JPWO2019163657A1 - Embedding agent - Google Patents

Abstract

Provided is an embedding agent having a low background when a specimen prepared by slicing from a frozen block is stained. An embedding agent for preparing frozen sections, the embedding agent can solve the problem with an embedding agent containing a frozen embedding agent and synthetic collagen.

Description

The present disclosure relates to embedding agents, and in particular to embedding agents containing synthetic collagen.

In recent years, in the medical field, it is required to collect living tissue during surgery and quickly inspect whether it is benign or malignant, and whether it is infiltrated into the excised margin or has lymph node metastasis. By performing the examination during the operation, the surgical procedure can be changed and the excision range can be appropriately determined during the operation, and the accuracy of the operation can be improved.

Examination of living tissue during surgery is generally performed by preparing a frozen tissue specimen and observing it under a microscope because it is excellent in rapidity and retention of antigenicity. Frozen tissue specimens may be described as "freezing block" in which the collected living tissue is embedded with a freezing embedding agent → frozen (hereinafter, the living tissue is embedded with a freezing embedding agent and frozen. ) → Thinly sliced → Dyed. As the freeze embedding agent, those using polymers such as polyvinyl alcohol (PVA) and polyethylene glycol (PEG) are known, and are commercially available as freeze embedding agents.

However, when sliced using a commercially available freeze-embedding agent, wrinkles, tears, etc. are likely to occur, and it may be difficult to obtain a clean thin film (section). In particular, in many hospitals and research institutes, O.D. C. T-compound is used, but the biological tissue is O.D. C. If it is not familiar with the T compound, there is a problem that it is difficult to slice it. Further, a frozen tissue specimen is prepared by adhering a sliced piece of biological tissue to a slide glass, but when a conventional freeze-embedding agent is used, there is a problem that it is easily peeled off from the slide glass.

The present inventors use gelatin as an embedding agent for sample preparation, which is in a liquid state at 15 ° C. to 25 ° C. and a solid state at 4 ° C. when made into an aqueous solution, or the gelatin is used as a known freeze embedding agent. It has been found that the above problem can be solved by adding the above-mentioned substance, and a patent application has been filed (see Patent Document 1).

International Publication No. 2015/199195

By the way, since gelatin is an animal protein, it is easily compatible with the biological tissue in which the specimen is prepared. In addition, gelatin is sticky. Therefore, the embedding agent made of gelatin described in Patent Document 1 or the embedding agent obtained by adding gelatin to the frozen embedding agent leaves gelatin that has entered the living tissue as a residue, so that the curable base material does not penetrate. When preparing a specimen, there is an advantage that the adhesion of living tissue to a support is improved. However, when the curable substrate non-penetrating sample is stained with hematoxylin, eosin (HE) or the like, gelatin remaining on the support as a residue is also stained. Therefore, when the stained sample is analyzed by image analysis software, there is a problem that the analysis accuracy is lowered because the stained gelatin is also analyzed.

The disclosure in the present specification has been made in order to solve the above-mentioned conventional problems, and as a result of diligent research, (1) when synthetic collagen is used as an additive to a known freeze-embedding agent, it freezes. The thinness of the block is improved, and (2) Synthetic collagen is a liquid at 0 ° C to room temperature, so it is easy to wash off even if the temperature of the water at the time of sample preparation is low, and the background when the biological tissue is stained. It was newly found that it is possible to reduce.

That is, an object of the disclosure in the present specification is to provide an embedding agent that can improve the thinness of a known frozen embedding agent and has a small background when a living tissue is stained.

The disclosure herein relates to the embedding agents shown below.

[1] An embedding agent for preparing a frozen section, the embedding agent is
Frozen embedding agent and
With synthetic collagen
Including embedding agent.
[2] Carboxylic acid for improving sliceability of frozen sections,
The embedding agent according to the above [1], further comprising.
[3] The synthetic collagen is represented by the following formula (1).
-(Gly-XY) n- (1)
The embedding agent according to the above [1] or [2].
[4] The synthetic collagen is represented by the following formula (2).
-(Pro-Z-Gly) n- (2)
The embedding agent according to the above [1] or [2].
[5] Z in the formula (2) is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp).
The embedding agent according to the above [4].

The embedding agent disclosed in the present specification can improve the sliceability of a frozen block prepared by using the embedding agent by adding synthetic collagen. In addition, since the synthetic collagen can be washed away with water after the biological tissue is attached on the support, the background at the time of staining is reduced.

FIG. 1 is a drawing substitute photograph, and FIG. 1A is a photograph of a frozen block prepared by using the embedding agents of Examples 1 and 2 and Comparative Example 1. In addition, FIG. 1B is a photograph of a frozen section obtained by slicing a frozen block. FIG. 2 is a drawing substitute photograph, and FIG. 2A is a photograph of a frozen block prepared by using the embedding agents of Examples 3 and 4 and Comparative Example 2. In addition, FIG. 2B is a photograph of a frozen section obtained by slicing a frozen block. FIG. 3 is a drawing substitute photograph, and FIG. 3A is a photograph of the HE-stained specimen prepared in Example 5. FIG. 3B is a photograph of the HE-stained specimen prepared in Comparative Example 3. FIG. 4 is a drawing substitute photograph, FIG. 4A is a photograph of the frozen block prepared in Example 6, and FIG. 4B is a photograph of a frozen section obtained by slicing the frozen block.

The embodiments of the embedding agent will be described in detail below.

The embedding agents disclosed herein include at least a frozen embedding agent and synthetic collagen. In addition, in this specification, "synthetic collagen" means non-animal-derived high molecular weight collagen synthesized using only amino acids produced by fermentation or synthesis.

It is said that it is very difficult to deodorize animal-derived collagen obtained by extracting from animals such as cows, pigs, and fish because it contains impurities such as odorous components. Therefore, the ratio of impurities and the like differs depending on the difference in raw materials, and it is difficult to homogenize the quality between products. On the other hand, since synthetic collagen is produced by synthesis, impurities such as odorous components are not mixed. In addition, by making the synthetic conditions constant, it is possible to obtain synthetic collagen having a desired molecular weight.

In addition, animal-derived materials may be contaminated with contaminants. Therefore, when a specimen is prepared using an embedding agent containing an animal-derived material, contaminants may also be stained during staining and become a background. On the other hand, since there is no risk of impurities being mixed in the synthetic collagen, it is possible to suppress the generation of background due to the embedding agent at the time of staining.

As described above, the synthetic collagen is not particularly limited as long as it is a non-animal-derived high molecular weight collagen synthesized using only amino acids produced by fermentation or synthesis. For example, the type 1 collagen molecule has a primary structure containing repetitions of the three amino acid residues represented by the formula (1). It is known that this polypeptide has the tertiary structure of a triple helix formed by gathering three of them in the same direction.
-(Gly-XY) n- (1)

In the above formula (1), X and Y are independently selected from the following amino acid residues.
Ala: L-alanine residue,
Arg: L-arginine residue Asn: L-asparagin residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue His: L-histidine residue Hyp: L-hydroxyproline residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L-proline residue Sar: Sarcosin residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue

As described above, X and Y are any combination, but it is preferable that X is Pro and Y is Hyp. The synthetic collagen represented by the formula (1) may be produced by a known production method. For example, it can be synthesized by subjecting a peptide oligomer containing a repeating unit of − (Gly—XY) − to a condensation reaction in dimethyl sulfoxide, an aqueous solvent containing ethylenediamine, or the like. When the condensation reaction is carried out in an aqueous solvent, the molecular weight can be adjusted by adjusting the phosphate ion concentration in the solvent (see JP-A-2017-8094). When the side chain of synthetic collagen has an amino group, it may be succinylated, phthalylated, or acetylated by a known method, if necessary. By eliminating the free amino group, synthetic collagen is less likely to clot.

Further, as synthetic collagen other than the formula (1), one consisting of repeating three amino acid residues represented by the following formula (2) is also known.
-(Pro-Z-Gly) n- (2)

In the above formula (2), Z is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp). That is, the synthetic collagen represented by the above formula (2) is
Poly (Pro-Pro-Gly), consisting of (Pro-Pro-Gly) units,
Poly (Pro-Hyp-Gly), consisting of (Pro-Hyp-Gly) units,
Poly (Pro-Pro or Hyp-Gly) in which the (Pro-Pro-Gly) unit and the (Pro-Hyp-Gly) unit are condensed in any order,
Aspects are included.

The number n of repetitions of the formulas (1) and (2) can be appropriately set according to the target molecular weight and the coexisting sequences. For example, the lower limit of the number of repetitions n is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, and the upper limit is 50,000 or less, 5000 or less, 1000 or less, 750 or less, 500 or less, 250 or less. And so on.

The synthetic collagen may be produced by a known method, or a commercially available one may be used. Examples of commercially available synthetic collagen include hybrid collagen (manufactured by Unix Co., Ltd.) and pure collagen (manufactured by JNC Corporation).

As the freeze embedding agent contained in the embedding agent disclosed in the present specification, a known freeze embedding agent may be used. For example, OCT compound (manufactured by Sakura Finetech), White Tish Coat (manufactured by UI Kasei), FSC22 (manufactured by Leica), Cliomount (low viscosity and high viscosity; manufactured by Muto Chemical Co., Ltd.), Cliomatrix (Thermo Fisher Scientific), SCAM (SECTION-LAB) and the like.

By adding synthetic collagen to the freeze-embedding agent, the sliceability of the frozen block can be improved. Further, after the frozen section is prepared from the frozen block, the synthetic collagen in the frozen section can be easily flushed with water. Therefore, the amount of synthetic collagen added to the freeze-embedding agent is not particularly limited as long as the desired slicing performance can be obtained and the freeze-embedding function of the freeze-embedded agent is not lost. Although not particularly limited, for example, about 0.001 to 1.0% by weight of synthetic collagen may be added to 100 parts by weight of the freeze-embedding agent.

By embedding a living tissue using the embedding agent disclosed in the present specification, freezing and slicing it, a "curable base material penetrating specimen" and a "curable base material non-penetrating specimen" can be prepared. .. A "curable substrate-penetrated specimen" is fixed with a known fixative such as formalin, paraformaldehyde, or glutaaldehyde in order to permanently fix the tissue on the specimen so that it will not be damaged even after long-term storage. This means a specimen obtained by permeating a curable base material such as paraffin, epoxy resin, methacrylate, or alkaline polyester into the inside of the tissue to cure the biological tissue after degreasing / dehydrating the living tissue. A paraffin specimen is a typical example. The specimen may be prepared by a known method.

The "curable base material non-penetrating specimen" means a specimen prepared without using the above-mentioned "curable base material". For example, a specimen obtained by slicing a biological tissue immobilized with the above-mentioned fixative or an unfixed biological tissue to have hardness using an embedding agent or the like and attaching a slice obtained to the support is obtained. Can be mentioned. A typical example is a frozen specimen.

As an example of performing a pathological examination using a non-penetrating specimen of a curable substrate, (1) washing the non-penetrating specimen of a curable substrate with water washes away the embedding agent adhering to the support, and (2) ) Staining by a known method such as a staining method such as HE staining or fat staining, or an immunostaining method such as an enzyme antibody method (DAB) or a fluorescent antibody method, and (3) observing under a microscope. In the procedure (1), when gelatin was used as an embedding agent (or a thin-slicing improving agent), there was a problem that gelatin that had adhered as a residue could not be washed with water and was stained. On the other hand, synthetic collagen is liquid at 0 ° C. to room temperature. Therefore, even if the temperature of water is low, the amount of synthetic collagen adhering as a residue on the support is reduced, and the background during staining can be lowered.

When preparing a "curable base material non-penetrating sample", a slice obtained by slicing may be attached to a support, and then the biological tissue may be fixed with a fixative. At that time, when an aldehyde-based fixative (for example, formalin) that crosslinks free amino groups was used as the fixative, gelatin was also fixed, and gelatin could not be washed away after the immobilization step. On the other hand, when synthetic collagen having no free amino group is used, it is not fixed with an aldehyde-based fixative. Therefore, if synthetic collagen having no free amino group is used, the synthetic collagen can be washed away after the immobilization step even when the biological tissue is immobilized with an aldehyde-based fixative in a state containing the synthetic collagen. The background of dyeing can be lowered.

In order to obtain synthetic collagen having no free transamination group, the free transamination group may be succinylated, phthalylated, or acetylated as described above. Alternatively, the synthetic collagen of the formula (2) may be used. When a fixative that does not crosslink free amino groups is used as the fixative, such as alcohol fixation, synthetic collagen having free amino groups may be used. Therefore, although synthetic collagen can be used regardless of the presence or absence of a free amino group, it is preferable to use synthetic collagen having no free amino group from the viewpoint of being compatible with various sample preparation methods.

Water-soluble substances may be added to the embedding agents disclosed herein, if necessary. For example, a water-soluble substance selected from the group consisting of sugars, salts, extracts, vitamins, pH adjusters, pigments, and surfactants can be mentioned.

More specifically, the water-soluble substance is described as a sugar (for example, agarose, sucrose, sorbitol, multitor, water candy, lactose, fructose, oligosaccharide, etc.); processed starch (for example, roasted dextrin, enzyme). Dextrin such as modified dextrin); Salts (eg, sodium chloride, calcium chloride, magnesium chloride, sodium sulfate, etc.); Extracts (eg, extracts derived from cows, pigs, shellfish, vegetables, etc.); Vitamin (eg, vitamin C) , Vitamin C sodium, vitamin B1 hydrochloride, etc.); pH adjusters (eg, citric acid, sodium citrate, sucrose, phthalic acid, hydrochloric acid, sulfuric acid, acetic acid, malic acid, tartrate, etc.); pigments (eg, tartaric acid, etc.) Red Nos. 2, 3, 102, 105, 106, Yellow Nos. 4, 5, Blue No. 2, etc.); Surfactants (eg, sucrose fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, propylene glyco -Lu fatty acid ester, etc.), and each may be used alone or in combination of two or more.

The support for preparing the specimen is not particularly limited as long as it can be observed under a microscope by attaching sliced pieces of biological tissue, and examples thereof include slide glass and a light-transmitting resin film.

The pathological examination using the prepared non-penetrating base material specimen is as described above. When pathological examination is performed using the prepared curable substrate penetrating specimen, a staining method such as HE staining, PAS staining, Alcian blue staining, or immunostaining such as enzyme antibody method (DAB) or fluorescent antibody method is performed. It may be stained by a known method such as a method and observed under a microscope.

Further, when the sample for sample preparation is adipose tissue, it is necessary to obtain good sliceability of the frozen block even when cooled to a temperature at which the adipose tissue freezes. In that case, carboxylic acid may be added to the embedding agent, if necessary. Examples of the carboxylic acid compound include monocarboxylic acid compounds such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid; dicarboxylic acid compounds such as oxalic acid, malonic acid, succinic acid, and glutaric acid; and tricarboxylic acids such as citric acid. Compounds; aromatic carboxylic acid compounds such as benzoic acid and phthalic acid; hydroxy acid compounds such as lactic acid and malic acid; carboxylic acid compounds substituted with halogen such as chloroacetic acid; and the like. The amount of the carboxylic acid compound added is not particularly limited as long as it improves the sliceability, and may be appropriately adjusted according to the type of embedding agent or carboxylic acid compound.

Hereinafter, embodiments of the embedding agent will be specifically described with reference to examples, but the embodiments are provided merely as a reference for specific embodiments of the embodiments. These examples do not represent limiting or limiting the scope of the invention.

[Preparation of embedding agent]
<Example 1>
As a freeze embedding agent, O.D. C. A T compound (manufactured by Sakura Finetech) was used, and 0.5% hybrid collagen (manufactured by Unix) was used as synthetic collagen. Then, a frozen embedding agent: synthetic collagen was mixed at a ratio of 9: 1 to prepare an embedding agent. The amount of synthetic collagen contained in 0.5% hybrid collagen is 0.5% by weight. Therefore, O. C. The amount of synthetic collagen added to the T compound is 9: 0.005, and the amount of synthetic collagen added to 100 parts by weight of the freeze-embedding agent is 0.056 weight.

<Example 2>
An acetic acid-added embedding agent was prepared by adding 30 μl of acetic acid (manufactured by Wako) to 1 g of the embedding agent prepared in Example 1.

<Example 3>
O. C. An embedding agent was prepared in the same procedure as in Example 1 except that White Tish Coat FL (manufactured by Ui Kasei Co., Ltd.) was used instead of T compound (manufactured by Sakura Finetech Co., Ltd.).

<Example 4>
An acetic acid-added embedding agent was prepared by adding 30 μl of acetic acid (manufactured by Wako) to 1 g of the embedding agent prepared in Example 3.

<Comparative example 1>
O. without the addition of synthetic collagen. C. Only the T compound was used as the embedding agent of Comparative Example 1.

<Comparative example 2>
Only White Tishcoat FL to which synthetic collagen was not added was used as the embedding agent of Comparative Example 2.

[Preparation of frozen sections]
Next, using the embedding agents of Examples 1 to 4 and Comparative Examples 1 and 2, frozen sections were prepared by the following procedure.
(1) In advance, a copper block (70 × 110 × 30 mm), a metal chuck for preparing frozen sections, and a metal weight are cooled at −50 ° C. in an aluminum block ultra-low temperature layer Micro Cool MC-100 (manufactured by TOMY). did.
(2) Human fat was used as a sample, and the prepared embedding agent was blended into the sample so as not to contain air bubbles.
(3) The hollowed-out bottom of Cliomold No. 3 (manufactured by Sakura Finetech Japan Co., Ltd.) was placed on a copper block, and the sample and embedding agent of (2) above were placed in Cliomold No. 3. Next, a metal chuck for preparing a frozen section was covered, a metal weight was further placed, and a Micro Cool lid was placed to prepare a frozen block of a sample and an embedding agent.
(4) The metal weight and the cryomold were removed, and the frozen block was sliced using a cryostat microtome (CM3050 manufactured by Leica) to obtain a frozen section having a thickness of about 5 μm.

FIG. 1A is a photograph of a frozen block prepared by using the embedding agents of Examples 1 and 2 and Comparative Example 1. In addition, FIG. 1B is a photograph of a frozen section obtained by slicing a frozen block.

FIG. 2A is a photograph of a frozen block prepared using the embedding agents of Examples 3 and 4 and Comparative Example 2. In addition, FIG. 2B is a photograph of a frozen section obtained by slicing a frozen block.

As is clear from the photographs of FIGS. 1B and 2B, the frozen sections prepared only with the freeze-embedding agents of Comparative Examples 1 and 2 had many omissions in the sample portion. On the other hand, the frozen sections prepared by using the embedding agents of Examples 1 to 4 to which synthetic collagen was added showed less sample omission than those of Comparative Examples 1 and 2. Furthermore, when synthetic collagen and acetic acid were added to the frozen embedding agent, the sample was less likely to come off and the frozen sections were less wrinkled. Based on the above results, the addition of synthetic collagen to the freeze-embedding agent improves the thinness of the frozen block, especially the thinness of the sample portion, and the addition of acetic acid further improves the thinness of the sample portion. It became clear that the sex was further improved.

[Comparison of stainability]
<Example 5>
Using mouse kidney as a sample and the embedding agent of Example 1 as an embedding agent, a frozen block was prepared with isopentane cooled in liquid nitrogen, and then sliced with a cryostat to obtain a frozen section. Next, staining was performed according to the following procedure.
(A) A curable base material non-penetrating sample was prepared by pressing and attaching a slide glass to the obtained frozen section.
(B) The obtained curable base material non-penetrating sample was rinsed with tap water to wash away the synthetic collagen on the slide glass. Then, HE staining was performed using hematoxylin and eosin (manufactured by Merck & Co., Inc.). FIG. 3A is a photograph of the HE-stained specimen prepared in Example 5.

<Comparative example 3>
As an embedding agent, MAX-F (manufactured by Nippi Co., Ltd.) aqueous solution (concentration: 5% by weight) of gelatin derived from fish and O.D. C. A HE-stained curable substrate non-penetrating specimen was prepared in the same procedure as in <Example 5> above, except that a mixture of T-compounds at a ratio of 1: 1 was used. FIG. 3B is a photograph of the HE-stained specimen prepared in Comparative Example 3.

As is clear from FIG. 3B, the HE-stained specimen prepared by using the embedding agent containing gelatin had the tissue periphery stained with HE. This is considered to be due to the effect of gelatin remaining without flowing in water when the curable base material non-penetrating specimen was rinsed with tap water. On the other hand, as is clear from FIG. 3A, in the HE-stained specimen prepared by using the embedding agent containing synthetic collagen, the background of HE-staining was not confirmed around the tissue.

Based on the above results, synthetic collagen, like gelatin, has the effect of improving sliceability, including the biological tissue portion, when preparing frozen sections from frozen blocks by adding it to a freeze-embedding agent. Became clear. On the other hand, when rinsing with tap water after a non-penetrating sample of a curable base material, synthetic collagen is much easier to flow than gelatin. As a result, it was clarified that the addition of synthetic collagen to the freeze-embedding agent has a remarkable effect of improving the sliceability of the frozen block and reducing the background due to staining.

[Oxalic acid-based embedding agent]
<Example 6>
9.5 g of White Tish Coat FL (manufactured by Ui Kasei Co., Ltd.) as a freeze embedding agent, 0.5 g of 0.5% hybrid collagen (manufactured by Unix Co., Ltd.) as synthetic collagen, 10% oxalic acid (Katayama Chemical Industry Co., Ltd.) An embedding agent was prepared by mixing 300 μl of (manufactured by the company). Next, a frozen section was prepared by using human adipose tissue as a living tissue and using the prepared embedding agent in the same procedure as in the above-mentioned [Preparation of frozen section]. FIG. 4A is a photograph of a frozen block, and FIG. 4B is a photograph of a frozen section obtained by slicing a frozen block. As is clear from FIG. 4B, even when oxalic acid was used, frozen sections having good sliceability of the biological sample portion were obtained. From the results of Examples 2, 4, and 6, frozen sections were frozen even when the temperature of the freezing block was lowered to the temperature at which the adipose tissue was frozen by further adding the carboxylic acid compound in addition to the freeze-embedding agent and synthetic collagen. It was confirmed that the sliceability of collagen can be improved.

The embedding agents disclosed herein reduce the background when the prepared specimens are stained. Therefore, it is useful not only for rapid pathological diagnosis during surgery, but also for preparation of curable base material non-penetrating specimens and curable base material penetrating specimens at medical institutions, research institutes such as university medical schools, general hospitals, and the like.

Claims (5)

An embedding agent for preparing frozen sections, the embedding agent is
Frozen embedding agent and
With synthetic collagen
Including embedding agent. Carboxylic acid to improve sliceability of frozen sections,
The embedding agent according to claim 1, further comprising. The synthetic collagen is represented by the following formula (1).
-(Gly-XY) n- (1)
The embedding agent according to claim 1 or 2. The synthetic collagen is represented by the following formula (2).
-(Pro-Z-Gly) n- (2)
The embedding agent according to claim 1 or 2. The embedding agent according to claim 4, wherein Z in the formula (2) is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp).