Tissue decoloring, deblood and decalcification composition and application thereof

Abstract

The invention relates to a tissue decoloring, blood-removing and decalcifying composition and application thereof. The tissue decoloring, blood-removing and decalcifying composition comprises: a) a divalent cation chelating agent; b) an imidazole reagent, wherein the imidazole reagent is imidazole with amino alkyl; and c) water. The tissue decoloring, deblood and decalcification composition can realize decalcification, deblood and decoloring of biological tissues at the same time, and can realize decalcification and decoloring effects better than those of an existing decalcification reagent and an existing decoloring reagent respectively.

Description

Technical Field

[0001] This invention relates to the field of tissue processing, and more specifically to a tissue decolorization, deblemishing, and decalcification composition and its application. Background Technology

[0002] Novel three-dimensional biological technologies combine thick tissue sectioning, thick tissue fluorescence labeling, tissue clearing, and three-dimensional fluorescence imaging to obtain microscopic three-dimensional images of biological tissues. Compared to two-dimensional histology, which observes a cross-section only a few micrometers thick, three-dimensional histology examines a large number of tissue cells from a comprehensive and realistic perspective. This allows for a more accurate and comprehensive assessment of histopathological mechanisms and a more detailed study of drug mechanisms and physiological phenomena. Therefore, the development and optimization of three-dimensional histological imaging technology has immense value in clinical diagnosis, scientific research, and drug development.

[0003] In order to perform procedures such as clearing sections of bone-containing or pathologically calcified tissues, decalcification is usually required. Existing decalcification agents typically perform decalcification under neutral conditions using calcium ion chelating agents, and can only proceed to the next step after the bone tissue has softened. The decalcification efficiency and effectiveness are limited.

[0004] On the other hand, human specimens or animal spleens, red bone marrow, and other blood-containing tissues cannot be perfused, or perfusion cannot remove the red blood cells. These samples with severe blood staining must undergo deblooding to achieve good tissue transparency or reduce the strong interference of blood autofluorescence on fluorescence imaging. Existing deblooding reagents have limited efficacy and cannot quickly and effectively remove the pigments from clotted blood in these tissues.

[0005] In addition, tissues such as myocardium and skeletal muscle contain myoglobin, while tissues such as liver and kidneys contain more pigment components such as flavin and lipofuscin. These components can also affect the transparency of tissues and the effect of fluorescence imaging.

[0006] Therefore, it is necessary to develop a reagent that can comprehensively achieve decalcification, decolorization, and dehemidation of tissues, which has great application prospects in bioimaging, especially three-dimensional histological imaging. Summary of the Invention

[0007] To overcome the aforementioned problems of the prior art, the main objective of this invention is to provide a novel tissue decolorization, dehemidation, and decalcification composition that can simultaneously achieve decalcification, dehemidation, and decolorization of biological tissues, thereby greatly improving processing efficiency and achieving better decalcification and decolorization effects than existing decalcification and decolorization reagents, respectively. The inventors of this invention unexpectedly discovered that by combining a divalent cationic chelating agent with an imidazole reagent containing an aminoalkyl group, it is possible to simultaneously achieve decalcification, dehemidation, and decolorization of biological tissues, and even achieve better effects than existing decalcification and decolorization reagents, thus completing this invention. Using the tissue decolorization, dehemidation, and decalcification composition of this invention to decalcify, dehemidate, and decolorize biological tissue materials is more conducive to imaging the treated biological tissues, especially three-dimensional histological imaging.

[0008] Therefore, in one aspect, the present invention provides a tissue decolorization, debledening, and decalcification composition comprising: a) a divalent cationic chelating agent; b) an imidazole reagent having an aminoalkyl imidazole; and c) water.

[0009] In some embodiments, the divalent cation chelating agent is a calcium ion chelating agent comprising at least one selected from ethylenediaminetetraacetic acid, ethylenediaminesuccinic acid, iminodisuccinic acid, ethylenediamine di-o-phenylacetic acid, glutamic acid diacetic acid, methylglycine diacetic acid, hypozinotriacetic acid, and their biologically acceptable salts, preferably at least one selected from ethylenediaminetetraacetic acid, sodium ethylenediaminetetraacetic acid, ethylenediaminesuccinic acid, and sodium ethylenediaminesuccinate.

[0010] In some embodiments, the aminoalkyl group can be located on the nitrogen atom at the 1-position of the imidazole ring or on the carbon atom at the 2-position. In a preferred embodiment, the imidazole reagent is an imidazole having a C1-6 aminoalkyl group. The imidazole reagent may further comprise an alkyl group, preferably a C1-6 alkyl group, such as methyl, ethyl, or propyl. When the aminoalkyl group is located on the nitrogen atom at the 1-position of the imidazole ring, the alkyl group can be located on the carbon atom at the 2-position; when the aminoalkyl group is located on the carbon atom at the 2-position, the alkyl group can be located on the nitrogen atom at the 1-position.

[0011] In a preferred embodiment, the imidazole reagent is selected from at least one of 1-(3-aminopropyl)imidazolium, 1-(2-aminopropyl)imidazolium, 1-(2-aminoethyl)imidazolium, 1-(aminomethyl)imidazolium, 2-(3-aminopropyl)imidazolium, 2-(2-aminopropyl)imidazolium, 2-(2-aminoethyl)imidazolium, 2-(aminomethyl)imidazolium, 1-(3-aminopropyl)-2-methyl-imidazolium, 1-(2-aminopropyl)-2-methyl-imidazolium, 1-(2-aminoethyl)-2-methyl-imidazolium, 1-(aminomethyl)-2-methyl-imidazolium, 1-methyl-2-(aminomethyl)imidazolium, 1-methyl-2-(2-aminoethyl)imidazolium, 1-methyl-2-(3-aminopropyl)imidazolium, and 1-methyl-2-(2-aminopropyl)imidazolium.

[0012] In some embodiments, the tissue decolorization, debledening, and decalcification composition has a pH in the range of 7.5 to 11.0, preferably 7.5 to 10.5, and more preferably 8 to 10.

[0013] In some embodiments, the content of the divalent cationic chelating agent in the tissue decolorization, deblemishing, and decalcification composition is 5% to 20% by w / v, for example, 5% to 10%, 5% to 15%, 10% to 15%, 10% to 20%, 15% to 20%, preferably 10% to 20%, and more preferably 15% to 20%.

[0014] In some embodiments, the imidazole reagent in the tissue decolorization, deblemishing, and decalcification composition is 5% to 40% w / v, for example 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, preferably 10% to 35%, and more preferably 25% to 35%.

[0015] In some embodiments, the tissue decolorization, debledening, and decalcification composition further comprises an additive selected from at least one of pH adjusters, permeation enhancers, and antioxidants. In a preferred embodiment, the additive is present in the tissue decolorization, debledening, and decalcification composition at a concentration of 5% w / v or less, more preferably 2% w / v or less.

[0016] In some embodiments, the tissue decolorization, debledening, and decalcification composition comprises a pH adjuster selected from at least one of 4-hydroxyethylpiperazine ethanesulfonic acid, triethanolamine, and tris-HCl. In some embodiments, the concentration of the pH adjuster is from 0.001M to 0.1M.

[0017] In some embodiments, the tissue decolorization, debledening, and decalcification composition comprises at least one penetration enhancer selected from sodium deoxycholate and Triton X-100. In some embodiments, the concentration of the penetration enhancer is from 0.1% w / v to 8% w / v. The penetration enhancer is used to improve the permeability of the composition to tissues or cells, thereby enabling other components in the composition to effectively penetrate into tissues and cells, improving the decolorization, debledening, and decalcification effects.

[0018] In some embodiments, the tissue decolorization, debleaching, and decalcification composition comprises at least one antioxidant selected from potassium metabisulfite, mercaptoethanol, and mercaptoethylamine. In some embodiments, the concentration of the antioxidant is from 0.02% w / v to 2% w / v. The antioxidant serves to prevent the other components of the composition from being affected by oxygen in the air.

[0019] In general, the concentration / content of each component in this invention is a mass-volume percentage concentration, defined as the percentage of the mass (g) of the substance contained in a unit volume (mL) of solution. For example, the concentration of physiological saline is 0.9% w / v, which means that every 100mL of solution contains 0.9g of NaCl.

[0020] In another aspect, the present invention also provides a method for decolorizing, debleaching, and decalcifying biological tissues, comprising the following steps:

[0021] 1) Obtain biological tissue samples from the donor; and

[0022] 2) Treat the biological tissue sample with the tissue decolorization, debledation, and decalcification composition of the present invention.

[0023] The treatment in step 2) refers to immersing the biological tissue sample in the tissue decolorization, deblemishing, and decalcification composition.

[0024] In some embodiments, step 2) is performed at a pH greater than 7 and less than or equal to 11, preferably 7.5 to 10.5, more preferably 8 to 10.

[0025] In some implementations, the processing time in step 2) is 30 minutes to 24 hours, preferably 2 hours to 24 hours, and more preferably 4 hours to 24 hours.

[0026] This invention achieves simultaneous decalcification, dehemidation, and decolorization of biological tissues through a combination of imidazole reagents with aminoalkyl groups and divalent cationic chelating agents. Compared with existing decalcification and decolorization reagents, it achieves better decalcification and decolorization effects, and has great application prospects in bioimaging, especially three-dimensional histological imaging. Attached Figure Description

[0027] The accompanying drawings described in this invention are only for illustrating selected embodiments of the invention, and not all possible embodiments, and are not intended to limit the scope of the invention.

[0028] Figure 1 The images show photographs of mouse spinal cord tissue and spleen before and after treatment with different compositions of the same ingredients in Example 1 of the present invention;

[0029] Figure 2 The OD values ​​of the decolorizing solutions of mouse spleen tissue soaked in the composition of different components in Example 1 of the present invention are shown.

[0030] Figure 3 The images shown are before and after processing mouse spinal tissue and spleen using the decolorizing, deblemishing, and decalcifying composition of the present invention in Example 2.

[0031] Figure 4 The images shown are before (A) and after (B) treatment of mouse spinal tissue with the decolorizing, deblemishing, and decalcifying composition according to an embodiment of the present invention, and a radiograph of a large tissue block after the decolorizing, deblemishing, and decalcifying treatment (C).

[0032] Figure 5 The images show photographs of mouse organs before (top view) and after (bottom view) treatment with the decolorizing, deblemishing, and decalcifying composition according to an embodiment of the present invention. Detailed Implementation

[0033] In the following description, embodiments of the invention will be presented in more detail to aid in understanding the invention. It should be understood that the description of these embodiments is for illustrative purposes only and is not intended to limit the scope of protection claimed by the invention in any way. Various modifications and variations can be made by those skilled in the art without departing from the scope and spirit of the invention, and such modifications and variations also fall within the scope of the invention.

[0034] Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0035] Example

[0036] Materials and methods

[0037] Unless otherwise specified, all reagents used in this invention are commercially available or prepared by methods known in the art, and all operations can be performed according to conventional methods in the art or other methods known to those skilled in the art.

[0038] All animal experiments were conducted in strict accordance with the animal welfare and ethical guidelines established by the Institute of Laboratory Animal Management and Use (IACUC) of Tsinghua University. The C57BL / 6 mice were sourced from Vital River and housed in a standard environment with adequate food and water.

[0039] Example 1. Determination of the components of the decolorizing, deblemishing, and decalcifying composition.

[0040] To improve the decolorization, debleaching, and decalcification effects, the inventors formulated a composition of the following four different ingredients:

[0041] 1) EDTA (ethylenediaminetetraacetic acid): 15% w / v EDTA dissolved in 0.01M PBS, pH adjusted to 7.0-8.0 with sodium hydroxide.

[0042] 2) EDTA-imidazole: 15% w / v EDTA + 20% w / v imidazole, soluble in water.

[0043] 3) EDTA-alkylimidazole: 15% w / v EDTA + 20% w / v 1-methylimidazole, soluble in water.

[0044] 4) EDTA-aminoalkylimidazolium: 15% w / v EDTA + 20% w / v 1-(3-aminopropyl)imidazolium, soluble in water.

[0045] Mice were perfused with 20 mL of pre-cooled physiological saline and 20 mL of pre-cooled 4% PFA (paraformaldehyde). The spinal cord tissue (including ribs) and spleen were then removed. Both tissues were immersed in the above four-component solution for 24 h at 37°C using a shaker. After treatment, the mice were rinsed three times with PBS, each time for at least two hours. Photos were taken before and after treatment to compare the decolorization and debleaching effects. The results are as follows: Figure 1 As shown.

[0046] During the treatment process, EDTA-aminoalkylimidazole began to show decolorization and debledation effects within 30 minutes, demonstrating superior treatment efficiency compared to other compositions. Figure 1It can be seen that for spinal tissue, EDTA-imidazole, EDTA-alkylimidazole, and EDTA-aminoalkylimidazole are all superior to EDTA alone in decolorization and debledation, especially the spinal tissue treated with EDTA-aminoalkylimidazole, which is nearly colorless. For spleen containing blood, EDTA-alkylimidazole and EDTA-aminoalkylimidazole are superior to EDTA-imidazole in decolorization and debledation, and all three combinations are significantly superior to EDTA alone, with the spleen treated with EDTA-aminoalkylimidazole showing the lightest color. Therefore, the EDTA-aminoalkylimidazole combination has the best decolorization and debledation effect. In practical applications, an appropriate treatment time can be selected based on the pigment or blood content of the tissue. For tissues with low pigment or blood content, the treatment time can be shortened, for example, to 2-12 hours.

[0047] To quantify the pigment removal effect, the absorbance of the depigmenting solution after soaking the spleen tissue was measured at 430 nm. The results are as follows: Figure 2 As shown in the figure, it can be seen that, compared with EDTA alone, the EDTA-imidazolium composition, the EDTA-alkylimidazolium composition, and the EDTA-aminoalkylimidazolium composition can remove more pigments, especially the EDTA-aminoalkylimidazolium composition, which removes about twice as much pigment as the EDTA-imidazolium composition and the EDTA-alkylimidazolium composition.

[0048] To determine the decalcification effect of the above composition, vertebral tissue was extracted at different soaking time points, and the state of bone decalcification was assessed by cutting and squeezing. The results are shown in Table 1.

[0049] Table 1. Decalcification effect of different compositions

[0050]

[0051] As can be seen, when spinal tissue was treated with EDTA, there was no significant change in hardness after 4 hours, the bone softened significantly after 8 hours, and it could only be cut with a blade after 24 hours. When spinal tissue was treated with EDTA-imidazole and EDTA-alkylimidazole compositions, there was no significant change in hardness after 2 hours, the bone softened significantly after 4 hours, it could be cut with a blade after 12 hours, and the bone was very soft, indistinguishable from muscle, after 24 hours. However, when spinal tissue was treated with EDTA-aminoalkylimidazole compositions, the bone softened significantly after 2 hours, it could be cut with a blade after 4 hours, and the bone was very soft, indistinguishable from muscle, after 8 hours. Therefore, EDTA-aminoalkylimidazole compositions can significantly improve the decalcification effect. In practical applications, an appropriate treatment time can be selected based on the calcium content of the tissue. For tissues with low calcium content, the treatment time can be less than 8 hours, for example, 4-6 hours.

[0052] Example 2. Effects of different decolorization, debloodization, and decalcification compositions

[0053] The inventors formulated compositions containing different divalent cationic chelating agents and aminoalkylimidazol, as shown in Table 2, and determined their decolorization, dehemidation, and decalcification effects on spinal tissue and spleen according to the method shown in Example 1.

[0054] Table 2. Composition of the decolorization, dehemification, and decalcification composition

[0055]

[0056] The results showed that the decolorizing, debleaching, and decalcifying compositions in this embodiment all had good decolorizing and debleaching effects (e.g., Figure 3 As shown in the figure, the bone of the spine softens significantly after 2 hours, can be cut with a blade after 4 hours, and becomes very soft, indistinguishable from muscle, after 8 hours.

[0057] Example 3. Destaining, debleaching, and decalcifying treatment and imaging of mouse spinal tissue

[0058] Ethylenediaminetetraacetic acid (EDTA) and 1-(3-aminopropyl)imidazole were added to water and mixed thoroughly to prepare the decolorizing, dehemidating, and decalcifying composition of the present invention, which contains 10% w / v EDTA and 20% 1-(3-aminopropyl)imidazole, and the pH was adjusted to approximately 8.0. To verify the decolorizing, dehemidating, and decalcifying effect of this composition, mouse spinal tissue was subjected to decolorizing, dehemidating, and decalcifying treatment as follows:

[0059] 1) The mice were perfused with 20 mL of pre-cooled physiological saline and 20 mL of pre-cooled 4% PFA (paraformaldehyde).

[0060] 2) Remove the mouse spine (including back muscles and ribs). This tissue contains a large amount of pigment (myoglobin in muscles and cytosine in bone marrow), as well as hard bone (vertebrae, ribs, and vertebral arch). Immerse the entire tissue in 20 mL of a decolorizing, deblemishing, and decalcifying composition and keep it at 37°C for 24 hours. The bone is completely softened, and the pigment in the pigmented parts is removed.

[0061] 3) The processed spinal tissue samples were sectioned or imaged using a Zeiss Lightsheet 7 light sheet microscope.

[0062] Figure 4 The images show photographs of mouse spinal tissue before (A) and after (B) treatment with the decolorizing, deblemishing, and decalcifying composition according to embodiments of the present invention, as well as a radiograph of a large tissue mass after the decolorizing, deblemishing, and decalcifying treatment (C). Figure 4 As shown in (A), the muscles of the mouse's complex spinal cord tissue appear deep red after fixation. In contrast, as... Figure 4As shown in (B), after treatment with the decolorizing, debledening, and decalcifying composition of the present invention, followed by a clearing treatment, the tissue exhibits high overall transparency, uniform transparency, and no visible spines, ribs, or fragments thereof, or obvious red pigment, demonstrating excellent decalcification, debledening, and decolorization effects. Furthermore, as... Figure 4 As shown in (C), large tissue block light imaging of mouse spinal tissue treated with the decolorizing, debledening, and decalcifying composition according to an embodiment of the present invention clearly shows the vascular structure, demonstrating the excellent effect and application potential of the decolorizing, debledening, and decalcifying composition of the present invention in tissue processing for three-dimensional histological imaging.

[0063] Example 4. Decolorization, debloodening, and decalcification treatment of different organs in mice.

[0064] Ethylenediaminetetraacetic acid (EDTA), 1-(3-aminopropyl)imidazole, and sodium deoxycholate were added to water and mixed evenly to prepare the decolorizing, debledraining, and decalcifying composition of the present invention, which contains 10% w / v EDTA, 20% 1-(3-aminopropyl)imidazole, and 4% w / v sodium deoxycholate, with the pH adjusted to approximately 9.0. To further illustrate the decolorizing, debledraining, and decalcifying effects of the decolorizing, debledraining, and decalcifying composition of the present invention, mouse organs were subjected to decolorizing, debledraining, and decalcifying treatment as follows:

[0065] 1) Mice were perfused with 20 mL of pre-cooled physiological saline and 20 mL of pre-cooled 4% PFA (paraformaldehyde) via cardiac perfusion, and various organs were removed by dissection;

[0066] 2) Soak the above-mentioned mouse organs in the decolorizing, debleaching, and decalcifying composition for 24 hours to perform whole-organ decolorization and debleaching;

[0067] 3) Perform organ sectioning and subsequent histological processing, or perform whole-organ tissue transparency and whole-organ imaging.

[0068] Figure 5 The images show photographs of mouse organs before (top) and after (bottom) treatment with the decolorizing, deblemishing, and decalcifying composition according to an embodiment of the present invention. As shown in the top image, the various mouse organs contain significant amounts of pigment (reproductive system, pancreas, thymus, liver) and may also contain blood (spleen, lungs), and these organs remain noticeably stained even after fixation. The bottom image shows the corresponding mouse organs treated with the decolorizing, deblemishing, and decalcifying composition of the present invention, and after a transparentizing process; the organs are highly transparent, significantly lighter in color, and virtually free of blood staining.

[0069] In summary, the tissue decolorization, debledation, and decalcification composition provided by this invention can simultaneously decalcify, debleden, and decolorize biological tissues. Compared with existing decalcification and decolorization reagents, it achieves better decalcification and decolorization effects and significantly improves processing efficiency. Therefore, it has great application prospects in biological imaging, especially three-dimensional histological imaging.

Claims

1. A tissue decolorization, debleaching, and decalcification composition, comprising: a) Divalent cationic chelating agents; b) Imidazole reagents, wherein the imidazole reagents are imidazoles having an aminoalkyl group; and c) Water.

2. The tissue decolorization, debleaching, and decalcification composition according to claim 1, wherein, The divalent cationic chelating agent is selected from at least one of ethylenediaminetetraacetic acid, ethylenediaminesuccinic acid, iminodisuccinic acid, ethylenediamine di-o-phenylacetic acid, glutamic acid diacetic acid, methylglycine diacetic acid, hypozinotriacetic acid, and their biologically acceptable salts.

3. The tissue decolorization, debleaching, and decalcification composition according to claim 1, wherein, The aminoalkyl group is located on the nitrogen atom at position 1 or the carbon atom at position 2 of the imidazole ring. Preferably, the aminoalkyl group is a C1-6 aminoalkyl group.

4. The tissue decolorization, debleaching, and decalcification composition according to claim 3, wherein, The imidazole reagent is selected from at least one of 1-(3-aminopropyl)imidazolium, 1-(2-aminopropyl)imidazolium, 1-(2-aminoethyl)imidazolium, 1-(aminomethyl)imidazolium, 2-(3-aminopropyl)imidazolium, 2-(2-aminopropyl)imidazolium, 2-(2-aminoethyl)imidazolium, and 2-(aminomethyl)imidazolium.

5. The tissue decolorization, debleaching, and decalcification composition according to claim 1, wherein, The imidazole reagent further comprises an alkyl group, preferably a C1-6 alkyl group, preferably, one of the aminoalkyl group and the alkyl group is located on the nitrogen atom at the 1 position of the imidazole ring, and the other is located on the carbon atom at the 2 position.

6. The tissue decolorization, debleaching, and decalcification composition according to claim 5, wherein, The imidazole reagent is selected from at least one of 1-(3-aminopropyl)-2-methyl-imidazolium, 1-(2-aminopropyl)-2-methyl-imidazolium, 1-(2-aminoethyl)-2-methyl-imidazolium, 1-(aminomethyl)-2-methyl-imidazolium, 1-methyl-2-(aminomethyl)imidazolium, 1-methyl-2-(2-aminoethyl)imidazolium, 1-methyl-2-(3-aminopropyl)imidazolium, and 1-methyl-2-(2-aminopropyl)imidazolium.

7. The tissue decolorization, debleaching, and decalcification composition according to any one of claims 1 to 6, wherein, The pH of the tissue decolorization, deblemishing, and decalcification composition is in the range of 7.5 to 11.

0.

8. The tissue decolorization, debleaching, and decalcification composition according to any one of claims 1 to 6, wherein, The content of the divalent cationic chelating agent in the tissue decolorization, deblemishing, and decalcification composition is 5% to 20% w / v, preferably 10% to 20%.

9. The tissue decolorization, debleaching, and decalcification composition according to any one of claims 1 to 6, wherein, The content of the imidazole reagent in the tissue decolorization, debledening, and decalcification composition is 5% to 40% w / v, preferably 10% to 35%.

10. The tissue decolorization, debleaching, and decalcification composition according to any one of claims 1 to 6, wherein, The tissue decolorization, debledening, and decalcification composition further comprises an additive selected from at least one of pH adjusters, permeation enhancers, and antioxidants. Preferably, the content of the additive in the tissue decolorization, debledening, and decalcification composition is less than 5% w / v.

11. The tissue decolorization, debleaching, and decalcification composition according to claim 10, wherein, The tissue decolorization, debledening, and decalcification composition comprises a pH adjuster selected from at least one of 4-hydroxyethylpiperazine ethanesulfonic acid, triethanolamine, and tris-HCl, preferably, the concentration of the pH adjuster is from 0.001M to 0.1M.

12. The tissue decolorization, debleaching, and decalcification composition according to claim 10, wherein, The tissue decolorization, debledination, and decalcification composition comprises at least one permeation enhancer selected from sodium deoxycholate and Triton X-100, preferably, the concentration of the permeation enhancer is from 0.1% w / v to 8% w / v.

13. The tissue decolorization, debleaching, and decalcification composition according to claim 10, wherein, The tissue decolorization, deblemishing, and decalcification composition comprises at least one antioxidant selected from potassium metabisulfite, mercaptoethanol, and mercaptoethylamine, preferably, the concentration of the antioxidant is from 0.02% w / v to 2% w / v.

14. A method for decolorizing, debleaching, and decalcifying biological tissue, comprising the following steps: 1) Obtain biological tissue samples from the donor; and 2) Treat the biological tissue sample with the tissue decolorization, deblemishing, and decalcification composition according to any one of claims 1 to 13, preferably, the treatment time in step 2) is 30 minutes to 24 hours.

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