JP7092294B2 - A material for coating a biological tissue made of an ultra-thin film, and a biological tissue coated with the material. - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Advanced Materials (2017), Vol. 29, DOI: 10.1002 / adma, published on August 11, 2017. 201703139

The present invention relates to a material for coating a biological tissue made of an ultrathin film and a biological tissue coated with the material.

Imaging technology using a microscope is constantly evolving, and it is an indispensable observation method for live visualization of life phenomena and obtaining raw information. As the development of two-photon excitation microscope, total internal reflection fluorescence microscope, and super-resolution microscope is mentioned as an example, the development of the hard surface (microscope body and observation accuracy) is remarkable. Recently, reagents (Non-Patent Documents 1 and 2) that make biological tissues such as organs transparent have been developed, enabling deep imaging of specific proteins in biological tissues, and imaging whole biological tissues that could not be done until now. Needs are increasing rapidly.
On the other hand, in the preparation of the observation sample (soft surface), the biological tissue to be observed is placed on a glass substrate, a buffer solution for preventing drying is dropped, and then covered with a cover glass or embedded with a hydrogel such as agarose. Is a conventional method (Non-Patent Document 3).

H. Hama, et al., Nat. Neurosci., 14, 1481 (2011) H. Hama, et al., Nat. Neurosci., 18, 1518 (2015) A. M. Glauert, Fixation, Dehydration and Embedding of Biological Specimens, Elsevier Science, Amsterdam, North Holland, 73-110 (1975)

In microscopic observation, when the biological tissue to be observed is covered with a cover glass, the biological tissue may be destroyed or the biological tissue may be shaken by the inertial force that moves the stage. Further, when the living tissue is embedded with a hydrogel such as agarose, the gel gets into the space between the glass substrate and the living tissue, and a high-precision and high-resolution image cannot be obtained deep into the gel. Furthermore, in the latter case, even if the living tissue is made transparent, the hydrogel or buffer solution invades the tissue and returns to the original opaque tissue, so that the hydrogel cannot be used when observing the transparent tissue. There is. As described above, in the prior art, it is not possible to prevent the living tissue from drying out and shaking during observation, and it is not possible to observe the living tissue deep into the living tissue with high accuracy and high resolution for a long time.
The present invention has been made in view of such a situation, and an object of the present invention is to provide a material for coating a living tissue made of an ultrathin film that can be produced by a simple method, and to provide a living tissue covered with the material.

As a result of diligent studies to solve the above problems, the present inventors can prevent the living tissue from drying and shaking during observation by covering the living tissue with an ultrathin film manufactured from a water-repellent resin. I found. Further, as the water-repellent resin, a resin having a refractive index substantially the same as that of water may be used in order to realize high-precision and high-resolution observation up to the deep part of the living tissue in microscopic observation. I found it. The present invention is as follows.

[1] A material for coating a biological tissue, which comprises an ultrathin film containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less.
[2] The material according to [1], wherein the water-repellent resin is a perfluoro (1-butenyl vinyl ether) polymer.
[3] A biological tissue coated with the material according to [1] or [2].
[4] The biological tissue according to [3], wherein the biological tissue is transparent.
[5] A biological tissue embedding body in which the biological tissue according to [4] is further coated with hydrogel.
[6] The material according to [1] or [2], which is stretched on a frame.
[7] A biological tissue covering kit containing the material according to [1] or [2] and a base material.
[8] The kit according to [7], further comprising a biological tissue not coated with the material.
[9] The kit according to [7] or [8], wherein the base material is a glass substrate.

According to the present invention, it is possible to provide a material for coating a biological tissue made of an ultrathin film that can be produced by a simple method, and to provide a biological tissue coated with the material. By covering the living tissue with the living tissue covering material, it is possible to prevent the living tissue from drying and shaking during observation, and it is possible to observe the living tissue deeply with high accuracy and high resolution for a long time.
That is, the material for coating a living tissue made of an ultra-thin film of the present invention suppresses changes due to drying of the living tissue to be observed, and also causes positional deviation or blurring of the living tissue to be observed due to the movement of each part of the microscope during observation. Since it is possible to hold down the tissue, it is possible to perform accurate observation from the tissue surface to the deep part for a longer time than before. In addition, the biological tissue coated with the biological tissue covering material is not only used in advanced life research for elucidation of biological phenomena, but also for specialized inspection and analysis of biological tissue collected at medical institutions such as hospitals. Even when observing and evaluating after transporting to an institution, the state at the time of collection can be maintained for at least 24 hours, which enables highly accurate inspection and analysis, and only doctors who make a diagnosis based on the inspection results can do so. However, it is also meaningful for patients.

It is a figure which shows the flow of the manufacturing example 1 of an ultrathin film. It is an image showing the water repellency of the ultrathin film produced in Production Example 1 (photograph substitute for drawing). It is a graph which shows the relationship between the concentration of a water-repellent resin and the film thickness of an ultrathin film in Production Example 1. It is a figure which shows the transmittance of the ultraviolet-visible region (200-800 nm) of the ultra-thin film produced in Production Example 1. It is a figure which shows the production example of the hydrogel coated with the ultra-thin film which concerns on Example 1. FIG. It is a graph which shows the relationship between the time after coating of a hydrogel by the ultra-thin film which concerns on Example 1 and the water retention rate of a hydrogel. It is the result of Example 1 and Comparative Example 1-1. It is an image of the hydrogel 24 hours after covering the hydrogel with an ultra-thin film (photograph substitute for drawing). (i) is when not coated, (ii) is when coated with a film thickness of 43 nm, (iii) is when coated with a film thickness of 133 nm, and (iv) is when coated with a film thickness of 294 nm. .. It is a visible light image and a fluorescent image of the biological tissue of Example 2-1, Example 2-2, Comparative Example 2-1 and Comparative Example 2-2 (photograph substitute for drawing). It is a confocal microscope image of the living tissue of Example 2-1, Example 2-2, and Comparative Example 2-2 (photograph substitute for drawing).

The present invention is a living tissue covering material composed of an ultrathin film containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less.
As used herein, the term "coating" refers to an ultrathin film covering all or part of a living tissue.

The ultra-thin film according to the present invention is a self-supporting ultra-thin film whose thickness is controlled to the nano-order (a state that does not require the support of a base material), exhibits high adhesiveness peculiar to nano-thickness, and is a reactive functional group. It can be attached to various interfaces (glass, plastics, biological tissues, etc.) only by physical adsorption without using adhesives.
The ultrathin film according to the present invention contains a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less, whereby the coated biological tissue can be prevented from drying and shaking during observation. It will be possible to observe living tissue deeply with high accuracy and high resolution.
The total content of the water-repellent resin in the ultrathin film according to the present invention is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more.

The resin is insoluble in a culture solution or a buffer solution used when observing a living tissue, and is preferably insoluble in a liquid medium for dissolving a sacrificial layer at the time of production, as will be described later. Examples of the liquid medium include an aqueous medium. Further, the resin does not affect the living tissue, and is preferably not, for example, a resin that gives a biological stimulus or a resin that is toxic to the living tissue.
The water repellency of the resin can be evaluated by the water contact angle, and can be measured according to a conventional method using, for example, a contact angle meter. The water repellency of the resin is such that the water contact angle measured with a contact angle meter is usually 90 ° or more, preferably 95 ° or more, more preferably 100 ° or more, while it is usually 130 ° or less, preferably 130 ° or less. It is 125 ° or less, more preferably 120 ° or less.
The resin is preferably a perfluoro (1-butenyl vinyl ether) polymer, and CYTOP (registered trademark) manufactured by AGC Asahi Glass Co., Ltd. is distributed as a product.

The film thickness of the ultrathin film according to the present invention is usually 20 nm or more, preferably 30 nm or more, more preferably 40 nm or more, while it is usually 200 nm or less, preferably 180 nm or less, more preferably 150 nm or less. The "ultra-thin film" according to the present invention refers to a thin film within the film thickness range.
When the film thickness is 20 nm or more, the ultra-thin film is easy to handle, while when the film thickness is 200 nm or less, the adhesiveness of the ultra-thin film is improved.
The film thickness can be appropriately adjusted from the conditions at the time of film formation. For example, it can be appropriately adjusted by adjusting conditions such as a solvent for dissolving the water-repellent resin, the concentration of the water-repellent resin, the number of rotations and the rotation time when forming a film by spin coating. Examples of the solvent for dissolving the water-repellent resin include perfluorotributylamine (PFTBA) and the like.
The film thickness can be measured by a known method and is not particularly limited. For example, a method of scraping a part of the surface of an ultrathin film manufactured on a silicon wafer with tweezers to expose the silicon wafer and measuring using a stylus type step meter can be mentioned.

The ultrathin film according to the present invention is used to coat a living tissue. The living tissue includes any tissue collected from the living body, for example, skin, muscle, bone, adipose tissue, cerebral nervous system, sensory system, circulatory system such as heart and blood vessel, lung, liver, spleen, pancreas, kidney. , Gastrointestinal system, thoracic gland, lymph, etc., as well as their cultures. The living tissue may contain body fluids such as blood (eg, whole blood, serum, plasma), lymph, saliva, urine, ascites, and sputum. Of these, biological tissues such as the cranial nerve system, sensory organ system, circulatory system, bone, and muscle, which often utilize fluorescent imaging using a fluorescent dye or the like, are preferable.

Next, an example of manufacturing the ultrathin film according to the present invention will be described, but the present invention is not limited thereto.
For example, first, a polymer film serving as a sacrificial layer is developed on a substrate having a smooth surface, and a layer containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less is developed on the polymer film.
Examples of the material of the base material include carbon materials such as silicon, silicon rubber, silica, glass, mica and graphite, polymer materials such as polyethylene, polypropylene, cellophane and elastomer, and calcium compounds such as apatite. The preferred material is silicon, and the preferred substrate is a silicon wafer. Commercially available products can be used in either case.

As the polymer to be the sacrificial layer, as will be described later, after developing the layer containing the water-repellent resin on the polymer, the sacrificial layer is dissolved by immersing it in a liquid medium, so that the polymer is soluble in the liquid medium at that time. If there is, there is no particular limitation. For example, when the liquid medium is an aqueous medium, polymer electrolytes such as polyacrylic acid, polymethacrylic acid and polystyrene sulfonic acid; polyethylene glycol, polyacrylamide, polyvinyl alcohol (PVA); polysaccharides such as starch and cellulose acetate, etc. Examples include nonionic water-soluble polymers. Commercially available products can be used in either case.
There are no particular restrictions on the method of developing the polymer film as the sacrificial layer and the method of developing the layer containing the water-repellent resin on the polymer film, and for example, a conventional method such as spin coating can be followed. Further, the film thickness can be appropriately adjusted from the conditions at the time of film formation. For example, it can be appropriately adjusted by adjusting conditions such as the concentration and the number of rotations and the rotation time when the film is formed by spin coating.

After developing the layer containing the water-repellent resin, the sacrificial layer is dissolved and only the layer containing the water-repellent resin is obtained by immersing the layer in a liquid medium in which only the sacrificial layer is dissolved. The layer containing the water-repellent resin is the ultrathin film according to the present invention. At this time, the immersion in the liquid medium may be performed before or after separating both films from the substrate.
As the liquid medium, for example, an aqueous medium is preferable, and examples thereof include water, distilled water, water in which a salt is dissolved, water in which a surfactant is dissolved, and a buffer solution. When the sacrificial layer is PVA, water or distilled water is preferable.

The obtained ultrathin film can be stored in the above-mentioned liquid medium, and can also be dried and stored. The drying method is not particularly limited, and examples thereof include natural drying, freeze drying, and vacuum drying, all of which can be carried out by a conventional method. As a further specific example, it can be attached to a silicon wafer and stored by drying in a desiccator.

The ultra-thin film according to the present invention may also include an embodiment in which it is stretched on a frame. For the explanation of the ultra-thin film, the above-mentioned explanation is used.
If the ultra-thin film according to the present invention is stretched on the frame, the frame can be grasped with tweezers or the like, so that it is not necessary to directly touch the ultra-thin film, and the living tissue can be obtained without damaging the ultra-thin film. Can be covered. Further, the ultrathin film according to the present invention may be in a state of being stretched without being twisted or twisted, but a state of being stretched without being twisted is preferable.
Further, it is preferable that the frame is provided with a grip handle. This is because the biological tissue can be covered with an ultra-thin film by grasping the grip handle by hand without using tweezers or the like.

The shape of the frame is not particularly limited, and examples thereof include a circle, a substantially circular shape, a square shape, a substantially square shape, and the like, and can be appropriately set based on the shape and size of the biological tissue to be covered.
The size of the frame is not particularly limited, but can be appropriately set based on the size of the biological tissue to be covered.
The material of the frame is not particularly limited as long as the properties of the ultrathin film stretched on the frame are not changed. For example, metal and plastic can be mentioned, and in the case of metal, aluminum, iron, copper, brass, stainless steel and the like can be mentioned.

The method of stretching the ultrathin film according to the present invention on the frame is not particularly limited, and examples thereof include a method of directly scooping and stretching the ultrathin film obtained by immersing it in a liquid medium in which the sacrificial layer dissolves. .. Further, the frame and the engaging ring to be fitted to the outer peripheral surface of the frame may be prepared, and the ultrathin film may be stretched between the frame and the engaging ring.

Another embodiment of the present invention is a biological tissue coated with a biological tissue covering material composed of the ultrathin film. For the explanations regarding ultra-thin films and biological tissues, the above-mentioned explanations are used.
The method of covering the living tissue with the ultra-thin film is not particularly limited, but the ultra-thin film is floated on a medium or a buffer solution, the living tissue is placed on the ultra-thin film, and a force is applied from above with a cover glass or a slide glass. It is preferable to submerge the living tissue in a medium or the like so that the living tissue is covered with the ultra-thin film together with the cover glass because air does not enter between the ultra-thin film and the living tissue.

The biological tissue coated with the ultra-thin film is preferably a transparent biological tissue, and is preferably made transparent before being coated with the ultra-thin film. The method of transparency is not limited, and examples thereof include the Scale method and the CUBIC method.

It is preferable that the biological tissue coated with the ultrathin film is further coated with hydrogel so as to be coated in the order of ultrathin film and hydrogel from the viewpoint of the biological tissue. This makes it possible to prevent drying for a longer period of time.
In addition, when the living tissue is transparent, if it is coated with hydrogel without being covered with an ultrathin film, the hydrogel permeates the living tissue and the living tissue returns to the original opaque living tissue. However, when viewed from the biological tissue, when the biological tissue is coated in the order of ultrathin film and hydrogel, the biological tissue is originally opaque because the liquid medium in the hydrogel or hydrogel does not penetrate into the biological tissue due to the presence of the ultrathin film. It will be possible to observe the living tissue for a long time with high accuracy and high resolution up to the deep part without returning to the living tissue.

The hydrogel is not particularly limited as long as it is used for microscopic observation of living tissue. Agarose gel is preferable, but examples of the polymer as a raw material thereof include alginic acid, collagen, hyaluronic acid, chitosan, gelatin and the like.
The method of further coating the biological tissue with hydrogel is not particularly limited. For example, there is a method of coating (embedding) the biological tissue coated with the ultrathin film with hydrogel in the same manner as the conventional method of coating (embedding) the biological tissue not coated with the ultrathin film with hydrogel. Specifically, a biological tissue coated with an ultrathin film on a cover glass, a slide glass, or the like is placed on the bottom surface of the culture dish, and the biological tissue is further subjected to a hydrogel having a temperature that does not affect the cells in the biological tissue. Examples include a method of covering (embedding).

Another embodiment of the present invention is a biological tissue covering kit including the living tissue covering material composed of the ultrathin film and a base material. For the explanations regarding ultra-thin films and biological tissues, the above-mentioned explanations are used.
Further, the kit according to the present embodiment may further include a biological tissue not covered with the biological tissue covering material composed of the ultrathin film.
The base material is not particularly limited as long as it can come into contact with the biological tissue before coating and can be coated with the ultrathin film together with the biological tissue. For example, a glass substrate, a plastic substrate and the like can be mentioned.
The shape and size of the base material are not particularly limited, and can be appropriately set based on the shape and size of the biological tissue to be coated, the shape and size of the ultrathin film, and the like.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

[Manufacturing Example 1]
An ultrathin film was produced by the flow shown in FIG. A case where a polymethylmethacrylate resin (PMMA, Mw: 120 kDa, manufactured by Sigma-Aldrich) was used instead of the perfluoro (1-butenyl vinyl ether) polymer was used as a comparative production example.
First, a PVA aqueous solution (10 mg / mL, Mw: 22 kDa, manufactured by Kanto Chemical Co., Inc.) was spin-coated on a silicon (SiO ₂ ) substrate at 4000 rpm for 20 seconds. Next, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, or 90 mg / mL perfluoro (1-butenyl vinyl ether) polymer (CYTOP® manufactured by AGC Asahi Glass Co., Ltd.) )) Solution (solvent: perfluorotributylamine (PFTBA), manufactured by AGC Asahi Glass Co., Ltd., CT-Solv.180) was spin-coated under the same conditions. Then, the entire substrate was immersed in pure water to rapidly dissolve the PVA sacrificial layer, and a transparent and smooth ultrathin film maintaining the shape of the substrate was obtained.

The water contact angle of the surface of the ultrathin film produced at a concentration of 40 mg / mL of perfluoro (1-butenyl vinyl ether) polymer was 111 ± 1 °, confirming that it was water repellent (Fig. 2). It was also confirmed that the film thickness of the ultrathin film increases depending on the concentration of the perfluoro (1-butenyl vinyl ether) polymer solution at the time of spin coating, and the film thickness can be arbitrarily controlled (about 18 to 687 nm). (Fig. 3).
In addition, when the transmittance of the ultra-thin film was measured with a spectrophotometer, no light absorption was observed in the ultraviolet / visible region (200 to 800 nm) (Fig. 4), and it was confirmed that the ultra-thin film showed transparency. did it.

(Drying prevention effect)
[Example 1]
A biological tissue model coated with an ultra-thin film was produced by the flow shown in FIG. In the production of ultrathin films, the concentration of perfluoro (1-butenyl vinyl ether) polymer (solvent: PFTBA, manufactured by AGC Asahi Glass Co., Ltd., CT-Solv.180) is 10 mg / mL, 20 mg / mL, 40 mg /. The concentration was adjusted to mL, 60 mg / mL, or 90 mg / mL, and spin-coated at 4000 rpm for 20 seconds in the same manner as in Production Example 1.
A small amount of blue dextran (Mw: 2000 kDa, manufactured by Sigma-Aldrich) was added to an aqueous solution of sodium alginate (20 mg / mL, manufactured by Kanto Chemical Co., Inc.), and an aqueous solution of calcium chloride (20 mg / mL) was added for gelation. (Room temperature, 12 h). The obtained hydrogel was cut into columns (diameter: 10 mm, thickness: 5 mm) and used as a biological tissue model.
The produced ultra-thin film is floated on distilled water, a hydrogel made of alginic acid is placed on it, and force is applied from above with a cover glass so that the hydrogel is covered with the ultra-thin film together with the cover glass and submerged in distilled water. , A biological tissue model coated with an ultra-thin film was prepared. This was allowed to stand under constant temperature and humidity (room temperature, RH: 40%), and the change in weight over time was measured with an electronic balance and converted into a water retention rate to examine the effect of preventing drying.

[Comparative Example 1-1]
The same procedure as in Example 1 was carried out except that the hydrogel made of alginic acid was not coated with an ultrathin film.

[Comparative Example 1-2]
The same procedure as in Example 1 was carried out except that a polymethylmethacrylate resin (PMMA) having a concentration of 60 mg / mL or 90 mg / mL was used instead of the perfluoro (1-butenyl vinyl ether) polymer. Chloroform (manufactured by Wako Pure Chemical Industries, Ltd., 035-02616) was used as the solvent.

[result]
In Example 1, as the film thickness increased, the gel could be remarkably suppressed from drying, and a clear effect of preventing drying was observed (except for the control of “CYTOP” in FIG. 6 and the film thickness of 18 nm). Images 24 hours after covering are shown in FIGS. 7 (ii), (iii), and (iv).
In Comparative Example 1-1, the gel gradually shrank with the evaporation of water, and after 10 hours, it was completely dried (control of "CYTOP" in FIG. 6, FIG. 7 (i)).
The anti-drying effect could not be confirmed in the ultra-thin film (water contact angle: 68 ± 1 °) made of PMMA with high transparency in Comparative Example 1-2 (“PMMA” in FIG. 6).

(Biological tissue imaging)
[Example 2-1]
A living tissue that was made transparent and covered with an ultra-thin film was prepared. The ultrathin film was produced in the same manner as in Example 1. Brain section (thickness: 250 μm) of a genetically modified mouse (Thy1-YFP-H) (source: Institute of Electronic Chemistry, Hokkaido University) in which some nerve cells were labeled with yellow fluorescent protein (EYFP) as a living tissue. ), Using ScaleS (prepared according to Non-Patent Document 2) as a clearing reagent, and clearing according to the manual. The clarified brain section was covered with an ultrathin film in the same manner as in Example 1 and observed under a microscope (FIG. 8). In confocal microscope observation (objective lens 60x, numerical aperture 1.49, oil immersion), the thickness of the brain section is 1 mm, the z direction is 40 to 44 μm at 1 μm intervals, and the x and y directions are 761 ×. A 756 μm tying shot (4 x 4 shots) was taken. It took about 2 hours to shoot this wide field of view as before. The confocal microscope image is shown in FIG.

[Example 2-2]
The transparent, ultrathin film-coated biological tissue produced in Example 2-1 was further coated with an agarose gel (2.5 wt%, dissolved in phosphate buffer (pH 7.4)) and observed under a microscope (Fig.). 8). The confocal microscope image is shown in FIG.

[Comparative Example 2-1]
Brain sections that did not clear the living tissue and were not covered with an ultrathin film were observed under a microscope (Fig. 8).

[Comparative Example 2-2]
The living tissue was made transparent in the same manner as in Example 2-1 but the brain section not covered with the ultrathin film was observed under a microscope (FIG. 8). The confocal microscope image is shown in FIG.

[Comparative Example 2-3]
The living tissue was made transparent in the same manner as in Example 2-1 but the brain section coated with agarose gel (2.5 wt%) in the same manner as in Example 2-2 without being coated with an ultrathin film was observed under a microscope. (Fig. 8).

[result]
In Comparative Example 2-1 it was opaque even under visible light irradiation and fluorescence irradiation.
In Comparative Example 2-2, the transparency of the living tissue was maintained, but the living tissue became dry after about 2 hours. In addition, confocal microscopy also showed unclear artifacts in which the nerve cell bodies and axons were fully extended in the z-direction, although they were in focus in the x- and y-direction imaging. This is due to the dryness of the brain section during imaging.
In Comparative Example 2-3, the living tissue was made transparent, but when coated with agarose, the buffer solution permeated the living tissue and returned to the original opaque living tissue.
In Example 2-1 the transparency of the living tissue was maintained, and the living tissue could be observed without drying even after 2-3 hours had passed since the coating. Also, by observing with a confocal microscope, clear images could be obtained in all directions of x, y, and z.
Even in Example 2-2, the transparency of the living tissue is maintained, and the living tissue can be observed without drying even after at least 12 hours have passed since the coating, which is longer than that of Example 2-1. there were.

Claims (5)

A material for coating a biological tissue stretched between a frame and an engaging ring that fits on the outer peripheral surface of the frame, which is made of an ultrathin film containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less. The material according to claim 1, wherein the water-repellent resin is a perfluoro (1-butenyl vinyl ether) polymer. A biological tissue covering kit comprising the material according to claim 1 or 2 and a base material. The kit of claim 3 , further comprising a biological tissue not coated with the material. The kit according to claim 3 or 4 , wherein the substrate is a glass substrate.

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