Improved method for cavity organ tissue cryoembedding

Abstract

This invention relates to the field of tissue processing technology and discloses an improved method for cryoembedding hollow organ tissues. The method includes the following steps: pretreatment of the ex vivo tissue; after ex vivo, the tissue is soaked in a buffer solution and gently shaken to clean off any adhering dirt, mucus, secretions, or necrotic material; then, the cleaned tissue is placed on multiple layers of absorbent paper to fully absorb excess water. Through a rapid gradient cooling process, combined with the low-temperature coagulation effect of water-soluble mixtures such as polyethylene glycol and polyvinyl alcohol, the tissue can be rapidly fixed, minimizing the formation of ice crystals and preventing tissue rupture. This also helps preserve the most accurate and detailed morphological structure of hollow organs rich in connective tissue, improving the accuracy of subsequent pathological diagnosis and research. Furthermore, for basic research, it is suitable for experiments such as immunofluorescence, spatial proteomics, and spatial transcriptomics.

Description

Technical Field

[0001] This invention relates to the field of tissue processing technology, specifically to an improved method for cryo-embedding hollow organ tissues. Background Technology

[0002] Cryo-embedding is a commonly used technique for embedding fresh tissue in clinical and research work. Its purpose is to rapidly fix tissue specimens and effectively preserve their spatial structure, genomics, and proteomics information. The main principle is to use a cryostat or dry ice to provide a low-temperature environment, and then use OCT embedding agents or glues—mediums that exhibit solidification at low temperatures—to fix the tissue, facilitating subsequent sectioning, staining, and protein or gene-related experiments.

[0003] Current clinical methods for cryoemulation utilize cryostats to create temperatures as low as -20°C, causing the embedding medium to solidify. Dry ice is commonly used in laboratories to achieve even better low-temperature effects. However, both methods are ineffective for tissues with high water content, especially hollow organs, and fail to effectively control ice crystal formation, a key factor affecting section quality. Ice crystallization of liquid components in tissues leads to increased interstitial volume and deformation, impacting pathological diagnosis. Ice crystals can also puncture cells, causing the loss of genetic material and proteins. The maximum ice crystal formation zone is -2 to -5°C. To minimize the ice crystal effect, this temperature range must be traversed as quickly as possible. Liquid nitrogen, at -196°C, is the optimal method for rapid, deep cooling, allowing tissues at room temperature to pass through the ice crystal formation zone within 2-3 seconds, making it a good choice for controlling the ice crystal effect. However, the sudden temperature drops and rises can easily cause thermal stress and thermal shock, leading to tissue fragmentation. Therefore, this method is rarely used for tissue fixation in clinical settings or laboratories.

[0004] In clinical pathology, rapid frozen sectioning is a routine technique. Due to its advantage of quickly fixing tissue specimens and making pathological diagnoses, it is often used as an important means of determining whether radical resection of tumors has been achieved during surgery. However, because cryostats are commonly used in clinical practice to freeze and fix tissues, the resulting ice crystal effect can alter tissue structure to varying degrees. This is especially true for hollow organs rich in loose connective tissue, blood vessels, and lymphatic vessels, which are most affected by structural deformation during routine freezing procedures. This often leads to misjudgments of whether cancerous lesions have been completely removed during cancer surgery.

[0005] On the other hand, in basic life science research, the requirements for fresh frozen tissue sections are becoming increasingly stringent. Cutting-edge scientific research technologies such as immunofluorescence staining, in situ hybridization, spatial proteomics, and spatial transcriptomics sequencing place high demands on the cryofixation of fresh specimens. However, the current conventional cryoembedding process cannot meet the precise requirements of specimen preparation due to its technical deficiencies, which greatly lags behind the progress of cutting-edge scientific research. Therefore, those skilled in the art have proposed an improved cryoembedding method for hollow organ tissues to solve the aforementioned technical problems. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an improved method for cryoembedding hollow organ tissues, which solves the problem that current conventional cryoembedding procedures cannot meet the precise requirements of specimen preparation due to their technical deficiencies.

[0007] To achieve the above objectives, the present invention provides the following technical solution: an improved method for cryopreservation of hollow organ tissues, comprising the following processing steps:

[0008] S1: Pretreatment of ex vivo tissues

[0009] After the tissue is removed from the body, it is soaked in a buffer solution and gently shaken to clean off any dirt, mucus, secretions or necrotic material attached to the tissue. Then, the cleaned tissue is placed on multiple layers of absorbent paper to fully absorb the moisture.

[0010] S2: Initial Organizational Processing

[0011] After the tissue has been processed in step S1, slowly immerse it into a transparent embedding cassette containing OCT (optimal cutting temperature compound) embedding medium. Use pointed forceps to adjust the orientation of the tissue and observe its position from multiple angles to ensure that the pre-cut surface of the tissue is facing down and the entire tissue is close to the bottom of the embedding cassette.

[0012] S3: Gradient Cooling of Tissue

[0013] Place the embedded cassette processed in step S2 on crushed ice for pre-cooling and keep it horizontal. Then take another pre-cooled empty transparent embedded cassette of the same size and slowly stack it on top of the previous embedded cassette. Avoid the formation of air bubbles during the stacking process. Then slowly and gently press the two layers of embedded cassettes together. You can see the embedding agent overflowing from around the groove. After confirming that there is no obvious change in the tissue surface, you can start the next step.

[0014] S4: Gradient Cooling of Tissue II

[0015] Use long-handled tweezers to hold and fix the double-layer embedding box after the treatment in step S3, and keep it at a horizontal liquid level. Then quickly immerse it in liquid nitrogen for 10-12 seconds. Carefully observe that the embedding agent quickly whitens.

[0016] S5: Freezing and embedding completed

[0017] Once the whitening effect is achieved, quickly drain the tissue specimen and place it in a dry ice or -80℃ environment. After the tissue temperature stabilizes for 5 minutes, it can be slided and subjected to subsequent experimental operations.

[0018] Preferably, in step S1, the buffer solution is either physiological saline or phosphate buffer solution, and the amount of buffer solution used is 10-15 times the tissue volume.

[0019] Preferably, in step S1, the tissue after being removed from the body should not come into contact with liquid solvents. If liquid cleaning is necessary, when placing the tissue on multiple layers of absorbent paper to absorb moisture, blunt-tipped tweezers should be used to assist in preventing the tissue from sticking to the absorbent paper and being forcibly moved, which could lead to tissue damage.

[0020] Preferably, in step S1, the overall operation time is within 20 minutes. During the tissue processing in step S1, a disposable transparent plastic embedding box is prepared, the embedding groove is filled with frozen embedding agent, and after the embedding groove is filled, it is placed on a horizontal surface. Air bubbles in the embedding agent are picked out or aspirated with a syringe. In this processing step, the OCT embedding agent can be replaced with office glue.

[0021] Preferably, in step S3, the environment during pre-cooling of the embedding cassette is 2-8°C, and the pre-cooling time of the embedding cassette is kept horizontal for 2-3 minutes.

[0022] Preferably, in step S4, the time for placing the double-layer embedding box into liquid nitrogen is 10-12 seconds, and the depth of the double-layer embedding box sinking into the liquid nitrogen surface is 5-10 cm.

[0023] Preferably, in step S5, after the tissue specimen has been drained, if the operating environment cannot be met, the temperature of the tissue specimen can be kept below -20°C, and the temperature stabilization time is 5 minutes.

[0024] This invention provides an improved method for cryopreservation of hollow organ tissues. It has the following beneficial effects:

[0025] 1. This invention utilizes a rapid gradient cooling process, combined with the low-temperature coagulation effect of water-soluble mixtures such as polyethylene glycol and polyvinyl alcohol, to quickly fix tissues, minimizing the formation of ice crystals and preventing tissue frostbite. It also helps preserve the most authentic and detailed morphological structure of hollow organs rich in connective tissue, which is beneficial for improving the accuracy of subsequent pathological diagnosis and research by medical personnel.

[0026] 2. This invention pre-cools the tissue, thereby shortening the time for the tissue to immerse in liquid nitrogen and cross the ice crystal formation zone. Furthermore, by using upper and lower embedding cassettes to enclose and isolate the embedding agent, the strong thermal shock effect of liquid nitrogen on the embedding agent during cooling can be minimized, thus avoiding tissue breakage. After achieving the desired cooling and fixation effect, the tissue is transferred to a low-temperature environment, avoiding the risk of tissue breakage due to a sudden temperature rise. This operation method not only improves the quality of sample preparation but also avoids the risk of sample damage.

[0027] 3. The improved cryoembedding technique of this invention is particularly suitable for hollow organ tissues with high water content. For clinical pathological diagnosis, it can be adapted to commonly used techniques such as frozen HE staining and in situ hybridization staining with immunohistochemical staining agents. For basic scientific research, it can be adapted to experiments such as immunofluorescence, spatial proteomics and spatial transcriptomics. Overall, it has high adaptability. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the processing method in step S1 of the present invention;

[0029] Figure 2 This is a schematic diagram of the processing method in step S2 of the present invention;

[0030] Figure 3 This is a schematic diagram of the pre-processing method in step S3 of the present invention;

[0031] Figure 4 This is a schematic diagram of the latter part of the processing method in step S3 of the present invention;

[0032] Figure 5 This is a schematic diagram of the processing method in step S4 of the present invention;

[0033] Figure 6 This is a schematic diagram of the processing method in step S5 of the present invention;

[0034] Figure 7 This is a schematic diagram of the tissue before and after fixation according to the present invention;

[0035] Figure 8 This is a schematic diagram illustrating the HE staining effect after the improved frozen embedding section according to the present invention;

[0036] Figure 9This is a schematic diagram illustrating the immunofluorescence staining effect after the improved frozen embedding section of the present invention;

[0037] Figure 10 This is a schematic diagram illustrating the spatial transcriptome sequencing results of the present invention. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example:

[0040] Please see the appendix Figure 1 - Appendix Figure 10 This invention provides an improved method for cryopreservation of hollow organ tissue, comprising the following processing steps:

[0041] S1: Pretreatment of ex vivo tissues

[0042] After the tissue is removed from the body, it is soaked in a buffer solution and gently shaken to clean off any dirt, mucus, secretions or necrotic material attached to the tissue. Then, the cleaned tissue is placed on multiple layers of absorbent paper to fully absorb the moisture.

[0043] S2: Initial Organizational Processing

[0044] After the tissue has been processed in step S1, slowly immerse it into a transparent embedding cassette containing OCT (optimal cutting temperature compound) embedding medium. Use pointed forceps to adjust the orientation of the tissue and observe its position from multiple angles to ensure that the pre-cut surface of the tissue is facing down and the entire tissue is close to the bottom of the embedding cassette.

[0045] S3: Gradient Cooling of Tissue

[0046] Place the embedded cassette processed in step S2 on crushed ice for pre-cooling and keep it horizontal. Then take another pre-cooled empty transparent embedded cassette of the same size and slowly stack it on top of the previous embedded cassette. Avoid the formation of air bubbles during the stacking process. Then slowly and gently press the two layers of embedded cassettes together. You can see the embedding agent overflowing from around the groove. After confirming that there is no obvious change in the tissue surface, you can start the next step.

[0047] S4: Gradient Cooling of Tissue II

[0048] Use long-handled tweezers to hold and fix the double-layer embedding box after the treatment in step S3, and keep it at a horizontal liquid level. Then quickly immerse it in liquid nitrogen for 10-12 seconds. Carefully observe that the embedding agent quickly whitens.

[0049] S5: Freezing and embedding completed

[0050] Once the whitening effect is achieved, quickly drain the tissue specimen and place it in a dry ice or -80℃ environment. After the tissue temperature stabilizes for 5 minutes, it can be slided and subjected to subsequent experimental operations.

[0051] Please see the appendix Figure 1 As shown, in step S1, the buffer solution is either physiological saline or phosphate buffer solution, and the amount of buffer solution used is 10-15 times the tissue volume.

[0052] In step S1, the excised tissue should not come into contact with liquid solvents. If liquid cleaning is necessary, when placing the tissue on multiple layers of absorbent paper to absorb moisture, use blunt-tipped forceps to assist in preventing the tissue from sticking to the absorbent paper and being forcibly moved, which could damage the tissue.

[0053] In step S1, the entire operation should take no more than 20 minutes to prevent protein and RNA degradation. During tissue processing in step S1, prepare a disposable transparent plastic embedding cassette, fill the embedding chamber with frozen embedding medium, and place the chamber on a level surface after filling. Use a syringe to remove or aspirate air bubbles from the embedding medium. Office glue can be used as a substitute for OCT embedding medium in this step.

[0054] Please see the appendix Figure 2 As shown, when immersing tissue in embedding medium, the tissue should be placed as close as possible to the bottom of the embedding cassette. Air bubbles attached to the tissue can cause fragmentation during frozen sectioning and should be thoroughly removed or expelled from the tissue in this step.

[0055] In step S3, the environment during pre-cooling of the embedding cassette is 2-8℃, and the embedding cassette is kept horizontal for 2-3 minutes during pre-cooling.

[0056] Please see the appendix Figure 3-4 As shown, during the pre-cooling of the embedding cassette, the liquid appears slightly whitish. This step is the first step of the gradient cooling process, which helps to further reduce the ice crystal effect. It should be noted that the fluid resistance within the liquid will increase after cooling. If the tissue orientation is incorrect, it can be adjusted slightly with pointed forceps to avoid air bubbles forming, as removing air bubbles after cooling will be more difficult.

[0057] When stacking embedding cassettes, the contact surfaces should be tilted appropriately so that the bottom of the upper embedding cassette gradually contacts the liquid surface of the lower embedding agent from the edge to avoid air bubble retention. The purpose of this step is to encapsulate the embedding agent in the embedding cassette and prevent the embedding agent from directly contacting the liquid nitrogen, which is a key step in preventing tissue cracking.

[0058] If tissue tilting occurs significantly due to improper operation in this step, it is often because of excessive embedding medium or excessive tissue thickness. To address this, reduce the amount of embedding medium, decrease the tissue thickness, or use a deeper embedding cassette. The embedding cassette depth should ideally be 2-2.5 times the tissue thickness. Air bubbles remaining in the interlayer do not need to be treated, as this surface is the fixing surface for sectioning. The air bubbles themselves do not affect the sectioning process, but they will somewhat affect the stability of the tissue on the microtome tray. To avoid air bubbles, adjust the amount of embedding medium to 1-3 mm below the embedding groove level.

[0059] If significant tissue tilting is observed at this stage, the tissue position, thickness, and embedding agent dosage need to be readjusted. Otherwise, it is recommended not to adjust the specimen further, and note that all operations should be performed in an environment of 2-8℃.

[0060] In step S4, the double-layer embedding box is placed in liquid nitrogen for 10-12 seconds, and the double-layer embedding box sinks to a depth of 5-10 cm into the liquid nitrogen surface.

[0061] Please see the appendix Figure 5-6 As shown, after the double-layer embedding cassette is immersed in liquid nitrogen, it can be clearly observed that the embedding agent whitens rapidly. If the time is insufficient, the tissue cannot be fully whitened with the embedding agent, and if the time is too long, there is a risk of tissue breakage.

[0062] In step S5, after the tissue specimen has been drained, if the operating environment cannot be met, the tissue specimen temperature can be kept below -20℃, and the tissue temperature stabilization time is 5 minutes.

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An improved method for cryo-embedding hollow organ tissues, characterized in that, The following processing steps are included: S1: Pretreatment of ex vivo tissues After the tissue is removed from the body, it is soaked in a buffer solution and gently shaken to clean off any dirt, mucus, secretions or necrotic material attached to the tissue. Then, the cleaned tissue is placed on multiple layers of absorbent paper to fully absorb the moisture. In step S1, the buffer solution is either physiological saline or phosphate buffer solution, and the amount of buffer solution used is 10-15 times the tissue volume. In step S1, the tissue after being removed from the body should not come into contact with liquid solvents. If liquid cleaning is necessary, when placing the tissue on multiple layers of absorbent paper to absorb moisture, blunt-tipped tweezers should be used to assist in preventing the tissue from sticking to the absorbent paper and being forcibly moved, which could cause tissue damage. S2: Initial Organizational Processing After the tissue has been processed in step S1, slowly immerse it into a transparent embedding cassette containing OCT (optimal cutting temperature compound) embedding medium. Use pointed forceps to adjust the orientation of the tissue and observe its position from multiple angles to ensure that the pre-cut surface of the tissue is facing down and the entire tissue is close to the bottom of the embedding cassette. S3: Gradient Cooling of Tissue Place the embedded cassette processed in step S2 on crushed ice for pre-cooling and keep it horizontal. Then take another pre-cooled empty transparent embedded cassette of the same size and slowly stack it on top of the previous embedded cassette. Avoid the formation of air bubbles during the stacking process. Then slowly and gently press the two layers of embedded cassettes together. You can see the embedding agent overflowing from around the groove. After confirming that there is no obvious change in the tissue surface, you can start the next step. S4: Gradient Cooling of Tissue II Use long-handled tweezers to hold and fix the double-layer embedding box after the treatment in step S3, and keep it at a horizontal liquid level. Then quickly immerse it in liquid nitrogen for 10-12 seconds. Carefully observe that the embedding agent quickly whitens. S5: Freezing and embedding completed Once the whitening effect is achieved, quickly drain the tissue specimen and place it in a dry ice or -80℃ environment. After the tissue temperature stabilizes for 5 minutes, it can be slided and subjected to subsequent experimental operations.

2. The improved method for cryopreservation and embedding of hollow organ tissue according to claim 1, characterized in that, In step S1, the entire operation takes less than 20 minutes. During the tissue processing in step S1, a disposable transparent plastic embedding box is prepared, and the embedding groove is filled with frozen embedding agent. After the embedding groove is filled, it is placed on a horizontal surface, and air bubbles in the embedding agent are picked out or aspirated with a syringe. In this process, OCT embedding agent can be replaced with office glue.

3. The improved method for cryopreservation and embedding of hollow organ tissue according to claim 1, characterized in that, In step S3, the environment during pre-cooling of the embedding cassette is 2-8℃, and the pre-cooling time of the embedding cassette is kept horizontal for 2-3 minutes.

4. The improved method for cryopreservation and embedding of hollow organ tissue according to claim 1, characterized in that, In step S4, the time for placing the double-layer embedding box into liquid nitrogen is 10-12 seconds, and the depth of the double-layer embedding box sinking into the liquid nitrogen surface is 5-10 cm.

5. The improved method for cryopreservation and embedding of hollow organ tissue according to claim 1, characterized in that, In step S5, after the tissue specimen has been drained, if the operating environment cannot be met, the tissue specimen temperature can be kept below -20℃, and the tissue temperature stabilization time is 5 minutes.

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