Preparation method of tissue slice patch based on tissue chip

Abstract

The invention provides a tissue slice patch preparation method based on a tissue chip. The tissue slice patch preparation method comprises the following steps: mixing raw materials to prepare a receptor wax block, collecting a tissue core and transferring the tissue core to the receptor wax block to form a fused wax block, cooling the fused wax block, storing the fused wax block, and carrying out operations of pre-cooling correction, slice unfolding and slice baking. According to the tissue chip-based tissue slice patch preparation method, the tissue chip can be better protected, and the integrity of the tissue chip is ensured.

Description

Technical Field

[0001] The present invention relates to the application field of tissue cores, and in particular to a method for preparing a tissue slice patch based on a tissue chip. Background Art

[0002] In the traditional tissue chip production process, due to the limitations of manual operation and errors in naked eye observation, invalid points often appear when constructing medium and high density tissue chips, that is, some tissue cores are not accurately sampled or missed. The increase of these invalid points not only reduces the quality of tissue chips, but also may affect the accuracy and reliability of subsequent experimental results. In order to solve this problem, a tissue slice patch preparation method based on tissue chips came into being.

[0003] This emerging technology makes full use of the high resolution and magnification capabilities of digital scanners to obtain clear magnified images by scanning paraffin sections. These images not only provide researchers with more intuitive and detailed sample information, but also make microscopic marking accurate and efficient. On this basis, researchers can accurately locate and mark the required tissue areas, thereby greatly reducing the generation of invalid points when constructing tissue chips.

[0004] Compared with traditional technologies, this tissue chip-based tissue slice patch preparation method significantly improves the core quality and accuracy of tissue chips. It not only optimizes the sample processing flow and improves operational efficiency, but also ensures that the information extracted from each tissue sample is highly representative and accurate.

[0005] However, during the wax block production process, poor control of operating parameters or poor stability of the wax block itself will also affect the quality and accuracy of the tissue core.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a tissue slice patch based on a tissue chip, which improves the quality and accuracy of the tissue core by optimizing the wax block and the operation process.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The present invention provides a method for preparing a tissue slice patch based on a tissue chip, comprising the following steps: mixing raw materials to prepare a receptor wax block, collecting tissue cores and transferring them to the receptor wax block to form a fused wax block, cooling the fused wax block and then storing it, and performing operations of pre-cooling correction, slicing, spreading, and baking.

[0010] Preferably, as a further feasible scheme, the receptor wax block is prepared by using the following raw materials: by mass, 100-110 parts of lycra paraffin, 3-5 parts of beeswax, 2-6 parts of graphene, 1-2 parts of rapeseed oil, and 0.5-1 part of fiber filaments.

[0011] Preferably, as a further feasible solution, 105 parts of Lycra paraffin, 4 parts of beeswax, 5 parts of graphene, 1.3 parts of rapeseed oil, and 0.7 parts of fiber filaments.

[0012] In the process of preparing the acceptor wax block, in addition to using lycra paraffin and beeswax as the basic components of the wax block, graphene, rapeseed oil and fiber filaments are also added for matching, because the addition of graphene and rapeseed oil can increase the binding property of paraffin with the tissue core, increase lubricity and the addition of fiber filaments can also provide a certain degree of support, because the subsequent tissue core is transferred to the acceptor wax block, it is necessary to ensure the support and stability of the wax block and also to ensure that the tissue core and the wax block can be better combined, so in order to meet the above requirements, the present invention specially optimizes the preparation components of the acceptor wax block to meet the needs of the wax block. Of course, the addition amount of each component should also be noted in the dosage of each component, especially graphene and fiber filaments, which mainly play an auxiliary role, and the addition amount does not need to be too large, because if the hardness of the wax block itself is too large or the lubricity is too high, it will also affect the stability when the tissue core is loaded, so the dosage of each component needs to be noted in the process of compatibility.

[0013] The fiber filaments used can be carbon fiber, of course, other chemical fibers can also be selected, and the amount added will not be too large. Of course, try to choose fiber filaments with better support. In order to facilitate adding, the fibers can be chopped in advance.

[0014] The process of preparing the receptor wax block is as follows:

[0015] Material mixing: Take the raw materials of the acceptor wax block and mix them to make a blank wax block with a length of 40-44 mm, a width of 22-24 mm, and a height of 8-10 mm. Design (11-12)×(7-8) point organization array within the range of (21-22) mm×(18-19) mm of the wax block;

[0016] Punching: Use ATA-27 fully automatic tissue chip spotter to punch holes with a diameter of 1.10-1.12 mm on blank wax blocks.

[0017] And before building the tissue core, there are the following steps:

[0018] Planned number of samples: Before constructing the tissue array, arrange 80-100 tissue samples confirmed by HE sectioning on the slide;

[0019] Mark representative points: Design the melting temperature of the receptor wax block so that it can combine smoothly with the tissue core during spotting. Design the sample interval to be 0.9-1.22mm. Reasonable intervals help maintain the independence and integrity of the samples and ensure the accuracy and reliability of the experimental results to prevent the tissue cores from interfering with or fusing with each other. Reserve a left edge area for writing or labeling in the wax block design.

[0020] In the above operation mode, by designing the microarray in advance, researchers can arrange a suitable number of samples on a limited slide to improve sample utilization, and reasonable sample layout helps reduce cross-contamination and experimental errors, thereby improving the reliability of the experiment.

[0021] Preferably, as a further feasible scheme, during the process of collecting the tissue core and transferring it to the recipient wax block, an aldehyde-free fixative is used for fixation, wherein the aldehyde-free fixative is mainly composed of the following components: in parts by mass, 2-3 parts of epichlorohydrin, 4-7 parts of toluene diisocyanate, 1-2 parts of ethanol, and 3-4 parts of glycidol.

[0022] Preferably, as a further feasible scheme, in parts by mass, epichlorohydrin is 2.2-2.8 parts, toluene diisocyanate is 5-6 parts, ethanol is 1.2-1.8 parts, and glycidol is 3.2-3.8 parts.

[0023] The process of collecting tissue cores and transferring them to the recipient wax block is as follows:

[0024] Collecting tissue cores: Use a fine needle with a diameter of 1.0 mm to extract the cylindrical tissue sample marked on the left edge of the donor wax block. This fine needle can accurately extract the tissue core of the required size while reducing damage to the surrounding tissue. This step needs to be performed slowly in a sterile environment to avoid damaging the donor wax block or tissue core.

[0025] Transfer and fix: inject the fine needle extracted tissue sample into the wells of neatly arranged recipient wax blocks and fix the tissue core with aldehyde-free fixative;

[0026] Fusion: Place the recipient wax block that receives the tissue core in an incubator and heat it so that the tissue core and the recipient wax block become one.

[0027] The formaldehyde-free fixative in the above steps is mainly used for fixing. It is a type of fixative that does not contain formaldehyde and is used for the preparation and preservation of biological tissue samples. Its components include alcohols, ketones or other cross-linking agents. The present invention can better fix the biological tissue samples by combining specific components with each other without causing any damage to the samples. In particular, the added ethanol and propylene oxide also have a good protective effect on the samples. Therefore, the addition of ethanol and propylene oxide needs to be carried out according to the scheme of the present invention to ensure that the fixative of the present invention can not only play a certain fixing role, but also play a protective role on the samples at the same time.

[0028] Preferably, as a further feasible solution, a staged heating mode is adopted during the fusion process, and the acceptor wax block receiving the tissue core is first heated at 55-60°C for 2-3 minutes, and then heated at 70-80°C for 4-5 minutes. The reason for adopting the staged heating process is that if the temperature is too high at the beginning, it will have a certain destructive effect on the tissue core if the two are not well fused. Therefore, through two-stage temperature control, it can ensure the smooth fusion without damage. And through practice, it is found that keeping in this temperature range can ensure that the tissue core and the acceptor wax block are smoothly combined during spotting, and the structure and composition of the tissue sample will not be damaged due to excessive temperature.

[0029] During the sample collection process, it is best to preheat the donor wax block at 37-40°C before collecting the sample. The preheated paraffin becomes softer and more pliable, thereby reducing the breakage of the paraffin during the punching process. It also makes it easier to insert the punching needle into the preheated paraffin, reducing the risk of bending of the punching needle and ensuring the accuracy of fine needle sampling.

[0030] Generally, when performing subsequent operations, the fused tissue core wax block is taken out of the incubator. This step requires careful operation to avoid displacement or damage of the tissue core due to improper operation. It is cooled to room temperature. This process requires patience to ensure that the temperature inside and outside the wax block is uniform to avoid internal stress caused by excessive temperature difference, which will affect the stability of the wax block and the quality of subsequent sectioning. After complete cooling, the wax block is placed in a 4-6℃ refrigerator for future use. Refrigerated storage can maintain the appropriate hardness of the wax block, prevent softening or deformation caused by temperature changes, and ensure the smooth progress of the subsequent sectioning process.

[0031] Preferably, as a further implementable solution, the precooling process is: precooling in an environment of 1°C-3°C for 3-4 hours, the temperature fluctuation range during the entire precooling process cannot exceed 0.5-1°C, and correction is performed after precooling. Generally, the precooled wax block is clamped on a slicer for correction until all tissues are completely exposed. During correction, the slicer and operating environment are kept under sterile conditions. In order to ensure the integrity of the tissue during the precooling process, the temperature fluctuation cannot be too large, so it is best that the temperature fluctuation range does not exceed 0.5-1°C.

[0032] The quality of slices can be improved by precooling and correcting the wax block. Precooling makes the hardness of the wax block uniform and reduces compression and deformation during the slicing process. Correction ensures that all tissue cores can be cut completely, avoiding the problem of missing or incomplete slices. The combined effect of precooling and correction makes the slices obtained smoother and more continuous.

[0033] Preferably, as a further feasible solution, the following freezing operation is required after precooling before slicing: freezing at a temperature of -20°C to -18°C for 9-11 minutes.

[0034] Preferably, as a further implementable solution, the process of slicing and spreading the slices is: slicing is performed in a temperature environment maintained at -20°C to -15°C;

[0035] Place the slices in ice water at -5℃ to 0℃ to unfold them naturally, use the temperature of cold water to help the slices flatten, and then transfer them to warm water at 40-45℃ for 3-4 minutes to fully unfold the slices through the temperature difference, and finally stick them on a sheet soaked with APES slice adhesive to dry, to prevent the slices from falling off during subsequent processing.

[0036] After removal, continuous sectioning was performed using a cryo-ultra-thin microtome with a thickness of 3.5-3.6 μm. A heat preservation film was used next to the cryo-microtome for low temperature protection to maintain the sectioning at a temperature of -20°C to -15°C.

[0037] Preferably, as a further implementable solution, the method for baking the slices comprises the following steps: baking the slices at 60-65°C for 3-4 minutes, pausing for 2.0-2.5 minutes, and then baking the slices at 58-60°C for 16-17 hours, and finally taking out the slices and storing them at -20°C to -18°C for later use.

[0038] Specifically, place the slides in a slide drying machine at 60-65°C for baking, set the baking time to 3-4 minutes, pause the slide drying machine for 2.0-2.5 minutes, then set the temperature of the slide drying machine to 58-60°C and continue baking the slides for 16-17 hours. Finally, take out the slices and store them in a sterile refrigerator at -20°C to -18°C for future use. The storage temperature of the slices is between -20°C and -18°C.

[0039] During the baking process, the temperature and time need to be closely monitored to ensure that each slide is heated evenly. At the same time, avoid overheating to avoid destroying the antigen structure in the tissue and affecting the results of subsequent experiments. Finally, take out the slices and store them in a sterile refrigerator at -20℃ to -18℃ for standby use. The storage temperature of the slices is between -20℃ and -18℃. This temperature can effectively prevent the degradation of tissue samples and maintain their biological characteristics. In addition, low-temperature storage also helps to reduce the potential contamination of samples by bacteria or other microorganisms, so that the processed slices can be used for subsequent staining, immunohistochemistry and other experiments.

[0040] The roles of the baking and preservation steps in the tissue chip preparation process are as follows: baking helps the tissue sections to adhere firmly to the slides, while the appropriate preservation temperature can maintain the integrity and biological activity of the samples, thus providing reliable guarantees for subsequent experiments, helping to improve the success rate of the experiments and avoiding potential sample damage and experimental errors.

[0041] Compared with the prior art, the solution proposed by the present invention has the following beneficial effects:

[0042] 1. By arranging a suitable number of samples on a limited slide, the utilization rate of the samples is improved, and reasonable sample layout helps to reduce cross contamination and experimental errors, thereby improving the reliability of the experiment.

[0043] 2. The added aldehyde-free fixative can effectively fix tissue samples and maintain the morphological and molecular biological characteristics of the tissue. Therefore, the use of aldehyde-free fixative and appropriate fixation methods can reduce the degeneration and damage of the tissue core and maintain the integrity of its cell structure and biological characteristics, which is helpful for accurate analysis of tissue samples in subsequent experiments.

[0044] 3. The present invention can improve the quality of slices through the steps of precooling and correcting the wax block. Precooling makes the hardness of the wax block uniform and reduces compression and deformation during the slicing process, while correction ensures that all tissue cores can be cut completely, avoiding the problem of missing or incomplete slices. Therefore, the combined effect of precooling and correction is to make the slices smoother and more continuous. During the entire operation process, it can solve the problem of invalid points in high-density tissue chips during the traditional tissue chip production process. DETAILED DESCRIPTION

[0045] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0046] Example 1

[0047] Step 1: Plan the number of samples: Before constructing the tissue chip, arrange 80-100 tissue samples confirmed by HE sectioning on the slide, including tumor tissues at different development stages and corresponding normal tissues;

[0048] Step 2: Take 105g of Lycra paraffin, 4g of beeswax, 5g of graphene, and 1.3g of rapeseed oil as raw materials and heat to melt, 1.2g / cm 3 0.7g of carbon fiber was chopped into powder and added to make a receptor wax block. According to the sample requirements, the corresponding mass was taken out to make a blank wax block with a length of 40mm, a width of 24mm, and a height of 8mm. A 12×8 point tissue array was designed within the range of 21mm×19mm in the wax block. A hole with a diameter of 1.10 was punched on the blank wax block using an ATA-27 fully automatic tissue chip spotter.

[0049] Step 3: Use a fine needle with a diameter of 1.0 mm to extract the marked cylindrical tissue sample from the left edge area of ​​the donor wax block;

[0050] Step 4: first prepare an aldehyde-free fixative, using raw materials 2.2g of epichlorohydrin, 6g of toluene diisocyanate, 1.2g of ethanol, and 3.8g of glycidol to prepare an aldehyde-free fixative for use, inject the tissue sample extracted by a fine needle into the holes of the neatly arranged receptor wax block in step 2, and then take out part of the prepared aldehyde-free fixative to fix the tissue core;

[0051] Step 5: Place the recipient wax block that receives the tissue core in an incubator, heat it at 55°C for 2 minutes, and then heat it at 70°C for 4 minutes to fuse the tissue core and the recipient wax block together;

[0052] Step 6: Place the wax block containing the tissue core in a sterile refrigerator at 1°C for 3 hours, and clamp the precooled wax block on the slicer for correction until all tissues are completely exposed;

[0053] Step 7: Place the corrected wax block in a sterile refrigerator and freeze it at -18°C for 11 minutes. After taking it out, use a cryo-ultra-thin slicer to perform continuous slicing with a slice thickness of 3.5 μm. Use a heat preservation film next to the cryo-slicer for low-temperature protection to maintain the temperature of -20°C for slicing.

[0054] Step 8: Place the slices in -5°C ice water to unfold naturally, use the temperature of the cold water to help the slices flatten, and transfer them to 40°C warm water in sequence to unfold the slices for 3 minutes, and finally stick them on a slide soaked in APES slice adhesive to dry.

[0055] Step nine: Place the slides in a 60°C slide dryer for baking, set the baking time to 3 minutes, pause the dryer for 2.5 minutes, then set the temperature of the dryer to 58°C and continue baking for 16 hours. Finally, take out the slices and store them in a sterile refrigerator at -18°C for later use. The storage temperature of the slices is between -20°C and -18°C.

[0056] Example 2

[0057] Step 1: Plan the number of samples: Before constructing the tissue chip, arrange 80-100 tissue samples confirmed by HE sectioning on the slide, including tumor tissues at different development stages and corresponding normal tissues;

[0058] Step 2: Take 100g of Lycra paraffin, 5g of beeswax, 2g of graphene, and 2g of rapeseed oil as raw materials and heat to melt, 1.2g / cm 3 1g of carbon fiber was chopped into powder and added to make a receptor wax block. According to the sample requirements, the corresponding mass was taken out to make a blank wax block with a length of 44mm, a width of 22mm and a height of 10mm. An 11×7 point tissue array was designed within the range of 22mm×18mm in the wax block. ATA-27 fully automatic tissue chip spotter was used to punch holes with a diameter of 1.12mm on the blank wax block.

[0059] Step 3: Use a fine needle with a diameter of 1.0 mm to extract the marked cylindrical tissue sample from the left edge area of ​​the donor wax block;

[0060] Step 4: first prepare an aldehyde-free fixative, using raw materials 2g of epichlorohydrin, 7g of toluene diisocyanate, 1g of ethanol, and 4g of glycidol to mix and react to cross-link to prepare an aldehyde-free fixative for use, inject the tissue sample extracted by a fine needle into the holes of the neatly arranged receptor wax block in step 2, and then take out part of the prepared aldehyde-free fixative to fix the tissue core;

[0061] Step 5: Place the recipient wax block that receives the tissue core in an incubator, heat it at 60°C for 3 minutes, and then heat it at 80°C for 5 minutes to fuse the tissue core and the recipient wax block together;

[0062] Step 6: Place the wax block containing the tissue core in a sterile refrigerator at 3°C ​​for 4 hours. The temperature fluctuation during the precooling process should not exceed 0.5°C. Clamp the precooled wax block on the slicer for correction until all tissues are completely exposed.

[0063] Step 7: Place the corrected wax block in a sterile refrigerator and freeze it at -20°C for 9 minutes. After taking it out, use a cryo-ultra-thin slicer to perform continuous slicing with a slice thickness of 3.6 μm. Use a heat preservation film next to the cryo-slicer for low-temperature protection to maintain the temperature of -15°C for slicing.

[0064] Step 8: Place the slices in 0°C ice water to unfold naturally, use the temperature of the cold water to help the slices flatten, and transfer them to 45°C warm water in sequence to unfold the slices for 4 minutes. Finally, stick them on a slide soaked in APES slice adhesive and let them dry.

[0065] Step nine: Place the slides in a 65°C slide dryer for baking, set the baking time to 4 minutes, pause for 2.0 minutes, then set the temperature of the slide dryer to 60°C and continue baking for 17 hours. Finally, take out the slices and store them in a sterile refrigerator at -20°C for later use. The storage temperature of the slices is between -20°C and -18°C.

[0066] Example 3

[0067] Step 1: Plan the number of samples: Before constructing the tissue chip, arrange 80-100 tissue samples confirmed by HE sectioning on the slide, including tumor tissues at different development stages and corresponding normal tissues;

[0068] Step 2: Take 110g of Lycra paraffin, 3g of beeswax, 6g of graphene, and 1g of rapeseed oil as raw materials and heat to melt, 1.3g / cm 3 0.5g of carbon fiber was chopped into powder and added to make a receptor wax block. According to the sample requirements, the corresponding mass was taken out to make a blank wax block with a length of 44mm, a width of 22mm, and a height of 10mm. An 11×7 point tissue array was designed within the range of 22mm×18mm in the wax block. ATA-27 fully automatic tissue chip spotter was used to punch holes with a diameter of 1.12mm on the blank wax block.

[0069] Step 3: Use a fine needle with a diameter of 1.0 mm to extract the marked cylindrical tissue sample from the left edge area of ​​the donor wax block;

[0070] Step 4: first prepare an aldehyde-free fixative, using raw materials 3g of epichlorohydrin, 4g of toluene diisocyanate, 2g of ethanol, and 3g of glycidol to mix and react to cross-link to prepare an aldehyde-free fixative for use, inject the tissue sample extracted by a fine needle into the holes of the neatly arranged receptor wax block in step 2, and then take out part of the prepared aldehyde-free fixative to fix the tissue core;

[0071] Step 5: Place the recipient wax block that receives the tissue core in an incubator, heat it at 57°C for 3 minutes, and then heat it at 75°C for 5 minutes to fuse the tissue core and the recipient wax block together;

[0072] Step 6: Precool the wax block containing the tissue core in a sterile refrigerator at 2°C for 4 hours. The temperature fluctuation during the precooling process should not exceed 1°C. Clamp the precooled wax block on the slicer for correction until all tissues are completely exposed.

[0073] Step 7: Place the corrected wax block in a sterile refrigerator and freeze it at -19°C for 10 minutes. After taking it out, use a cryo-ultra-thin slicer to perform continuous slicing with a slice thickness of 3.6 μm. Use a heat preservation film next to the cryo-slicer for low-temperature protection to maintain the temperature of -17°C for slicing.

[0074] Step 8: Place the slices in -2°C ice water to unfold naturally, use the temperature of the cold water to help the slices flatten, and transfer them to 42°C warm water in sequence to unfold the slices for 4 minutes. Finally, stick them on a slide soaked in APES slice adhesive and let them dry.

[0075] Step nine: Place the slides in a 62°C slide dryer for baking, set the baking time to 4 minutes, pause for 2.2 minutes, then set the temperature of the slide dryer to 59°C and continue baking for 17 hours. Finally, take out the slices and store them in a sterile refrigerator at -19°C for later use. The storage temperature of the slices is between -20°C and -18°C.

[0076] Example 4

[0077] The specific operation steps are the same as those in Example 3, except that 105 g of Lycra paraffin, 4 g of beeswax, 5 g of graphene, and 1.3 g of rapeseed oil (1.3 g / cm 3 0.7 g of carbon fiber was used as raw material and heated and melted to make an acceptor wax block.

[0078] Example 5

[0079] The specific operation steps are consistent with those of Example 3, except that 2.8 g of epichlorohydrin, 5 g of toluene diisocyanate, 1.8 g of ethanol, and 3.2 g of glycidol are mixed and cross-linked to prepare a formaldehyde-free fixing agent for use.

[0080] Example 6

[0081] The specific operation steps are the same as those in Example 3, except that rapeseed oil is not added.

[0082] Example 7

[0083] The specific operation steps are the same as those in Example 3, except that the amount of carbon fiber added is 1.5 g.

[0084] Example 8

[0085] The specific operation steps are the same as those in Example 3, except that glycidol is not added.

[0086] Example 9

[0087] The specific operation steps are the same as those in Example 3, except for step 5: placing the acceptor wax block receiving the tissue core in an incubator and heating it at 57° C. for 8 minutes to fuse the tissue core and the acceptor wax block into one.

[0088] Comparative Example 1

[0089] The specific operation steps are the same as those in Example 3, except that the correction is performed directly without precooling.

[0090] Experimental Example 1

[0091] The states of the acceptor wax block after the tissue core melted and the final product of the above embodiment and the comparative example are compared, and the specific experimental results are shown in Table 1 below:

[0092] Table 1 Test results

[0093]

[0094] In the data in the above table, the data in the first two columns compare the performance of the recipient wax block during the fusion process of the tissue core, and the data in the last three columns compare the specific performance of the final slices and the final prepared products.

[0095] As can be seen from Table 1, the wax blocks of Examples 1 to 5 have excellent performances. However, when rapeseed oil is not added when preparing the wax block in Example 6, it cannot be compatible with graphene, which will affect the bonding between the wax block and the sample, and also affect the stability of the wax block itself, so its performance will be affected to a certain extent. Similarly, in Example 7, the strength of the prepared wax block is too high due to the large amount of carbon fiber added, which is also not conducive to slicing and the stability of the wax block itself.

[0096] In Example 8, since no glycidol is added, the protective effect on the sample will be affected.

[0097] Since Example 9 does not adopt a segmented fusion temperature control process, it is still impossible to avoid the risk of liquefaction of the wax block and the morphological damage of the tissue structure.

[0098] Since no precooling operation was performed in Comparative Example 1, it should be noted that precooling makes the hardness of the wax block uniform and reduces compression and deformation during slicing. Correction ensures that all tissue cores can be cut completely, avoiding the problem of missing or incomplete slices. The combined effect of precooling and correction makes the obtained slices smoother and more continuous. In Comparative Example 1, direct correction was performed, and the wax block without precooling was too soft, which would cause deformation of the wax block during slicing and affect its stability.

[0099] Finally, it is understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a tissue slice patch based on a tissue chip, characterized in that: The steps include: The raw materials are mixed to prepare a receptor wax block, tissue cores are collected and transferred to the receptor wax block to form a fused wax block, the fused wax block is cooled and stored, and the operations of pre-cooling correction, slicing, spreading and baking are performed.

2. The method for preparing a tissue slice patch according to claim 1, characterized in that: The receptor wax block is prepared by using the following raw materials: by weight, 100-110 parts of lycra paraffin, 3-5 parts of beeswax, 2-6 parts of graphene, 1-2 parts of rapeseed oil, and 0.5-1 part of fiber.

3. The method for preparing a tissue slice patch according to claim 2, characterized in that: 105 parts of Lycra paraffin, 4 parts of beeswax, 5 parts of graphene, 1.3 parts of rapeseed oil, and 0.7 parts of fiber.

4. The method for preparing a tissue slice patch according to claim 1, characterized in that: During the process of collecting the tissue core and transferring it to the recipient wax block, an aldehyde-free fixative is used for fixation, wherein the aldehyde-free fixative is mainly composed of the following components: 2-3 parts of epichlorohydrin, 4-7 parts of toluene diisocyanate, 1-2 parts of ethanol, and 3-4 parts of glycidol, by mass.

5. The method for preparing a tissue slice patch according to claim 4, characterized in that: In terms of mass, epichlorohydrin is 2.2-2.8 parts, toluene diisocyanate is 5-6 parts, ethanol is 1.2-1.8 parts, and glycidol is 3.2-3.8 parts.

6. The method for preparing a tissue slice patch according to any one of claims 1 to 5, characterized in that: During the fusion process, a staged heating mode is adopted, and the recipient wax block receiving the tissue core is first heated at 55-60°C for 2-3 minutes, and then heated at 70-80°C for 4-5 minutes.

7. The method for preparing a tissue slice patch according to any one of claims 1 to 5, characterized in that: The precooling process is: precooling for 3-4 hours in an environment of 1°C-3°C, during the whole precooling process, the temperature fluctuation range cannot exceed 0.5-1°C, and correction is performed after precooling.

8. The method for preparing a tissue slice patch according to claim 6, characterized in that: After precooling, the following freezing operation is required before slicing: Freeze at -20℃ to -18℃ for 9-11 minutes.

9. The method for preparing a tissue slice patch according to claim 6, characterized in that: The process of slicing and spreading the slices is as follows: slicing is performed in an environment where the temperature is maintained at -20°C to -15°C; The slices were spread naturally in ice water at -5°C to 0°C, and transferred to warm water at 40-45°C in sequence for 3-4 minutes, and finally attached to a sheet soaked in APES slice adhesive and allowed to dry.

10. The method for preparing a tissue slice patch according to claim 6, characterized in that: The method for baking slices comprises the following steps: baking the slices at 60-65° C. for 3-4 minutes, pausing for 2.0-2.5 minutes, and then baking the slices at 58-60° C. for 16-17 hours, and finally taking out the slices and storing them at -20° C. to -18° C. for future use.

Images