Tumor tissue digestive juice in organoid construction as well as preparation method and application thereof

Abstract

The invention discloses a tumor tissue digestive juice in organoid construction as well as a preparation method and application thereof. The tumor tissue digestive juice is prepared from the following components: an Advanced DMEM / F12 culture medium, 0.5 to 1.5 X of mycillin / amphotericin B three-antibody mother liquor, 0.5 to 1.5 X of glutamine, 4 to 6 unit / ml of deoxyribonuclease I, 8 to 12 mu of MY-27632 mother liquor and 900 to 1100 U / ml of IV type collagenase. The preparation method of the tumor tissue digestive juice comprises the following steps: weighing required IV type collagenase, putting the IV type collagenase into a centrifugal tube, adding an Advanced DMEM / F12 culture medium, sequentially sucking mycillin, amphotericin B three-antibody mother liquor, glutamine mother liquor, mother liquor of a deoxyribonuclease I solution and Y-27632 mother liquor, adding the mother liquor into a mixed solution, and uniformly mixing to finally obtain the tumor tissue digestive juice. The invention discloses application of tumor tissue digestive juice in organoid construction. The viscosity of the obtained digestive juice is greatly reduced, and a large number of cells and cell clusters are digested, so that the success rate of organoid modeling is obviously improved.

Description

Technical Field

[0001] The patent of this invention belongs to the field of biotechnology, and specifically relates to tumor tissue digestive fluid, preparation method and application in organoid construction. Background Art

[0002] Whether the organoids are successfully constructed, the digestion of tumor tissue by digestive fluid is very important. During the research process, the inventors found that the existing digestive fluids have the following problems in the construction of organoids: 1. During the initial digestion process of tumor organoid construction, tumor cells or cell clusters are difficult to be digested by collagenase; 2. The digested cells or cell clusters show apoptosis and decreased viability during the digestion with collagenase and the 30min~2h digestion process; 3. Since the DNA released by dead cells in the digestion solution makes the solution too viscous, most of the DNA will attach to tissues and cells. The free DNA will cause the cells to aggregate into clumps, affecting the digestion efficiency of other collagenases. In addition, cell agglomeration is not conducive to the passage of cells through the filter, resulting in low recovery rates of cells and cell clusters, and a reduced success rate of library construction.

[0003] Based on the above points, it can be seen that whether the digestive effect of tumor tissue digestive fluid can obtain a large number of cells and cell clusters with high viability is one of the reasons for the successful construction of organoids.

[0004] Therefore, this article proposes a tumor tissue digestion fluid, preparation method, and application in organoid construction. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a tumor tissue digestion fluid in organoid construction, a preparation method and an application thereof, which can improve the success rate of organoid modeling.

[0006] In order to achieve the above technical effects, the present invention provides a tumor tissue digestion solution in organoid construction, including Advanced DMEM / F12 culture medium, 0.5-1.5X penicillin-streptomycin / amphotericin B triple antibody stock solution, 0.5-1.5X glutamine, 4-6unit / ml deoxyribonuclease I, 8-12uMY-27632 stock solution, and 900-1100U / ml type IV collagenase; The Y-27632 mother solution is prepared by adding DMSO to Y-27632 and completely dissolving it by ultrasonication to obtain the Y-27632 mother solution.

[0007] The present invention also provides a method for preparing tumor tissue digestion fluid in organoid construction, and the method specifically comprises the following steps: S1. Weigh the required type IV collagenase and put it into a centrifuge tube, then add Advanced DMEM / F12 culture medium; S2, aspirate penicillin-streptomycin and amphotericin B triple antibody mother solution and add it into the mixed solution obtained in S1 and mix well; S3, taking the glutamine mother solution and adding it into the mixed solution obtained in S2 and mixing well; S4, taking the mother solution of deoxyribonuclease I solution and adding it into the mixed solution obtained in S3 and mixing well; S5, taking the Y-27632 mother solution and adding it into the mixed solution obtained in S4 and mixing well; S6. Use a sterile disposable medical syringe to extract the mixed solution obtained in S5, install a disposable needle filter and filter it into a new centrifuge tube. This is the tumor tissue digestion fluid.

[0008] The present invention also provides an application of tumor tissue digestion fluid in organoid construction, including using the tumor tissue digestion fluid of the present invention, and the application specifically comprises the following steps: S1. Tumor tissue pretreatment: After disinfection, the selected tumor tissue was placed in three culture dishes containing PBS to wash and separate impurities, then placed in a centrifuge tube and the tissue blocks were cut into pieces; Furthermore, the tumor tissue pretreatment method in S1 specifically comprises the following steps: S101, obtain the corresponding tumor tissue from the operating room, sterilize it and place it on the clean bench, prepare three culture dishes, and pour PBS with 1% antibiotics into them; S102, then immerse the tumor tissue in the first culture dish, shake it thoroughly to wash it, use tweezers to remove dead cells, fat tissue, muscle tissue, and some blood cells, and after they are completely removed, transfer the sample to the second culture dish; S103. Cut the tissue into pieces the size of rice grains in a second culture dish and wash the tissue thoroughly with PBS containing double antibodies; S104, continue to transfer the small tumor tissue block to the third culture dish, continue to fully wash the tissue block; and then put the tissue block into a centrifuge tube; S105. Use sterile scissors to cut the tissue blocks in the centrifuge tube into small pieces as much as possible, and cut the tissue blocks into a viscous state without large particles; S2. Transfer the chopped tissue pieces into the tumor tissue digestion solution, blow and resuspend the sample, quickly transfer to a shaker, and digest at 37°C for 50 min to 90 min. Use a pipette to beat once every 10 min and take out 10 ul to observe the digestion status until a large number of tumor cells are digested and there are no large tumor tissue pieces. S3. Termination of digestion: After digestion, quickly add FBS to the digestion solution to make the final concentration of FBS 4% to terminate digestion, mix the liquid, and finally obtain cells and cell clusters that can be used for organoid construction.

[0009] Compared with the prior art, the beneficial effects of the present invention are mainly: The present invention optimizes the digestive fluid of tumor tissue in the process of organoid construction, and by adding suitable collagenase to the digestive fluid, more tumor cells and cell clusters can be separated from tumor tissue in a short time; substances necessary for maintaining cell life are added, and reagents for inhibiting cell apoptosis are added to improve the viability of cells separated under the action of digestive enzymes and during digestion, greatly reducing cell death and apoptosis; DNA enzyme is added to reduce the viscosity of the digestive fluid, reduce the situation of digested cell agglomeration, and increase the number of cells and cell clusters separated; antibiotics are added to better inhibit bacterial growth. Under the joint action of these reagents, the viscosity of the digestive fluid of the present invention is greatly reduced, and a large number of cells and cell clusters are digested out, and when the digestive fluid is applied in organoid modeling, the success rate of organoid modeling is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for describing the embodiment are briefly introduced below. Those skilled in the art can also obtain other drawings based on these drawings without paying any creative work.

[0011] Figure 1 This is a graph analyzing the survival rate of cells digested by the digestion solution of the present invention; Figure 2 This is a graph showing the survival rate of cells digested from Bozhen’s organoid digestion fluid; Figure 3 The sample pictures of tumor tissue and digestive fluid during digestion in the embodiment of the present invention are shown in FIG. The tube marked with IV is the digestive fluid of the present invention, and the tube marked with II is the digestive fluid of the organoid of Bozhen; Figure 4 This is an analysis diagram of cells and cell clusters digested by the digestion solution of the present invention; Figure 5 This is an analysis diagram of cells and cell clusters digested from the digestion fluid of the organoid of Bozhen of the present invention; DETAILED DESCRIPTION

[0012] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0013] This is because the existing digestion fluid has the following problems for organoid construction: in the initial digestion process of tumor organoid construction, tumor cells or cell clusters are difficult to be digested by collagenase; the digested cells or cell clusters undergo apoptosis and reduced viability during the digestion of collagenase and the 30min~2h digestion process; and because the DNA released by the dead cells in the digestion fluid makes the solution too viscous, most of the DNA will attach to tissues and cells. Free DNA will cause cells to aggregate into clusters, affecting the digestion efficiency of other collagenases, and cell clusters are not conducive to cells passing through the filter, resulting in low recovery rates of cells and cell clusters and reduced success rates in library construction.

[0014] Therefore, the inventors provide a tumor tissue digestion solution for organoid construction, including Advanced DMEM / F12 culture medium, 0.5-1.5X penicillin-streptomycin / amphotericin B triple antibody stock solution, 0.5-1.5X glutamine, 4-6unit / ml deoxyribonuclease I, 8-12uMY-27632 stock solution, and 900-1100U / ml type IV collagenase; The Y-27632 mother solution is prepared by adding DMSO to Y-27632 and completely dissolving it by ultrasonication to obtain the Y-27632 mother solution.

[0015] The present invention also provides a method for preparing tumor tissue digestion fluid in organoid construction, and the method specifically comprises the following steps: S1. Weigh the required type IV collagenase and put it into a centrifuge tube, then add Advanced DMEM / F12 culture medium; S2, aspirate penicillin-streptomycin and amphotericin B triple antibody mother solution and add it into the mixed solution obtained in S1 and mix well; S3, taking the glutamine mother solution and adding it into the mixed solution obtained in S2 and mixing well; S4, taking the mother solution of deoxyribonuclease I solution and adding it into the mixed solution obtained in S3 and mixing well; S5, taking the Y-27632 mother solution and adding it into the mixed solution obtained in S4 and mixing well; S6. Use a sterile disposable medical syringe to extract the mixed solution obtained in S5, install a disposable needle filter and filter it into a new centrifuge tube. This is the tumor tissue digestion fluid.

[0016] The present invention also provides an application of tumor tissue digestion fluid in organoid construction, including using the tumor tissue digestion fluid of the present invention, and the application specifically comprises the following steps: S1. Tumor tissue pretreatment: After disinfection, the selected tumor tissue was placed in three culture dishes containing PBS to wash and separate impurities, then placed in a centrifuge tube and the tissue blocks were cut into pieces; Furthermore, the tumor tissue pretreatment method in S1 specifically comprises the following steps: S101, obtain the corresponding tumor tissue from the operating room, sterilize it and place it on the clean bench, prepare three culture dishes, and pour PBS with 1% antibiotics into them; S102, then immerse the tumor tissue in the first culture dish, shake it thoroughly to wash it, use tweezers to remove dead cells, fat tissue, muscle tissue, and some blood cells, and after they are completely removed, transfer the sample to the second culture dish; S103. Cut the tissue into pieces the size of rice grains in a second culture dish and wash the tissue thoroughly with PBS containing double antibodies; S104, continue to transfer the small tumor tissue block to the third culture dish, continue to fully wash the tissue block; and then put the tissue block into a centrifuge tube; S105. Use sterile scissors to cut the tissue blocks in the centrifuge tube into small pieces as much as possible, and cut the tissue blocks into a viscous state without large particles; S2. Transfer the chopped tissue pieces into the tumor tissue digestion solution, blow and resuspend the sample, quickly transfer to a shaker, and digest at 37°C for 50 min to 90 min. Use a pipette to beat once every 10 min and take out 10 ul to observe the digestion status until a large number of tumor cells are digested and there are no large tumor tissue pieces. S3. Termination of digestion: After digestion, quickly add FBS to the digestion solution to make the final concentration of FBS 4% to terminate digestion, mix the liquid, and finally obtain cells and cell clusters that can be used for organoid construction. Example

[0017] Based on the above technical solution, the inventor conducted the following specific experiments: Because each organoid library construction only requires 10 ml of tumor tissue digestion fluid, the inventors used the technical solution of the present invention to configure 10 ml of tumor tissue digestion fluid. At the same time, the inventors purchased the mainstream product in the prior art: Bozhen's organoid digestion fluid, and conducted comparative tests based on the construction of breast cancer organoids, and recorded data images of the digestion of breast cancer tumor tissue.

[0018] The specific implementation of the technical solution is as follows: 1. The reagents required for tumor tissue digestion fluid in organoid construction are as follows:

[0019] 2. Required experimental consumables: 0.22um disposable needle filter, 20ml syringe, 1 / 1000 electronic balance, medicine spoon, 15ml centrifuge tube made of PET material, 1ml pipette, 1ml pipette tip, 200ul pipette, 200ul pipette tip, 20ul pipette, 20ul pipette tip, small ultrasonic cleaner 3. Experimental steps (10ml digestion solution preparation steps are as follows): 3.1. Type IV collagenase: Calculate the mass of type IV collagenase required for 10 ml based on the enzyme activity unit of collagenase (the enzyme activity unit of each batch can be obtained according to the specifications produced by the manufacturer) and the required 1000 units / ml, weigh it with a 1 / 1000 balance, and put it into a 15 ml centrifuge tube for later use. For example: the purchased type IV collagenase is 310 units / mg, so weigh 32.3 mg from 10 ml of digestion solution for later use.

[0020] 3.2. Preparation of Y-27632 mother solution: Add 2.022 ml of DMSO to 10 mg of Y-27632 and dissolve it completely by ultrasonication to obtain 20 mM Y-27632 mother solution.

[0021] 3.3. Preparation steps of organoid tumor tissue digestion fluid: 3.3.1. Weigh 10 ml of the required type IV collagenase and put it into a 15 ml centrifuge tube. Add 9.775 ml of Advanced DMEM / F12 medium. 3.3.2. Pipette 100ul of penicillin-streptomycin and amphotericin B triple antibody stock solution and add it into the mixed solution in 3.3.1 and mix well; 3.3.3. Pipette 100ul of glutamine mother solution and add it into the mixed solution in 3.3.2 and mix well; 3.3.4. Pipette 20ul of DNase I solution and add it into the mixed solution prepared in 3.3.3 and mix well. 3.3.5. Pipette 5ul of the mother solution of Y27632 and add it into the mixed solution in 3.3.4 and mix well; 3.4.6. Use a sterile 20ml disposable medical syringe to extract the mixed solution in 3.3.5, and filter it into a new 15ml centrifuge tube using a 0.22um disposable needle filter. This is the tumor tissue digestion solution.

[0022] According to the above experimental steps, the inventors applied them to the process of organoid construction: 1. Prepare 10 ml of tumor tissue digestion solution for organoid construction in a 15 ml centrifuge tube; 2. Obtain the corresponding tumor tissue from the operating room and transport it to the laboratory at 4°C; 3. After registration, the tumor tissue is transferred to the clean laboratory and placed on the clean bench for experiment after disinfection; 4. Prepare three 6 cm culture dishes and pour PBS with 1% antibiotics into them; 5. Use tweezers to take out the tissue block. A 1cm3 tumor tissue block can be used to construct an organoid. Soak 1cm3 of tumor tissue in the first dish, shake it thoroughly to wash it, and use tweezers to remove dead cells, fat tissue, muscle tissue, and some blood cells. After removing them, transfer the sample to the second dish. 6. Cut the tissue into pieces the size of rice grains in a second dish and wash the tissue thoroughly with PBS containing double antibodies; 7. Transfer the small tumor tissue block to the third culture dish and continue to wash the tissue block thoroughly; then place the tissue block into a 1.5ml centrifuge tube; 8. Use sterile scissors to cut the tissue blocks in the EP tube into small pieces as much as possible, and cut the tissue blocks into a viscous state without large particles; 9. Transfer the chopped tissue pieces to 10 ml of tumor tissue digestion solution configured by the present invention, blow and resuspend the sample, quickly transfer to a shaker, and digest at 37°C for 50 min to 90 min (depending on the situation of different tissues). Beat once with a 1 ml pipette every 10 min and take out 10 ul to observe the digestion situation until a large number of tumor cells are digested out and there are no large amounts of tumor tissue pieces, then proceed to the next step; 10. Termination of digestion: After digestion, quickly add FBS to the digestion solution to make the final concentration of FBS 4% to terminate digestion, mix the liquid, and finally obtain cells and cell clusters that can be used for organoid construction.

[0023] Based on the above embodiments, the inventors also conducted the following tests: 1. Cell survival rate: like Figure 1 to Figure 2 As shown, the digestion solution configured in this method evenly distributes the chopped tumor tissue to two tubes of digestion solution for digestion. After 1 hour, the cell viability obtained by this method is 85.8%; that of Bozhen's organoid digestion solution is 83.2%; at the same time, the digested cells are counted, and 964,000 cells can be obtained by this method, and there are a large number of cells and cell clusters that are easy to construct organoids; 625,000 cells were obtained by Bozhen's organoid digestion solution, and the cells were relatively loose, and most of them were single cells.

[0024] 2. Viscosity of digestive juice: like Figure 3As shown in the figure, the digested tumor tissue is observed during the digestion process. The tube marked as IV is the formula of the present invention, and the tube marked as II is the organoid digestion fluid of Bozhen. It can be seen that during the digestion process of the formula of the present invention, the digestion fluid is relatively clear and the viscosity of the digestion fluid is relatively low, while during the digestion process of Bozhen's organoid digestion fluid, the tissues are adhered together, which is not conducive to collecting a large number of cells or cell clusters, and greatly reduces the success rate of organoid construction.

[0025] Number of cells after digestion: like Figures 4 to 5 As shown in the figures, both are pictures in which the inventors filter the digested tumor tissue after the digestion with the digestive fluid is completed, collect cells and cell clusters for organoid culture. It can be seen from the above figures that the present invention can obtain a large number of cells and cell clusters for the construction of organoids.

[0026] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0027] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Tumor tissue digestion fluid in organoid construction, characterized by: The components of the tumor tissue digestion fluid include Advanced DMEM / F12 culture medium, 0.5-1.5X penicillin-streptomycin / amphotericin B triple antibody stock solution, 0.5-1.5X glutamine, 4-6unit / ml deoxyribonuclease I, 8-12uMY-27632 stock solution, and 900-1100U / ml type IV collagenase.

2. The tumor tissue digestion fluid in organoid construction according to claim 1, characterized in that: The Y-27632 mother solution is prepared by adding DMSO to Y-27632 and completely dissolving it by ultrasonication to obtain the Y-27632 mother solution.

3. A method for preparing tumor tissue digestion fluid in organoid construction, characterized in that: The specific steps include: S1. Weigh the required type IV collagenase and put it into a centrifuge tube, then add Advanced DMEM / F12 culture medium; S2, aspirate penicillin-streptomycin and amphotericin B triple antibody mother solution and add it into the mixed solution obtained in S1 and mix well; S3, taking the glutamine mother solution and adding it into the mixed solution obtained in S2 and mixing well; S4, taking the mother solution of deoxyribonuclease I solution and adding it into the mixed solution obtained in S3 and mixing well; S5, taking the Y-27632 mother solution and adding it into the mixed solution obtained in S4 and mixing well; S6. Use a sterile disposable medical syringe to extract the mixed solution obtained in S5, install a disposable needle filter and filter it into a new centrifuge tube. This is the tumor tissue digestion fluid.

4. The method for using the tumor tissue digestion fluid in organoid construction according to any one of claims 1 to 3, comprising using the tumor tissue digestion fluid of the present invention, characterized in that: The application specifically comprises the following steps: S1. Tumor tissue pretreatment: After disinfection, the selected tumor tissue was placed in three culture dishes containing PBS to wash and separate impurities, then placed in a centrifuge tube and the tissue blocks were cut into pieces; S2. Transfer the chopped tissue pieces into the tumor tissue digestion solution, blow and resuspend the sample, quickly transfer to a shaker, and digest at 37°C for 50 min to 90 min. Use a pipette to beat once every 10 min and take out 10 ul to observe the digestion status until a large number of tumor cells are digested and there are no large tumor tissue pieces. S3. Termination of digestion: After digestion, quickly add FBS to the digestion solution to make the final concentration of FBS 4% to terminate digestion, mix the liquid, and finally obtain cells and cell clusters that can be used for organoid construction.

5. The use of tumor tissue digestion fluid in organoid construction according to claim 4, characterized in that: The tumor tissue pretreatment method in S1 specifically comprises the following steps: S101, obtain the corresponding tumor tissue from the operating room, sterilize it and place it on the clean bench, prepare three culture dishes, and pour PBS with 1% antibiotics into them; S102, then immerse the tumor tissue in the first culture dish, shake it thoroughly to wash it, use tweezers to remove dead cells, fat tissue, muscle tissue, and some blood cells, and after they are completely removed, transfer the sample to the second culture dish; S103. Cut the tissue into pieces the size of rice grains in a second culture dish and wash the tissue thoroughly with PBS containing double antibodies; S104, continue to transfer the small tumor tissue block to the third culture dish, continue to fully wash the tissue block; and then put the tissue block into a centrifuge tube; S105. Use sterile scissors to cut the tissue blocks in the centrifuge tube into small pieces as much as possible, and cut the tissue blocks into a viscous state without large particles.

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