Tissue preserving fluid in organoid construction as well as preparation method and application of tissue preserving fluid

Abstract

The invention discloses a tissue preservation solution in organoid construction as well as a preparation method and application thereof. The tissue preservation solution is prepared from the following components: an Advanced DMEM / F12 culture medium, a mycillin / amphotericin B three-antibody mother solution, Glutamax-1, an HEPES buffer solution and a Y-27632 mother solution. The preparation method of the tissue preserving fluid comprises the following steps: adding an Advanced DMEM / F12 culture medium into a centrifugal tube on a super clean bench; then sequentially adding a mycillin / amphotericin B three-antibody mother solution, Glutamax-1, an HEPES buffer solution and Y-27632, and uniformly mixing, so as to obtain the tissue preservation solution. After uniformly mixing, filtering and sterilizing by using a disposable needle type filter, and collecting the preserving fluid into a centrifugal tube; the invention also discloses an application of the tissue preserving fluid in organoid construction. According to the tissue preserving fluid obtained by the invention, in the transportation process, the cell viability in the tissue is improved, the cell damage is reduced, and the success rate of primary cell adherent culture and organoid construction is increased.

Description

Technical Field

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

[0002] In the construction of primary adherent cells and organoids, tumor tissue needs to be stored in the corresponding preservation fluid after being removed from the body, and then transported to the laboratory for subsequent primary adherent cell culture or tumor organoid culture. During the transportation process, the ability of the tissue preservation fluid to preserve the tumor tissue or normal tissue after removal from the body is one of the key steps in the successful construction of primary cells or organoids. Existing preservation fluids have the following problems: 1. The preservation fluid has a weak ability to preserve cells in the tissue. When the tissue is transported to the laboratory, most of the cells in the tissue die or apoptosis; 2. The storage time is short, generally 24h~48h; beyond this time point, the cell mortality rate decreases linearly with the extension of time.

[0003] Therefore, this article proposes a tissue preservation fluid, preparation method, and application in organoid construction. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a tissue preservation solution for organoid construction, which can improve the viability of tissue cells during transportation, and a preparation method and application thereof.

[0005] In order to achieve the above technical effects, the present invention provides a tissue preservation solution for organoid construction, including Advanced DMEM / F12 culture medium, penicillin-streptomycin / amphotericin B triple antibody mother solution, Glutamax-1, HEPES buffer, and Y-27632 mother solution; The working final concentration of the penicillin-streptomycin / amphotericin B triple antibody stock solution is 0.5-1.5X; The final working concentration of Glutamax-1 is 0.5-1.5X; The final working concentration of the HEPES buffer is 4-6 unit / ml; The final working concentration of the Y-27632 stock solution is 8-12uM.

[0006] The present invention also provides a method for preparing a tissue preservation solution for organoid construction, the method specifically comprising the following steps: S1. Add Advanced DMEM / F12 medium to the centrifuge tube on the clean bench. S2, then add penicillin-streptomycin / amphotericin B triple antibody mother solution, Glutamax-1, HEPES buffer, and Y-27632 in sequence and mix well, which is the tissue preservation solution; S3. After mixing, filter and sterilize with a disposable needle filter, and collect the preservation solution in a centrifuge tube.

[0007] Furthermore, the preservation solution collected in the centrifuge tube in S3 can be stored in a 4°C refrigerator and used up within 2 weeks, and can be stored at -20°C for 6 months.

[0008] The present invention also provides an application of a tissue preservation solution in organoid construction, including using the tissue preservation solution of the present invention, wherein the application specifically comprises the following steps: S1. Preparation of preservation solution: Prepare tissue digestion solution in a centrifuge tube on a clean bench in the laboratory, filter the preservation solution with a disposable needle filter, and finally collect it in a centrifuge tube; S2. Bring the tissue preservation solution to the operating room in an ice box or a 4°C low-temperature transport box. Take the tissue sample in the operating room and place it in the preservation solution. Furthermore, the tissue sample in S2 needs to be cut into 1 cm pieces with scissors. 3 Place 3 to 5 tissue blocks in the preservation solution to ensure that the tissue can be completely immersed in the preservation solution; S3. Insert the centrifuge tube containing the tissue and preservation solution into an ice box, or place it vertically in a low-temperature transport box to ensure that the tissue will not be exposed to the air during transportation. At the same time, the transportation temperature is strictly controlled at 4~8℃.

[0009] Furthermore, the transportation time for transporting the tissue back to the laboratory in S3 must be less than 4 days.

[0010] Compared with the prior art, the beneficial effects of the present invention are mainly: The present invention optimizes the tumor tissue preservation solution during the transportation of tumor tissue: by adding an appropriate concentration of Glutamax-1 to the preservation solution, the substances necessary for the cell life of the tumor tissue during transportation are maintained; Y-27632 with a final concentration of 10uM is added to inhibit the death and apoptosis of cells in the tumor tissue during transportation; HEPES with an appropriate concentration is added to maintain the stability of the preservation solution to avoid damage to cells caused by an over-acid and over-alkaline environment during transportation; antibiotics are added to inhibit the growth of bacteria; through the above combination, after 72 hours, the survival rate of primary cells digested from the tissue preserved by the preservation solution of the present invention is more than 85%, and the contamination rate is low. Under the joint action of a certain concentration of these reagents, the cell survival rate in the tissue is improved during transportation, the cell damage is reduced, and the success rate of primary cell adherent culture and organoid construction is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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.

[0012] Figure 1 The cell survival rate analysis diagram of the umbilical cord tissues of the present invention, brand 1 and brand 2 respectively stored for 96 hours and then digested; Figure 2 The left picture shows cells digested after 96 hours of storage of umbilical cord tissue. The middle picture shows cells digested after 96 hours of storage using a certain brand 1 storage solution. The right picture shows cells digested after 96 hours of storage using a certain brand 2 storage solution. Figure 3 The diagram shows the cell status after umbilical cord tissue was stored for 96 hours and digested and cultured for 3 days. The left picture shows the cell status after 96 hours of digestion and cultured for 3 days by this method. The middle picture shows the cell status after 96 hours of digestion and cultured for 3 days by a certain brand 1 preservation solution. The right picture shows the cell status after 96 hours of digestion and cultured for 3 days by a certain brand 2 preservation solution. Figure 4 The cell survival rate analysis diagram of the tumor tissues preserved for 96 hours and digested using the present invention and a certain brand 1; Figure 5 The left picture is a picture of the inoculation of organoids after digestion, collection and culture of breast cancer tumor tissue preserved in the preservation solution for 72 hours; the left picture is a picture of the inoculation of organoids after digestion, collection and culture of breast cancer tumor tissue preserved in the preservation solution of the present invention for 72 hours on day 0, and the right picture is a picture of the inoculation of organoids after digestion, collection and culture of breast cancer tumor tissue preserved in a preservation solution of a certain brand 1 for 72 hours on day 0; Figure 6 The left picture is a picture of the growth of organoids from a breast cancer tumor tissue bank preserved in the preservation solution for 72 hours; the right picture is a picture of the growth of organoids from a breast cancer tumor tissue bank preserved in the preservation solution of a certain brand 1 preservation solution for 72 hours on the 7th day. DETAILED DESCRIPTION

[0013] 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.

[0014] This is because the existing preservation fluid has the following problems for the construction of organoids: the preservation fluid has a weak ability to preserve cells in the tissue, and most of the cells in the tissue die or apoptosis when the tissue is transported to the laboratory; the preservation time is short, generally 24h~48h; beyond this time point, the cell mortality rate decreases linearly with the extension of time.

[0015] Therefore, the inventors provide a tissue preservation solution for organoid construction, including Advanced DMEM / F12 culture medium, penicillin-streptomycin / amphotericin B triple antibody stock solution, Glutamax-1, HEPES buffer, and Y-27632 stock solution; The working final concentration of the penicillin-streptomycin / amphotericin B triple antibody stock solution is 0.5-1.5X; The final working concentration of Glutamax-1 is 0.5-1.5X; The final working concentration of the HEPES buffer is 4-6 unit / ml; The final working concentration of the Y-27632 stock solution is 8-12uM.

[0016] The present invention also provides a method for preparing a tissue preservation solution for organoid construction, the method specifically comprising the following steps: S1. Add Advanced DMEM / F12 medium to the centrifuge tube on the clean bench. S2, then add penicillin-streptomycin / amphotericin B triple antibody mother solution, Glutamax-1, HEPES buffer, and Y-27632 in sequence and mix well, which is the tissue preservation solution; S3. After mixing, filter and sterilize with a disposable needle filter, and collect the preservation solution in a centrifuge tube.

[0017] Furthermore, the preservation solution collected in the centrifuge tube in S3 can be stored in a 4°C refrigerator and used up within 2 weeks, and can be stored at -20°C for 6 months.

[0018] The present invention also provides an application of a tissue preservation solution in organoid construction, including using the tissue preservation solution of the present invention, wherein the application specifically comprises the following steps: S1. Preparation of preservation solution: Prepare tissue digestion solution in a centrifuge tube on a clean bench in the laboratory, filter the preservation solution with a disposable needle filter, and finally collect it in a centrifuge tube; S2. Bring the tissue preservation solution to the operating room in an ice box or a 4°C low-temperature transport box. Take the tissue sample in the operating room and place it in the preservation solution. Furthermore, the tissue sample in S2 needs to be cut into 1 cm pieces with scissors. 3 Place 3 to 5 tissue blocks in the preservation solution to ensure that the tissue can be completely immersed in the preservation solution; S3. Insert the centrifuge tube containing the tissue and preservation solution into an ice box, or place it vertically in a low-temperature transport box to ensure that the tissue will not be exposed to the air during transportation. At the same time, the transportation temperature is strictly controlled at 4~8℃.

[0019] Furthermore, the transportation time for transporting the tissue back to the laboratory in S3 must be less than 4 days. Example 1

[0020] Based on the above technical solution, the inventor conducted the following specific experiments: For each tumor tissue or normal tissue, 10 ml of preservation solution should be prepared and stored at 4°C.

[0021] 1. The reagents and configurations required for tumor / normal tissue preservation solution are as follows: Serial number Reagents Working final concentration Volume required for 10ml system 1 Advanced DMEM / F12 9.70ml 2 Penicillin-streptomycin, amphotericin B triple antibody (100X) 1X 100ul 3 Glutamax-1 (100X) 1X 100ul 4 HEPES (concentration: 1M) 10mM 100uL 5 Y-27632 (concentration: 10 mM) 10uM 1ul 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, 2.5ul pipette, 2.5ul pipette tip, small ultrasonic cleaner; 3. In a clean bench, add 9.7 ml of DMEM / F12 basal medium to a 15 ml centrifuge tube, then add 100 ul of 100X penicillin-streptomycin B, 100 ul of 100X Glutamax-1, 100 ul of 1M HEPES, and 1 ul of 10 mM Y-27632. Mix well, filter and sterilize with a 0.22 um disposable needle filter, collect the preservation solution in a 15 ml centrifuge tube, and store it in a 4 °C refrigerator for a short time. Generally, use it up within 2 weeks and store it at -20 °C for 6 months. 4. The functions of each reagent in the technical solution are as follows: 4.1 Advanced DMEM / F12 (1X): As the main component of the reagent configuration, it contains amino acids required to maintain cell activity and supports the normal physiological metabolism of cells during tissue transport; 4.2 Penicillin-streptomycin / amphotericin B triple antibiotic: Penicillin is a β-lactam antibiotic that kills bacteria by blocking the synthesis of bacterial cell walls, causing them to swell, rupture, and autolyze; streptomycin is an aminoglycoside antibiotic that can inhibit bacterial protein synthesis; amphotericin B is a polyene antifungal drug that can irreversibly bind to the sterols of fungal cell membranes to form transmembrane channels that destroy the integrity of the membrane, causing small molecules or nucleotides, amino acids, etc. in the cell to leak out of the membrane, destroying the normal metabolism of the fungus and causing death, and is used to inhibit fungal and yeast contamination. The combination of the three ingredients can effectively inhibit the reproduction of bacteria, fungi, and yeast and the contamination of tumor cells; 4.3 Glutamax-1: It is a conditionally essential amino acid that provides important energy and nitrogen source for cell metabolism. The appropriate concentration of glutamine in the process of tumor tissue transportation can maintain the normal physiological and metabolic functions of cells; 4.4 Y-27632: a small molecule inhibitor of the Rho-associated protein kinase p160ROCK, which can effectively prevent cell apoptosis during tumor tissue digestion; 4.5 HEPES: A non-ionic amphoteric buffer with good buffering capacity, no toxicity to cells, and the ability to maintain a constant pH range for a long time and maintain the stability of the solution. Example 2

[0022] Based on the above embodiments, the inventors conducted experiments based on the construction of primary adherent cells from umbilical cord tissue and the construction of breast cancer organoids, as follows: 1. Configuration of preservation solution: 1.1 In advance, prepare 10 ml of tissue digestion solution in a 15 ml centrifuge tube in the laboratory clean bench according to the reagents in the table below, and then filter the preservation solution with a 0.22 uM disposable needle filter. After collection, it can be stored in a 4 ° C refrigerator for two weeks and -20 ° C for 6 months; Serial number Reagents Working final concentration Volume required for 10ml system 1 Advanced DMEM / F12 9.70ml 2 Penicillin-streptomycin, amphotericin B 1X 100ul 3 Glutamax-1 (100X) 1X 100ul 4 HEPES 5unit / ml 100ul 5 Y-27632 10uM 1ul 1.2 Use an ice box or a 4℃ low-temperature transport box to bring the tissue preservation solution to the operating room. Take the tissue sample in the operating room and cut it into 1cm3 tissue blocks with scissors. Place 3 to 5 tissue blocks in the preservation solution to ensure that the tissue can be completely immersed in the preservation solution. 1.3 Insert the centrifuge tube containing the tissue and preservation solution into an ice box, or place it vertically in a low-temperature transport box to ensure that the tissue is not exposed to the air during transportation. At the same time, the transportation temperature is strictly controlled at 4-8°C. It is best to transport the tissue back to the laboratory within 3 days.

[0023] 2. Enzyme digestion and culture of umbilical cord tissue: 2.1 Bring the tissue block back to the laboratory, register the sample information, and then transfer it to the laboratory clean bench for subsequent operations; 2.2 Use HBSS to prepare collagenase type VI digestion solution with an enzyme activity of 200 U / ml. The volume ratio of digestion solution to tissue block is about 1:10; 2.3 Wash the umbilical cord tissue in the tissue preservation solution with PBS several times until it turns white, and remove the blood vessels and capsule in the umbilical cord; 2.4 Cut the tissue blocks into 1mm3 pieces on an ice box, add the prepared type IV collagenase digestion solution, blow and mix, and place in a 37℃ shaker for digestion for 3-5 hours; 2.5 After no obvious tissue is found, add appropriate amount of DMEM basal medium to dilute the digestion mixture, filter it with a 100uM cell sieve, collect the filtered solution, and centrifuge it at 500g for 5 minutes; 2.6 Discard the supernatant, add DMEM basal medium to wash the precipitate, aspirate 90ul of the liquid and add 10u of table blue to count the cells and calculate the cell viability, centrifuge the remaining liquid at 500g for 5min to collect the precipitate; repeat this step once; 2.7 Discard the supernatant and add the precipitate to a T25 bottle containing complete culture medium containing 10% FBS. Incubate and observe in a 37°C, 5% CO2 incubator.

[0024] 3. Digestion and culture of tumor organoids: 3.1 Bring the tissue block back to the laboratory, register the sample information, and then transfer it to the laboratory clean bench for subsequent operations; 3.2 Prepare three 6 cm culture dishes and pour PBS with 1% antibiotics into them; 3.3 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; 3.4 In the second dish, cut the tissue into pieces the size of rice grains and wash the tissue thoroughly with PBS containing double antibodies; 3.5 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; 3.6 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; 3.7 Transfer the chopped tissue pieces to 10 ml of 1000U / ml type IV collagenase digestion solution, and resuspend the sample by blowing. Transfer to a shaker, and digest at 37°C, 80 rpm for 50-90 min (depending on the situation of different tissues). Use a pipette to blow once every 10 min and take out 10ul to observe the digestion situation until a large number of tumor cells are digested and there are no large tumor tissue pieces. Then proceed to the next step; 3.8 Termination of digestion: After digestion, quickly add FBS to the digestion solution to make the final concentration of FBS 4% to terminate digestion and mix the liquid.

[0025] At the same time, based on the above experiments, the inventor compared the phenomena existing in a certain brand and the patent of this invention, as follows: 1. Preservation of umbilical cord tissue: The ex vivo umbilical cord tissue was obtained in the operating room and cut into pieces with sterile scissors on ice, about the size of soybeans, and then placed in tissue preservation solution with a volume ratio of tissue to preservation solution of about 1:10. After being stored in a 4°C transport box for 96 hours, the tissue was lysed with enzymes, stained with Benzodiac Blue to calculate the cell viability and take pictures. Figure 1 and Figure 2 As shown, the cell viability of the cells digested after 96 hours of storage in the preservation solution of this method is 80.7%; that of a certain brand 1 is 25.2%; and that of a certain brand 2 is 75.5%.

[0026] At the same time, the digested cells were cultured for adhesion. After 24 hours of culture, the cell viability was much higher than that of the preservation solution on the market after 96 hours of preservation using this method. At the same time, the digested cells were cultured for 24 hours, and the cells adhered to the wall, showing the spindle-shaped state of umbilical cord stem cells, while the cell adhesion state of a certain brand was poor, such as Figure 3 shown.

[0027] 2. For tumor tissues used to build breast cancer organoid libraries: The ex vivo tumor tissue was obtained from the operating room and cut into pieces the size of soybeans with sterile scissors on ice. It was divided into two equal parts and then placed in tissue preservation solution with a volume ratio of tissue to preservation solution of approximately 1:10. It was stored in a 4°C transport box for 72 hours and decomposed with tissue digestion solution. After dyeing with pan blue, the cell survival rate was calculated and photographed. Figure 4 and Figure 6 As shown, the cell viability of cells digested after 72 hours of storage in the preservation solution of this method is 88.2%; that of a certain brand 1 is 85.7%.

[0028] At the same time, if Figure 5 Kazuto Figure 6As shown, cells and cell fragments digested from tissues of the same size were used to construct breast cancer organoids, and the cells were embedded in matrix gel and then seeded. The preservation solution formula of the present invention can obtain relatively more living cells and cell clusters after 72 hours of tissue preservation, and the organoids have a good growth condition after 7 days of culture.

[0029] 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.

[0030] 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. A tissue preservation solution for organoid construction, characterized in that: Including Advanced DMEM / F12 culture medium, penicillin-streptomycin / amphotericin B triple antibody stock solution, Glutamax-1, HEPES buffer, Y-27632 stock solution; The working final concentration of the penicillin-streptomycin / amphotericin B triple antibody stock solution is 0.5-1.5X; The final working concentration of Glutamax-1 is 0.5-1.5X; The final working concentration of the HEPES buffer is 4-6 unit / ml; The final working concentration of the Y-27632 stock solution is 8-12uM.

2. A method for preparing a tissue preservation solution for organoid construction, characterized in that: The method specifically comprises the following steps: S1. Add Advanced DMEM / F12 medium to the centrifuge tube on the clean bench. S2, then add penicillin-streptomycin / amphotericin B triple antibody mother solution, Glutamax-1, HEPES buffer, and Y-27632 in sequence and mix well, which is the tissue preservation solution; S3. After mixing, filter and sterilize with a disposable needle filter, and collect the preservation solution in a centrifuge tube.

3. The method for preparing tissue preservation solution for organoid construction according to claim 2, characterized in that: The preservation solution collected in the centrifuge tube in S3 can be stored in a 4°C refrigerator and used up within 2 weeks. It can be stored at -20°C for 6 months.

4. Use of the tissue preservation solution according to any one of claims 1 to 3 in organoid construction, comprising using the tissue preservation solution of the present invention, characterized in that: The application specifically comprises the following steps: S1. Preparation of preservation solution: Prepare tissue digestion solution in a centrifuge tube on a clean bench in the laboratory, filter the preservation solution with a disposable needle filter, and finally collect it in a centrifuge tube; S2. Bring the tissue preservation solution to the operating room in an ice box or a 4°C low-temperature transport box. Take the tissue sample in the operating room and place it in the preservation solution. S3. Insert the centrifuge tube containing the tissue and preservation solution into an ice box, or place it vertically in a low-temperature transport box to ensure that the tissue will not be exposed to the air during transportation. At the same time, the transportation temperature is strictly controlled at 4~8℃.

5. The use of the tissue preservation solution according to claim 4 in organoid construction, characterized in that: The tissue samples in S2 need to be cut into 1 cm3 tissue blocks with scissors, and 3 to 5 tissue blocks are placed in the preservation solution to ensure that the tissue can be completely immersed in the preservation solution.

6. The use of the tissue preservation solution in organoid construction according to claim 4, characterized in that: The transportation time for transporting the tissue back to the laboratory in S3 must be less than 4 days.

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