JP7493945B2 - Observation method, culture method, evaluation method and culture device for cultured tissue - Google Patents

Description

The present invention relates to tissue culture technology.

A method is widely used in which candidate substances are exposed to cultured cells, and the dynamic changes in the cultured cells are used as an indicator to screen for substances that could be used as active ingredients in medicines, cosmetics, and health foods (see, for example, Patent Document 1). Screening methods using cultured cells have the advantage of being easy to carry out, but also have the disadvantage that the test environment is significantly different from the actual in vivo environment.

A method using tissue is known to perform screening in an environment as close as possible to the in vivo environment. By exposing tissue taken from a living body or artificially produced tissue to a candidate substance, screening in an environment closer to the in vivo environment becomes possible (for example, Patent Document 2).

There is a known method for culturing cells or tissues using an instrument called a culture insert. This is a method in which cells are seeded on a culture platform with micropores and culture medium is supplied to the cells through the micropores. The use of a culture insert allows tissue culture (air-liquid interface culture) in a state where the top surface of the tissue is exposed to air and the bottom surface is exposed to the culture medium, making it particularly effective for culturing skin tissue.

Patent Document 3 discloses a technique for culturing tissue on a cell culture sheet with micropores. Patent Document 4 describes a method for culturing skin tissue using a culture insert and evaluating the state of dryness.

JP 2019-007759 A JP 2017-169540 A JP 2014-147342 A JP 2012-247284 A

When observing tissues cultured using a culture insert with an inverted microscope, the optical path of the microscope passes through the culture platform of the culture insert. However, there is a problem in that the light is scattered due to the influence of the micropores, decreasing the light transmittance. Although there is a tendency for the light transmittance to improve by increasing the pore diameter of the micropores as in Patent Document 3, it has not been possible to achieve 100% light transmittance over the entire wavelength range.

In addition, when culturing a culture by storing culture medium in a culture dish and placing the cultured tissue there, the cultured tissue drifts and moves in the culture medium, making it very difficult to observe specific locations in the tissue over time.

The problem that the present invention aims to solve is to provide a new observation and evaluation system for tissue culture.

The present invention, which solves the above problems, provides a method for observing a culture target that is a biological tissue and/or a cell-containing solid composition, comprising:
A culture step of placing the culture object on a culture platform having a through hole penetrating in a front-back direction and culturing the culture object by contacting a culture solution with the culture object through the through hole;
An observation step according to the following (a) or (b);
The observation method is characterized by comprising:

(a) The culture target placed on the culture platform is observed through the through-hole using an inverted microscope.
(b) Light from a light source is irradiated from below onto the culture object placed on the culture platform through the through-hole, and the culture object is observed using an upright microscope.

The present invention is characterized in that the culture platform is provided with a through-hole that runs from front to back. A culture medium is supplied to the culture object through this through-hole. In addition, when observing the culture object with an inverted microscope or an upright microscope, the light path passes through the through-hole without being blocked by the culture platform, so the culture object can be observed more clearly.

In a preferred embodiment of the present invention, in the culturing step, the culture object is cultured while maintaining the position of the culture object on the culture platform,
The observation step is carried out over time.
By culturing the culture object while maintaining its position in this manner, the culture object does not drift away in the culture solution and move, making it possible to observe specific positions of the culture object over time and making it easier to evaluate its morphological changes, etc.

In a preferred embodiment of the present invention, the through hole has a diameter of 1 mm or more.
By making the diameter of the through hole 1 mm or more, a sufficient optical path for the microscope can be secured.

In a preferred embodiment of the present invention, the culture process is a process of placing a culture object on the culture stand provided inside a culture medium storage container, and culturing the culture object such that the culture medium stored in the culture medium storage container comes into contact with the culture object through the through hole.
By constructing the culture platform in this way, so to speak, in a structure similar to that of a culture insert, simple culture and observation become possible.

In a preferred embodiment of the present invention, a co-culture target, which is a cell and/or a biological tissue, is cultured in the culture medium stored in the culture medium storage container.
According to this embodiment, the influence of the co-culture subject on the culture subject can be evaluated.

In a preferred embodiment of the present invention, the culture subject is skin tissue.
The present invention is suitable for culturing skin tissue and for an observation and evaluation system thereof.

In a preferred embodiment of the present invention, the culture subject is a gel composition in which cells and/or biological tissues are embedded.
The present invention is suitable for observing cells or biological tissues embedded in a gel composition.

In a preferred embodiment of the present invention, in the culturing step, the culture target is cultured in a state in which at least the upper surface of the culture target is not in contact with the culture medium.
This form is so-called air-liquid interface culture, which is suitable for culturing tissues that are in contact with the air and liquid phases with polarity, such as skin tissue, and for observing and evaluating the culture.

In a preferred embodiment of the present invention, the method further comprises a staining step of staining the culture object while the culture object is placed on the culture table,
The observation step is carried out after the staining step,
In the observation step, a stained image of the culture subject is observed.
According to this embodiment, the culture subject can be easily stained and observed.

In a preferred embodiment of the present invention, in the staining process, the culture table on which the culture object is placed is placed in a container containing a staining solution, and the staining solution is brought into contact with the culture object through the through hole, thereby staining the culture object.
By adopting such a form, it becomes possible to stain the cultured object more easily.

The present invention also relates to a method for evaluating the effect of a test subject on a culture subject by the above-mentioned observation method.
The evaluation method of the present invention includes a step of applying a test subject to the culture subject, the culture step, and the observation step,
After the application step, the culture step is carried out for an arbitrary period of time, and then the observation step is carried out, and the effect of the test subject on the culture subject is evaluated based on the observation results in the observation step.

In a preferred embodiment of the present invention, in the culturing step, the culture object is cultured while the position of the culture object on the culture platform is held by a holding means,
The observation step is also carried out before or immediately after the application step,
The effect of the test subject on the culture subject is evaluated based on the observation results in the observation step before or immediately after the application step and the observation results in the observation step after the culture step for the arbitrary period of time.

According to this embodiment of the present invention, a specific position of the culture subject can be observed over time through the through-holes in the culture platform, allowing for more accurate evaluation.

In a preferred embodiment of the present invention, the culture subject is skin tissue,
In the applying step, the test subject is applied to the skin tissue.
According to this aspect of the present invention, screening under conditions close to those for application in living bodies becomes possible, allowing candidate substances to be narrowed down with greater precision.

The present invention also relates to a culture method.
The culture method of the present invention is a method for culturing a culture target that is a biological tissue and/or a cell-containing solid composition, comprising:
The method is characterized in that the culture object is placed on a culture platform having a through hole passing through from the front to the back, and culture is performed by bringing the culture medium into contact with the culture object through the through hole.

The present invention also relates to a culture device.
The culture instrument of the present invention is characterized by comprising a culture platform having a through hole passing through from the front to the back, on which a culture subject, which is a biological tissue and/or a cell-containing tissue, is placed and cultured.

In a preferred embodiment of the present invention, a holding means for holding the position of the culture object on the culture platform is provided.
In this embodiment of the present invention, the culture subject can be cultured while being maintained in position on the culture platform, which is advantageous for a culture system that requires continuous observation.

In a preferred embodiment of the present invention, the through hole has a diameter of 1 mm or more.
By making the diameter of the through hole 1 mm or more, a sufficient optical path can be ensured during microscope observation.

According to the present invention, the optical path of the microscope does not pass through the culture stage, and the culture object can be observed through the through-hole, allowing for clearer observation and evaluation of the culture object.

FIG. 1 is a schematic diagram of a culture device of the present invention. FIG. 2 is a cross-sectional view of the culture device of the present invention. FIG. 1 is a cross-sectional view showing an embodiment of a culture device of the present invention having a protrusion for placing a culture platform on a culture medium storage container. FIG. 11 is a cross-sectional view showing an embodiment of the culture instrument of the present invention having a holding means for holding a culture target by a protrusion provided around the periphery of a through-hole. FIG. 1 is a cross-sectional view showing an embodiment of the culture instrument of the present invention that includes a weight for holding a culture target. 1 is a schematic cross-sectional view showing the optical path (white arrow) when a culture object cultured in the culture instrument of the present invention is observed with an epi-illumination inverted microscope. 1 is a schematic cross-sectional view showing the optical path (white arrow) when a culture object cultured in the culture instrument of the present invention is observed with a transmission type inverted microscope. FIG. 1 is a schematic cross-sectional view showing the optical path (white arrow) when a culture object cultured in the culture instrument of the present invention is observed with a transmission upright microscope. FIG. 1 is a flowchart showing an embodiment of an observation method of the present invention including a staining step. 1 is a flow chart showing an embodiment of the evaluation method of the present invention, in which A represents an embodiment in which the observation step is performed before the application step, and B represents an embodiment in which the observation step is performed immediately after the application step. 3D stained images of adipose tissue co-cultured with adipose-derived stem cells are shown. The upper image is a stained image obtained in the observation step before the culture step, and the lower image is a stained image obtained in the observation step after the culture step. The left image shows a stained image of fibronectin, and the right image shows a stained image of type I procollagen. 3D stained images of comparative adipose tissue cultured without co-culture with adipose-derived stem cells are shown. The upper row shows a stained image obtained in the observation step before the culture step, and the lower row shows a stained image obtained in the observation step after the culture step. The left shows a stained image of fibronectin, and the right shows a stained image of type I procollagen. FIG. 1 is a schematic cross-sectional view showing the light path (white arrow) when observing a culture subject cultured in a conventional culture insert with an epi-illumination inverted microscope. FIG. 1 is a schematic cross-sectional view showing the light path (white arrow) when observing a culture subject cultured in a conventional culture insert with a transmission inverted microscope. FIG. 1 is a schematic cross-sectional view showing the light path (white arrow) when observing a culture subject cultured in a conventional culture insert with a transmission upright microscope.

Hereinafter, embodiments of the present invention will be described with reference to Figures 1 to 10. However, it goes without saying that the technical scope of the present invention is not limited to the specific embodiments described below.
13 to 15 are schematic diagrams of a tissue culture technique using a culture insert, which is a known technique, and will be described in comparison with the present invention as appropriate.

<1> Culture Instrument The culture instrument of the present invention includes a culture platform 10. The culture platform 10 is provided with a through hole 11. The through hole 11 is for observation through a microscope, and has a hole diameter that is completely different from that of the microholes provided in the culture platform (membrane) of a conventional culture insert.

Specifically, the diameter of the through hole 11 is preferably 0.1 mm or more, more preferably 0.3 mm or more, more preferably 0.5 mm or more, more preferably 0.8 mm or more, even more preferably 1 mm or more, even more preferably 1.5 mm or more, even more preferably 2 mm or more, even more preferably 2.5 mm or more.

The upper limit of the hole diameter of the through-hole 11 is not particularly limited as long as the culture target 3 placed on it does not fall off. For example, a guideline is 3 cm or less, more preferably 1 cm or less.

The number of through holes 11 provided in the culture platform 10 is not limited, and one or two or more can be selected as desired.

The culture platform 10 must have a configuration in which the through-hole 11 is provided, but other configurations are not particularly limited.
For example, the culture platform 10 may be provided with a plurality of microholes in addition to the through-hole 11. The diameter of the microholes is preferably 0.01 to 20 μm, and more preferably 0.1 to 10 μm.
The culture platform 10 of this embodiment can also be manufactured by drilling through-holes 11 in a commercially available culture insert.

It is preferable to provide a wall 12 around the periphery of the culture platform 10 (Figs. 1 to 5). This prevents the culture medium 4 from overflowing from the periphery of the culture platform 10 and washing away the culture target 3.

The culture platform 10 is preferably provided inside the culture medium storage container 2 (FIGS. 1 to 5). The shape of the culture medium storage container 2 is not particularly limited as long as it can store the culture medium 4.
When a co-culture subject is cultured inside the culture medium storage container 2 described below, the culture medium storage container 2 preferably has a form suitable for cell culture.
As the culture medium storage container 2, a culture dish used in normal culture operations or a culture plate equipped with multiple wells may be used.

As shown in FIG. 1, it is preferable that the culture table 10 and the culture medium storage container 2 are configured to be removable. Since commercially available culture dishes and culture plates are standardized, if the size of the culture table 10 is adjusted to match the standard, the culture table 10 can be sold separately from the culture medium storage container 2.

In the embodiment shown in Figures 1 and 2, legs 13 are provided on the bottom of the culture platform 10. This ensures the height from the bottom of the culture medium storage container 2 to the through-hole 11, making it possible to store the culture medium 4.

In an embodiment, protrusions 14 may be provided on the edge of the wall 12 of the culture platform 10 instead of the legs 13. By suspending the protrusions 14 over the edge of the culture medium storage container 2 and suspending the culture platform 10 horizontally over the culture medium storage container 2, the height from the bottom of the culture medium storage container 2 to the through-hole 11 is ensured, making it possible to store the culture medium 4 (Figure 3).

It is also preferable to provide an adjuster extending in the vertical direction on the inner wall surface of the culture medium storage container 2, and to engage the protrusion 14 with the adjuster (not shown). By configuring the protrusion 14 to be able to slide in the vertical direction along the adjuster in this way, the height from the bottom of the culture medium storage container 2 to the through hole 11 can be adjusted as desired.

Both the culture platform 10 and the culture medium storage container 2 are preferably made of a transparent material. Examples include glass and synthetic resin. Examples of synthetic resin include ETFE (tetrafluoroethylene-ethylene copolymer), FEP (tetrafluoroethylene-hexafluoropropylene copolymer), HDPE (high density polyethylene), LDPE (low density polyethylene), PC (polycarbonate), PETG (glycol-modified polyester), PFA (tetrafluoroethylene-perfluoroalkoxyethylene copolymer), PMMA (polymethylmethacrylate), PMP (polymethylpentene), PP (polypropylene), PS (polystyrene), PSF (polysulfone), PUR (polyurethane), rigid PVC (vinyl chloride), flexible PVC (vinyl chloride), PVDF (polyvinylidene fluoride), silicone, TPE (thermoplastic elastomer), and PTFE (polytetrafluoroethylene).

The culture solution storage container 2 may be provided with a perfusion mechanism for the culture solution 4. That is, a culture solution supplying means for supplying the culture solution 4 into the culture solution storage container 2 and a culture solution discharging means for discharging the culture solution 4 from the culture solution storage container 2 may be provided (not shown).
In this case, it is preferable that the perfusion mechanism is automatically controlled.

The culture instrument may have a holding means for holding the position of the culture target 3 on the culture platform 10 .
The specific embodiment of the holding means is not particularly limited, and a configuration in which it is integrated with the culture platform 10 may be adopted, or a member separate from the culture platform 10 may be adopted.

Figure 4 shows an embodiment in which the culture platform 10 itself is equipped with a holding means. In the culture platform 10 in Figure 4, a convex portion 151 is provided around the through-hole 11 at a certain distance. The convex portion 151 limits the movement of the culture object 3 in the planar direction.

Figure 5 shows an embodiment in which a holding means is provided that is separate from the culture platform 10. The holding means shown in Figure 5 is a weight 152 for placing on the culture object 3. The weight 152 limits the movement of the culture object 3, allowing it to be cultured while maintaining its position on the culture platform 10.

As long as the culture subject 3 can be held, the shape of the weight 152 is not particularly limited.
In order to enable the culture subject 3 to be observed by an upright microscope or a transmission type inverted microscope, it is preferable that the weight 152 is in a form that does not block the optical path of the microscope. From the viewpoint of ensuring the optical path of the microscope, it is preferable that the weight 152 is made of a transparent material or has a through hole.
When performing gas-liquid interface culture, it is preferable that the weight 152 has a through hole in order to ensure contact between the culture subject 3 and the gas phase.
Specific examples of the structure of the weight 152 having a through hole include a doughnut-shaped structure and a net-like structure.

<2> Culture method <2-1> Culture subject The culture subject 3 of the present invention is a living tissue and a cell-containing solid composition. The present invention can be applied to any of the tissues derived from human, animal, and plant species.
The culture medium 4 is appropriately selected depending on the type of the culture subject 3.

A biological tissue is a collection of human, animal, or plant cells. It includes both tissues taken from living organisms and artificially produced tissues.
Examples of human or animal biological tissues to which the present invention can be applied include epithelial tissue, connective tissue, muscle tissue, and nervous tissue, and more specifically, examples of such tissues include skin tissue, bone tissue, and cartilage tissue.
Since the present invention is suitable for air-liquid interface culture, it is preferable to apply it to the culture of skin tissue.

The cell-containing solid composition refers to a composition that contains human, animal, or plant cells or biological tissues and is in a solid form. Specifically, it includes a gel composition in which cells or biological tissues are embedded in gelatin or agar.
The cells to be embedded in the gel composition include cultured cells and cells collected from a living body, and the type of cells is not limited. The biological tissue to be embedded in the gel composition includes both tissue collected from a living body and artificially produced tissue.

The size of the culture target 3 is not particularly limited as long as it does not fall through the through-hole 11. Specifically, the maximum diameter of the culture target 3 needs to be larger than the diameter of the through-hole 11.
The thickness of the culture subject 3 can also be selected arbitrarily as long as it does not interfere with microscopic observation.

<2-2> Cultivation method The culturing method of the present invention is characterized in that the culture solution 4 is supplied to the culture target 3 through the through-hole 11 (FIGS. 1 to 5). In order to bring the culture solution 4 into contact with the culture target 3, it is necessary that the liquid level of the culture solution 4 reaches at least the upper opening surface of the through-hole 11.
In the embodiment shown in FIGS. 1 to 5, the above-mentioned configuration can be easily realized by adjusting the amount of culture medium 4 stored in the culture medium storage container 2.

Since the culture medium 4 is supplied to the culture target 3 through the through hole 11, it is preferable to place the culture target 3 directly above the through hole 11.

In the embodiment shown in FIGS. 1 to 5, the upper opening surface of the through-hole 11 is flush with the liquid surface of the culture medium 4 in the culture medium storage container 2 .
The present invention is not limited to this embodiment, and the liquid level of the culture solution 4 in the culture solution storage container 2 may be located higher than the upper opening surface of the through-hole 11. In this case, since the culture solution 4 overflows onto the culture platform 10 from the through-hole 11, it is preferable to place a weight 152 on the culture target 3 as shown in FIG.

The co-culture subject may be cultured (not shown) in the culture medium 4 stored in the culture medium storage container 2. The co-culture subject may be either cells or biological tissue.
The combination of the co-culture subject 3 and the co-culture subject is not particularly limited. Any combination that is known to have an effect of the secretion of the co-culture subject on the morphological change, differentiation, and culture/growth promotion of the culture subject 3 may be adopted.
It is also possible to adopt a combination in which the influence of the co-culture subject on the culture subject 3 or the influence of the culture subject 3 on the co-culture subject is not publicly known. In this case, the influence of the co-culture subject on the culture subject 3 or the influence of the culture subject 3 on the co-culture subject can be observed and evaluated by the evaluation method of the present invention.

The culture mode of the co-culture target in the culture medium storage container 2 is not particularly limited. It may be in the form of adherent culture on the bottom surface of the culture medium storage container 2, or in the form of suspension culture in the culture medium 4 stored in the culture medium storage container 2. In the case of an embodiment in which suspension culture is performed, stirring blades can also be provided inside the culture medium storage container 2.

<3> Observation method The observation method of the present invention includes a culture step and an observation step. The explanation of the embodiment of the culture method described above is applicable to the embodiment of the culture step as it is.

The observation step is carried out using an inverted microscope or an upright microscope. Specifically, the observation step is carried out in the following form (a) or (b).
(a) The culture target placed on the culture platform is observed through the through-hole using an inverted microscope.
(b) Light from a light source is irradiated from below onto the culture object placed on the culture platform through the through-hole, and the culture object is observed using an upright microscope.

(a) Examples of embodiments of the present invention that use an inverted microscope include an embodiment that uses an epi-illumination microscope (Figure 6) and an embodiment that uses a transmission microscope (Figure 7).

In a configuration using an inverted epi-illumination microscope, light from a light source 51 is irradiated onto the culture target 3, and the light reflected from the culture target 3 is captured and observed by an objective lens 50 (Figure 6). In the case of an inverted epi-illumination microscope, excitation light from a light source 51 is irradiated onto the culture target 3, and the fluorescence emitted from the culture target 3 is captured and observed by an objective lens 50 (Figure 6).

When the culture target 3 on a conventional culture insert is observed with an inverted incident-light microscope, as shown in FIG. 13, the light from the light source 51 and the reflected light or fluorescence from the culture target 3 pass through the membrane 6. In other words, since the membrane 6 is present in the light path related to the microscope observation, the clarity of the microscope image depends on the light transmittance of the membrane 6. As described in Patent Document 3, the light transmittance tends to increase as the pore diameter of the micropores formed in the membrane 6 increases, but it is not possible to achieve 100% light transmittance in the entire wavelength range.

On the other hand, in the observation method of the present invention, since the culture platform 10 has a through hole 11, the culture platform 10 is not present in the optical path of the light from the light source 51 or the reflected light or fluorescent light from the culture subject 3 (Fig. 6). In other words, the light loss is significantly reduced compared to the conventional culture insert (Fig. 13), and a clearer microscope image can be obtained.

This effect is also obtained when the present invention is applied to observation with a transmission type inverted microscope (Figure 7). As shown in Figure 7, in an embodiment of the present invention in which observation is performed with a transmission type inverted microscope, light from a light source 51 passes through a through hole 11 and reaches an objective lens 50. In other words, there is no culture platform 10 on the optical path of the light source 51. Therefore, compared to the conventional form in which a culture target 3 on a culture insert is observed with an incident-light inverted microscope (Figure 14), light loss is significantly reduced, and a clearer microscope image can be obtained.

Next, an embodiment of the present invention using an upright microscope (b) will be described (FIG. 8). The microscope in this embodiment is a transmission type upright microscope.
As shown in Fig. 8, in the embodiment of the present invention in which observation is performed with a transmission type upright microscope, light from a light source 51 passes through a through hole 11 and reaches an objective lens 50. In other words, a culture platform 10 does not exist on the optical path of the light source 51. Therefore, compared with the conventional form in which a culture target 3 on a culture insert is observed with a transmission type upright microscope (Fig. 15), light loss is significantly reduced, and a clearer microscope image can be obtained.

As described above, by carrying out the observation process in the form (a) (using an inverted reflected light microscope or an inverted transmitted light microscope) or the form (b) (using an upright transmitted light microscope), it is possible to obtain the advantageous effect of obtaining a clearer microscopic image than in the form of observing the culture subject 3 cultured on a conventional culture insert.
There is no problem if the culture subject 3 cultured by the culture method of the present invention is observed with an incident-light upright microscope.

In normal tissue culture, the culture target is placed in a culture dish containing culture medium. In this case, the culture target floats and moves in the culture medium, making it very difficult to keep the culture target in a fixed position during the culture process. Therefore, it is very difficult to focus on a specific part of the culture target and observe it over time.
In order to solve such problems, it is preferable to carry out the observation method of the present invention using the culture instrument of the embodiment having the above-mentioned holding means (FIGS. 4 and 5).

If the culture subject 3 is cultured while being held in position on the culture platform 10 by the holding means, it is possible to focus on specific locations of the culture subject 3 and observe them over time. By photographing specific locations of the culture subject 3 under a microscope at multiple different points in time during the culture process and comparing the microscope images at each point in time, it is possible to evaluate with extremely high precision how the culture subject 3 has changed, or has not changed, during the culture process.

There are no particular limitations on the method of comparing microscopic images taken over time. For example, any characteristic part that appears in common in microscopic images taken at different times is set as a reference point, and multiple microscopic images are overlaid so that the reference points match. By overlaying multiple microscopic images in this way so that the reference points match, and comparing them, a more accurate evaluation can be achieved.

The method for setting the reference point is not particularly limited.
Preferably, two or more reference points are set, and more preferably, three or more reference points are set.
It is also preferable to obtain a three-dimensional microscopic image (Z-stack image) by reconstructing microscopic images captured by gradually shifting the focal plane, and to set multiple reference points distributed three-dimensionally rather than on the same plane.
The superposition of multiple microscope images after matching the reference points can be easily performed using known microscope image processing software.

The present invention preferably includes a staining step of staining the culture object 3. In this case, in the observation step, a stained image of the culture object 3 is observed.
The staining method is not particularly limited, and any known method can be used. Either in vitro staining for staining non-living tissues or in vivo staining for staining living tissues can be used.
When observing the culture subject 3 undergoing the culture step over time, it is preferable to employ in vivo staining.
Since any antigen can be stained by selecting an antibody, it is preferable to employ immunostaining.

A preferred embodiment is one in which the culture object 3 is stained while placed on the culture table 10.
For example, there is an embodiment in which the culture platform 10 on which the culture target 3 is placed is removed from the culture medium storage container 2 and placed inside a container storing a staining solution to stain the culture target 3. In this case, the staining solution comes into contact with the culture target 3 through the through-hole 11.

In this case, it is preferable to add a staining agent to the culture solution 4 and use this as the staining solution. For example, in the case of immunostaining, the culture solution 4 to which an antibody for staining has been added is used as the staining solution, and the culture subject 3 is stained by contacting this with the culture subject 3. This embodiment is preferable from the viewpoint of staining the culture subject 3 while it is still alive (i.e., in vivo staining).

The cleaning of the culture target 3 during the staining process can also be performed following the above example. Specifically, the culture platform 10 is placed inside a container that contains a cleaning solution such as PBS, and the cleaning solution is brought into contact with the culture target 3 through the through-hole 11 to clean it.

An example of an embodiment including a staining step is shown in Fig. 9. In this embodiment, the staining step is first performed, and a stained image is obtained in the observation step. After that, after a culture step for an arbitrary period of time, the staining step is performed again, and a stained image is obtained in the observation step (Fig. 9).
FIG. 9 shows a configuration in which the incubation step is carried out again after the second observation step, and the observation step is carried out three or more times, but it is of course possible to limit the observation step to two.

As shown in Figure 9, by observing the culture target 3 multiple times at different points in time, it is possible to accurately observe and evaluate the changes in the culture target 3 during the culture process.

6 to 8 show a form in which the culture stage 10 is placed in the culture medium storage container 2 used in the culture step and observed under a microscope, but the form is not limited to this. During the observation step, the culture stage 10 may be transferred to a container for observation other than the culture medium storage container 2.
The observation vessel is preferably made of a transparent material. In the observation step, the culture medium 4 may be stored in the observation vessel (substantially the embodiment shown in Figs. 6 to 8).

<4> Evaluation method The present invention also relates to a method for evaluating the effect of a test subject on a culture subject. The evaluation method of the present invention can be applied to, for example, screening of active ingredients of medicines, cosmetics, and health foods, toxicity tests of specific substances, and pure biological and medical research.

In this specification, the term "test subject" includes both tangible and intangible objects.
For example, a "test subject" includes, without limitation, a single compound or a mixture of compounds, such as a chemically synthesized compound, a compound extracted from a natural product, an artificially produced composition, or an extract composition extracted from a natural source.
Additionally, invisible things such as light, gas, electrical stimuli, and physical stimuli can also be used as "test subjects."
Furthermore, the co-culture subject in the above-mentioned co-culture, more specifically, a secretion secreted from the co-culture subject, may also be included in the "test subject".

The evaluation method of the present invention includes an application step, a culture step, and an observation step. The explanation of the culture method of the present invention described above is applicable to the embodiment of the culture step. The explanation of the observation method of the present invention described above is applicable to the embodiment of the observation step.

The application step is a step of applying a test subject to a culture subject. The application method can be appropriately selected depending on the test subject to be applied.

When the test subject is a tangible substance such as a compound or a mixture, it may be directly applied to the culture subject 3 or indirectly applied to the culture subject 3 by adding it to the culture solution 4.
Examples of the direct application include a method of applying, spraying, or injecting the test subject onto the culture subject 3. When the culture subject 3 is a skin tissue, a preferred example is a method of applying the test subject or a composition containing the test subject onto the culture subject 3.

When the test subject is light, it is preferable to apply it by directly irradiating it on the culture subject 3. In this case, the wavelength and intensity of the irradiated light are appropriately set according to the purpose of the test. This embodiment can be applied to, for example, an evaluation test of the effects of ultraviolet light.

When the test subject is a gas, it is preferable that the gas phase of the culture system is composed of the gas that is the test subject. This form of the invention makes it possible to evaluate, for example, the effect of humidity or dryness on the culture subject 3, the effect of the oxygen concentration in the gas phase of the culture system on the culture subject 3, and the effect (pharmacological action, toxicity, etc.) of a specific gas component on the culture subject 3.

When the test subject is an electrical or physical stimulus, it is preferable to apply the stimulus directly to the culture subject 3.

When the test subject is a co-culture subject or a secretion secreted from a co-culture subject, it can be said that "the test subject has been applied to the culture subject" by placing a culture platform 10 on which a culture subject 3 is placed inside a culture medium storage container 2 in which culture medium 4 in which a co-culture subject is present is stored.
Of course, an embodiment in which another test subject is added to the culture subject 3 while co-culture is being performed is also included within the technical scope of the present invention.

FIG. 10 is a flow chart showing an embodiment of the evaluation method of the present invention.
FIG. 10A shows an embodiment in which the culture object 3 is first observed through an observation process, the initial state of the culture object 3 is confirmed, and then the application process is performed, and the shape of the culture object 3 after a culture process for an arbitrary period of time is confirmed through the observation process.
FIG. 10B shows an embodiment in which an observation step is performed immediately after the application step, the initial state of the culture object 3 is confirmed, and then a culture step is performed for an arbitrary period of time, and the shape of the culture object 3 thereafter is confirmed by the observation step.
In either form of FIG. 10A or FIG. 10B, the influence of the test subject on the culture subject 3 can be evaluated.

Figures 10A and B show a configuration in which the incubation process is performed again after the second observation process, and the observation process is performed three or more times, but of course the observation process may be limited to two times.

The observation step may be performed by observing a stained image of the culture subject 3. That is, the above-mentioned staining step may be performed before the observation step in FIG. 10.

<1> Preparation of Adipose Tissue-Containing Gel Composition A 5% agarose solution was heated and dissolved, then poured into a 12-well plate and kept warm in an incubator.
Adipose tissue was excised from commercially available human skin tissue and added to a 5% agarose solution that had been kept warm in an incubator. A 12-well plate was then sandwiched between ice packs from above and below to cool and solidify.
The cooled and solidified gel composition was cut with a razor and molded into a flat shape with the adipose tissue exposed on the surface, to prepare an adipose tissue-containing gel composition.

<2> Observation step (1)
A culture table 10 having a wall portion 12 and a leg portion 13 as shown in FIG. 1 was prepared.
The through-hole 11 provided in the culture platform 10 has a diameter of 3.0 mm. The wall 12, the legs 13, and the culture platform 10 are each made of transparent synthetic resin. The culture platform 10 is a porous membrane, and in addition to the through-hole 11, micropores with a diameter of about 1 μm are provided throughout the entire platform.

The prepared adipose tissue-containing gel composition was placed directly above the through-hole 11 of the culture platform 10 with the surface on which the adipose tissue was exposed facing down. A sterilized metal weight 152 was placed on top of the adipose tissue-containing gel composition as shown in FIG. 5.

The culture platform 10 was placed on a 10 cm dish containing a staining solution prepared by diluting primary antibodies (anti-fibronectin antibody and anti-type I procollagen antibody) with culture solution 4 (DMEM). The staining solution was brought into contact with the adipose tissue-containing gel composition through the through-holes 11, and the mixture was incubated for 16 hours.
After washing the adipose tissue-containing gel composition with PBS, the culture platform 10 was placed on a 10 cm dish containing a staining solution prepared by diluting fluorescently labeled secondary antibodies against the above-mentioned two types of primary antibodies in culture solution 4 (DMEM). The staining solution was brought into contact with the adipose tissue-containing gel composition through the through-hole 11, and the composition was incubated for 2 hours.
After washing the adipose tissue-containing gel composition with PBS, the culture platform 10 was placed inside a glass bottom plate containing culture medium 4 (DMEM), and a three-dimensional stained image of the adipose tissue-containing gel composition was photographed from below through the through hole 11 using an epi-illumination inverted confocal fluorescence microscope (see Figure 6).

The culture platform 10 was placed on a 6-well plate (culture solution storage container 2) in which a culture solution 4 (DMEM) was stored and in which adipose-derived stem cells (ADMSCs) were seeded in advance as co-culture subjects. The culture was performed in an incubator for 7 days in a state in which the culture solution 4 was in contact with the adipose tissue-containing gel composition through the through-holes 11 (the state shown in FIG. 5 ).
In addition, in order to comparatively evaluate the effects of co-culture, the culture platform 10 was also placed on a 6-well plate in which the adipose-derived stem cells to be co-cultured had not been seeded, and the adipose tissue-containing gel composition was similarly cultured.

<4> Observation step (2)
The adipose tissue-containing gel composition was stained in the same manner as in the observation step (1) described above, and the three-dimensional stained image was observed.

<5> Results A stained image of the adipose tissue co-cultured with the adipose-derived stem cells is shown in Figure 11, and a stained image of the adipose tissue cultured without co-culture is shown in Figure 12. As shown in Figures 11 and 12, the stained images are extremely clear.

Furthermore, as is clear from a comparison of the stained images in the upper and lower rows of Figures 11 and 12, the stained images before and after culture are of the same location in the adipose tissue-containing gel composition that is the subject of culture. In other words, the present invention makes it possible to perform time-dependent observation focusing on a specific location of the tissue, which was previously difficult.

Comparing the stained images in the upper and lower rows of Figure 11, it is easy to see that the expression levels of fibronectin and type I procollagen have increased significantly. This is an effect that arises from the fact that the present invention makes it possible to observe the same location over time. If different locations on the culture subject are observed before and after culture, it is difficult to make such a clear evaluation.

Furthermore, by comparing Figures 11 and 12, it is easy to see that when co-culture with adipose-derived stem cells is not performed, there is no increase in the expression levels of fibronectin and type I procollagen in the adipose tissue, or the increase is weaker than when co-culture is performed. Such a clear evaluation is possible because it is possible to observe the same location over time.

As described above, the present invention makes it possible to obtain clear microscopic images and to observe the same location of cultured tissue over time.

10 Culture platform 11 Through hole 12 Wall 13 Leg 14 Projection 151 Convex 152 Weight 2 Culture fluid storage container 3 Culture target 4 Culture fluid 50 Objective lens 51 Light source 521 Dichroic mirror 522 Light source mirror 6 Membrane

Claims (15)

A method for observing a culture target which is a biological tissue and/or a cell-containing solid composition, comprising:
A culture step of placing the culture object on a culture platform having a through hole penetrating in a front-back direction and culturing the culture object by contacting a culture solution with the culture object through the through hole;
An observation step according to the following (a) or (b);
Including,
The culture platform has a flat surface on which the culture object is placed , and the through hole is provided so as to penetrate the flat surface in a front-back direction,
In the culturing step, the culture object is placed on the flat surface and directly above the through-hole;
An observation method, characterized in that the diameter of the through-hole is 1 mm or more and is of a size such that the culture object placed on the flat surface will not fall off.
(a) The culture target placed on the culture platform is observed through the through-hole using an inverted microscope.
(b) Light from a light source is irradiated from below onto the culture object placed on the culture platform through the through-hole, and the culture object is observed using an upright microscope. In the culturing step, the culture object is cultured while maintaining the position of the culture object on the culture platform;
The observation method according to claim 1 , wherein the observation step is carried out over time. The observation method according to claim 1 or 2, characterized in that the culture step is a step of placing a culture object on the culture stand provided inside a culture medium storage container, and culturing the culture object so that the culture medium stored in the culture medium storage container contacts the culture object through the through-hole. The culturing step comprises:
The culture subject;
A co-culture subject to be co-cultured with the culture subject;
Cultivating the
The culture object is placed on the culture table and cultured,
The co-culture subject is cultured in a culture solution stored in the culture solution storage container,
The observation method according to claim 3 , wherein the co-culture subject is a cell and/or a biological tissue. The observation method according to any one of claims 1 to 4, characterized in that the culture subject is skin tissue. The observation method according to any one of claims 1 to 4, characterized in that the culture subject is a gel composition in which cells and/or biological tissues are embedded. The observation method according to any one of claims 1 to 6, characterized in that in the culturing step, the culture object is cultured in a state in which at least the upper surface of the culture object is not in contact with the culture medium. A staining step of staining the culture object while the culture object is placed on the culture table,
The observation step is carried out after the staining step,
The observation method according to any one of claims 1 to 7, wherein in the observation step, a stained image of the culture object is observed. The observation method according to claim 8, characterized in that in the staining step, the culture table on which the culture object is placed is placed in a container in which a staining solution is stored, and the culture object is stained by contacting the staining solution with the culture object through the through-hole. A method for evaluating an effect of a test subject on a culture subject by the observation method according to any one of claims 1 to 9,
The method includes a step of providing a test subject to the culture subject, the culture step, and the observation step,
The evaluation method is characterized in that after the application step, a culture step is performed for an arbitrary period of time, and then the observation step is performed, and the effect of the test subject on the culture subject is evaluated based on the observation results in the observation step. In the culturing step, the culture object is cultured while the position of the culture object on the culture platform is held by a holding means;
The observation step is also carried out before or immediately after the application step,
The evaluation method according to claim 10, characterized in that an influence of the test subject on the culture subject is evaluated based on an observation result in the observation step before or immediately after the application step and an observation result in the observation step after the culture step for the arbitrary period of time. The culture subject is a skin tissue,
The evaluation method according to claim 10 or 11, wherein the test subject is applied to the skin tissue in the applying step. A method for culturing a culture target that is a biological tissue and/or a cell-containing solid composition, comprising:
The culture object is placed on a culture platform having a through hole penetrating the front and back directions, and the culture object is cultured by contacting a culture solution with the culture object through the through hole,
The culture platform has a flat surface on which the culture object is placed , and the through hole is provided so as to penetrate the flat surface in a front-back direction,
The culture target is placed on the flat surface and directly above the through-hole,
A culture method, characterized in that the diameter of the through hole is 1 mm or more and is of a size such that the culture object placed on the flat surface will not fall off. A culture platform having a through hole extending from front to back for placing and culturing a culture target which is a biological tissue and/or a cell-containing tissue,
The culture platform has a flat surface on which the culture object is placed , and the through hole is provided so as to penetrate the flat surface in a front-back direction,
The culture object is configured to be placed on the flat surface and directly above the through-hole,
A culture device, characterized in that the through-hole has a diameter of 1 mm or more and is of a size such that a culture target placed on the flat surface will not fall off. The culture device according to claim 14, characterized in that it is provided with a holding means for holding the position of the culture object on the culture platform.