Method for shaping intestinal tissues and application of intestinal tissues in preparation of Swiss roll embedding form

Abstract

The invention provides an intestinal tract tissue shaping method and application of the intestinal tract tissue in preparation of a Swiss roll embedding form, and relates to the technical field of biologication.The intestinal tract tissue shaping method comprises the steps that sheet-shaped intestinal tract tissue is flatly laid on the surface of a supporting layer, the flatly laid intestinal tract tissue and the supporting layer are curled in the long axis direction of the intestinal tract tissue at the same time, and the intestinal tract tissue in the Swiss roll form is obtained; the supporting layer effectively prevents direct and tight attachment or overlapping of intestinal tissues, and clear intervals between the layers are ensured; the pulling force directly applied to the soft sheet-shaped intestinal tissues in the curling operation is reduced, the stress is effectively buffered, and the denatured damages such as stretching and tearing of the tissues are reduced, so that the curling operation is easier to carry out, the form is controlled, and the preparation efficiency is improved; looseness or tissue distortion and denaturation during placement are prevented. The technical problems that in the prior art, due to the fact that intestinal tract tissue is curled and is not prone to sizing, tissue overlapping and unclear separation are caused after embedding, tissue deformation and damage affect the accuracy of observation results, and raw tissue segmentation and deconvolution are solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for shaping intestinal tissue and its application in preparing Swiss roll embedding morphologies. Background Technology

[0002] In histopathological and molecular biological studies, tubular tissues such as the intestine are often prepared into a "Swiss roll" shape before cryopreservation and sectioning to facilitate observation of long, continuous intestinal structures on a single cross-section. However, current techniques for directly curling and embedding intestinal tissue have the following significant drawbacks: 1. Overlapping and unclear separation of tissues: During the curling process, the layers of the intestine tend to adhere closely or even overlap, making it difficult to clearly distinguish the structures of adjacent intestinal segments or intestinal wall layers in subsequent sections.

[0003] 2. Tissue deformation and damage: Intestinal tissue is soft and easily deformable. During direct rolling operations, it is easily subjected to tensile forces, which can lead to tissue tearing, stretching deformation or structural damage, affecting the accuracy of observation results.

[0004] 3. High operational difficulty: Without effective support, intestinal tissue is not easy to shape, the curling process is difficult to control, and the success rate is low.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for shaping intestinal tissue, in order to solve the technical problems in the prior art where poor shaping of intestinal tissue due to its curling leads to unclear tissue overlap and separation after embedding, and tissue deformation and damage affect the accuracy of observation results, bioinformatics segmentation, and deconvolution.

[0007] The second objective of this invention is to provide the application of the above-mentioned shaping method in the preparation of Swiss roll embedded morphology.

[0008] The third objective of this invention is to provide a method for embedding intestinal tissue in the form of a Swiss roll.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for shaping intestinal tissue, comprising laying sheet-like intestinal tissue flat on the surface of a support layer, and simultaneously rolling the flat intestinal tissue and the support layer along the long axis of the intestinal tissue to obtain intestinal tissue in the shape of a Swiss roll. The thickness of the support layer is 0.1~2mm; The support layer includes any one of the following: an encapsulating agent cryopreservation support layer, an encapsulating agent drying film support layer, or a PVA composite film support layer.

[0010] Furthermore, the method for preparing the embedding agent cryopreservation support layer includes placing the pre-support layer, which has been rapidly frozen at low temperature, at 0~4℃ for 5~10 seconds to rewarm it.

[0011] Furthermore, the method for preparing the pre-supported layer includes placing a liquid embedding agent in a low-temperature quick-freezing and shaping process.

[0012] Preferably, the temperature for the low-temperature quick-freezing and shaping is ≤-20℃; Preferably, the thickness of the embedding agent cryopreservation support layer is 0.5~1mm; Preferably, the embedding agent is a water-soluble tissue embedding medium; Preferably, the embedding agent includes any one of OCT embedding agent, Tissue Freezing Medium, or Leica Cryo-Gel Embedding Medium.

[0013] Furthermore, the method for preparing the embedding agent drying film support layer includes spreading the embedding agent flat and then drying it.

[0014] Furthermore, the thickness of the embedding agent drying film support layer is 0.1~1mm; Preferably, the drying temperature is 37°C.

[0015] Furthermore, the PVA composite film support layer includes an adhesive layer and a PVA base layer covering the adhesive layer; Preferably, the adhesive layer comprises 1-5% sodium alginate, 1-10% glycerol, 0.1-0.5% sodium carboxymethyl cellulose and 0.05-0.2% calcium chloride by mass, and the solvent is DEPC-PBS; Preferably, the PVA base layer comprises 5-10% PVA, 1-10% glycerol and 0.01-0.05% n-octanol by mass, and the solvent is NF-Water; Preferably, the thickness of the PVA composite film support layer is 0.1~2mm; Preferably, the thickness ratio of the PVA base layer to the adhesive layer is 1:1 to 10:1.

[0016] Furthermore, the preparation method of the PVA composite film support layer includes the following steps: A. Preparation of PVA base layer: The PVA base layer solution prepared according to the formula is obtained by scraping and gradient drying. B. Prepare the adhesive liquid layer. The adhesive solution prepared according to the formula is used to form an adhesive liquid layer by scraping. The PVA base layer obtained in step A is covered on the adhesive liquid film layer, and the PVA composite film support layer is obtained by roll pressing. Preferably, the roll-pressed composite is followed by freeze-drying and storage.

[0017] Furthermore, the sheet-like intestinal tissue is pre-treated sheet-like intestinal tissue; The pretreatment includes removing contents, mucus, or excess surface moisture.

[0018] Secondly, the present invention provides the application of the above-described shaping method in the preparation of Swiss roll embedded morphology.

[0019] Thirdly, the present invention provides a method for embedding intestinal tissue in the form of a Swiss roll, wherein intestinal tissue in the form of a Swiss roll is prepared by the above-mentioned shaping method and embedded in an embedding mold.

[0020] This invention provides a method for shaping intestinal tissue. By setting a support layer, which exists between adjacent intestinal tissue layers during curling, it acts as a physical separator, effectively preventing the intestinal tissue from directly adhering or overlapping, and ensuring clear intervals between layers. The support layer has a certain thickness to maintain its rigidity, which can significantly reduce the tensile force directly applied to the soft sheet-like intestinal tissue during the curling operation, effectively buffering stress and minimizing tissue stretching, tearing, and other degenerative damage, making the curling operation easier, controlling the morphology, and improving preparation efficiency. At the end of the curling, the support layer provides initial support and shape memory for the soft intestinal tissue, preventing the curled shape from loosening or the tissue from twisting and degenerating during placement. This solves the technical problems in the prior art where poor shaping of intestinal tissue leads to unclear tissue overlap and separation after embedding, and tissue deformation damage affects the accuracy of observation results, bioinformatics segmentation, and deconvolution. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is an image showing the effect of embedding intestinal tissue in Swiss roll form as provided in Embodiment 6 of the present invention. Figure 2 This is an image showing the effect of embedding intestinal tissue in Swiss roll form as provided in Embodiment 7 of the present invention; Figure 3 This is an image showing the effect of embedding intestinal tissue in Swiss roll form as provided in Embodiment 8 of the present invention; Figure 4 This is an image showing the effect of embedding intestinal tissue in Swiss roll form as provided in Embodiment 9 of the present invention. Figure 5This is an image showing the effect of embedding intestinal tissue in Swiss roll form as provided in Embodiment 10 of the present invention. Figure 6 This is an image showing the effect of embedding intestinal tissue in Swiss roll form, as provided in Comparative Example 7 of this invention. Figure 7 This is an image showing the effect of embedding intestinal tissue in Swiss roll form, as provided in Comparative Example 8 of this invention. Figure 8 This is an image showing the effect of embedding intestinal tissue in Swiss roll form, as provided in Comparative Example 9 of this invention. Figure 9 This is an image showing the effect of embedding intestinal tissue in Swiss roll form, as provided in Comparative Example 10 of the present invention. Figure 10 This is an image showing the effect of embedding intestinal tissue in Swiss roll form, as provided in Comparative Example 11 of the present invention. Figure 11 This is an image showing the effect of embedding intestinal tissue in Swiss roll form, as provided in Comparative Example 12 of the present invention. Detailed Implementation

[0023] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0024] Unless otherwise stated, the methods and techniques of the present invention are generally based on conventional methods well known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0025] The present invention provides a method for shaping intestinal tissue, comprising laying sheet-like intestinal tissue flat on the surface of a support layer, and simultaneously rolling the flat intestinal tissue and the support layer along the long axis of the intestinal tissue to obtain a Swiss roll-shaped intestinal tissue; the thickness of the support layer is 0.1~2mm; the support layer includes any one of an embedding agent cryopreservation support layer, an embedding agent drying film support layer, or a PVA composite film support layer.

[0026] By incorporating a support layer, which exists between adjacent intestinal tissue layers during curling, it acts as a physical separator, effectively preventing direct and tight adhesion or overlap of the intestinal tissue itself and ensuring clear intervals between layers. The support layer has a certain thickness to maintain its rigidity, significantly reducing the tensile force applied directly to the soft, sheet-like intestinal tissue during curling, effectively buffering stress, and minimizing tissue stretching, tearing, and other degenerative damage. This makes curling easier, allows for better shape control, and improves preparation efficiency. At the curling endpoint, the support layer provides initial support and shape memory for the soft intestinal tissue, preventing the curled shape from loosening or the tissue from twisting and degenerating during placement. This solves the technical problems in existing technologies where poor shaping of curled intestinal tissue leads to unclear tissue overlap and separation after embedding, and tissue deformation damage affects the accuracy of observation results, bioinformatics segmentation, and deconvolution.

[0027] The thickness of the support layer can be, but is not limited to, 0.1mm, 0.2mm, 0.4mm, 0.6mm, 0.8mm, 1.0mm, 1.2mm, 1.4mm, 1.6mm, 1.8mm or 2.0mm, or any value between 0.1mm and 2.0mm.

[0028] In some specific embodiments, the method for preparing the embedding agent cryopreservation support layer includes placing a pre-support layer that has been rapidly frozen at low temperature at 0-4°C for 5-10 seconds to rewarm it. The resulting cryopreservation support layer is surface-softened but internally unmelted, with no free liquid on the surface, thus maintaining its basic shape and support strength.

[0029] In some specific embodiments, the preparation method of the pre-support layer includes placing a liquid embedding agent in a low-temperature rapid freezing and shaping environment; placing a support carrier mold carrying the liquid embedding agent in a low-temperature environment to allow the embedding agent to be fully and rapidly frozen and shaped, forming a solid pre-support thin layer. In some specific embodiments, the low-temperature rapid freezing and shaping temperature is ≤-20℃; a refrigerator or low-temperature freezing table with a temperature of -20℃ or below is acceptable. In some specific embodiments, the thickness of the embedding agent frozen support layer is 0.5~1mm. Specifically, the spreading thickness of the liquid embedding agent is controlled within the range of approximately 0.5mm to 1mm. If it is too thin (<0.5mm), the mechanical strength of the support layer is insufficient, making it difficult to shape and prone to breakage; if it is too thick (>1mm), the flexibility of the support layer is reduced, which is not conducive to subsequent curling operations, and will also significantly increase the volume of non-target tissue in the final embedded block.

[0030] In some specific embodiments, the preparation method of the embedding agent drying film support layer includes spreading the embedding agent flat and then drying it. Specifically, a film scraper is used to spread the embedding agent body flat on a glass plate and then dry it to obtain a transparent and tough transparent embedding agent drying film as the support layer. In some specific embodiments, the drying temperature is 37°C.

[0031] In some specific embodiments, the embedding agent is a water-soluble tissue embedding medium; in some specific embodiments, the embedding agent includes any one of OCT embedding agent, tissue freezing medium (e.g., brand: Leica, product number: 14020108926) or Leica Cryo-Gel Embedding Medium (e.g., brand: Leica, product number: 14020108926).

[0032] In some specific embodiments, the thickness of the embedding agent drying film support layer is 0.1~1mm.

[0033] The thickness of the embedding agent drying film support layer can be, but is not limited to, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm or 1.0 mm, or any value between 0.1 and 1 mm.

[0034] In some specific embodiments, the PVA composite membrane support layer includes an adhesive layer and a PVA base layer covering the adhesive layer; the PVA composite membrane has good water solubility, flexibility and biocompatibility, and can be used to adhere intestinal tissue after activation with PBS, making it suitable for intestinal roll-up embedding operations.

[0035] In some specific embodiments, the adhesive layer comprises 1-5% sodium alginate, 1-10% glycerol, 0.1-0.5% sodium carboxymethyl cellulose and 0.05-0.2% calcium chloride by mass, and the solvent is DEPC-PBS.

[0036] The mass percentage of sodium alginate in the adhesive layer can be, but is not limited to, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, or any value between 1% and 5%, preferably 2%.

[0037] The mass percentage of glycerin in the adhesive layer can be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or any value between 1% and 10%, preferably 5%.

[0038] The mass percentage of sodium carboxymethyl cellulose in the adhesive layer can be, but is not limited to, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, or any value between 0.1% and 0.5%, preferably 0.5%.

[0039] The mass percentage of calcium chloride in the adhesive layer can be, but is not limited to, 0.05%, 0.07%, 0.09%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, or 0.2%, or any value between 0.05% and 0.2%, preferably 0.1%.

[0040] In some specific embodiments, the adhesive layer comprises 2% sodium alginate, 5% glycerol, 0.5% nanocellulose and 0.1% calcium chloride by mass, and the solvent is DEPC-PBS.

[0041] In some specific embodiments, the PVA base layer comprises 5-10% PVA, 1-10% glycerol and 0.01-0.05% n-octanol by mass, with water as the solvent.

[0042] The mass percentage of PVA in the PVA base layer can be, but is not limited to, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%, or any value between 5% and 10%, preferably 7.35%. The mass percentage of glycerol in the PVA base layer can be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, or any value between 1 and 10%, preferably 1.27%.

[0043] The mass percentage of n-octanol in the PVA base layer can be, but is not limited to, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%, or any value between 0.01% and 0.05%, preferably 0.02%.

[0044] In some specific embodiments, the PVA base layer comprises 7.35% PVA, 1.27% glycerol and 0.02% n-octanol by mass, with water as the solvent.

[0045] In some specific embodiments, the thickness of the PVA composite film support layer is 0.1~2mm; in some specific embodiments, the thickness ratio of the PVA base layer to the adhesive layer is 1:1~10:1.

[0046] In some specific embodiments, the method for preparing the PVA composite film support layer includes the following steps: A. Preparation of PVA base layer: The PVA base layer solution prepared according to the formula is obtained by scraping and gradient drying. B. Prepare the adhesive liquid layer. The adhesive solution prepared according to the formula is used to form an adhesive liquid layer by scraping. The PVA base layer obtained in step A is covered on the adhesive liquid film layer, and the PVA composite film support layer is obtained by roll pressing.

[0047] Before use, it needs to be stored in a light-proof and sealed container. It can be cut into strips of appropriate size according to specific needs, such as strips of 1×8cm.

[0048] In some specific embodiments, the roll-pressed composite is then frozen and stored.

[0049] In some specific embodiments, the sheet-like intestinal tissue is pre-treated sheet-like intestinal tissue; the pretreatment includes gently rinsing the inner surface of the intestinal tissue with physiological saline or a suitable buffer solution to remove contents and mucus; placing the washed intestinal tissue on a sterile absorbent material (such as sterile gauze or dust-free filter paper) to gently absorb and remove excess moisture from the tissue surface, so that the tissue is moderately moist but without obvious free liquid.

[0050] The above-described shaping method is applicable to various intestinal tissue samples requiring the preparation of a "Swiss roll" morphology. According to another aspect of the invention, the application of the above-described shaping method in the preparation of Swiss roll embedding morphologies is also provided.

[0051] According to another aspect of the present invention, a method for embedding intestinal tissue in the form of a Swiss roll is also provided, wherein intestinal tissue in the form of a Swiss roll is prepared by the above-described shaping method and embedded in an embedding mold.

[0052] Specifically, the process involves transferring Swiss roll-shaped intestinal tissue into a pre-cooled embedding mold, injecting liquid embedding agent to completely cover the tissue, and then rapidly freezing and embedding it in an ultra-low temperature environment until the embedding agent is completely solidified, forming an embedding block that can be used for sectioning. Pre-cooling can be achieved by placing it on crushed ice or in an environment of 0–4°C, while the ultra-low temperature environment can be achieved by placing it on dry ice.

[0053] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0054] In Examples 1 and 2, a frozen mold measuring 10 cm in length, 1 cm in width, and 0.2 cm in height was used as the support carrier mold, and subsequent operations such as rewarming and sausage rolling were performed on this mold. This mold was also used in Examples 3 and 4 to maintain and control the directionality of the sausage rolls and to keep the tissues on the same plane.

[0055] Intestinal tissue in Examples 1-4: Small intestinal tissue of 8-week-old C57 mice, with a length of 5-8 cm, was selected for embedding experiment testing.

[0056] Example 1 A method for shaping intestinal tissue, specifically following these steps: 1. Preparation of the embedding agent cryopreservation support layer 1) In this embodiment, OCT compound embedding agent (manufacturer: SAKUEA, product number: 4583) is selected. Take an appropriate amount of liquid embedding agent and spread it evenly on a smooth, inert, and peelable support carrier mold. In this embodiment, silicone is selected as the support carrier mold. The mold is 10cm long, 1cm wide, and 0.2cm high. The length, width, and height of the mold can be determined according to the sample size in the application.

[0057] 2) Control the spreading thickness within the range of approximately 0.5mm to 1mm.

[0058] 3) Place the support carrier mold containing the liquid embedding agent in a low-temperature freezing table at a temperature of -20℃ to allow the embedding agent to freeze and solidify quickly, forming a solid pre-support layer.

[0059] 2. Intestinal tissue pretreatment 1) Take a sample of the target intestinal tissue, cut it longitudinally along its long axis to form a sheet.

[0060] 2) Gently rinse the intestinal lumen with physiological saline or a suitable buffer solution to remove contents and mucus.

[0061] 3) Place the cleaned intestinal tissue on sterile gauze and gently absorb and remove excess moisture from the tissue surface, so that the tissue is moderately moist but without obvious free liquid.

[0062] 3. Tissue Placement and Temperature Control 1) The pre-treated sheet of intestinal tissue is naturally laid flat on the surface of the pre-supported layer of embedding agent prepared in step 1, which is in a frozen and fixed state.

[0063] 2) Together with the support carrier mold below, the composite containing the intestinal tissue and support layer is quickly transferred to the ice plate and placed in an environment of 0-4℃.

[0064] 3) Precisely control the rewarming time to approximately 5 to 10 seconds. Continuously observe to ensure the embedding agent freeze support layer reaches a specific physical state: its surface begins to soften, gaining sufficient plasticity and viscosity, but the internal matrix has not yet melted, and it can still maintain its basic shape and support strength.

[0065] 4. Rolling and shaping When the optimal softening state is reached in step 3, the plasticity and viscosity of the embedding agent freeze support layer are immediately utilized to gently and smoothly roll the flat intestinal tissue together with the pre-support layer below it into a "Swiss roll" shape along the long axis of the intestinal tissue.

[0066] Example 2 A method for shaping intestinal tissue, specifically following these steps: 1. Intestinal tissue pretreatment 1) Take a sample of the target intestinal tissue, cut it longitudinally along its long axis to form a sheet.

[0067] 2) Gently rinse the intestinal lumen with physiological saline or a suitable buffer solution to remove contents and mucus.

[0068] 3) Place the cleaned intestinal tissue on sterile gauze and gently absorb and remove excess moisture from the tissue surface, so that the tissue is moderately moist but without obvious free liquid.

[0069] 2. Preparation of the embedding agent cryopreservation support layer 1) In this embodiment, OCT compound embedding agent (manufacturer: SAKUEA, product number: 4583) is selected. Take an appropriate amount of liquid embedding agent and spread it evenly on a smooth, inert, and peelable support carrier mold. In this embodiment, silicone is selected as the support carrier mold. The mold is 10cm long, 1cm wide, and 0.2cm high. The length, width, and height of the mold can be determined according to the sample size in the application.

[0070] 2) Control the spreading thickness within the range of approximately 0.5mm to 1mm.

[0071] 3) Place the support carrier mold containing the liquid embedding agent in a low-temperature freezing table at -20℃ to allow the embedding agent to freeze and solidify quickly, forming a solid pre-support layer.

[0072] 4) Transfer the solid pre-supported layer to an ice plate and place it in an environment of 0-4℃. Precisely control the warming time to approximately 5 to 10 seconds. Continuously observe to allow the embedding agent to reach a specific physical state: its surface begins to soften, gaining sufficient plasticity and viscosity, but the internal matrix has not yet melted, and it can still maintain its basic shape and support strength.

[0073] 3. Organization, Laying out, and Rolling The pre-treated sheet of intestinal tissue is laid flat on the surface of the cryopreservation support layer prepared in step 1, which has been precisely heated. Immediately, taking advantage of the plasticity and viscosity of the cryopreservation support layer, the flat intestinal tissue, together with the pre-support layer below it, is gently and steadily rolled into a "Swiss roll" shape along the long axis of the intestinal tissue.

[0074] Example 3 The difference from Example 1 is that the support layer was replaced with an embedding agent drying film support layer for subsequent experimental operations. The embedding agent was an OCT compound embedding agent (manufacturer: SAKUEA, catalog number: 4583).

[0075] 1. Preparation of the support layer for the embedding agent drying film The embedding agent was spread evenly on a glass plate using a 500 μm speckle scraper and dried overnight at 37°C to obtain a transparent and tough OCT membrane. The prepared OCT membrane was then cut into appropriate sizes for later use.

[0076] 2. Intestinal tissue pretreatment is the same as in Example 1. 3. Tissue placement: The pre-treated sheet-like intestinal tissue is naturally laid flat on the OCT drying membrane support layer prepared in step 1.

[0077] 4. Rolling and shaping By utilizing the plasticity and adhesiveness of the embedding agent drying film support layer, the flat intestinal tissue, together with the pre-support layer below it, is gently and smoothly rolled into a "Swiss roll" shape along the long axis of the intestinal tissue.

[0078] Example 4 The difference from Example 1 is that the support layer was replaced with a PVA composite film support layer for subsequent experimental operations.

[0079] 1. Preparation of PVA composite film support layer 1) Preparation of PVA base solution: PVA powder (pre-dried at 60°C for 2 hours), about 1.27% glycerol, and about 0.02% n-octanol, accounting for about 7.35% of the total mass of the solution, are added to about 89.36% of NF-Water preheated at 85°C. The solution is stirred and dissolved at 60°C to 95°C for 2.5 hours. In this example, the temperature is set to 95°C to obtain a homogeneous solution. After the solution is dispensed, it is centrifuged at 5000 rpm for 15 minutes to remove bubbles, or n-octanol is added dropwise and the solution is allowed to stand for 10 minutes to remove bubbles.

[0080] The PVA powder was sourced from Sinopharm Chemical Reagent Co., Ltd., product number: 30153160.

[0081] 2) Film coating and gradient drying: Take an appropriate amount of degassed PVA solution and coat it on a substrate at 25℃ with a 500 μm gap and a coating speed of 8 mm / s to form a wet film. Then, it is sequentially leveled at 25℃ and 80% RH for 20 minutes, initially dried by forced air at 30℃ and 60% RH for 3 hours, mainly dried at 35℃ and 45% RH for 6 hours, and finally dried under vacuum at 40℃, 30% RH and -50kPa for 1 hour to form a PVA base film with a thickness of 0.45~0.5mm.

[0082] 3) Preparation of adhesive solution: In a clean environment at 4℃, add about 2.0% sodium alginate (by mass of the total adhesive solution) to about 80% pre-cooled DEPC-PBS, and stir magnetically at 300 rpm for 45 minutes until transparent. Let stand for 30 minutes to defoam. Add about 5.0% glycerol and stir at 600 rpm for 10 minutes. Disperse about 0.5% nanocellulose in about 10% DEPC-PBS, sonicate on ice for 5 minutes (40 kHz), and then add to the main solution. Stir at 400 rpm for 20 minutes. Make up the remaining volume with DEPC-PBS. Before use, dissolve about 0.1% calcium chloride (by mass of the total solution) in a small amount of DEPC-PBS and quickly mix it into the solution. Sterilize by filtering through a 0.22 μm filter membrane.

[0083] 4) Composite film preparation: Take an appropriate amount of adhesive solution and drop it onto a pre-cooled glass plate at 4℃. Coat the film with a 100μm gap and a coating speed of 10cm / s. Immediately cover it with a PVA base film. After roller lamination at 4℃ and 0.5MPa, freeze-dry at -20℃ for 20 minutes. Finally, cut it into 1×8cm strips, seal it in a light-proof place, and store it at -20℃ for later use. The thickness of the PVA composite film support layer is 0.55~0.6 mm.

[0084] 2. The intestinal tissue pretreatment is the same as in Example 1.

[0085] 3. Tissue placement: The pre-treated sheet-like intestinal tissue is naturally laid flat on the PVA composite membrane support layer prepared in step 1. Before use, a small amount of PBS can be sprayed on to activate it.

[0086] 4. Rolling and shaping By utilizing the plasticity and adhesiveness of the PVA composite membrane support layer, the flat intestinal tissue, together with the pre-support layer below it, is gently and smoothly rolled into a "Swiss roll" shape along the long axis of the intestinal tissue.

[0087] Example 5 The difference from Example 3 is that the sheet-like intestinal tissue is the whole intestinal segment of the small intestine of 8-week-old C57 mice.

[0088] Comparative Example 1 The difference from Example 1 is that, in step 2) of preparing the embedding agent cryopreservation support layer, the spreading thickness is controlled to be in the range of about 0.3~0.5mm, less than 0.5mm, and the actual thickness is about 0.5mm.

[0089] Comparative Example 2 The difference from Example 1 is that, in step 2) of preparing the embedding agent cryopreservation support layer, the spreading thickness is controlled to be in the range of about 1 to 1.3 mm, which is greater than 1 mm and the actual thickness is about 2 mm.

[0090] Comparative Example 3 The difference from Example 1 is that the rewarming time of the embedding agent cryopreservation support layer is precisely controlled to be about 3 to 4 seconds during preparation.

[0091] Comparative Example 4 The difference from Example 1 is that the rewarming time is precisely controlled to be about 12 to 15 seconds during the preparation of the embedding agent cryopreservation support layer.

[0092] Comparative Example 5 The difference from Example 3 is that when preparing the embedding agent drying film support layer, a 100μm specification film scraper is used to spread the embedding agent body flat on the glass plate.

[0093] Comparative Example 6 The difference from Example 3 is that when preparing the embedding agent drying film support layer, a 2000μm speckled film scraper is used to spread the embedding agent body flat on the glass plate.

[0094] Examples 6-10 A method for embedding intestinal tissue in the form of a Swiss roll, wherein the "Swiss roll" tissue prepared in Examples 1-5 is used as the basis for embedding.

[0095] 1. Transfer the rolled-up "Swiss roll" tissue (along with its surrounding pre-support layer) as a whole to a standard tissue embedding mold that has been pre-cooled on crushed ice.

[0096] 2. Inject sufficient liquid embedding agent (using OCT embedding agent) into the mold to ensure complete coverage of the tissue.

[0097] 3. Perform final rapid freezing and embedding on dry ice at ultra-low temperatures until the embedding medium is completely solidified, forming a standard embedding block suitable for sectioning. The embedding effect is as follows: Figures 1-5 As shown.

[0098] Comparative Examples 7-12 The difference from Example 6 is that the "Swiss roll" tissues prepared in Comparative Examples 1-6 were used as the basis for embedding, and the embedding effect is as follows: Figures 6-11 As shown.

[0099] The results showed that the "Swiss roll" prepared in Examples 6-10 had clear intervals between the layers after embedding, and there was no tight adhesion or overlap. Compared to Example 6, in Comparative Example 7, the cryopreservation support layer of the embedding machine was too thin, causing almost the entire layer to melt after the intestine was laid flat. This resulted in some areas having too small gaps after embedding, leading to intestinal degeneration and preventing the intestine from being rolled into an ideal shape. In Comparative Example 8, the cryopreservation support layer of the embedding machine was too thick, increasing resistance during the rolling process and preventing the sample from being rolled into a Swiss roll shape, resulting in excessively large gaps between samples. In Comparative Example 9, the short warming time caused the cryopreservation support layer of the embedding agent to become too rigid, causing tissue breakage and inability to be rolled into shape. In Comparative Example 10, the excessively long warming time caused the cryopreservation support layer of the embedding agent to melt, failing to provide support and preventing the intestinal tissue from maintaining a Swiss roll shape during the rolling process, resulting in tissue bending and deformation. In Comparative Example 11, the dry film of the embedding agent was too thin, causing some of the support sides to melt and deform during the adhesion process, preventing a uniform rolling state and resulting in tissue deformation. In Comparative Example 12, the dry film support layer of the embedding agent was too thick and too rigid, making it impossible to control the flow direction of the embedded tissue, leading to tissue deformation and distortion.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for shaping intestinal tissue, characterized in that, This includes laying sheet-like intestinal tissue flat on the surface of a support layer, and simultaneously rolling the flattened intestinal tissue and the support layer along the long axis of the intestinal tissue to obtain intestinal tissue in the shape of a Swiss roll. The thickness of the support layer is 0.1~2mm; The support layer includes any one of the following: an encapsulating agent cryopreservation support layer, an encapsulating agent drying film support layer, or a PVA composite film support layer.

2. The shaping method according to claim 1, characterized in that, The method for preparing the embedding agent cryopreservation support layer includes placing a pre-support layer that has been rapidly frozen at low temperature at 0~4℃ and then rewarming it for 5~10 seconds.

3. The shaping method according to claim 2, characterized in that, The method for preparing the pre-supported layer includes placing a liquid embedding agent in a low-temperature quick-freezing and shaping process. Preferably, the temperature for the low-temperature quick-freezing and shaping is ≤-20℃; Preferably, the thickness of the embedding agent cryopreservation support layer is 0.5~1mm; Preferably, the embedding agent is a water-soluble tissue embedding medium; Preferably, the embedding agent includes any one of OCT embedding agent, Tissue Freezing Medium, or Leica Cryo-Gel Embedding Medium.

4. The shaping method according to claim 1, characterized in that, The method for preparing the embedding agent drying film support layer includes spreading the embedding agent flat and then drying it.

5. The shaping method according to claim 4, characterized in that, The thickness of the embedding agent drying film support layer is 0.1~1mm; Preferably, the drying temperature is 37°C.

6. The shaping method according to claim 1, characterized in that, The PVA composite film support layer includes an adhesive layer and a PVA base layer covering the adhesive layer; Preferably, the adhesive layer comprises 1-5% sodium alginate, 1-10% glycerol, 0.1-0.5% sodium carboxymethyl cellulose and 0.05-0.2% calcium chloride by mass, and the solvent is DEPC-PBS; Preferably, the PVA base layer comprises 5-10% PVA, 1-10% glycerol and 0.01-0.05% n-octanol by mass, and the solvent is NF-Water; Preferably, the thickness of the PVA composite film support layer is 0.1~2mm; Preferably, the thickness ratio of the PVA base layer to the adhesive layer is 1:1 to 10:

1.

7. The shaping method according to claim 6, characterized in that, The method for preparing the PVA composite film support layer includes the following steps: A. Preparation of PVA base layer: The PVA base layer solution prepared according to the formula is obtained by scraping and gradient drying. B. Prepare the adhesive liquid layer. The adhesive solution prepared according to the formula is used to form an adhesive liquid layer by scraping. The PVA base layer obtained in step A is covered on the adhesive liquid film layer, and the PVA composite film support layer is obtained by roll pressing. Preferably, the roll-pressed composite is followed by freeze-drying and storage.

8. The shaping method according to any one of claims 1 to 7, characterized in that, The sheet-like intestinal tissue is pre-treated sheet-like intestinal tissue; The pretreatment includes removing contents, mucus, or excess surface moisture.

9. The application of the shaping method according to any one of claims 1 to 8 in the preparation of Swiss roll embedded morphology.

10. A method for embedding intestinal tissue in the form of a Swiss roll, characterized in that, Swiss roll-shaped intestinal tissue was prepared using the shaping method described in any one of claims 1 to 8 and then embedded in an embedding mold.

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