Freezing protection liquid for tumor tissues and preparation and use methods thereof

Abstract

The invention provides a cryoprotectant for tumor tissues and a preparation and use method of the cryoprotectant. The cryoprotectant is prepared by limiting selection of types and dosage ratios of components such as bovine serum albumin, polyvinylpyrrolidone, cane sugar, dimethyl sulfoxide, ethylene glycol and a phosphate buffer solution; according to the method, the cryoprotectant and the phosphate buffer solution are prepared into a plurality of protective solutions with different proportions, and the proportion of the cryoprotectant to the phosphate buffer solution in each step, the size of a tumor tissue slice and the treatment time of each step are controlled, so that the moisture in the tissue can be gradually reduced on the basis of reducing the cytotoxicity, and the tumor tissue can be effectively prevented from being damaged. Formation of ice crystals is reduced, cell damage in the freezing process is reduced to the maximum extent, and the activity and integrity of tissues are kept; according to the invention, the cryoprotection liquid and the serum preservation liquid are further combined for use, so that the cytotoxicity can be remarkably reduced, the activity, survival rate and multiplication capacity of tumor cells are increased, and a synergistic interaction effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue cryopreservation, and in particular to a cryoprotectant for tumor tissue and a preparation and use method thereof. Background Art

[0002] Living tissue, especially living tumor tissue, has irreplaceable value in medical research and clinical treatment. For scientific research applications, living tissue preservation technology is particularly important. At present, the preservation of living tumor tissue mainly relies on the establishment of human-derived tumor tissue xenograft models (PDX, Patient-Derived Xenograft) and living tumor tissue cryopreservation technology.

[0003] Cryopreservation of live tumor tissue means storing the tissue in a low-temperature environment to temporarily remove the tissue from the growth state and preserve the characteristics of the tumor tissue. This preservation method is worry-free and labor-saving, easy to operate, and low-cost. When needed, the tissue can be revived and transplanted to proliferate in the form of primary tumors, which well preserves the original mutations of the tumor and obtains comprehensive tumor information data at multiple levels such as molecules, cells, tissues, and in vivo.

[0004] However, the existing method of freezing live tumor tissue is similar to cell freezing, which is generally programmed cooling and has some limitations. Tissue freezing fluid is usually composed of DMSO, serum and cell culture fluid, and needs to be prepared on demand. Due to the instability of serum components, the shelf life of the freezing fluid is short and the storage conditions are harsh. In addition, tumor tissues frozen using existing freezing fluids have very low activity during resuscitation, only a very small number can be successful, and it is difficult to proliferate in the form of primary tumors. The original mutations of the tumor are easily lost, which limits the application value and scientific research transformation capabilities of tumor tissue samples.

[0005] In this regard, vitrification technology has attracted widespread attention as a method for rapid freezing of cells or tissues. This technology has the advantages of fast freezing speed, small freeze-thaw damage, simple operation, etc., and can improve the survival rate after freezing and thawing.

[0006] Although vitrification technology has many advantages, it still faces technical obstacles in the practice of human tissue freezing. Cells are easily damaged by the formation of ice crystals due to water during the cooling process. In order to reduce this damage, it is necessary to add tissue freezing solution (TFS, Tissue Freeze Solution) during the cooling process to improve the motility of cells during freezing. In addition, different cell tissues have different cell morphology and functions, and their activity and survival rates after freezing treatment with the same formula of cryopreservation solution are different. This is why there are still a lot of research on the formulation of cryopreservation solution despite the fact that there are many similar cryopreservation solution formulations in the prior art. So far, there are few reports on vitrification cryopreservation solutions specifically for tumor tissues in the prior art.

[0007] Based on this, there is an urgent need to develop a cryoprotectant for tumor tissue and a method for its preparation and use, which can act on tumor cells in a targeted manner, improve the freezing effect, quickly freeze cells or tissues, reduce the production of cell ice crystals, and reduce cytotoxicity, so as to improve the activity, survival rate and primary tumor proliferation ability of cells after recovery. Summary of the invention

[0008] The present invention aims to provide a cryoprotectant for tumor tissue and a preparation and use method thereof to solve the above problems.

[0009] The present invention is achieved through the following technical solutions:

[0010] A cryoprotectant for tumor tissues, comprising the following components: bovine serum albumin, polyvinyl pyrrolidone, sucrose, dimethyl sulfoxide, ethylene glycol and phosphate buffer. Specifically, the contents of the components in the cryoprotectant are: 1.5-2.5 w / v bovine serum albumin, 2-3 w / v polyvinyl pyrrolidone, 30-40 w / v sucrose, 8-12 v / v dimethyl sulfoxide, 20-30 v / v ethylene glycol, and the balance is phosphate buffer.

[0011] Furthermore, the contents of the components in the cryoprotectant solution are: bovine serum albumin 2 w / v%, polyvinyl pyrrolidone 2.5 w / v%, sucrose 34.23 w / v%, dimethyl sulfoxide 10 v / v%, ethylene glycol 26 v / v%, and the balance is phosphate buffer.

[0012] Furthermore, the phosphate buffer does not contain calcium and magnesium ions; the PBS buffer does not contain calcium and magnesium ions, which can avoid the formation of precipitation during the freezing process and affect cell activity.

[0013] It should be noted that, in the cryoprotectant solution for tumor tissue described in the present invention, by selecting the above-mentioned various components and limiting their usage ratios, the cryoprotectant solution is more suitable for freezing tumor tissue, avoiding tumor cells from rupturing due to dehydration or excessive water absorption, significantly reducing the production of cell ice crystals during the cooling process, and increasing the activity and survival rate of cells after recovery.

[0014] It should be noted that:

[0015] Bovine serum albumin (BSA) acts as a protein stabilizer, helping to prevent damage to cells and tissues during freezing and providing nutrients, helping to maintain the osmotic pressure of the solution and reducing ice crystal formation during cell freezing;

[0016] Polyvinylpyrrolidone (PVP) acts as a membrane protectant, helping to protect the integrity of the cell membrane and prevent rupture during freezing, helping to lower the freezing point and reduce the formation of ice crystals;

[0017] Sucrose, as an osmotic protectant, helps maintain the osmotic balance inside and outside the cell and prevents the cell from dehydrating or swelling during freezing. The concentration of sucrose reaches 1M to provide sufficient osmotic pressure, which can cause cell dehydration by generating extracellular osmotic pressure, reduce ice crystal formation in the cell, and stabilize the structure of the cell membrane.

[0018] Dimethyl sulfoxide (DMSO) is an osmotic protective agent that can penetrate the cell membrane, lower the freezing point of the intracellular solution, and reduce the formation of ice crystals, which helps protect cells from freezing damage;

[0019] Ethylene glycol also acts as an osmotic protectant, helping to reduce dehydration of cells during freezing. Ethylene glycol can also increase the viscosity of the solution, combining these two aspects to reduce the formation of ice crystals.

[0020] Phosphate buffered saline (PBS) is used to provide a stable pH environment, which helps maintain the physiological activity of cells and tissues. PBS without calcium and magnesium ions can avoid the formation of precipitation during the freezing process, which affects cell activity.

[0021] The cryoprotectant solution prepared by mixing various cryoprotectant ingredients in specific proportions is beneficial to the synergistic effect of cryoprotectants of different properties and improving the freezing effect. It is also beneficial to reduce the concentration of each component and reduce the cytotoxicity of a single cryoprotectant, so as to improve the survival rate and activity of cells after recovery.

[0022] The present invention also provides a method for preparing a cryoprotectant for tumor tissue, comprising the following steps:

[0023] Bovine serum albumin, polyvinyl pyrrolidone and sucrose were fully dissolved in phosphate buffer under the action of a magnetic stirrer, and then dimethyl sulfoxide and ethylene glycol were added to the above system and mixed thoroughly, and the volume was made up to 1000 ml with phosphate buffer, and then filtered with a filter element and stored for later use.

[0024] Furthermore, the dosage of each component in the preparation method is: bovine serum albumin 1.5-2.5 w / v, polyvinyl pyrrolidone 2-3 w / v, sucrose 30-40 w / v, dimethyl sulfoxide 8-12 v / v, ethylene glycol 20-30 v / v, and the balance is phosphate buffer.

[0025] Furthermore, the dosage of each component in the preparation method is: bovine serum albumin 2 w / v%, polyvinyl pyrrolidone 2.5 w / v%, sucrose 34.23 w / v%, dimethyl sulfoxide 10 v / v%, ethylene glycol 26 v / v%, and the balance is phosphate buffer.

[0026] Furthermore, the phosphate buffer does not contain calcium and magnesium ions, and the filter element specification is 0.22um.

[0027] It should be noted that in the method for preparing a cryoprotectant for tumor tissue described in the present invention, by selecting the various components mentioned above and limiting their usage ratios, the cryoprotectant is made more suitable for freezing tumor tissue, thereby avoiding rupture of tumor cells due to dehydration or excessive water absorption, significantly reducing the production of cell ice crystals during the cooling process, and increasing the activity and survival rate of cells after recovery.

[0028] The present invention also provides a method for using a cryoprotectant for tumor tissue, comprising the following steps:

[0029] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0030] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into tissue slices with surgical scissors;

[0031] 3) Quickly use sterile gauze to absorb as much physiological saline as possible on the tissue slices, immediately transfer to culture dish 1, fully contact with TFS1 solution therein, and treat at room temperature for a period of time;

[0032] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution therein, and treat it at room temperature for a period of time;

[0033] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution therein, and treat it at room temperature for a period of time;

[0034] 6) Use sterile gauze to quickly remove the TFS3 solution on the surface of the tissue slice treated in step 5) as much as possible, transfer it to the cryopreservation rack, spread it evenly, and transfer the cryopreservation rack containing the tissue to sterile liquid nitrogen;

[0035] 7) Transfer the fully frozen tissue and cryopreservation rack into the labeled cryopreservation tube pre-cooled with liquid nitrogen, tighten the cryopreservation tube and transfer it to the liquid nitrogen tank and keep records.

[0036] Furthermore, the phosphate buffer in step 1) does not contain calcium and magnesium ions; the PBS buffer does not contain calcium and magnesium ions to avoid the formation of precipitation during the freezing process, which affects the cell activity.

[0037] Furthermore, the thickness of the tissue slice in step 2) is 0.5-1.5 mm, preferably 1 mm.

[0038] It should be noted that the thickness of the tumor tissue slice may affect the penetration of the cryoprotectant, thereby affecting the protective effect of the cryoprotectant. At the same time, when the tumor tissue slice is put into liquid nitrogen, a layer of steam will be formed on the surface of the tissue slice due to the boiling of liquid nitrogen, blocking heat transfer, slowing down the cooling rate in the tissue, and more likely to cause ice crystals and cell damage. Therefore, reducing the thickness of the tumor tissue slice can reduce the thickness of the steam layer formed on the surface of the tumor tissue slice, increase the penetration efficiency of the cryoprotectant, speed up the heat conduction rate, and reduce freezing damage; however, the tumor tissue slice cannot be too thin, so as to avoid causing too many tumor cells to be broken and lost during the cutting process and produce many useless cell fragments.

[0039] Furthermore, the treatment time at room temperature in step 3) is 3 to 7 minutes, preferably 5 minutes;

[0040] Furthermore, the treatment time at room temperature in step 4) is 3 to 7 minutes, preferably 5 minutes;

[0041] Furthermore, the treatment time at room temperature in step 5) is 1 to 3 minutes, preferably 1 minute;

[0042] It should be noted that by strictly controlling the ratio of cryoprotectant to phosphate buffer in each step, as well as the processing time of tumor tissue slices, it is possible to gradually reduce the water content in the tissue and the formation of ice crystals while reducing cytotoxicity, thereby minimizing cell damage during the freezing process and maintaining tissue activity and integrity.

[0043] It should be noted that in step 6), the dehydrated tissue samples are quickly transferred to liquid nitrogen for rapid freezing to minimize cell damage; in step 7), the frozen tissue samples are transferred to a liquid nitrogen tank to provide a long-term stable low-temperature environment to maintain tissue activity.

[0044] Furthermore, the present invention also provides another method for using a cryoprotectant for tumor tissue by combining TFS cryoprotectant with serum preservation solution, comprising the following steps:

[0045] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0046] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into tissue slices with surgical scissors;

[0047] 3) Quickly use sterile gauze to absorb as much physiological saline as possible on the tissue slices, immediately transfer to culture dish 1, fully contact with TFS1 solution therein, and treat at room temperature for a period of time;

[0048] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution therein, and treat it at room temperature for a period of time;

[0049] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution therein, and treat it at room temperature for a period of time;

[0050] 6) Take out the tissue from the TFS3 solution and quickly transfer it to a cryopreservation rack. Transfer the cryopreservation rack with the tissue to cryopreservation tube 1 containing serum preservation solution, place it in a cryopreservation box, and transfer the cryopreservation box to a -80°C refrigerator overnight.

[0051] 7) Transfer the cryotube 1 containing the tissue to the labeled cryotube 2 pre-cooled with liquid nitrogen, tighten the cryotube 2, transfer it to the liquid nitrogen tank and keep a record.

[0052] Furthermore, the phosphate buffer in step 1) does not contain calcium and magnesium ions;

[0053] Furthermore, the thickness of the tissue slice in step 2) is 0.5 to 1.5 mm, preferably 1 mm;

[0054] Furthermore, the treatment time at room temperature in step 3) is 3 to 7 minutes, preferably 5 minutes;

[0055] Furthermore, the treatment time at room temperature in step 4) is 3 to 7 minutes, preferably 5 minutes;

[0056] Furthermore, the treatment time at room temperature in step 5) is 1 to 3 minutes, preferably 1 minute;

[0057] Furthermore, the serum preservation solution in step 6) comprises the following components: 90 v / v% serum, 10 v / v% dimethyl sulfoxide; the preparation method of the serum preservation solution is: take 900 ml serum, add 100 ml DMSO, mix thoroughly and store for later use.

[0058] By combining the cryoprotectant in the present invention with the serum preservation solution, treating tumor tissue slices with three concentrations of cryoprotectant, placing the tissue slices in a cryovial filled with the serum preservation solution, freezing them slowly overnight at -80°C, and then freezing them in liquid nitrogen, the defects of the TFS cryoprotectant and serum preservation solutions are overcome while the advantages of the two are fully combined, so that the combined use method can reduce the damage of cells during the freezing process by lowering the freezing point and reducing the formation of ice crystals through the TFS cryoprotectant, thereby playing a role in antifreeze and protecting cell membranes; and can also contribute to the long-term preservation of cells and the rapid resumption of growth after recovery through the nutrients and growth factors contained in the serum preservation solution; the protein in the serum preservation solution can also help maintain the structural integrity of the extracellular matrix, reduce and repair tissue collapse and cell separation caused by the osmotic effect of the osmotic components during the freezing process, and improve the success rate of cell transplantation.

[0059] In summary, the sequential use of TFS cryoprotectant and serum preservation solution provides a comprehensive method to protect the integrity and activity of tumor tissue during freezing and recovery, which helps to improve the application value and scientific research transformation capabilities of tumor tissue samples.

[0060] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0061] 1. The present invention provides a cryoprotectant for tumor tissue and a preparation method thereof. By selecting the above-mentioned various components and limiting their dosage ratios, the types and dosages of the components are further improved on the basis of the general cryopreservation solution, so that the cryoprotectant is more suitable for freezing tumor tissue, can fully avoid tumor cells from rupturing due to dehydration or excessive water absorption, significantly reduce the generation of cell ice crystals during the cooling process, and increase the activity, survival rate and proliferation ability of cells after recovery, thus overcoming the defects of the prior art that there are few vitrification cryopreservation solutions for targetedly improving the cryopreservation effect and activity of tumor tissue cells;

[0062] 2. The present invention provides a method for using a cryoprotectant for tumor tissues, wherein the cryoprotectant and phosphate buffer are configured into a plurality of cryoprotectant solutions in different ratios, gradient concentration treatment is performed, and the ratio of the cryoprotectant to the phosphate buffer in each step, as well as the size of the tumor tissue slices and the processing time of each step are strictly controlled, thereby gradually reducing the water content in the tissue and the formation of ice crystals on the basis of reducing cytotoxicity, improving the adaptability of cells to changes in the processing environment, minimizing cell damage during the freezing process, and maintaining the activity and integrity of the tissue;

[0063] 3. The present invention further combines TFS cryoprotectant with serum preservation solution in succession. After treating tumor tissue slices with three concentrations of cryoprotectant, the tissue slices are placed in a cryopreservation tube filled with serum preservation solution, and then slowly frozen at -80°C overnight, and then placed in liquid nitrogen for freezing. While overcoming the defects of TFS cryoprotectant and serum preservation solution, the advantages of both are fully combined, so that the combined use method can not only reduce freezing damage and protect cell membranes by lowering the freezing point and reducing the formation of ice crystals through TFS cryoprotectant; but also improve the long-term preservation effect of cells and the ability to quickly resume growth after resuscitation through the nutrients and growth factors contained in the serum preservation solution, maintain the structural integrity of the extracellular matrix, reduce and repair tissue collapse and cell separation caused by the freezing process, and improve the success rate of cell transplantation. The combination of TFS cryoprotectant and serum preservation solution can significantly reduce cytotoxicity, increase the activity, survival rate and proliferation ability of tumor cells, and play a synergistic role. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0065] Figure 1 This is a tissue slice image after processing in step 2) of Example 2;

[0066] Figure 2 The tissue slice image after the processing in step 3) of Example 2;

[0067] Figure 3 The tissue slice image after the processing in step 4) of Example 2;

[0068] Figure 4 The tissue slice image is a tissue slice image on a different color background after processing in step 5) in Example 2;

[0069] Figure 5The tumor distribution diagram of the tumor tissues inoculated into mice in Examples 2 and 5 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in combination with the embodiments and drawings. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention; the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

[0071] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0072] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0073] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0074] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0075] The invention provides a cryoprotective solution for tumor tissue, which is composed of the following components: bovine serum albumin, polyvinyl pyrrolidone, sucrose, dimethyl sulfoxide, ethylene glycol and phosphate buffer.

[0076] In a specific embodiment, the contents of the components in the cryoprotectant solution are: 1.5-2.5 w / v bovine serum albumin, 2-3 w / v polyvinyl pyrrolidone, 30-40 w / v sucrose, 8-12 v / v dimethyl sulfoxide, 20-30 v / v ethylene glycol, and the remainder is phosphate buffer.

[0077] Example 1

[0078] A cryoprotectant for tumor tissues, wherein each 1000 ml of the cryoprotectant consists of the following components: 20 g of bovine serum albumin, 25 g of polyvinyl pyrrolidone, 342.3 g of sucrose, 100 ml of dimethyl sulfoxide, 26 v / v% of ethylene glycol, and the remainder being a phosphate buffer solution free of calcium and magnesium ions.

[0079] The preparation method of the cryoprotectant solution for tumor tissue is as follows:

[0080] 20 g of bovine serum albumin, 25 g of polyvinyl pyrrolidone, and 342.3 g of sucrose were fully dissolved in 400 ml of phosphate buffer containing no calcium or magnesium ions under the action of a magnetic stirrer, and then 100 ml of dimethyl sulfoxide and 260 ml of ethylene glycol were added to the above system and mixed thoroughly, and the volume was made up to 1000 ml with phosphate buffer containing no calcium or magnesium ions, and then filtered with a 0.22 μm filter element and stored for later use.

[0081] The present invention provides a method for using a cryoprotectant for tumor tissue: preparing cryoprotectants of three concentrations; placing a tumor tissue slice after removing the surface blood vessels and capsule in low, medium and high concentration cryoprotectants for a period of time, preferably, first treating in a low concentration cryoprotectant for 3 to 7 minutes, then treating in a medium concentration cryoprotectant for 3 to 7 minutes, and then treating in a high concentration cryoprotectant for 1 to 3 minutes; and then vitrifying and freezing the tumor tissue slices treated in the three cryoprotectants. Embodiment 2 to Embodiment 4 are three specific embodiments, the main difference between which is the different treatment time in the cryoprotectant.

[0082] Example 2

[0083] A method for using a cryoprotectant for tumor tissue comprises the following steps:

[0084] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0085] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into 1mm thick tissue slices with surgical scissors ( Figure 1 );

[0086] 3) Quickly use sterile gauze to absorb as much saline as possible from the tissue slices, and immediately transfer them to culture dish 1, fully contacting them with the TFS1 solution, and treat them at room temperature for 5 minutes ( Figure 2 );

[0087] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution, and treat it at room temperature for 5 minutes ( Figure 3 );

[0088] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution, and treat it at room temperature for 1 minute ( Figure 4 );

[0089] 6) Use sterile gauze to quickly remove the TFS3 solution on the surface of the tissue slice treated in step 5) as much as possible, transfer it to the cryopreservation rack, spread it evenly, and transfer the cryopreservation rack containing the tissue to sterile liquid nitrogen;

[0090] 7) Transfer the fully frozen tissue and cryopreservation rack into the labeled cryopreservation tube pre-cooled with liquid nitrogen, tighten the cryopreservation tube and transfer it to the liquid nitrogen tank and keep records.

[0091] The cryoprotectant used in this example is the cryoprotectant prepared in Example 1.

[0092] Depend on Figure 1-Figure 4 It can be seen that after the treatment in step 5) of this embodiment, the tissue slice has undergone three steps of dehydration and has become relatively transparent, which reflects the penetration effect of the cryoprotectant on the tumor tissue.

[0093] Example 3

[0094] A method for using a cryoprotectant for tumor tissue comprises the following steps:

[0095] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0096] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into 1 mm thick tissue slices with surgical scissors;

[0097] 3) Quickly use sterile gauze to absorb as much saline as possible from the tissue slices, and immediately transfer them to culture dish 1, fully contact them with the TFS1 solution, and treat them at room temperature for 3 minutes;

[0098] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution, and treat it at room temperature for 3 minutes;

[0099] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution therein, and treat it at room temperature for 2 minutes;

[0100] 6) Use sterile gauze to quickly remove the TFS3 solution on the surface of the tissue slice treated in step 5) as much as possible, transfer it to the cryopreservation rack, spread it evenly, and transfer the cryopreservation rack containing the tissue to sterile liquid nitrogen;

[0101] 7) Transfer the fully frozen tissue and cryopreservation rack into the labeled cryopreservation tube pre-cooled with liquid nitrogen, tighten the cryopreservation tube and transfer it to the liquid nitrogen tank and keep records.

[0102] The cryoprotectant used in this example is the cryoprotectant prepared in Example 1.

[0103] Example 4

[0104] A method for using a cryoprotectant for tumor tissue comprises the following steps:

[0105] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0106] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into 1 mm thick tissue slices with surgical scissors;

[0107] 3) Quickly use sterile gauze to absorb as much saline as possible from the tissue slices, and immediately transfer them to culture dish 1, fully contact them with the TFS1 solution, and treat them at room temperature for 7 minutes;

[0108] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution, and treat it at room temperature for 7 minutes;

[0109] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution, and treat it at room temperature for 3 minutes;

[0110] 6) Use sterile gauze to quickly remove the TFS3 solution on the surface of the tissue slice treated in step 5) as much as possible, transfer it to the cryopreservation rack, spread it evenly, and transfer the cryopreservation rack containing the tissue to sterile liquid nitrogen;

[0111] 7) Transfer the fully frozen tissue and cryopreservation rack into the labeled cryopreservation tube pre-cooled with liquid nitrogen, tighten the cryopreservation tube and transfer it to the liquid nitrogen tank and keep records.

[0112] The cryoprotectant used in this example is the cryoprotectant prepared in Example 1.

[0113] The present invention also provides another method for using a cryoprotectant for tumor tissue, wherein the cryoprotectant of the present invention is used in combination with a serum preservative solution, which is different from the previous method for using a cryoprotectant in that: after treating tumor tissue slices with three concentrations of cryoprotectant, the tissue slices are placed in a cryopreservation tube filled with a serum preservative solution, and then slowly frozen at -80°C overnight, and then placed in liquid nitrogen for cryopreservation. Examples 5 and 6 are two specific examples, the main difference between which is the different treatment times in the cryoprotectant solution.

[0114] Example 5

[0115] A method for using a cryoprotectant for tumor tissue comprises the following steps:

[0116] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0117] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into 1 mm thick tissue slices with surgical scissors;

[0118] 3) Quickly use sterile gauze to absorb as much saline as possible from the tissue slices, and immediately transfer them to culture dish 1, fully contact them with the TFS1 solution, and treat them at room temperature for 5 minutes;

[0119] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution therein, and treat it at room temperature for 5 minutes;

[0120] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution, and treat it at room temperature for 1 minute;

[0121] 6) Take out the tissue from the TFS3 solution and quickly transfer it to a cryopreservation rack. Transfer the cryopreservation rack with the tissue to a cryopreservation tube filled with serum preservation solution, place it in a cryopreservation box, and transfer the cryopreservation box to a -80℃ refrigerator overnight.

[0122] 7) Transfer the cryotube 1 containing the tissue to the labeled cryotube 2 pre-cooled with liquid nitrogen, tighten the cryotube 2, transfer it to the liquid nitrogen tank and keep a record.

[0123] The cryoprotectant solution in this embodiment is the cryoprotectant solution prepared in Example 1; the preparation method of the serum preservation solution is: take 900 ml of serum, add 100 ml of DMSO, mix thoroughly and store for later use.

[0124] Example 6

[0125] A method for using a cryoprotectant for tumor tissue comprises the following steps:

[0126] 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:3 and transfer to culture dish 1 to obtain TFS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer without calcium and magnesium ions in a ratio of 1:1 and transfer to culture dish 2 to obtain TFS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain TFS3 solution, and equilibrate to room temperature;

[0127] 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into 1 mm thick tissue slices with surgical scissors;

[0128] 3) Quickly use sterile gauze to absorb as much saline as possible from the tissue slices, and immediately transfer them to culture dish 1, fully contact them with the TFS1 solution, and treat them at room temperature for 4 minutes;

[0129] 4) Use sterile gauze to quickly remove the TFS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the TFS2 solution, and treat it at room temperature for 6 minutes;

[0130] 5) Use sterile gauze to quickly remove the TFS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to culture dish 3, fully contact it with the TFS3 solution therein, and treat it at room temperature for 2 minutes;

[0131] 6) Take out the tissue from the TFS3 solution and quickly transfer it to a cryopreservation rack. Transfer the cryopreservation rack with the tissue to a cryopreservation tube filled with serum preservation solution, place it in a cryopreservation box, and transfer the cryopreservation box to a -80℃ refrigerator overnight.

[0132] 7) Transfer the cryotube 1 containing the tissue to the labeled cryotube 2 pre-cooled with liquid nitrogen, tighten the cryotube 2, transfer it to the liquid nitrogen tank and keep a record.

[0133] The cryoprotectant used in this example is the cryoprotectant prepared in Example 1; the preparation method of the serum preservation solution in step 6) is: take 900 ml of serum, add 100 ml of DMSO, mix thoroughly and store for later use.

[0134] Comparative Example 1

[0135] Serum freezing method:

[0136] 1) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish filled with physiological saline, and then quickly cut it into 1 mm thick slices with surgical scissors.

[0137] 2) Transfer the cut tissue to a freezing rack, transfer the freezing rack with tissue to a cryovial containing serum preservative solution, place it in a freezing box, and transfer the freezing box to a -80°C refrigerator overnight.

[0138] 3) Transfer the cryotube containing the tissue to a labeled cryotube pre-cooled with liquid nitrogen, tighten the cryotube, transfer it to the liquid nitrogen tank and keep a record.

[0139] The preparation method of the serum preservation solution is as follows: take 900 ml of serum, add 100 ml of DMSO, mix thoroughly and store for later use.

[0140] Comparative Example 2

[0141] Direct freezing in liquid nitrogen:

[0142] 1) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish filled with physiological saline, and then quickly cut it into 1 mm thick slices with surgical scissors.

[0143] 2) Transfer the cut tissue to the freezing rack, transfer the freezing rack with tissue to the labeled liquid nitrogen pre-cooled cryotube, tighten the cryotube and transfer it to the liquid nitrogen tank and keep records.

[0144] The frozen tissues in Example 2, Example 5 and Comparative Example 1-2 were taken respectively, and the temperature was quickly thawed and raised to 37°C. After discarding the serum, the tissues frozen by TFS were subjected to gradient water immersion treatment to restore the tissue cell ion concentration to normal. The resuscitated tumor cell tissues were taken of the same size and inoculated subcutaneously into immunodeficient mice. The tumor cell tissues of each example were inoculated at 4 sites, and the tumor growth status of the mice was observed after 21 days.

[0145] like Figure 5 As shown (for ease of observation, Figure 5 The closed curve is used to depict the tumor contour). Figure 5 The mice were inoculated with the tumor tissues cryopreserved in Example 5, Example 2, Comparative Example 1, and Comparative Example 2, respectively, from left to right.

[0146] In the mice in Comparative Example 1 that were only cryopreserved with serum and in Comparative Example 2 that were directly cryopreserved, it was difficult to observe the protrusions in the tumor cell group by naked eyes and touch, indicating that the tumor tissues treated with serum preservation solution and directly cryopreserved did not grow and proliferate in the inoculated mice. In Example 5, TFS cryopreservation solution was used in combination with serum preservation solution, and in Example 2, mice that were transplanted after being treated with TFS cryopreservation solution were observed to have obvious tumor mass growth. In particular, the mice inoculated with cryopreserved tissue in Example 2 had obvious aggregated growth of tumor tissue observed at one point, and the size of the tumor was calculated to be 8.26 mm after measurement with a vernier caliper. 3 In Example 5, after the mice were inoculated with frozen tissue, obvious aggregated growth of tumor tissue was observed at two points, and the volumes were calculated by vernier caliper to be 90.125 mm 3 、13.95mm 3 It can be seen that the TFS cryoprotectant and its use method provided by the present invention can significantly improve the activity and proliferation ability of tumor cells after recovery, and obvious aggregated tumor tissue blocks can be observed in mice after transplantation; among them, the activity and proliferation ability of tumor tissue treated by the method of using TFS cryoprotectant in combination with serum preservation solution are stronger, and the number of obvious aggregated tumor tissue blocks obtained by transplanting it into mice is more, and the size of the tumor blocks is also larger, which confirms that the combination of TFS cryoprotectant and serum preservation solution can significantly reduce cytotoxicity, increase the activity, survival rate and proliferation ability of tumor cells, and achieve a synergistic effect.

[0147] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cryoprotectant for tumor tissue, characterized in that: The invention is composed of the following components: 1.5-2.5 w / v bovine serum albumin, 2-3 w / v polyvinyl pyrrolidone, 30-40 w / v sucrose, 8-12 v / v dimethyl sulfoxide, 20-30 v / v ethylene glycol, and the balance is phosphate buffer; the phosphate buffer does not contain calcium and magnesium ions.

2. The cryoprotectant for tumor tissue according to claim 1, characterized in that: The contents of the components in the cryoprotectant solution are as follows: bovine serum albumin 2 w / v, polyvinyl pyrrolidone 2.5 w / v, sucrose 34.23 w / v, dimethyl sulfoxide 10 v / v, ethylene glycol 26 v / v, and the balance is phosphate buffer.

3. A method for preparing a cryoprotectant solution for tumor tissue according to any one of claims 1 to 2, characterized in that: include: Bovine serum albumin, polyvinyl pyrrolidone and sucrose were fully dissolved in phosphate buffer under the action of a magnetic stirrer, and then dimethyl sulfoxide and ethylene glycol were added to the above system and mixed thoroughly, and the volume was made up to 1000 ml with phosphate buffer, and then filtered with a filter element and stored for later use.

4. A method for using the cryoprotectant solution for tumor tissue according to any one of claims 1 to 2, comprising the following steps: 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:3 and transfer to culture dish 1 to obtain FTS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:1 and transfer to culture dish 2 to obtain FTS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain FTS3 solution, and equilibrate to room temperature; 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into tissue slices with surgical scissors; 3) Quickly use sterile gauze to absorb the saline on the tissue slice as much as possible, immediately transfer it to culture dish 1, fully contact it with the FTS1 solution, and treat it at room temperature for a period of time; 4) Use sterile gauze to quickly remove the FTS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the FTS2 solution therein, and treat it at room temperature for a period of time; 5) Use sterile gauze to quickly remove the FTS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to the culture dish 3, fully contact it with the FTS3 solution therein, and treat it at room temperature for a period of time; 6) Use sterile gauze to quickly remove the FTS3 solution on the surface of the tissue slice treated in step 5) as much as possible, transfer it to a cryopreservation rack, spread it evenly, and transfer the cryopreservation rack containing the tissue to sterile liquid nitrogen; 7) Transfer the fully frozen tissue and cryopreservation rack into the labeled cryopreservation tube pre-cooled with liquid nitrogen, tighten the cryopreservation tube and transfer it to the liquid nitrogen tank and keep records.

5. A method for using the cryoprotectant solution for tumor tissue according to any one of claims 1 to 2, comprising the following steps: 1) Use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:3 and transfer to culture dish 1 to obtain FTS1 solution; use a pipette to mix the cryoprotectant solution and phosphate buffer in a ratio of 1:1 and transfer to culture dish 2 to obtain FTS2 solution; pipette the cryoprotectant solution into culture dish 3 to obtain FTS3 solution, and equilibrate to room temperature; 2) Take the tumor tissue, remove the surface blood vessels and capsule in a culture dish 4 filled with physiological saline, and quickly cut it into tissue slices with surgical scissors; 3) Quickly use sterile gauze to absorb the saline on the tissue slice as much as possible, immediately transfer it to culture dish 1, fully contact it with the FTS1 solution, and treat it at room temperature for a period of time; 4) Use sterile gauze to quickly remove the FTS1 solution on the surface of the tissue slice treated in step 3) as much as possible, and immediately transfer it to culture dish 2, fully contact it with the FTS2 solution therein, and treat it at room temperature for a period of time; 5) Use sterile gauze to quickly remove the FTS2 solution on the surface of the tissue slice treated in step 4) as much as possible, and immediately transfer it to the culture dish 3, fully contact it with the FTS3 solution therein, and treat it at room temperature for a period of time; 6) Take out the tissue from the FTS3 solution and quickly transfer it to the cryopreservation rack. Transfer the cryopreservation rack with the tissue to cryopreservation tube 1 containing serum preservation solution, place it in the cryopreservation box, and transfer the cryopreservation box to a -80℃ refrigerator overnight. 7) Transfer the cryotube 1 containing the tissue to the labeled cryotube 2 pre-cooled with liquid nitrogen, tighten the cryotube 2, transfer it to the liquid nitrogen tank and keep a record.

6. The method for using the cryoprotectant solution for tumor tissue according to claim 5, characterized in that: The treatment time at room temperature in step 3) is 3 to 7 minutes; the treatment time at room temperature in step 4) is 3 to 7 minutes; and the treatment time at room temperature in step 5) is 1 to 3 minutes.

7. The method for using the cryoprotectant solution for tumor tissue according to claim 6, characterized in that: The treatment time at room temperature in step 3) is 5 minutes; the treatment time at room temperature in step 4) is 5 minutes; and the treatment time at room temperature in step 5) is 1 minute.

8. The method for using the cryoprotectant for tumor tissue according to any one of claims 5 to 7, characterized in that: The serum preservation solution in step 6) comprises the following components: serum 90 v / v%, dimethyl sulfoxide 10 v / v%.

9. The method for using the cryoprotectant solution for tumor tissue according to claim 8, characterized in that: The preparation method of the serum preservation solution in step 6) is: take 900 ml of serum, add 100 ml of DMSO, mix thoroughly and store for later use.

10. The method for using the cryoprotectant solution for tumor tissue according to claim 8, characterized in that: The thickness of the tissue slice in step 2) is 0.5-1.5 mm.

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