when there is not necessarily uniformity in one culture unit, it is uniform. By selecting only a cell mass, the quality between test units becomes uniform, and it can be expected to conduct a comparative test with high accuracy.

  In the method of the present invention, the shape of the cell mass remains as it is after it is initially processed, or is further processed, and a test unit can be provided. When leaving the cell mass in the original area, the test unit can be provided as it is without moving in the original area, or the test unit can be provided with the arrangement changed in the original area. Can also be provided. Whether moving to a new culture region or leaving it in the original culture region, it can be immediately transferred to the test process, or an appropriate stabilization period or culture period can be provided.

  In the test step in the method of the present invention, cell spreading is observed from the edge of the cell mass obtained by processing when cell culture is possible. In the method of the present invention, by using cell extension, for example, by exposing a cell mass obtained as a test unit to various chemical loads or physical loads, these loads can be evaluated. For example, in a chemical load, the efficacy or toxicity of the test substance can be detected by comparing the distance and area of cell extension in the absence of the test substance and in the presence of the test substance. Moreover, also in the physical load, the influence of each physical load can be detected by comparing the distance and area of cell extension similarly to the chemical load.

  In the test step in the method of the present invention, in addition to cell spreading, various evaluation indices that can analyze the state of cells, such as the number of cells, cell morphology, cell activity, the amount of cellular material taken up, the amount of cellular material The amount of excretion, the amount of mRNA transcribed, the enzyme activity, protein expression detected by a microscope, and function expression can be used. An evaluation system can be appropriately selected according to various chemical loads and physical loads, and types of cells to be used.

Moreover, the method of exposing the load to the cell mass can also be appropriately determined according to the evaluation system. For example, with respect to the chemical load, an arbitrary form can be selected from a liquid, a gas, a solid, or an intermediate thereof as the state at the time of exposure of the test substance. Exposure can be performed with the test substance alone or mixed with the culture medium. Examples of the physical load include incision, excision, friction, compression, temperature, pressure, vibration, electromagnetic waves, radiation, and gravity.
The exposure to the load can be selected at any time and frequency. For example, short-term exposure can be performed once a day, or multiple times at intervals, or long-term exposure can be performed in the same manner.

  The culture environment in which the cell mass is cultured can be changed from the test unit providing step or the test step following the processing step. Examples of changes in the culture environment include a culture vessel, a culture form, the amount of the culture solution, a culture solution composition, a carbon dioxide concentration, temperature, humidity, pressure, vibration, electromagnetic waves, radiation, and gravity. In the examples, in the test step, co-culture using a culture insert was performed as before the processing step, and air lift culture was performed to expose the cell surface to air. It is also possible to test with a culture method that fills so that it is sufficiently immersed. Examples of changes in the culture solution composition include salts, metals, dyes, serum, and substances that act positively or negatively on cell activity and proliferation. The culture environment does not necessarily have to be good for the cells. When the culture environment is poor, it is possible to easily detect the effect of the load to be exposed, or it is possible to detect an effect that cannot be detected under normal culture.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1
(1) Acquisition of porcine corneal epithelial cells Porcine eyeballs were purchased from Tokyo Shibaura Organ Co., Ltd. (2-7-19 Konan, Minato-ku, Tokyo). The cornea is separated from the eyeball, chopped into approximately 5 mm tissue, and DMEM / with 5% fetal calf serum and 10 ng / mL EGF on a collagen gel (pig derived, Type I; Nitta Gelatin Co., Ltd.). The cells were cultured in F12 medium (hereinafter referred to as medium). Cells proliferated from the tissue pieces and spread on the gel are considered to be derived from the corneal epithelium, separated and collected by enzymatic treatment, suspended in a cell cryopreservation cell banker (Nippon Zenyaku Kogyo Co., Ltd.) and stored frozen at -80 ° C did.

(2) Preparation of corneal epithelial cell sheet Porcine corneal epithelial cells that had been cryopreserved were thawed and suspended in a medium so as to be 5 × 10 ⁵ cells / mL. 1 mL of the culture was seeded on a collagen gel arranged as a substrate that can also be used as a substrate on the bottom of two 6-well plate culture inserts (Corning, hereinafter referred to as insert). The culture was started by adding 1 mL of the same medium outside the insert. From the day after the start, the medium was adjusted to 1 mL inside the insert and 2 mL outside the insert, and 3T3 cells were cultured outside the insert as a feeder.

  About one week after the start of the culture, the cells were cultured until they spread and proliferated on the entire surface in the insert. Subsequently, cell stratification was carried out in culture for another week. After confirming the progress of stratification with a phase-contrast microscope, in order to obtain a cell sheet having the same function as in vivo, the cell surface is exposed to air by removing the medium only in the insert (Air-Lift). Incubation was carried out for 3 days. This culture method is a common method for culturing corneal epithelial cells (Koizumi, N., NJ Fullwood, et al. (2000). "Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane." Invest Ophthalmol Vis Sci 41 (9): 2506-2513.).

(3) Preparation of test well (method using separated and transferred cell mass)
An incision is made with a 4 mm diameter trepan together with the insert-derived membrane that is the culture vessel in the layered cells, and a disc-shaped cell mass with the insert-derived membrane is obtained for 9 inserts per unit area. A total of 18 test units were obtained from the two inserts.
In this example, a collagen gel was placed on the bottom of the insert and cultured, then an incision was made in the insert-derived membrane with a trepan to obtain a disk-shaped cell mass. For example, as shown in FIG. 4 or FIG. It is also possible to obtain a disk-shaped cell mass by detaching the collagen gel from the insert after placing the collagen gel that has been previously separated or processed so as to be easily separated on the bottom of the insert and culturing. . An incision can also be made after separation of the collagen gel from the insert prior to separation.
After removing the membrane derived from the insert used for migration from each separated disc-shaped cell mass, 6 pieces per well are placed on a collagen gel prepared in a new 6-well culture insert. The sample was allowed to stand for 1 hour with no medium inside and outside the insert to form a 3-well test well.

(4) Preparation of test well (method using cell mass left after migration)
After the disc-shaped cell mass was separated in Example 1 (3), two cell masses having 9 holes left like a honeycomb remained. In order to obtain a cell mass having three holes per one, one cell mass having nine holes was cut into three, and a cell mass having a total of six holes was obtained. After removing the membrane derived from the insert from each cell mass, two cells per well are placed on a collagen gel prepared in a new 6-well culture insert. The test wells (3 wells in total) were prepared by allowing to stand for a period of time.

(5) Exposure test The test substance was added as an antiseptic to eye drops and cytotoxic to corneal epithelial cells (Burgalassi, S., P. Chetoni, et al. (2001). "Cytotoxicity of potential ocular permeation enhancers evaluated on rabbit and human corneal epithelial cell lines. "Toxicology letters 122 (1): 1-8 .; Huhtala, A., M. Mannerstrom, et al. (2002)." Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing. "Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics 18 (2): 163-175.) Ltd .; hereinafter referred to as BAK).
BAK was dissolved in PBS (pH 7.2; GIBCO) to 1% (w / v), and then filtered through a 0.45 μm filter. The resulting 1% solution by the dilution with ^PBS, the 10 -2% BAK-containing ^PBS, 10 -4% BAK-containing PBS was prepared and used in the following exposure test with PBS containing no BAK.

Test wells using the disk-shaped cell mass produced in Example 1 (3) (the sample was allowed to stand for 1 hour in the absence of medium inside and outside the insert), and the cell mass produced in Example 1 (4) Air lift culture using 3T3 cells as feeder cells was started for each of the test wells (same as above) using the cell mass with holes left after the separation. After 24 hours of airlift culture, three different BAK concentrations (containing 1:10 ⁻² % BAK, 2:10 ⁻⁴ % BAK, 3: BAK) in the insert with the cell mass placed on the collagen gel 100 μL of any of PBS (not containing) was added and allowed to stand for 1 minute. After the standing, the inside of the insert was thoroughly washed with PBS, and then airlift culture was continued.

  This exposure operation is performed 3 times a day (the interval between each operation is about 4 hours), and the cells that extend from the edge of the cell mass are exposed to the test substance once a day while performing a total of 4 days. Before starting, it was observed and photographed under a microscope. Based on the photographed images, the distance of cells extending from the edge of the cell mass was measured, and comparison was made between the three groups. The Mann-Whitney test was used for the significant difference test, and it was determined to be significant with p <0.05.

The results are shown in FIGS.
FIG. 6 is a photomicrograph showing the state of cell extension from one edge of the disk-shaped cell mass on the third day from the start of BAK exposure in a test well prepared with a disk-shaped cell mass. 10 ⁻² % BAK, 10 ⁻⁴ % BAK, and 0% BAK mean a PBS exposure group containing 10 ⁻² % BAK, a PBS exposure group containing 10 ⁻⁴ % BAK, and a PBS exposure group containing 0% BAK, respectively. For better understanding, the tip of the cell extension was traced and shown as a black line.
FIG. 7 is a graph showing the change over time of the cell extension distance from the edge of a hole in a test well made with a cell mass having a hole.
FIG. 8 is a graph showing the change over time of the cell extension distance from the edge of each hole in a test well prepared with a cell mass having holes.

In any of the methods using two types of cell clusters, no cell extension from the edge was observed throughout the observation period in the PBS exposure group containing 10 ⁻² % BAK. On the other hand, in the PBS exposure group containing 10 ⁻⁴ % or 0% BAK, cell spreading from the edge of the cell mass was observed throughout the observation period from the day after the start of exposure. The cell extension distance of the 10 ⁻⁴ % BAK-containing PBS exposed group was the same as that of the 0% BAK-containing PBS exposed group at each observation point.

  INDUSTRIAL APPLICABILITY The present invention can be used as a method for evaluating various loads on a living body, as a chemical load, for example, detection of the effect of a test substance on drug efficacy or toxicity, or physical load.

Claims (19)

(1) By culturing an immortalized cultured cell or a non-immortalized cell derived from a living tissue to a state of being reconstructed so as to mimic a monolayer or a multilayer, or a living body, Obtaining a cell mass ,
(2) a step of processing the cell mass of one unit region provided in the step (1) so that two or more test units can be provided;
(3) providing two or more test units with a cell mass moved from the original one unit region to a new culture region and / or a cell mass left in the original one unit region;
(4) the step (3) the cell mass exposed to chemical or physical load in each test unit provided by, viewed including the step of detecting the impact, the cell mass is derived from the cornea, the load Evaluation method.   As the processing and movement, an incision is made in the cell mass in one unit area, and all or part of the cell mass moved to a new culture area and / or all or part of the cell mass left in the original area 2. The method of claim 1, wherein two or more test units are provided.   As the processing and movement, cells are cultured in a state where one or more movable substrates are arranged in advance on the whole surface or a part of one unit region, and all or a part of the substrate is moved or moved. 2 or more test units are provided with all or part of the cell mass migrated to the new culture area and / or with all or part of the cell mass left in the original area The method according to 1.   The method according to claim 3, wherein the movable substrate is disposed on the entire surface of one unit region.   The method according to claim 3, wherein the movable substrate is disposed in a part of the unit region.   The method as described in any one of Claims 3-5 using only the base material which can culture a cell as a movable base material.   The method according to any one of claims 3 to 5, wherein only a substrate that is difficult to culture is used as the movable substrate.   The method according to any one of claims 3 to 5, wherein a substrate capable of cell culture and a substrate difficult to culture are used in combination as the movable substrate.   The method according to claim 3, wherein the entire movable substrate is moved.   The method according to claim 3, wherein a part of the movable substrate is moved.   The method according to claim 3, wherein the movable substrate is not moved.   The method according to any one of claims 3 to 11, wherein the entire surface of one unit region is capable of cell culture.   The method according to any one of claims 3 to 11, wherein the entire surface of one unit region is difficult to culture.   The method according to any one of claims 3 to 11, wherein a part of one unit region is capable of cell culture and the rest is difficult to culture.   15. The method according to any one of claims 1 to 14, wherein the test unit is provided only from cell masses that have migrated to a new culture area.   The method according to any one of claims 1 to 14, wherein the test unit is provided only from a cell mass left in one unit region.   The method according to any one of claims 1 to 14, wherein the test unit is provided from a cell mass moved to a new culture region and a cell mass left in one unit region. The method according to any one of claims 1 to 17 , wherein the cell mass is derived from a corneal epithelium. The evaluation after exposure to chemical or physical load, is analyzed in cell spreading from the cell mass, the method according to any one of claims 1 to 18.