WO2016052078A1 - Plastic container - Google Patents

Abstract

Provided is a plastic container in which cells or tissues can be cultured and an image of the cells or tissues can be taken in a living state with a phase contrast microscope. The plastic container (10) is a multi-well plate having at least two wells (11). The diameter R of the opening of each of the wells (11) is 0.5 mm or more and less than 2 cm, and the depth D of each of the wells (11) is 2 mm or more and less than 2 cm. The inside surface (11a) of each of the wells (11) has water repellency, and the bottom surface (11b) of each of the wells (11) has hydrophilicity.

Description

Plastic container

The present invention relates to a plastic container for culturing cells and tissues and observing the cultured cells and tissues.

In recent years, in the fields of drug discovery and regenerative medicine, attention has been focused on techniques for imaging living cells and tissues (hereinafter referred to as live cells) without staining. The phase contrast microscope is a typical device for imaging live cells, and includes a phase difference observation capacitor having a ring slit and a phase difference observation objective lens having a ring-shaped phase difference plate. Living cells are placed in a plastic container and placed between a phase difference observation capacitor and a phase difference observation objective lens and imaged. In recent years, in order to increase the throughput of screening, a plastic container used in a phase-contrast microscope also serves as a culture container for culturing living cells, and has a plurality of recesses (hereinafter referred to as culturing and observing living cells). , Well). Such a plastic container is called a microplate, a microtiter plate, a microwell plate, a multiwell plate, a well plate, or the like.

By the way, depending on the nature of the plastic container (or the inner wall of the well) with respect to the culture solution and the size of the opening of the well, the surface of the culture solution for culturing living cells may be curved in a concave or convex shape. That is, the liquid level of the culture solution may become a meniscus. When the liquid level of the culture solution is a meniscus, a phase difference occurs in the light passing through the culture solution between the vicinity of the well wall and the center of the well. In addition, the meniscus on the surface of the culture solution acts as a lens. Since the phase contrast microscope observes the phase difference by converting it into contrast, if the phase difference due to the position in the well occurs due to the meniscus on the liquid surface of the culture solution, it is impossible to image live cells normally. May be imaged, the image quality may not be good. For this reason, a method of flattening the liquid level of the culture solution by floating a transparent flat plate on the level of the culture solution placed in the well is known (Japanese Patent No. 3133786).

In addition, although not a plastic container suitable for use in a phase-contrast microscope as described above, plastic containers with various contrivances are known in the biological and biochemical fields. For example, by making the inner surface of the well water repellent (hydrophobic) and making the bottom surface hydrophilic, a plastic container that smoothly guides a hydrophilic solution such as DNA (DeoxyriboNucleic® Acid) to the bottom surface of the well Known (Japanese Patent Laid-Open No. 2002-065299). Similarly, in a plastic container for reagent analysis, a hydrophilic solution may be smoothly guided to the bottom surface of the well by making the inner surface of the well water-repellent and making the bottom surface hydrophilic (see Japanese translation 2002). -525573). Plastic containers that make the inner surface of the well water-repellent are also known to make it difficult for the aqueous solution in the well to overflow or to prevent contamination due to sample contamination between the wells (cross-contamination). JP 2003-066033, JP 2004-212359 A).

As in Japanese Patent No. 3133786, if a transparent flat plate is floated on the liquid level of the culture solution placed in the well, the liquid level of the culture solution can be flattened and meniscus can be reduced. The oxygen supply path is cut off. For this reason, if the meniscus is reduced by floating a transparent flat plate on the liquid surface of the culture solution, the living cells will die or give a lot of stress to the living cells. It is difficult to do.

That is, in order to cultivate live cells, image and observe in a live state, it is necessary to keep the well open. And in order to image a living cell with a phase-contrast microscope, it is necessary to reduce the meniscus which arises on the liquid level of a culture solution in the state which opened the well.

An object of the present invention is to provide a plastic container capable of culturing cells and tissues and imaging them with a phase-contrast microscope while they are alive.

The plastic container of the present invention has an equivalent circle diameter of an opening of 0.5 mm or more and less than 2 cm, a depth of 2 mm or more and less than 2 cm, an inner surface having water repellency, and a bottom surface having hydrophilicity. At least two wells are provided.

A water-repellent coat containing fluorine is provided on the inner side surface of the well, and the water-repellent coat preferably has a fluorine content of 0.01 mg / cm ² or more and less than 1.5 mg / cm ² .

The inner surface of the well is preferably provided with a diamond-like coating.

The inner surface of the well preferably has a contact angle with pure water of 75 ° to 100 °.

It is preferable that the bottom surface of the well has a contact angle with respect to pure water of 0 ° or more and 70 ° or less.

The difference between the contact angle of the inner surface of the well with pure water and the contact angle of the well bottom with pure water is preferably 5 degrees or more and 110 degrees or less.

The inner surface of the well is preferably roughened.

The inner surface of the well is preferably subjected to a surface treatment by a vapor phase method.

It is preferably formed of polystyrene, polyethylene terephthalate, or polycarbonate.

According to the plastic container of the present invention, cells and tissues can be cultured, and these can be imaged with a phase contrast microscope while they are alive.

It is an external view of a plastic container. It is sectional drawing of a plastic container. It is explanatory drawing which shows the contact angle of the pure water with respect to the inner surface of a well. It is explanatory drawing which shows the contact angle of the pure water with respect to the bottom face of a well. It is explanatory drawing which shows the liquid level of a culture solution. It is explanatory drawing of a phase-contrast microscope. It is explanatory drawing which shows the liquid level of the culture solution in the plastic container of a comparative example. It is explanatory drawing which shows the liquid level of the culture solution in the plastic container of a comparative example. It is a plastic container having a well whose inner surface is inclined. It is a plastic container having a well whose inner surface is inclined.

As shown in FIG. 1, a plastic container 10 is a container for culturing living cells such as living cells and tissues, and a container for imaging (or observing) the cultured living cells with a phase contrast microscope. is there. The plastic container 10 is a so-called multi-well plate, and has a plurality of wells 11 opened in a circular shape. The well 11 is a recess for storing a living cell to be imaged after culturing and a culture solution for culturing the living cell. In FIG. 1, a 96-well plate having 96 wells 11 in 8 rows and 12 columns is shown as an example, but the plastic container 10 only needs to have at least two wells 11. The number of is arbitrary. In addition to the 96 well plate, a multiwell plate having 6, 24, or 384 wells 11 is often used.

Suitable materials for the plastic container 10 include, for example, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE). ), Medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane acrylate and other acrylic materials, cellulose, glass and the like. In addition, a resin such as a biodegradable polymer such as polylactic acid, polyglycolic acid, polycaprolactan, or a copolymer thereof can be used. Among these, polyethylene terephthalate, polystyrene, and polycarbonate can be preferably used, and polystyrene can be particularly preferably used. This is because the cytotoxicity is low. Further, the surface of the plastic container 10 may be subjected to any surface treatment (for example, irradiation with plasma, corona, microwave, electron beam, ultraviolet light, etc.).

As shown in FIG. 2, the well 11 is a non-through hole and is opened on the surface of the plastic container 10. A living cell 13 to be cultured and a culture solution 12 (for example, serum solution) for culturing the living cell 13 are injected into the well 11 from the opening. When the plastic container 10 is used, since the well 11 is opened, the culture solution 12 injected into the well 11 is always in the vicinity of the opening of the well 11 during culturing of the living cells 13 and during imaging with a phase contrast microscope. Contact with air. For this reason, the cultured living cells 13 are imaged (observed) with a phase contrast microscope while alive.

In the plastic container 10, the well 11 has an opening diameter R of 0.5 mm or more and less than 2 cm, and a depth D of 2 mm or more and less than 2 cm. The reason why the diameter R of the opening of the well 11 is 0.5 mm or more and less than 2 cm is that it is preferable for obtaining data that converges the number of living cells 13 cultured in the same environment without variation. . The diameter R of the opening of the well 11 is more preferably 1 mm or more. When the diameter R of the opening of the well 11 is less than 2 cm, the meniscus of the liquid surface 12a of the culture solution 12 becomes prominent. Therefore, the present invention is particularly useful, and the diameter R of the opening of the well 11 is 1 cm or less. It is particularly suitable for the case. For example, in the case of a 96 well plate having 96 wells 11, the diameter of the opening of the well 11 is 6 mm, for example. In the case of a 384 well plate having 384 wells 11, the diameter of the opening of the well 11 is 3 mm, for example. is there.

The depth D of the well 11 is the height from the bottom surface 11b of the well 11 to the opening (the surface of the plastic container 10), and the depth D of the well 11 is formed to be 2 mm or more. This is because a sufficient medium for culturing 13 and a sufficient amount of the culture solution 12 for culturing the living cells 13 are secured. The depth D of the well 11 is formed to be less than 2 cm in order to prevent a decrease in the amount of light in the peripheral visual field due to vignetting when observing with a transmission microscope. If the depth D of the well 11 is in the range of 3 mm or more and 1 cm or less, it is particularly suitable for the culture and imaging of the living cells 13. Further, the amount of the culture solution 12 put into the well 11 is preferably ½ or less of the depth D of the well 11.

The inner surface 11a of the well 11 has water repellency. As shown in FIG. 3, the inner surface 11a of the well 11 is provided with, for example, a water-repellent coating 17 containing fluorine. When a minute droplet 16 of pure water is dropped, the droplet 16 and the well 11 11 has a water repellency such that the contact angle α with the inner side surface 11a is 75 degrees or more and 110 degrees or less. The inner surface 11a of the well 11 preferably has water repellency such that the contact angle α with the pure water droplet 16 is 80 degrees or more and 100 degrees or less, and the contact angle α with the pure water droplet 16 is 85. It is particularly preferable that the angle is not less than 95 degrees and not more than 95 degrees. The contact angle α can be measured with a commercially available contact angle meter by cutting out a part of the inner surface 11 a of the well 11 and dropping a pure water droplet 16. The fluorine content of the water-repellent coat 17 is preferably 0.01 mg / cm ² or more and less than 1.5 mg / cm ² . When the fluorine content of the water-repellent coat 17 is less than 0.01 mg / cm ² , there is a problem that pinholes are formed in the water-repellent coat 17 or the water-repellent coat 17 has low durability and thus dissolves. There is. When the fluorine content of the water repellent coat 17 is 1.5 mg / cm ² or more, the water repellent coat 17 becomes thick and the observation field of the well 11 may be narrowed.

The bottom surface 11b of the well 11 has hydrophilicity. The bottom surface 11b of the well 11 is subjected to a hydrophilic treatment on the surface of the material of the plastic container 10 by, for example, plasma treatment (IHI Technical Report Vol. 52 No. 4 (2012) p. 65) or corona treatment. As shown in FIG. 4, the contact angle β of the minute pure water droplet 16 with respect to the bottom surface 11 b of the well 11 is in the range of 0 degrees to 70 degrees. The reason why the bottom surface 11b of the well 11 is hydrophilic is that the living cells 13 are stably adsorbed on the bottom surface 11b of the well 11 and cultured (growth). In order to stably culture the living cells 13, it is particularly preferable that the bottom surface 11b of the well 11 has a hydrophilic property so that the contact angle β of the pure water droplet 16 is 10 degrees or more and 65 degrees or less. .

As described above, the plastic container 10 includes the well 11 in which the diameter R and the depth D of the opening are determined, the inner side surface 11a has water repellency, and the bottom surface 11b has hydrophilicity. For this reason, as shown in FIG. 5, when the living cells 13 and the culture solution 12 are injected into the well 11, the angle γ1 formed by the inner surface 11a of the well 11 and the liquid surface 12a of the culture solution 12 becomes approximately 90 degrees. The components of the culture solution 12 may change for each of the plurality of wells 11 (or for each measurement in which the contents of the well 11 are replaced), but in any case, the culture solution 12 is still an aqueous solution. If the plastic container 10 is used, an angle γ1 formed by the liquid surface 12a of the culture medium 12 with respect to the inner surface 11a of the well 11 can be obtained by simply using the plastic container 10 without floating a transparent flat plate on the culture liquid 12. It will be about 90 degrees.

After culturing the living cells 13 cultured in the plastic container 10, or in the process of culturing the living cells 13, the living cells 13 are set together with the plastic container 10 in a phase contrast microscope and imaged. For example, as shown in FIG. 6, the phase contrast microscope 20 includes a light source 21 that emits illumination light, a phase difference observation capacitor 22, a phase difference observation objective lens 26, and an image sensor 29. Is arranged between the phase difference observation capacitor 22 and the phase difference observation objective lens 26. In FIG. 6, the imaging sensor 29 is arranged at a position where the light from the phase difference observation objective lens 26 directly enters, but a mirror (not shown) is provided between the phase difference observation objective lens 26 and the imaging sensor 29. , And the light from the phase difference observation objective lens 26 may be bent 90 degrees by a mirror and incident on the image sensor 26.

The phase difference observation capacitor 22 includes a ring slit 23 provided with a slit on the ring and a condenser lens 24. The illumination light emitted from the light source 21 is linked by the ring slit 23, and the plastic is formed by the condenser lens 24. A specific well 11 of the container 10 is passed. The phase difference observation objective lens 26 includes an objective lens 27 and a ring-like phase difference plate 28, and the light passing through the specific well 11 is imaged on the image sensor 29 by the objective lens 27. In the process, the phase difference of the light passing through the specific well 11 is shifted by the phase difference plate 28. If there are no living cells 13 in the well 11, the light of all the optical paths passes through the phase difference plate 28, so that an image with uniform brightness is captured as a whole.

On the other hand, if there are living cells 13 in the well 11, a difference occurs in the optical path length between the light passing through the living cells 13 and the light passing through only the culture solution 12. For this reason, the phase difference plate 28 advances the phase by a quarter wavelength or delays the wavelength by a quarter wavelength to cause these lights to interfere with each other. The phase difference is imaged as contrast. That is, the imaging sensor 29 images the substantially transparent living cells 13 in the transparent culture solution 12.

As described above, when imaging the living cells 13 in the well 11 with the phase contrast microscope 20, the phase contrast microscope 20 images the phase difference of the diffracted light from the living cells 13 as a contrast. It is assumed that the surface 12 a is substantially perpendicular to the inner surface 11 a of the well 11 and no phase difference is caused by the culture solution 12. According to the plastic container 10, since the liquid surface 12a of the culture solution 12 is substantially perpendicular to the inner surface 11a of the well 11, the plastic container 10 that is a culture container for the living cells 13 is used as it is. With the phase-contrast microscope 20, the living cells 13 can be imaged with good image quality while being alive.

For example, as shown in FIG. 7, in the plastic container 110 of the comparative example in which the dimensions of the well 11 and the characteristics of the inner surface 11 a or the bottom surface 13 b do not satisfy the characteristics of the above embodiment, the liquid surface 12 a of the culture solution 12 is The angle γ2 formed with the inner surface 11a is larger than 90 degrees, and the liquid surface 12a of the culture solution 12 becomes a concave meniscus. Similarly, for example, as shown in FIG. 8, in the plastic container 120 of the comparative example, the angle γ3 formed by the liquid surface 12 a of the culture solution 12 and the inner surface 11 a of the well 11 is smaller than 90 degrees. The liquid surface 12a becomes a convex meniscus. As described above, when the liquid surface 12a of the culture solution 12 becomes a meniscus, the optical path length passing through the culture solution 12 differs depending on the position in the well 11, so that the light passing through the well 11 has a corresponding phase difference. . Further, the meniscus on the liquid surface 12a of the culture solution 12 acts as a lens. For these reasons, in the plastic containers 110 and 120 of the comparative example that do not satisfy the characteristics of the above embodiment, the live cells 13 cannot be imaged correctly or the image quality is poor, but the plastic container 10 of the above embodiment has the same effect. According to this, there is no such inconvenience, and the living cell 13 can be imaged correctly and with high image quality by the phase-contrast microscope 20 in a state where it is alive.

In the above embodiment, the inner surface 11a of the well 11 is substantially perpendicular to the front and back surfaces of the plastic container 10, but the well 11 has an inner surface 211a as in the plastic container 210 shown in FIG. The well 211 may be inclined and constricted from the opening to the bottom surface 211b. Further, like the plastic container 220 shown in FIG. 10, the well 221 may have a shape in which the inner side surface 221a is inclined and expands from the opening to the bottom surface 221b. In the case of forming a well having an inclined inner surface like the plastic containers 210 and 220, as long as the inclination of the inner surface of the well is about several degrees, similar to the well 11 of the plastic container 10 of the above embodiment. If formed, the liquid surface 12a of the culture solution 12 can be made substantially horizontal.

In the case of forming a well 221 having a shape that expands from the opening to the bottom surface 221b as in the plastic container 220 shown in FIG. 10, the plastic container 220 may be molded by injection integral molding, and the inner side surface 221a of the well 221 may be formed. The first plate 231 provided with the holes to be formed and the second plate 232 forming the bottom surface 221b of the well 221 may be separately molded and pasted together. Of course, also in the case of producing the plastic container 10 of the above embodiment and the plastic container 210 shown in FIG. 9, the first plate provided with holes for forming the inner surface of the well and the second plate for forming the bottom surface of the well. And may be created separately and bonded together.

In the above-described embodiment, the well 11 is opened in a circular shape, but the opening shape of the well 11 is arbitrary, and may be opened in an arbitrary shape such as a quadrangle. In this case, the diameter R of the opening of the well 11 in the above embodiment is a diameter when the area of the opening is converted into a circle (hereinafter referred to as an equivalent circle diameter). For example, in the case of a square with one side having a length p, R = 2 (p ² / π) ^1/2 .

In the above embodiment, the inner side surface 11a of the well 11 has water repellency by providing the water repellent coat 17, but instead of providing the water repellent coat 17, the inner side surface 11a of the well 11 is roughened by sandblasting or the like. It may be made to have water repellency by surfaceizing and providing fine irregularities on the surface. Further, the inner surface 11a of the well 11 may be provided with water repellency by surface treatment by a vapor phase method such as atmospheric pressure plasma treatment or ultraviolet irradiation treatment.

In the above embodiment, the water repellent coating 17 containing fluorine (so-called fluorine coating) gives the inner surface 11a of the well 11 water repellency. However, the inner surface 11a of the well 11 is water repellent by coating with a silicone resin. May be given.

In the above embodiment, the inner surface 11a of the well 11 is given water repellency by the water repellent coat 17 containing fluorine. However, instead of the water repellent coat 17 containing fluorine, a diamond-like coating is provided to provide the well 11 The inner side surface 11a may have water repellency. The diamond-like coating is formed by, for example, a combination of atmospheric pressure plasma treatment and carbon coating. The diamond-like coating formed in this case is diamond-like carbon. A diamond-like coating can be formed using silicon (Si) instead of a carbon coating. When using diamond-like coating, the meniscus shape of the liquid surface 12a of the culture solution 12 is particularly excellent in stability over time, and environmental safety is also high.

Further, instead of providing the water-repellent coating 17 containing fluorine, the inner surface 11 a of the well 11 may be given water repellency by exposing the base material of the plastic container 10 to the inner surface 11 a of the well 11. good. When the plastic container 10 is formed of polystyrene, if the hydrophilic treatment is performed by plasma treatment or corona treatment, the contact angle α of the pure water droplet 16 with respect to polystyrene becomes 70 degrees or less. Otherwise, the contact angle α of the pure water droplet 16 with respect to polystyrene is about 91 degrees. Therefore, if polystyrene that has not been subjected to hydrophilic treatment is exposed on the inner side surface 11a of the well 11, the inner side surface 11a of the well 11 can have the same water repellency as in the above embodiment.

The thickness of the water repellent coat 17 is preferably 0.001 μm or more and 1000 μm or less. Similarly, in the case where the above-described various treatments are applied to the inner side surface 11a of the well 11 instead of the water-repellent coat 17, the water-repellent layer and the layer subjected to various treatments for imparting water repellency (water-repellent layer) The thickness of the treatment layer is preferably 0.001 μm or more and 1000 μm or less. When the thickness of the water-repellent coat 17 or the like is less than 0.001 μm, the water-repellent coat 17 or the like is too thin and other conditions may not be obtained, and the necessary water-repellent property may not be obtained, and is thicker than 1000 μm. This is because the field of view of the well 11 is narrowed. In order for the water repellent coat 17 to have sufficient continuity, the thickness of the water repellent coat 17 is preferably 0.02 μm or more. Furthermore, in order to prevent the water-repellent coat 17 from having a sufficient rubbing resistance, practically preventing the water-repellent from being lowered immediately, and to withstand repeated use, the water-repellent coat 17 The thickness is particularly preferably 0.5 μm or more.

The water repellent coat 17 and various treatments performed in place of the water repellent coat 17 do not need to be performed on the entire inner surface 11 a of the well 11. The inner surface 11a of the well 11 only needs to have water repellency by the water-repellent coat 17 or the like in a range where at least the liquid surface of the culture solution 12 can contact the inner surface 11a of the well 11.

In the above embodiment, the water repellency of the inner surface 11a and the hydrophilicity of the bottom surface 11b of the well 11 can be set separately, but both the culture of the living cells 13 and the observation with the phase contrast microscope 20 are compatible. For this, the balance between the water repellency of the inner surface 11a of the well 11 and the hydrophilicity of the bottom surface 11b is important. Therefore, for example, the difference (| α−β |) between the contact angle α of the pure water droplet 16 with the inner surface 11 a of the well 11 and the contact angle β of the pure water droplet 16 with the bottom surface 11 b of the well 11 is It is preferably 5 degrees or more and 110 degrees or less, and more preferably 10 degrees or more and 80 degrees or less.

In addition, the image obtained by imaging the living cells 13 with the phase-contrast microscope 20 can be used, for example, to acquire the number of living cells 13 cultured by image processing. The number of living cells 13 can be determined by, for example, detecting cells from an image by counting the number of cells using a threshold separation method such as the Otsu method, a method using machine learning such as a Level Set method, or the like. Can be acquired.

Moreover, if the plastic container 10 of the said embodiment is used, since the living cell 13 can be imaged with the phase-contrast microscope 20 in the state of being alive, culture | cultivation of the living cell 13 is imaged after imaging with the phase-contrast microscope 20. After the continuation, the same well 11 can be imaged with the phase contrast microscope 20 again by the phase contrast microscope 20. For this reason, a cell phylogenetic tree can be acquired by performing image processing using a plurality of images obtained by repeating culture and imaging. The cell lineage tree can be obtained by the method of Kanade et al. (“Cell ら Image （Analysis: Algorithms, System and Applications” 2011 IEEE Workshopon Applications of Computer Vision, “WACV 2011”) or the method of Kurokawa et al. Software for precise tracking of cell proliferation "Biochemical and Biophysical Research Communications 417 (2012) 1080-1085).

In the above embodiment, the living cell 13 is imaged by the phase contrast microscope 20 using the plastic container 10, but the plastic container 10 is also used for imaging and observation using other microscopes such as a transmission microscope. Is preferred.

Claims (9)

1. The equivalent circle diameter of the opening is 0.5 mm or more and less than 2 cm, the depth is 2 mm or more and less than 2 cm, the inner side surface has water repellency, and the bottom surface has hydrophilicity at least two or more wells. Plastic container.
2. A water-repellent coat containing fluorine is provided on the inner surface of the well,
   The plastic container according to claim 1, wherein the fluorine content of the water-repellent coat is 0.01 mg / cm ² or more and less than 1.5 mg / cm ² .
3. The plastic container according to claim 1, wherein the inner surface of the well is provided with a diamond-like coating.
4. The plastic container according to claim 1, wherein the inner surface of the well has a contact angle with respect to pure water of 75 degrees or more and 100 degrees or less.
5. The plastic container according to claim 1, wherein the bottom surface of the well has a contact angle with respect to pure water of not less than 0 degrees and not more than 70 degrees.
6. The plastic container according to claim 1, wherein a difference between a contact angle of the inner surface of the well with pure water and a contact angle of the bottom surface of the well with pure water is 5 degrees or more and 110 degrees or less.
7. The plastic container according to claim 1, wherein the inner surface of the well is roughened.
8. The plastic container according to claim 1, wherein the inner surface of the well is subjected to a surface treatment by a vapor phase method.
9. The plastic container according to claim 1, which is made of polystyrene, polyethylene terephthalate, or polycarbonate.

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