CN118641745B - A method for preparing acellular matrix with full tissue three-dimensional structure based on thick-cut technology - Google Patents

Abstract

The invention provides a preparation method of a whole-tissue three-dimensional structure acellular matrix based on a thick cutting technology, which comprises the following steps: step one: embedding the tissue lacking endogenous vascular channels with an embedding agent; step two: performing thick cutting treatment on the embedded tissue to obtain a tissue sample with a certain slice thickness, wherein the slice thickness range of the tissue sample is 500-1000 mu m; step three: and carrying out chemical decellularization treatment on the tissue sample with a certain slice thickness through a chemical eluent, so that the chemical eluent can uniformly permeate the tissue sample, and an extracellular matrix with a liquid flow path inside and a three-dimensional stable cell-free component is obtained. The beneficial effects of the invention are as follows: according to the invention, the acellular treatment is carried out by the thickly cut tissue, and the soaking and treatment of the chemical eluent can be more uniformly carried out by combining with the accurate thickness control and embedding technology, and meanwhile, the specific chemical eluent is adopted, so that the more efficient acellular effect is realized.

Description

A method for preparing acellular matrix with full tissue three-dimensional structure based on thick-cut technology

Technical Field

The invention belongs to the field of biomedicine technology, and particularly relates to a method for preparing a three-dimensional decellularized matrix of a whole tissue based on thick-cut technology.

Background Art

Extracellular matrix (ECM) is a complex extracellular scaffold structure composed of collagen, fibronectin, glycoprotein and other molecules. It fills the intercellular space, provides support for cells, promotes cell-to-cell interactions, and regulates cell morphology and function. ECM plays a key role in the formation of local homeostasis of tissues and organs, and affects the development and repair of tissues in various ways. Taking tumors as an example, ECM can change the spatial structure and physical conformation by rearrangement, cross-linking, etc., thereby regulating the invasion and migration of tumors. In addition, ECM can also promote tumor proliferation by affecting signal transduction pathways. Not only that, ECM reduces the efficiency of drug diffusion and limits the activity of immune cells by forming a dense network structure. Therefore, ECM plays an important role in tumor growth, invasion, metastasis and treatment response. In-depth study of the interaction between ECM and tissue cells will help reveal the mechanism of tissue homeostasis formation, and the preparation of decellularized extracellular matrix (dECM) samples that retain three-dimensional structure and complete components is a prerequisite for studying the mechanical properties and biological functions of ECM.

The preparation of dECM samples involves tissue decellularization technology, which is to remove cells and antigen components in tissues through physical, chemical or biological methods while retaining ECM components. Physical decellularization mainly destroys cell membranes through cyclic freezing and thawing, but this method is easy to destroy the ECM structure. Biological methods use enzymes such as trypsin and collagenase to decellularize through enzymatic reactions, but long-term exposure to proteases will cause some ECM proteins to degrade, thereby destroying their structure and components. Chemical methods usually use decellularizing agents such as sodium deoxysulfate (SDS), Triton X-100 and sodium deoxycholate (SDC), which can dissolve structures such as cytoplasm and nuclear lipid membranes to achieve effective decellularization. Detergents are delivered to the tissue through intravascular channels in the tissue, thereby maximizing the removal of cells while maintaining the tissue structure. At present, the use of decellularizing agents can achieve better decellularization effects. However, local tissues (such as tumor tissues) lack obvious endogenous vascular channels, which makes it difficult to establish a complete fluid flow path. At present, the dECM acquisition method for local tissues mainly adopts a chemical washing method of in vitro immersion and shaking to achieve the decellularization effect. In 2020, Madeleine J. Oudin et al. immersed and shook breast cancer tissue in 0.1% SDS at room temperature for several days until the tissue became transparent to the naked eye, and then washed the decellularized tissue with pure water for 4 days. In 2022, Thomas R. Cox et al., based on their predecessors, decellularized breast cancer tissue by shaking it at room temperature for 16 hours in a 0.5% SDC solution, shortening the decellularization time. However, tumor cells are surrounded by dense tissue, making it difficult for detergents to fully penetrate the tissue in a short time. Long-term in vitro decellularization can easily lead to the degradation of matrix proteins. Therefore, how to efficiently remove cellular components without destroying the components, three-dimensional structure and mechanical properties of ECM has become the key to current technological improvements.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a three-dimensional decellularized matrix of a whole tissue based on thick-cut technology.

The object of the present invention is achieved through the following technical solution: A method for preparing a three-dimensional decellularized matrix of a whole tissue based on thick-cut technology, comprising the following steps:

Step 1: Obtain complete tissue, embed the tissue with embedding agent without destroying the overall structure of the tissue, and ensure that the rigidity of the embedding agent is consistent with that of the tissue;

Step 2: The embedded tissue is subjected to thick-cut processing to obtain tissue samples with a certain slice thickness, and the slice thickness of the tissue sample ranges from 500 μm to 1000 μm;

Step 3: chemically decellularize tissue samples of a certain slice thickness by using a decellularizing agent, so that the decellularizing agent can evenly penetrate the tissue sample to obtain a three-dimensional spatial structure with a stable cell-free extracellular matrix having a liquid flow path inside; wherein the decellularizing agent is a 0.1% - 0.5% by weight SDS mixed solution containing 0.1 - 0.5 mM ethylenediaminetetraacetic acid EDTA, 1 mM glutathione, 1-10 μM proteasome inhibitor and 0.1-1 mM phenylmethylsulfonyl fluoride protease inhibitor.

Furthermore, in step one, the embedding agent is agarose gel of different concentrations selected according to the stiffness of the tissue, and the concentration of agarose is adjusted according to the physical properties (stiffness) of the tissue to ensure that the stiffness of the embedding material is consistent with that of the tissue to avoid damaging the physical properties of the tissue.

Furthermore, in step 2, a vibrating slicer is used to perform thick-cut processing on the embedded tissue. Depending on the different tissue types and tissue stiffness, the vibration speed range is 1.00-1.50 mm/s and the vibration amplitude range is 0.50-1.50 mm to ensure that the slice thickness is uniform and the three-dimensional structure is not destroyed.

Furthermore, during the chemical decellularization process, the following conditions are maintained: at room temperature, the tissue sample with the added decellularizing agent is placed in a centrifuge tube, placed on a shaking shaker, and decellularized at 100-120 rpm.

Furthermore, during the chemical decellularization process, the following conditions were maintained: tissue samples of a certain slice thickness were placed in a microfluidic chip, equipped with an OxyGEN microfluidic system, and the SDS mixed solution was delivered at a pressure of 10-50 mbar, with a total volume of 100-250 ml.

Furthermore, during the chemical decellularization process, a balanced salt buffer PBS was used, containing 1% by weight of penicillin-streptomycin dual antibody solution and 2.5 μg/ml of amphotericin B.

Furthermore, the subsequent processing steps for the decellularized tumor tissue are as follows: removal of residual nucleic acid clusters: 125 units/ml super nuclease, 200 units/ml deoxyribonuclease I, balanced pH = 8, 37°C shaker, for 5 minutes.

Furthermore, specific analysis or detection is performed on the decellularized tumor tissue, such as imaging of collagen fibers using second harmonic generation microscopy with a two-photon microscope, and observation with a laser confocal microscope.

Furthermore, the present invention is applied to a variety of local tissues, including but not limited to various solid tumor tissues such as melanoma, liver cancer, lung cancer, intestinal cancer, etc.; various organ tissues such as skin tissue, liver tissue, brain tissue, muscle tissue, adipose tissue, etc.

The beneficial effects of the present invention are:

1. Applicable to local tissues: The present invention provides an effective solution to the problem that local tissues (such as tumor tissues, etc.) lack endogenous vascular channels. By performing decellularization through thick-cut tissues, combined with precise thickness control and embedding technology, the decellularization agent can be soaked and treated more evenly. At the same time, the use of specific decellularization agents overcomes the difficulties of traditional in vitro soaking and shaking methods in establishing liquid flow paths, thereby achieving a more efficient decellularization effect;

2. Preservation of three-dimensional structure: Traditional decellularization technology often destroys the three-dimensional structure and physical properties of tissues. This invention is the first to propose the decellularization of thick-cut tissues to study the extracellular matrix, which to a certain extent preserves the three-dimensional structure and physical properties of the extracellular matrix, providing researchers with more information on the spatial structure and physical properties of the extracellular matrix;

3. Precise thickness control: Using a vibrating slicer to slice tissue thickly can accurately control tissue thickness and achieve batch operation, thereby reducing experimental errors and improving research efficiency;

4. Improve decellularization efficiency: embed tissues with materials such as agarose, adjust the concentration of agarose according to the physical properties (rigidity) of the tissue, ensure that the embedding material is consistent with the rigidity of the tissue, and avoid destroying the physical properties of the tissue. This not only improves the success rate and accuracy of thick cutting operations, but also better maintains the three-dimensional structure and mechanical strength of the extracellular matrix.

5. Reduce protein degradation: The tissue is decellularized after thick cutting, which greatly improves the efficiency of decellularization, shortens the exposure time of extracellular matrix proteins at room temperature, and effectively reduces protein degradation;

6. After the thick-cut tissue is decellularized at room temperature, it is placed on ice and washed with PBS to further reduce protein degradation at room temperature;

7. High-quality decellularization effect: The decellularization quality of thick-cut tissue is higher, which can completely remove the cell components, thereby enriching the extracellular matrix proteins, which is beneficial for researchers to detect more functional extracellular matrix proteins in the body.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art or ordinary technicians, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a flow chart of continuous thick-section tumor tissue decellularization matrix;

FIG2 is a mass spectrometry identification result and immunofluorescence staining diagram of tumor matrix proteins after decellularization and tumor tissue matrix proteins before decellularization;

FIG3 is a schematic diagram 1 of three-dimensional spatial imaging of tumor matrix proteins after decellularization;

FIG4 is a schematic diagram 2 of three-dimensional spatial imaging of tumor matrix proteins after decellularization;

FIG5 is a schematic diagram 3 of three-dimensional spatial imaging of tumor matrix proteins after decellularization;

FIG6 is a schematic diagram 1 showing the comparison of the results of the tumor tissue decellularization method of the present invention and that of the literature;

FIG7 is a schematic diagram 2 showing the comparison of the results of the tumor tissue decellularization method of the present invention and that of the literature;

FIG8 is a schematic diagram of the protein silver staining results of thick-sectioned tumor tissue.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

The present invention provides a method for preparing a three-dimensional decellularized matrix of a whole tissue based on thick-cutting technology, which can maintain the three-dimensional structure and physical properties of dECM to a certain extent, and the duration of the entire decellularization process can be shortened to about 6-8 hours, as shown in FIG1 .

Taking tumor tissue as an example, the local tissue dECM preparation method involved in the present invention comprises the following steps:

1. Obtain tissue: Obtain fresh tumor tissue and rinse it with pre-cooled sterile balanced salt buffer to remove residual blood on the surface;

2. Embedding tissue: embed the intact tissue in an embedding box with 4% agarose (dissolved in sterile PBS);

3. Thick cut tissue:

1) Place the cooled and solidified embedded tissue into the buffer tray of the vibratome and fix it to ensure that the tissue is completely covered by the balanced salt solution;

2) Set the speed, amplitude and cutting thickness of the vibrating slice, adjust the cutting arm height, and the forward and backward movement position, and start the cutting mode;

3) Gently separate the sliced tissue from the agarose gel and place it in a pre-cooled balanced salt buffer solution;

4. Tissue decellularization:

1) Place the thick-cut tissue sections in a 50 ml centrifuge tube containing 0.5 mM EDTA and 1 mM glutathione 0.5% SDS solution, place on a decolorizing shaker at room temperature, replace with fresh SDS solution after 20 minutes, and repeat 3 times;

2) If the volume of the thickly cut tissue slices is small, the SDS solution can be transported by a microfluidic circulation pressure system to further improve the decellularization efficiency and achieve precise control;

3) SDS solution continues to decellularize until the tissue color becomes transparent; the time is 2-4 hours;

4) Wash the tissue treated in step 2) with balanced salt buffer, place on ice, and replace with fresh balanced salt buffer after 20 minutes on a decolorizing shaker. Repeat 3 times;

5) Add super nuclease and deoxyribonuclease I to the tissue treated in step 4), adjust the pH to 8, shake at 37°C for 5 minutes, and observe that the residual nucleic acid group disappears significantly;

6) Wash the tissue treated in step 2) with balanced salt buffer, place on ice, and replace with fresh balanced salt buffer after 20 minutes on a decolorizing shaker. Repeat this process three times;

5. Decellularized Tissue Preservation

1) Protein spectrum sample storage: Place the decellularized matrix on a sterile test paper, absorb the residual liquid, and quickly freeze the treated decellularized tissue in liquid nitrogen until the matrix is completely frozen. Take out the quick-frozen decellularized matrix and place it in a pre-cooled sterile container for subsequent protein spectrum testing;

2) Tissue immunofluorescence sample preservation: Place the decellularized matrix on a sterile test paper, absorb the residual liquid, place the tissue in paraformaldehyde, and conduct subsequent immunofluorescence experiments.

3) Vacuum freeze-drying sample storage: Place the decellularized matrix on a sterile test paper, absorb the residual liquid, and quickly transfer it to a freeze dryer. Set appropriate temperature and vacuum conditions for freeze drying to allow the moisture in the matrix to sublime under low temperature and low pressure conditions. After freeze drying, seal the matrix in a sterile package and store it in a dry, low-temperature environment to prevent moisture absorption and contamination.

Example: Preparation of decellularized matrix of pancreatic cancer tumor tissue

Preparation in Advance:

①Sterilized surgical instruments (ophthalmic scissors, tissue forceps, gauze, blades, hemostatic forceps); sterile test paper; balanced salt solution (PBS, 1% penicillin-streptomycin double antibody solution, 2.5 μg/ml amphotericin B); 4% agarose; 70% ethanol; 0.45μm filter; 50 ml syringe; 0.5% SDS solution; 0.5 mM EDTA; 1 mM glutathione; 1μM proteasome inhibitor; 1mM phenylmethylsulfonyl fluoride protease inhibitor; super nuclease; deoxyribonuclease I;

② The balanced salt solution should be prepared immediately before use and needs to be filtered before use;

1. Preparation of vibrating microtome:

①Disinfect the buffer tray with 70% ethanol and wipe it with sterile paper towels;

Insert the sterilized vibrating blade vertically into the cutting arm, making sure the blade is firmly fixed to the cutting arm to prevent it from falling off during vibration;

②Put the buffer plate into the vibrator and tighten it to install; adjust the cutting arm to the maximum available height;

2. Obtaining tumor tissue:

①Euthanize the mice by cervical dislocation and disinfect the skin surface of the mice with 70% ethanol solution;

② Fix the mouse on its back and its limbs on a dissecting board; make a vertical midline incision on the skin from the bottom of the abdominal cavity to above the diaphragm;

③ Pull the skin back and cut the connective tissue between the skin and the abdominal cavity to prevent the hair from falling into the abdominal cavity. Use a pin to clamp the skin outside the abdominal cavity.

④Use clean forceps and scissors to open the abdominal cavity;

⑤ Use tissue forceps to quickly separate the pancreatic tumor from the pancreatic tissue and cut the connecting connective tissue short;

Be careful not to damage the gastrointestinal tract to avoid microbial contamination of pancreatic tumor tissue;

⑥ Place the removed tumor tissue in a 10 cm sterile tissue culture dish filled with pre-cooled balanced salt buffer and keep it on ice throughout the operation;

⑦ Use tissue forceps and ophthalmic scissors to quickly trim the remaining pancreatic tissue and blood on the surface of the tumor tissue, and continuously rinse with pre-cooled balanced salt solution;

Be careful not to damage the tumor tissue;

3. Embedding tumor tissue:

① Use sterile gauze to absorb the residual liquid on the surface of the tumor tissue and place the tissue in the embedding box;

②4% agarose was heated and melted in a 70℃ constant temperature metal bath, then transferred to a 40℃ shaking constant temperature metal bath;

③ Cool to room temperature, embed the entire tumor tissue with agarose, and wait for the agarose to solidify completely;

4. Thick cut tumor tissue:

① Place the cooled and solidified embedded tissue into a vibrating buffer tray and fix it to ensure that the tissue is completely covered by the pre-cooled balanced salt solution;

② Set the vibration sectioning speed to 1.50 mm/s, amplitude to 1.00 mm, and cutting thickness to 500 - 700 μm;

③ Lower the cutting arm until it is at the working height above the embedded tissue;

④Fix the forward and backward movement position of the cutting blade and start the automatic cutting mode;

⑤ During the cutting process, carefully use tissue forceps to gently lift the tissue and separate it from the agarose gel, and place the tissue slices in pre-cooled balanced salt buffer solution and place on ice;

⑥ Use a blade to scrape off the cured glue and waste tissue on the buffer tray;

5. Tumor tissue decellularization:

① Prepare 0.5% SDS solution in advance and add 0.5 mM EDTA, 1 mM glutathione, 1 μM proteasome inhibitor and 1 mM phenylmethylsulfonyl fluoride protease inhibitor;

② Place the thick-cut tissue sections in a 50 ml centrifuge tube containing a 0.5% SDS mixed solution, place on a shaking shaker at room temperature at a frequency of 100-120 rpm, replace with fresh 0.5% SDS solution after 20 min, and repeat 3 times;

③ 0.5% SDS mixed solution continues to decellularize until the tissue color becomes transparent, which lasts for about 2-4 hours;

④ Replace the liquid with balanced salt buffer, place on ice, and shake on a decolorizing platform at a frequency of 100-120 rpm. Replace with fresh balanced salt buffer after 20 min, and repeat 3 times;

⑤Removal of residual nucleic acid: 125 units/ml super nuclease, 200 units/ml deoxyribonuclease I, equilibrium pH = 8, 37℃ shaker, 5 min, the residual nucleic acid can be seen to disappear significantly;

⑥ Replace the liquid with balanced salt buffer, place on ice, and shake on a decolorizing shaker at 100-120 rpm. Replace with fresh balanced salt buffer after 20 min, and repeat 3 times.

6. Decellularized Tissue Preservation

① Use sterile test paper to absorb the residual liquid after decellularization, freeze it quickly in liquid nitrogen, and store it at -80 for non-labeled quantitative protein spectrum identification;

② Use sterile test paper to absorb the residual liquid after decellularization, fix with paraformaldehyde at room temperature, and conduct subsequent tissue immunofluorescence experiments;

7. Results Analysis

① The results of non-labeled quantitative protein spectrum are shown in Figure 2: By performing non-labeled protein spectrum detection on the total protein of the extracted samples and matching the detected peptide sequences, it was found that 128 known extracellular matrix proteins can be detected in the decellularized samples. The results are shown in A and B in Figure 2. Compared with non-decellularized tissue samples, decellularization technology can amplify the proportion of extracellular matrix proteins in the total protein, achieving an enrichment effect, which makes it easier to be detected by protein spectrum;

② The immunofluorescence results are shown in Figure 2: The results of immunofluorescence (as shown in C, D, and E in Figure 2) show that compared with non-decellularized tissue samples, the decellularization efficiency of the technology of the present invention is very high, and non-matrix protein components such as cell nuclei (Hoechst staining positive) and cytoskeleton (Actin staining positive) can be fully removed, while the components of extracellular matrix proteins (taking collagen as an example) are greatly retained, achieving a better decellularization effect;

③ Three-dimensional imaging of the extracellular matrix is shown in Figures 3-5: The results show that, taking Collagen I as an example, in the decellularized tissue sample, the collagen molecules are tightly entangled together, maintaining an obvious triple helix structure. The collagen fibers are cross-linked to form a network structure, maintaining good mechanical strength (see Figure 3). This shows that the decellularization operation in the technology of the present invention retains the three-dimensional spatial conformation characteristics of the extracellular matrix (see Figures 4-5).

8. Compare the results with the tumor tissue decellularization methods in the literature:

① According to the study published in Nature Communications in 2022 by Thomas R.Cox et al. (Temporal profiling of the breast tumour microenvironment reveals collagenXII as a driver of metastasis), we used the tumor tissue decellularization method described in the article to decellularize pancreatic cancer tissue. These decellularized tissues were stained and compared with the tissues decellularized by the technical method in the patent of this invention, and three-dimensional imaging of immunofluorescence staining was performed (see Figures 6 and 7). According to the method in the literature, there are still a lot of cellular components remaining in the whole tumor tissue after 4 hours of decellularization, and the cellular components can only be completely eliminated after continuous treatment for 24 hours. In contrast, the thick-cut tumor tissue in the patent of this invention only needs to be treated for 4 hours to achieve the effect of completely eliminating the cellular components. In addition, compared with the 24-hour decellularization group in the literature, the patent of this invention better retains the spatial structure of the extracellular matrix components.

②Furthermore, the above two different decellularized tumor tissue proteins were subjected to protein silver staining experiments (see Figure 8), and it was found that compared with the whole tissue decellularization technology in the literature, the protein bands after thick tissue decellularization were clearer and the band distribution was obvious, indicating that this method enriched specific proteins without obvious degradation.

The innovation of the present invention:

1. Application of thick-cut tissue decellularization technology: Traditional decellularization technology usually minces the tissue or destroys the cell structure by freezing and repeatedly freezing and thawing with liquid nitrogen, thereby improving the efficiency of decellularization. However, this method will cause great damage to the spatial structure of the extracellular matrix. Repeated freezing and thawing of liquid nitrogen will cause the collagen fibers to be disordered and sparse, and the extracellular matrix fibronectin and chondroitin sulfate will be obviously dissolved and lost, resulting in structural destruction of the extracellular matrix and failing to reflect the true structure of the extracellular matrix. In the present invention, by using the thick-cut method of a vibrating slicer, efficient slicing can be performed by precisely controlling the thickness of the tissue without destroying the three-dimensional structure of the local tissue, and at the same time, it can ensure that the decellularizing agent can evenly penetrate the tissue slices, thereby effectively achieving the purpose of decellularization.

① Precision of slice thickness control: In the present invention, a vibrating slicer is used for thick tissue slices, and the slice thickness can be accurately controlled within the range of 500-1000 μm. Compared with traditional methods, this thick slice technology can ensure that the decellularizing agent penetrates evenly into the tissue, thereby achieving a more efficient decellularization effect.

② Application of vibration slicer: By controlling the parameter settings and operation steps of the vibration slicer, such as slicing speed, amplitude, cutting thickness, etc., the repeatability and stability of the operation can be ensured. This improvement can improve the decellularization efficiency while maintaining the three-dimensional structure of the tissue.

2. Advantages of retaining three-dimensional structure and physical properties: The three-dimensional structure and mechanical strength are maintained by selecting embedding materials and controlling key parameters in the thick-cutting step. In addition, the three-dimensional imaging data of tissues before and after decellularization and the mechanical strength test results prove the superiority of the present invention in maintaining the three-dimensional structure.

① Application of embedding technology: Select appropriate embedding materials based on the stiffness of local tissues to ensure that the tissue structure is not damaged during thick sectioning, greatly improve the success rate of thick sectioning and achieve precise control, avoiding the problems of tissue damage and uneven thickness in traditional methods; for example, liver tissue can be embedded with low-melting point glue, while pancreatic cancer tissue can be embedded with agarose.

② Control of key thick-cutting parameters: Accurately control key thick-cutting parameters based on the stiffness of local tissues, which can improve the decellularization effect while preserving the three-dimensional structure and mechanical strength of the tissue. For example, the vibration speed range of liver tissue can be controlled to be 1.00 mm/s, the vibration amplitude range can be 1.50 mm, and the vibration speed range of pancreatic cancer tissue can be controlled to be 1.50 mm/s, and the vibration amplitude range can be 1.00 mm.

③Realize the three-dimensional structure reconstruction of the extracellular matrix of the whole tissue: There is no mature technology to complete the three-dimensional structure reconstruction of the extracellular matrix of the whole tissue. The present invention first marks the tissue, and then through continuous thick cutting and optimized decellularization technology operation, combined with image processing technology, finally realizes the three-dimensional structure reconstruction of the extracellular matrix of the whole tissue.

3. Improved decellularization method:

① Use different decellularization agent combinations: During the decellularization process, a combination of 0.5% SDS solution and different catalysts is used. By adding EDTA, the calcium-dependent structures in the cell membrane and extracellular matrix are destroyed, and cell membrane rupture and protein cleavage are promoted. At the same time, glutathione is an important antioxidant in cells, which can help protect the components of the extracellular matrix during the decellularization process and provide the biocompatibility and functionality of the matrix. Among them, the super nuclease only needs to act for 5 minutes to remove residual nucleic acid clusters. By comparing with the decellularization technology reported in previous literature, it is found that the present invention significantly improves the efficiency of the decellularization process while maintaining the ECM components and shortens the time required for decellularization. In addition, compared with previous decellularization technologies, the use of different decellularization agent combinations makes the tissue decellularization process more uniform and the effect is better.

② Optimize the eluent delivery method: For small local tissue pieces, the SDS solution can be delivered by optimizing the circulating pressure system such as microfluidics. Compared with the traditional interval solution replacement, the continuous supply of fresh SDS solution through the microfluidic system can further significantly improve the decellularization efficiency, save manpower, and achieve precise control.

4. New application areas: The present invention is not only applicable to tumor tissues, but also to the preparation of decellularized matrices for non-tumor tissues, such as other tissue engineering projects in regenerative medicine, such as damaged tissue repair, organ transplantation, cartilage and bone repair, etc. The use of thick sections and decellularization technology can maintain the three-dimensional structure and mechanical properties of tissues and provide a good growth environment for cells. The decellularized matrix can adapt to the needs of different types of tissues by adjusting different chemical treatments and physical parameters, and has broad application prospects.

5. Innovative preservation methods: Traditional preservation methods for decellularized matrices include cryopreservation, freeze-drying, and chemical fixation. However, each of these methods has its own advantages and disadvantages. The present invention targets different experimental requirements and subsequent protein spectrum identification experiments. The use of liquid nitrogen quick freezing can effectively maintain the biological activity of the matrix and prevent protein denaturation and degradation of extracellular matrix components. In response to the experimental requirements of in vitro culture, vacuum freeze-drying can maximize the retention of the physical and chemical properties of the matrix and extend the shelf life. By adopting an innovative preservation method that combines liquid nitrogen quick freezing and vacuum freeze-drying, the present invention can extend the shelf life of the decellularized matrix while maintaining its structure and function, providing better support for subsequent applications.

6. Comprehensive processing steps: From thick cutting, decellularization process to subsequent washing with PBS on ice, each step has been optimized to ensure the improvement of the overall effect. The multi-step optimization design demonstrates systematic and comprehensive innovation;

7. Reduce experimental errors and protein degradation: Through thick-cut technology and optimized decellularization methods, the time of in vitro room temperature decellularization is effectively shortened. Combined with PBS washing on ice, it effectively reduces protein degradation during the experiment and improves the reliability of research results;

8. Repeatability and batch operation: Using a vibrating slicer for thick cutting achieves precise thickness control and batch operation, greatly improving the repeatability and efficiency of the experiment, and providing the possibility for large-scale application and promotion;

9. Novel decellularization method: By introducing thick cutting and embedding technology, the present invention provides a new and optimized decellularization method, expands the application scope of existing decellularization technology, and provides a basis for the future development of new biomaterials and regenerative medicine technologies.

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims (9)

1. A preparation method of a whole-tissue three-dimensional structure acellular matrix based on a thick cutting technology is characterized by comprising the following steps of: the method comprises the following steps:

step one: obtaining a complete tissue, and carrying out embedding treatment on the tissue by an embedding agent on the premise of not damaging the integral structure of the tissue, so as to ensure that the rigidity of the embedding agent is consistent with that of the tissue;

Step two: performing thick cutting treatment on the embedded tissue to obtain a tissue sample with a certain slice thickness, wherein the slice thickness range of the tissue sample is 500-1000 mu m;

Step three: the tissue sample with a certain slice thickness is subjected to chemical decellularization treatment through the decellularization agent, so that the decellularization agent can uniformly permeate the tissue sample, and an extracellular matrix with a liquid flow path and stable three-dimensional space and no cell component is obtained; wherein the decellularization agent adopts SDS mixed solution with the mass percentage of 0.1-0.5%, and contains 0.1-0.5 mM ethylenediamine tetraacetic acid EDTA, 1-mM glutathione, 1-10 mu M proteasome inhibitor and 0.1-1 mM phenylmethanesulfonyl fluoride protease inhibitor.

2. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to claim 1, which is characterized in that: in the first step, the embedding agent is agarose gel with different concentrations selected according to the rigidity of the tissue.

3. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to claim 1, which is characterized in that: in the second step, the embedded tissue is subjected to thick cutting treatment by adopting a vibration slicer, and the vibration speed range is 1.00-1.50 mm/s and the vibration amplitude range is 0.50-1.50 mm according to different tissue types and tissue rigidities so as to ensure that the slice thickness is uniform and the three-dimensional structure is not damaged.

4. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to claim 1, which is characterized in that: during the chemical decellularization treatment, the following conditions were maintained: placing the tissue sample of the added decellularizing agent into a centrifuge tube at room temperature, placing the centrifuge tube on an oscillating and uniformly mixing shaking table, and performing decellularization at the amplitude of 100-120 rpm.

5. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to claim 1, which is characterized in that: during the chemical decellularization treatment, the following conditions were maintained: and placing the tissue sample with a certain slice thickness into a microfluidic chip, carrying OxyGEN a microfluidic system, and conveying the SDS mixed solution at 10-50 mbar pressure, wherein the total volume is 100-250 ml.

6. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to claim 1, which is characterized in that: during the chemical decellularization treatment, a balanced salt buffer PBS was used, containing 1% penicillin-streptomycin diabody solution by mass, 2.5 μg/ml amphotericin B.

7. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to any one of claims 1 to 6, wherein the method comprises the following steps: subsequent treatment steps on tumor tissue after decellularization: removing the nucleic acid residue: 125 units/ml supernuclease, 200 units/ml deoxyribonuclease I, equilibrated ph=8, shaking at 37 ℃ for 5 min.

8. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to any one of claims 1 to 6, wherein the method comprises the following steps: tumor tissue after decellularization is analyzed or detected.

9. The method for preparing the acellular matrix of the whole tissue three-dimensional structure based on the thick cutting technology according to any one of claims 1 to 6, wherein the method comprises the following steps: is applied to various local tissues, including various solid tumor tissues or various organ tissues.