Frozen mixture and method for freezing tissue by using frozen mixture

Abstract

The invention discloses a frozen mixture and a method for freezing tissue by using the frozen mixture. The method comprises the following steps: placing an embedded tissue in a frozen mixture until an embedding medium is completely solidified; wherein the frozen mixture comprises dry ice, absolute ethyl alcohol and glycerol. According to the method provided by the invention, the disintegration of tissues can be reduced, good tissue immunogenicity is reserved, and the slice quality is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a freezing mixture and a method for freezing tissue using the same. Background Art

[0002] Tissue Cross-Reactivity (TCR) test is an in vitro test for detecting the binding of antibody drugs to antigenic epitopes on tissues. The purpose is to confirm the binding of the test antibody to the target tissue antigen epitope of the drug efficacy, and to detect whether the antibody binds to the non-target tissue antigen and causes serious adverse reactions, so as to provide a reference for determining the relevant animal species for non-clinical safety tests and predicting toxic target organs. Conducting TCR tests is an important part of the preclinical safety evaluation of antibody drugs, which is crucial for the research and development of antibody drugs and is also a requirement of domestic and foreign regulatory agencies. Immunohistochemistry (IHC) is the core technology of TCR tests. It uses fresh frozen or chemically fixed tissue specimens for experiments to locate and semi-quantitate the corresponding antigens.

[0003] The processing of tissue specimens is the premise of reliable tissue cross-test results. Chemical fixation of tissues will cause the loss of antigen epitopes and weak antigen immunoreactivity, so fresh tissues are generally directly frozen for subsequent sectioning and antigen-antibody reaction experiments. The embedding agent used for freezing is OCT (Optimal Cutting Temperature Compound, a water-soluble mixture of polyethylene glycol and polyvinyl alcohol). During operation, care should be taken to avoid slow freezing and repeated freezing and thawing. At present, the main method used at home and abroad is to directly immerse the tissue in liquid nitrogen for quick freezing and embedding. However, because the temperature of the tissue drops sharply from room temperature to liquid nitrogen, and a huge temperature difference will instantly occur inside and outside the tissue, it is very easy to cause the tissue to disintegrate or fragment during the quick freezing process, resulting in tissue morphology fragmentation, poor section quality, and serious detachment during sectioning. Problems, thereby interfering with the accuracy of the TCR test, causing adverse effects such as misjudgment of pathological diagnosis, and sometimes even leading to the inability to collect samples and subsequent tests. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the method of directly freezing tissue by immersing the tissue in liquid nitrogen in the prior art causes the tissue to disintegrate or fragment, resulting in the problem of broken tissue morphology, poor slice quality, and serious detachment during slicing. The present invention provides a method for collecting frozen tissue of cynomolgus monkeys for tissue cross-reaction tests. The method can well ensure the integrity of frozen tissue blocks, the antigenic immune activity of tissues, and improve the slice quality, and effectively collect cynomolgus monkey tissues for tissue cross-reaction tests.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for freezing tissue, which comprises: placing the embedded tissue in a freezing mixture until the embedding agent is completely solidified; wherein the freezing mixture comprises dry ice, anhydrous ethanol and glycerol.

[0007] In some embodiments of the present invention, the volume ratio of anhydrous ethanol to glycerol is 50-120:1, for example, 100:1; the volume ratio of dry ice to anhydrous ethanol is 0.4-0.7:1, for example, 0.4:1 or 0.7:1.

[0008] In some preferred embodiments of the present invention, the embedded tissue is placed into the freezing mixture after the freezing mixture is mixed uniformly for 5-10 minutes.

[0009] In some embodiments of the present invention, the method further comprises: placing the metal box containing the mixture in a low temperature environment.

[0010] In some embodiments of the invention, the method satisfies one or more of the following conditions:

[0011] The embedding medium is OCT;

[0012] The embedding is to bury the tissue in the embedding agent;

[0013] The tissue is selected from the group consisting of liver, heart, lung, spleen, kidney, ureter, stomach, pituitary, adrenal gland, thyroid, parathyroid gland, bladder, cerebellum, cerebral cortex, colon, small intestine, eyeball, lymph node, breast, pancreas, prostate, skin, muscle, testis, thymus, uterus, ovary, fallopian tube, bone marrow, vascular endothelium, blood cells and spinal cord;

[0014] The low temperature environment is a low temperature liquid.

[0015] In a specific embodiment of the invention, the tissue is liver.

[0016] In a specific embodiment of the present invention, the low temperature environment is liquid nitrogen.

[0017] In the present invention, the kidney comprises glomerulus and renal tubule; and the uterus comprises cervix and endometrium.

[0018] In some embodiments of the present invention, the method further comprises: a step of collecting tissue, wherein the collecting method is to remove the tissue after dissecting the organism.

[0019] In some preferred embodiments of the present invention, the organism is an animal.

[0020] In some more preferred embodiments of the present invention, the animal is a mammal.

[0021] In a specific embodiment of the invention, the mammal is a cynomolgus monkey.

[0022] The present invention also provides a frozen mixture, which is the frozen mixture as described above.

[0023] The present invention also provides a method for preparing the frozen mixture as described above, the method comprising: uniformly mixing dry ice, anhydrous ethanol and glycerol according to a certain proportion.

[0024] In some preferred embodiments of the present invention, the mixing is to firstly mix anhydrous ethanol and glycerol uniformly according to a certain proportion, and then add them into dry ice.

[0025] In some embodiments of the invention, anhydrous ethanol and / or glycerol is pre-chilled.

[0026] In some preferred embodiments of the present invention, the precooling temperature of anhydrous ethanol is -20°C; the precooling temperature of glycerol is 0°C.

[0027] The present invention also provides use of the freezing mixture as described above or the freezing mixture prepared by the method as described above in tissue freezing.

[0028] The present invention also provides use of the freezing mixture as described above or the freezing mixture prepared by the method as described above in preparing a tissue freezing reagent.

[0029] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0030] The reagents and raw materials used in the present invention are commercially available.

[0031] The positive and progressive effect of the present invention is that the tissue freezing method provided by the present invention can reduce tissue collapse, retain good tissue immunogenicity, and improve the quality of slices for use in antibody drug tissue cross-reaction tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a photo of the frozen tissue block in Example 1 of the present invention.

[0033] Figure 2 This is a photo of the frozen tissue block in Comparative Example 1.

[0034] Figure 3 This is a microscopic photograph of TCR test staining of frozen sections under a 100x microscope in Example 1 of the present invention.

[0035] Figure 4This is a micrograph of TCR staining of frozen sections under a 100x microscope in Comparative Example 1. DETAILED DESCRIPTION

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0037] The unembedded tissues in the following examples are all fresh liver tissues of cynomolgus monkeys.

[0038] The present invention is described in further detail below in conjunction with examples:

[0039] The method for collecting frozen tissue of cynomolgus monkey for tissue cross-reaction test of the present invention comprises the following steps:

[0040] Example 1

[0041] (1) Cynomolgus monkey dissection and collection of fresh tissues:

[0042] ① After the cynomolgus monkeys were anesthetized, the jugular vein was fully bled, and conventional dissection techniques were used to perform autopsies and remove fresh tissues from the cynomolgus monkeys, removing excess fibers and fat from the tissues to ensure the integrity and cleanliness of the tissue surface;

[0043] ② The order of dissection and organ collection is as follows: body surface, subcutaneous, abdominal cavity, pelvic cavity, oral cavity, neck, thoracic cavity, cranial cavity, spinal cord, muscle, peripheral nerve, bone;

[0044] ③ The collection is based on the 33 types of tissues required by the US Food and Drug Administration (FDA) for TCR testing;

[0045] ④ During the operation, attention should be paid to proceed with the subsequent steps immediately after collecting a single tissue, and the dissection and collection of the remaining tissues should be carried out after the operation is completed.

[0046] (2) Precooling of embedding reagents and tools:

[0047] Precool tissue embedding reagents and tools in advance to reduce the temperature difference when freezing tissue and reduce the probability of frozen tissue cracking. The specific method is as follows:

[0048] ①OCT embedding medium: Place the OCT embedding medium and packaging in an ice-water mixture (0°C) 30-60 min in advance for precooling, and take it out when it is used;

[0049] ②PBS solution: Place the PBS solution and packaging in an ice-water mixture (0°C) for precooling 30-60 min in advance and take it out when it is used;

[0050] ③ Anhydrous ethanol: Put the anhydrous ethanol and its packaging into a -20℃ refrigerator for precooling 6 hours in advance and take it out when it is used;

[0051] ④ Glycerol: Place the glycerol and its packaging in an ice-water mixture (0°C) for precooling 60 minutes in advance and take it out when it is used;

[0052] ⑤ Disposable cryoembedding mold (2.2 cm*2.2 cm*2.0 cm): Place in a foam box with sufficient dry ice 30 minutes in advance for precooling and take out when needed;

[0053] ⑥ Frozen metal plates: Place them in a foam box filled with sufficient dry ice 30 minutes in advance to pre-cool them, and take them out when they are needed;

[0054] ⑦ Frozen metal box (length, width and height are 40 cm*20 cm*4 cm respectively): Place it in a foam box filled with sufficient dry ice for 30 minutes in advance to pre-cool it, and take it out when needed.

[0055] (3) Preparation of the "dry ice-anhydrous ethanol-glycerol" triple system quick-freezing tissue fluid first invented by the present invention:

[0056] The invention firstly prepared the "dry ice-anhydrous ethanol-glycerol" triple system quick-freezing tissue fluid. The specific method is as follows:

[0057] ① Prepare a foam box with an inner diameter of 50 cm, length, width and height of 30 cm, and 10 cm, and pour about 8 cm of liquid nitrogen into it. Be sure to use thermal gloves to avoid frostbite;

[0058] ②Put the pre-cooled frozen metal box into the foam box and fix it appropriately so that it is suspended in the foam box but cannot move at will; then prepare a mixed solution of pre-cooled anhydrous ethanol and glycerol in a ratio of 100:1 and pour it into the frozen metal box. The depth of the mixed solution is about 1.0 cm;

[0059] ③ Slowly place the dry ice strips into the freezing metal box, so that the depth of the quick-freezing tissue fluid of the "dry ice-anhydrous ethanol-glycerol" triple system is between 1.4-1.7 cm. Once the solution is prepared, the tissue quick-freezing operation can be performed;

[0060] ④ During use, dry ice will continue to evaporate, causing the components to continue to decrease. When the tissue fluid is less than 1.4 cm deep, dry ice should be replenished in time to maintain the quick-frozen tissue fluid system.

[0061] (4) Full integration and OCT embedding and freezing of fresh tissues of cynomolgus monkeys:

[0062] Full integration of fresh tissue from cynomolgus monkeys:

[0063] ① Rinse away the remaining blood with pre-cooled PBS solution and gently cover the tissue with clean sterile gauze to absorb moisture;

[0064] ②Use surgical forceps to gently grasp the tissue that has been cleaned of PBS and place it horizontally in the pre-cooled OCT embedding medium, so that the surface of the tissue is evenly coated with OCT embedding medium to reduce the air bubble gap between the tissue and the embedding medium during embedding and freezing;

[0065] OCT embedding and freezing:

[0066] ③ Squeeze the pre-cooled OCT embedding medium into a disposable freezing embedding box (about 1 / 3 of the height of the embedding box), gently suck out the bubbles with a disposable syringe without a needle, and place it on ice to pre-cool slightly;

[0067] ④ Gently clamp the tissue wrapped with OCT embedding agent into the embedding box, add OCT embedding agent to make the tissue completely immersed, and the embedding agent liquid level is ≥3 mm above the tissue. Use a syringe to absorb the bubbles or use tweezers to push away the bubbles to avoid bubbles around the tissue;

[0068] ⑤ Clamp the edge of the embedding box with hemostats and transfer it to the "dry ice-anhydrous ethanol-glycerol" triple system quick-freezing tissue solution. When the embedding agent is completely solidified (about 3-5 minutes) and no longer transparent, use hemostats to remove the embedding box, use the temperature of your fingers to slightly heat the surrounding of the embedding box and demold it, turn it upside down and tap it gently to make the embedding block fall off. Note: For tissues that are easy to float, such as the lungs, use blunt-tipped forceps to fix the tissue when the embedding agent begins to solidify.

[0069] (5) Storage of frozen tissue specimens of cynomolgus monkeys:

[0070] ① Prepare a piece of tin foil of appropriate size (about 15 cm × 10 cm in length and width), and write the sample number, name, date and other information in the middle of the upper edge of the matte surface with a marker;

[0071] ② Use the smooth side of tin foil to wrap the demolded frozen tissue, with the information side facing up, and then quickly put it into dry ice to keep it frozen;

[0072] ③ After forming a steady state, transfer it into a storage box, affix a waterproof note with information such as species, number, name, date, etc. on the box surface, and store it in a -80℃ refrigerator for subsequent frozen sections, which are used for tissue cross-reaction tests;

[0073] (6) TCR test

[0074] The frozen tissue blocks in Example 1 and Comparative Example 1 were serially sectioned and immunohistochemically stained using an automatic staining machine to minimize human differences. Primary antibody: abcam CD31, 1:1000.

[0075] Comparative Example 1

[0076] (1) Non-precooling of reagents and tools for dissection and embedding of cynomolgus monkeys:

[0077] Normal and healthy cynomolgus monkeys were anesthetized and bled, and their tissues were removed in sequence using dissection techniques to remove excess fat and fiber from the tissues to ensure that the tissues were clean and complete, and to avoid the appearance of ice crystals during tissue slicing to facilitate the preparation of slices.

[0078] Place OCT embedding medium, PBS, anhydrous ethanol, freezing embedding mold, metal plate and metal box at room temperature for later use.

[0079] (2) Collection and full integration of fresh tissues of cynomolgus monkeys OCT:

[0080] Take a 1.5 cm × 1.5 cm × 3 mm size fresh liver tissue of cynomolgus monkey, rinse the residual blood with room temperature PBS and absorb the water with clean sterilized gauze. Put the tissue with PBS absorbed into room temperature OCT embedding medium, so that the surface of the tissue is evenly covered with OCT embedding medium to reduce the air bubble gap between the tissue and the embedding medium during embedding and freezing.

[0081] (3) OCT embedding and freezing of fresh tissues of cynomolgus monkeys:

[0082] Squeeze the room temperature OCT embedding medium into a disposable freezing embedding box (about 1 / 3 of the height of the embedding box), gently suck out the bubbles with a disposable syringe without the needle, and place it on ice to pre-cool slightly. Place the trimmed tissue in the embedding box, add OCT embedding medium, make the tissue completely immersed, and the embedding medium liquid level is ≥3 mm above the tissue. Use a syringe to suck out the bubbles or use tweezers to push away the bubbles to avoid bubbles around the tissue.

[0083] Place the embedding cassette directly into liquid nitrogen for quick freezing. When the embedding agent is completely solidified and no longer transparent, use hemostatic forceps to remove the embedding cassette. Use the temperature of your fingers to slightly heat the surrounding area of ​​the embedding cassette and demould it. Turn it upside down and tap it gently to make the embedded block fall off.

[0084] (4) Storage of frozen tissue specimens of cynomolgus monkeys:

[0085] Prepare a suitable tin foil of about 15 cm × 10 cm in length and width, and write the sample number, name, date and other information in the middle of the upper edge of the matte surface with a marker. Use the glossy side of the tin foil to wrap the demolded frozen tissue so that the information side is facing up, and then quickly put it in dry ice to keep it frozen. After forming a steady state, transfer it to a storage box, and affix a waterproof note with information such as species, number, name, date, etc. on the surface of the box, and store it in a -80℃ refrigerator for subsequent frozen sections, which are used for tissue cross-reaction tests.

[0086] result

[0087] The fresh liver frozen tissue blocks of cynomolgus monkeys in Example 1 were well preserved without any cracks ( Figure 1 ), the subsequent sections were not broken, the sections were intact, and the TCR test showed that the tissue antigen immunity was well preserved, the staining was clear, the vascular endothelial cells were obviously positive, the tissue edges were clear, and there was little non-specific staining ( Figure 3 ). Reduce the difficulty of slice evaluation and TCR test evaluation.

[0088] In control example 1, the tissue block collapsed without human intervention ( Figure 2 ), which increases the difficulty of tissue block preservation and slicing. The integrity of tissue slices is destroyed. The TCR test shows that the staining of the target area is acceptable, but the overall staining is not clear, and the tissue edge collapses, showing obvious non-specific staining ( Figure 4 ), which increases the difficulty of evaluating TCR test results.

[0089] In summary, the present invention uses pre-cooling reagents and embedding tools, and uses a dry ice-anhydrous ethanol-glycerol mixture to embed frozen tissues, which can effectively reduce the problems of tissue cracking and fragmentation caused by direct liquid nitrogen freezing embedding. The subsequent frozen tissue sections have good integrity, good tissue morphology, and high immunoretention of tissue surface antigens. This method can be used for the collection of cynomolgus monkey frozen tissues for tissue cross-reaction experiments.

Claims

1. A method for tissue freezing, characterized in that: The method comprises: placing the embedded tissue in a freezing mixture until the embedding agent is completely solidified; wherein the freezing mixture contains dry ice, anhydrous ethanol and glycerol.

2. The method according to claim 1, characterized in that The volume ratio of anhydrous ethanol to glycerol is 50-120:1, for example, 100:1; the volume ratio of dry ice to anhydrous ethanol is 0.4-0.7:1, for example, 0.4:1 or 0.7:1; Preferably, the embedded tissue is placed into the freezing mixture after the freezing mixture is mixed for 5-10 minutes.

3. The method according to claim 1, characterized in that The method further comprises placing the metal box containing the frozen mixture in a low temperature environment.

4. The method according to claim 3, characterized in that The method meets one or more of the following conditions: The embedding medium is OCT; The embedding is to bury the tissue in the embedding agent; The tissue is selected from the group consisting of liver, heart, lung, spleen, kidney, ureter, stomach, pituitary, adrenal gland, thyroid gland, parathyroid gland, bladder, cerebellum, cerebral cortex, colon, small intestine, eyeball, lymph node, breast, pancreas, prostate, skin, muscle, testis, thymus, uterus, ovary, fallopian tube, bone marrow, vascular endothelium, blood cells and spinal cord, for example, liver; The low temperature environment is a low temperature liquid, such as liquid nitrogen.

5. The method according to any one of claims 1 to 4, characterized in that: The method further comprises: a step of collecting tissue, wherein the collecting method is to remove the tissue after dissecting the organism; Preferably, the organism is an animal; More preferably, the animal is a mammal, such as a cynomolgus monkey.

6. A frozen mixture, characterized in that The frozen mixture is as claimed in claim 1 or 2.

7. A method for preparing the frozen mixture as claimed in claim 6, characterized in that: The method comprises: mixing dry ice, anhydrous ethanol and glycerin uniformly in proportion; Preferably, the mixing is to first mix anhydrous ethanol and glycerol evenly in proportion, and then add them into dry ice.

8. The method according to claim 7, characterized in that Anhydrous ethanol and / or glycerol are pre-cooled; Preferably, the precooling temperature of anhydrous ethanol is -20°C; the precooling temperature of glycerol is 0°C.

9. Use of the freezing mixture according to claim 6 or the freezing mixture prepared by the method according to claim 7 or 8 in tissue freezing.

10. Use of the freezing mixture according to claim 6 or the freezing mixture prepared by the method according to claim 7 or 8 in preparing a tissue freezing agent.

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