Efficient freezing slicing method suitable for large tissue and application of efficient freezing slicing method in MALDI-MSI imaging

Abstract

The invention discloses an efficient freezing slicing method suitable for large tissue and application of the efficient freezing slicing method in MALDI-MSI imaging, and the method comprises the following steps: pouring crushed dry ice into a first container, putting a second container into the first container, and filling a gap between the second container and the first container with the crushed dry ice; freezing liquid is poured into the second container, and the freezing liquid reaches a pre-cooled low-temperature state under the action of dry ice; pouring a pre-cooled CMC solution into the embedding container, putting the fresh tissue into the CMC solution, and enabling the CMC solution to submerge the fresh tissue; putting the embedding container into a freezing liquid for freezing treatment, so that the fresh tissues are preliminarily frozen and formed; then transferring into a refrigerator for storage; before slicing, the frozen and formed tissue is subjected to rewarming treatment and then is cut. According to the freezing method provided by the invention, the generation of ice crystals can be remarkably reduced, the slice quality is improved, and high-quality large-tissue frozen slices and accurate mass spectrum imaging are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of MALDI-MSI imaging, and particularly relates to a high-efficiency frozen section method suitable for large tissues and application thereof in MALDI-MSI imaging. BACKGROUND

[0002] Since the birth of frozen tissue section technology, it has played an irreplaceable role in the field of medical diagnosis and research. In 1818, the discovery of Dutch pathologist Pieter van der Riet that low-temperature rapid freezing of fresh biological tissues can make them harden for sectioning and reduce mechanical damage laid a solid theoretical foundation for this technology. In 1905, American pathologist Lewis Wilson completed the first frozen section diagnosis, marking the formal application of this technology in clinical pathological diagnosis, especially intraoperative rapid pathological diagnosis, which provided a key means for doctors to quickly judge the condition during surgery. In 1938, Danish pathologists Lindstrom Lang and Gensen invented the first frozen section machine, which realized precise control of sectioning at low temperature and greatly improved the sectioning quality and efficiency, becoming an important milestone in the development process. Since then, with the coordinated progress of microscopes and low-temperature technology in the 1950s-1960s, frozen section machine technology has been continuously innovated. In the 1970s, the application range of frozen section technology was further expanded, and the automation and control capability of the sectioning machine was also significantly improved. In the 21st century, the frozen section machine has made breakthrough progress in automation, sectioning quality and other key dimensions, such as intelligent control system can accurately set sectioning parameters, new sectioning knives and advanced refrigeration technology reduce the damage of ice crystals to tissues.

[0003] However, the existing frozen tissue section technology still has many bottlenecks in the freezing of large tissues. Due to its large volume and complex structure, large tissues are difficult to achieve uniform heat transfer during freezing, and large ice crystals are easily formed. These ice crystals can cause physical damage to cells and tissues, destroy tissue structure and cell function, and cause the tissue to lose its original physiological activity after thawing. Although existing cryoprotective agents can alleviate the problem of ice crystal damage to some extent, for large tissues, their penetration and distribution are difficult to be uniform, and some cryoprotective agents will be toxic to tissue cells at high concentrations, seriously affecting the subsequent application of the tissue. In addition, the freezing of large tissues has high requirements for equipment, and most existing freezing equipment cannot accurately control the complex cooling and heating rate curves required for large tissue freezing, and the operation process is complicated and prone to errors, which seriously restricts the development and application of large tissue freezing technology. Therefore, it is urgent to develop an innovative frozen tissue section method. SUMMARY

[0004] The application aims to provide a high-efficiency frozen section method suitable for large tissues and its application in MALDI-MSI imaging.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: A high-efficiency frozen section method suitable for large tissues, comprising the following steps: (1) pour crushed dry ice into a first container, then put a second container into the first container, and fill the gap between the second container and the first container with crushed dry ice; pour a freezing liquid into the second container, and the freezing liquid reaches a pre-cooled low-temperature state under the action of the crushed dry ice; (2) pour a pre-cooled CMC solution into an embedding container, put a fresh tissue into the CMC solution so that the CMC solution completely submerges the fresh tissue; put the embedding container into the freezing liquid pre-cooled in step (1) for freezing treatment, so that the fresh tissue is preliminarily frozen into a shape; then transfer to a low-temperature refrigerator at-80℃ for long-term storage; preferably, the embedding container is an embedding box, and tin foil paper is laid in the embedding box; the embedding container can be selected; the fresh tissue is a pig brain tissue or a human brain tissue.

[0006] (3) before sectioning, the frozen tissue is subjected to rewarming treatment, and then sectioning is performed; specifically, the frozen tissue is subjected to rewarming treatment for 2-3 hours by using a Leica frozen section machine CM3600XP, and then large tissue sectioning operation is performed by using a sectioning machine; As a preferred technical scheme, the first container is a foam box. The foam box has good heat preservation and insulation effect, and can keep the dry ice in the interior at a low-temperature environment for a long time; the second container is a stainless steel barrel. The stainless steel barrel has good heat transfer effect, and can effectively transfer the low temperature of the crushed dry ice to the freezing liquid.

[0007] As a preferred technical scheme, the freezing liquid is ethanol or ethylene glycol. The use of ethanol or ethylene glycol as the freezing liquid has low toxicity and reduces the harm to the operator.

[0008] As a preferred technical scheme, the CMC solution is prepared in water by using a sodium carboxymethyl cellulose solution. Further preferably, the mass ratio of the sodium carboxymethyl cellulose to water is 1:10.

[0009] The application also provides application of the high-efficiency frozen section method to MALDI-MSI imaging.

[0010] The application has the following beneficial effects: The method provided by the application can prevent the problem of tissue damage caused by air bubbles in the sectioning process by embedding the tissue with a CMC solution to discharge air contained in the tissue, and can shorten the freezing time and avoid the problem of ice crystals in the tissue caused by the conventional cryopreservation method by using a liquid freezing solution to preliminarily freeze the tissue into a shape. The application effectively solves the problem of uneven heat transfer in the freezing process of a large tissue by a unique freezing method, and realizes uniform freezing of the large tissue. The sectioning effect is significantly improved, and there is no crack or air bubble, so that the structure of the tissue can be completely preserved, thereby providing a high-quality sample for subsequent accurate mass spectrometry imaging.

[0011] The method provided by the application is simple to operate and has superior performance: the method has the characteristics of being fast and simple, does not require complex pretreatment steps, has a low detection limit, and has high sensitivity, and can intuitively and clearly present the spatial distribution of a substance in a large tissue, thereby providing strong technical support for medical research and diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The photos show the state of the tissue taken out after the pig brain tissue is frozen for 50 minutes and the effect of the trimming on the sectioning machine; Figure 2 The photos show the sectioning effect and imaging effect of the cortex region and the cerebellum region of the parasagittal pig brain; DETAILED DESCRIPTION The application will be further described below in conjunction with examples, so that those skilled in the art can better understand the application and implement it. However, the examples are not intended to limit the application. In addition, in the preparation process of the following examples, if not otherwise specified, the conventional means in the prior art are used, and thus, they will not be described in detail.

[0013] EMBODIMENT A high-efficiency frozen section method suitable for a large tissue, comprising the following steps: (1) a foam box is selected as a first container, a stainless steel barrel is selected as a second container, crushed dry ice is poured into the first container, then the second container is placed into the first container, and the gap between the second container and the first container is filled with the crushed dry ice; a freezing solution is poured into the second container, and the freezing solution is anhydrous ethanol, which is cooled to a low temperature state under the action of the crushed dry ice; (2) Prepare a CMC solution by mixing sodium carboxymethyl cellulose and water at a mass ratio of 1:10 and pre-cool it. Line the embedding cassette with tin foil and pour in the pre-cooled CMC solution. Place the fresh tissue into the CMC solution so that the CMC solution completely submerges the fresh tissue. Place the embedding cassette into a pre-cooled cryogenic solution for freezing treatment to pre-freeze and shape the fresh tissue. Then transfer it to a low-temperature freezer at -80°C for long-term storage. (3) Before slicing, the frozen tissue is thawed and then sliced. Specifically, a Leica CM3600XP cryostat can be used to thaw the frozen tissue for 2 hours, and then the large tissue is sliced ​​using the statostat. Taking large tissues of pig brain as an example, Figure 1 The image shows the state of the pig brain tissue after freezing for 50 minutes (left image) and the result after trimming on a microtome (right image). It can be seen that the freezing effect is very good and the section quality is high.

[0014] The fixation of large porcine brain tissue sections was validated using a spray test on a DHB matrix. Section imaging was performed on the sagittal cortical region and the cerebellar region of the porcine brain, respectively. Figure 2 As shown. Figure 2 Image A shows the sagittal cortical section and MALDI-MSI imaging results, while Image B shows the sagittal cerebellar section and MALDI-MSI imaging results. The imaging results clearly demonstrate that the freezing method of this invention not only meets the needs of large tissue freezing but also achieves a high standard in section quality, fully verifying the effectiveness and practicality of the method.

[0015] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for efficient cryosectioning of large tissue samples, comprising, The method comprises the following steps: (1) pouring crushed dry ice into a first container, then placing a second container into the first container, and filling the gap between the second container and the first container with crushed dry ice; pouring a freezing solution into the second container, and under the action of the crushed dry ice, the freezing solution reaches a low-temperature state of pre-cooling; (2) pouring a pre-cooled CMC solution into an embedding container, placing the fresh tissue into the CMC solution so that the CMC solution completely submerges the fresh tissue; placing the embedding container into the freezing solution pre-cooled in step (1) for freezing treatment, so that the fresh tissue is preliminarily frozen into a shape; then transferring to a refrigerator for long-term storage; (3) before slicing, the frozen tissue is subjected to rewarming treatment, and then slicing is performed.

2. The method of high efficiency cryosectioning of large tissues of claim 1, wherein, The first container is a foam box.

3. The method of high efficiency cryosectioning of large tissues of claim 1, wherein, The second container is a stainless steel barrel.

4. The method of high efficiency cryosectioning of large tissues of claim 1, wherein, The freezing solution is ethanol or ethylene glycol.

5. The method of high efficiency cryosectioning of large tissues of claim 1, wherein, The embedding container is lined with tin foil paper.

6. The method of high efficiency cryosectioning of large tissues of claim 1, wherein, The CMC solution is prepared in water.

7. The method of high efficiency cryosectioning of large tissues of claim 6, wherein, The mass fraction of sodium carboxymethyl cellulose in the CMC solution is 2-10%.

8. The method of high efficiency cryosectioning of large tissues of claim 1, wherein, The temperature of the refrigerator is -80℃.

9. A method for efficient cryosectioning of large tissues according to any one of claims 1 to 8, wherein, The fresh tissue is pig brain tissue or human brain tissue.

10. Use of the high-efficiency frozen section method according to any one of claims 1 to 9 in MALDI-MSI imaging.

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