Tissue preservation solution for organoid culture as well as preparation method and application of tissue preservation solution

Abstract

The invention relates to the technical field of tissue preservation, in particular to tissue preservation liquid for organoid culture as well as a preparation method and application of the tissue preservation liquid. The tissue preserving fluid provided by the invention takes an Advanced DMEM / F12 culture medium as a basic culture medium, and comprises the following components in concentration: 50-60 [mu] M of 2-mercaptoethanol, 10-30 g / L of bovine serum albumin, 100-200 [mu] g / ml of a primary cell antibiotic and 8-12 [mu] M of a ROCK inhibitor. By compounding the components with proper concentrations, the obtained tissue preserving fluid can preserve a tissue sample for organoid culture at low temperature for 72 hours, maintain tissue cell activity and reduce cell damage, does not influence organoid culture, and improves the organoid culture success rate.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue preservation, and in particular to a tissue preservation solution for organoid culture and a preparation method and application thereof. Background Art

[0002] Organoids are three-dimensional cultures of cells in vitro that contain some key characteristics of the organs from which they are derived. Organoids are usually cultured from tissue samples. After the tissue samples are removed from the body, in order to maintain the activity of the tissue and improve the success rate of organoid culture, they often need to be processed and cultured in a short period of time. Once the sample is removed from the body for more than 4 hours without any treatment, it is easy to cause culture failure. However, due to long transportation time and other practical reasons, the ex vivo tissue often cannot be processed immediately. Instead, the tissue is first immersed in the corresponding tissue preservation solution and then processed.

[0003] At present, the common tissue preservation fluids in vitro are phosphate buffer saline (PBS) and cell culture medium. The main components of PBS buffer include disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and potassium chloride, which have salt balance and adjustable pH buffering effect; the main components of cell culture medium include basal culture medium, bovine serum, antibiotics, etc., which can be used to maintain cell growth and proliferation. However, in actual use, it is found that PBS buffer has simple components but limited functions and cannot well maintain the biological activity of in vitro tissues; cell culture medium has complex components and high cost, so it is not the best choice as a tissue preservation fluid. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a tissue preservation solution for organoid culture and its preparation method and application. The tissue preservation solution provided by the present invention can preserve tissue samples for organoid culture at low temperatures for 72 hours, maintain tissue cell activity, reduce cell damage, and improve the success rate of organoid culture.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a tissue preservation solution for organoid culture. The tissue preservation solution uses Advanced DMEM / F12 culture medium as a basic culture medium and comprises the following components in concentrations: 50-60 μM 2-mercaptoethanol, 10-30 g / L bovine serum albumin (BSA), 100-200 μg / ml primary cell antibiotics and 8-12 μM ROCK inhibitor.

[0006] Preferably, the tissue preservation solution comprises components in the following concentrations: 55 μM 2-mercaptoethanol, 10 g / L bovine serum albumin, 100 μg / ml primary cell antibiotics and 10 μM ROCK inhibitor.

[0007] Preferably, the primary cell antibiotic comprises primocin.

[0008] Preferably, the ROCK inhibitor comprises Y-27632.

[0009] The present invention provides a method for preparing the tissue preservation solution described in the above technical solution, comprising the following steps: 2-mercaptoethanol, bovine serum albumin, primary cell antibiotics, ROCK inhibitor and Advanced DMEM / F12 culture medium were mixed in proportion, and filtered through a 0.22 μm filter membrane to obtain a filtrate as the tissue preservation solution.

[0010] The present invention provides the use of the tissue preservation solution described in the above technical solution or the tissue preservation solution prepared by the preparation method described in the above technical solution in the preservation and / or transportation of tissue samples for organoid culture.

[0011] Preferably, the tissue sample comprises one or more of a lung cancer tissue sample, a breast cancer tissue sample and a colon cancer tissue sample.

[0012] The present invention provides a method for preserving and / or transporting tissue for organoid culture, comprising the following steps: The tissue sample for organoid culture is placed in a tissue preservation solution and transported or stored at 4°C; the tissue preservation solution is the tissue preservation solution described in the above technical solution or the tissue preservation solution prepared by the preparation method described in the above technical solution.

[0013] Preferably, the transportation or storage time is ≤ 72 hours.

[0014] Preferably, the tissue sample comprises one or more of a lung cancer tissue sample, a breast cancer tissue sample and a colon cancer tissue sample.

[0015] Beneficial effects: The present invention provides a tissue preservation solution for organoid culture, which uses Advanced DMEM / F12 culture medium as a basic culture medium and includes the following components at the following concentrations: 2-mercaptoethanol 50-60 μM, BSA 10-30 g / L, primary cell antibiotics 100-200 μg / ml and ROCK inhibitor 8-12 μM. The 2-mercaptoethanol in the tissue preservation solution provided by the present invention is a biological antioxidant that can remove hydroxyl free radicals; BSA is the main protein component of serum, which replaces FBS as a nitrogen source for cell growth and maintenance of metabolic activities, and can reduce cell attachment to the wall of the centrifuge tube; primary cell antibiotics can inhibit bacteria-fungi-mycoplasma; ROCK inhibitors can inhibit cell apoptosis. The present invention compounded the components at appropriate concentrations, and the obtained tissue preservation solution can preserve tissue samples for organoid culture at low temperatures for 72 hours, maintain tissue cell activity, reduce cell damage, and does not affect the culture of organoids, thereby improving the success rate of organoid culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is a picture of lung cancer organoids preserved for 24 hours; Figure 2 This is a picture of lung cancer organoids preserved for 48 hours; Figure 3 This is a picture of lung cancer organoids preserved for 72 hours; Figure 4 This is a picture of breast cancer organoids preserved for 24 hours; Figure 5 This is a picture of breast cancer organoids stored for 48 hours; Figure 6 This is a picture of breast cancer organoids preserved for 72 hours; Figure 7 This is a picture of a colon cancer organoid preserved for 24 hours; Figure 8 This is a picture of intestinal cancer organoids preserved for 48 hours; Fig. 9 This is a picture of colorectal cancer organoids preserved for 72 hours. DETAILED DESCRIPTION

[0018] The invention provides a tissue preservation solution for organoid culture. The tissue preservation solution uses Advanced DMEM / F12 culture medium as a basic culture medium and comprises the following components in concentrations: 50-60 μM 2-mercaptoethanol, 10-30 g / L bovine serum albumin (BSA), 100-200 μg / ml primary cell antibiotics and 8-12 μM ROCK inhibitor.

[0019] As an embodiment, the tissue preservation solution uses Advanced DMEM / F12 medium as the basic medium, including the following components at the following concentrations: 55 μM 2-mercaptoethanol, 10 g / L bovine serum albumin, 100 μg / ml primary cell antibiotics, and 10 μM ROCK inhibitor.

[0020] As an embodiment, the primary cell antibiotic may be primocin.

[0021] As an embodiment, the ROCK inhibitor may be Y-27632.

[0022] The tissue preservation solution obtained by compounding the components at appropriate concentrations in the present invention can preserve tissue samples for organoid culture at low temperatures for 72 hours, maintain tissue cell activity, reduce cell damage, and does not affect the culture of organoids, thereby improving the success rate of organoid culture.

[0023] The tissue preservation solution of the present invention is stored for a long time at -20°C, thawed at 4°C before use, and stored at 4°C until use, thereby obtaining a usable tissue preservation solution for organoid culture.

[0024] The present invention provides a method for preparing the tissue preservation solution described in the above technical solution, comprising the following steps: After 2-mercaptoethanol, bovine serum albumin, primary cell antibiotics, ROCK inhibitor and Advanced DMEM / F12 culture medium are mixed in proportion, the mixture is filtered through a 0.22 μm filter membrane to obtain a filtrate as the tissue preservation solution. The present invention can remove bacteria from the tissue preservation solution by filtering through a 0.22 μm filter membrane.

[0025] Based on the above advantages, the present invention provides the use of the tissue preservation solution described in the above technical solution or the tissue preservation solution prepared by the preparation method described in the above technical solution in the preservation and / or transportation of tissue samples for organoid culture.

[0026] As an embodiment, the tissue sample includes one or more of a lung cancer tissue sample, a breast cancer tissue sample, and a colon cancer tissue sample.

[0027] Based on the above advantages, the present invention provides a method for tissue preservation and / or transportation for organoid culture, comprising the following steps: The tissue sample for organoid culture is placed in a tissue preservation solution and transported or stored at 4°C; the tissue preservation solution is the tissue preservation solution described in the above technical solution or the tissue preservation solution prepared by the preparation method described in the above technical solution.

[0028] As an embodiment, the transportation or storage time is ≤72 hours.

[0029] As an embodiment, the tissue sample includes one or more of a lung cancer tissue sample, a breast cancer tissue sample, and a colon cancer tissue sample.

[0030] In order to further illustrate the present invention, a tissue preservation solution for organoid culture provided by the present invention, a preparation method thereof and an application thereof are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 A tissue preservation solution for organoid culture, the sources of the components and working concentrations are shown in Table 1.

[0032] Table 1 Sources and working concentrations of components in tissue preservation solution

[0033] The preparation method of the tissue preservation solution is as follows: 1. According to the concentration requirements, take 5 mg of Y-27632 powder and dissolve it in 1.56 ml of DMSO to prepare a 10 mM Y-27632 stock solution; 2. According to the concentration requirements, dissolve 10g BSA powder in 100ml PBS to prepare a mother solution with a concentration of 10%; 3. Add 2-mercaptoethanol, Y-27632 stock solution, BSA stock solution and primocin to Advanced DMEM / F12 medium in the working concentrations listed in Table 1 to prepare tissue preservation solution, and then filter and sterilize using a 0.22 μm filter membrane.

[0034] Example 2 The tissue preservation solution prepared in Example 1 was divided into 3 groups, each with 3 tubes. Fresh human lung cancer, breast cancer, and intestinal cancer tissues were taken, with a tissue weight of 0.5 g each, placed in a 50 ml centrifuge tube, added with 20 ml of tissue preservation solution, and stored in a 4°C refrigerator for 24 hours, 48 ​​hours, and 72 hours, respectively. The tissue was sheared, added with tissue digestion solution, and digested in a 37°C water bath shaker for 15 to 60 minutes, dissociated into a cell suspension, and the cell survival rate was detected using a cell counter as follows: Resuspend the cell suspension with 1-3 ml HBSS, take 10 μl of the cell suspension and mix it with 10 μl AO / PI dye, then drop it into the cell counting plate, insert it into the cell counter for detection and counting, and read the counting results of the counter. The results are shown in Table 2.

[0035] After the cells were inoculated and cultured, the growth status of the organoids was observed. Figure 1 to Figure 9,in Figure 7~Figure 9 The scale bars in the lower right corner are all 200 μm.

[0036] Table 2 Cell survival rates of different tissues stored at different times

[0037] The results show that the tissue preserved by the tissue preservation solution provided by the present invention can maintain the activity of tissue cells within 72 hours, does not affect the culture of organoids, and improves the success rate of organoid culture.

[0038] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue preservation solution for organoid culture, characterized in that: Advanced DMEM / F12 medium was used as the basic medium, including the following components at the following concentrations: 2-mercaptoethanol 50-60 μM, bovine serum albumin 10-30 g / L, primary cell antibiotics 100-200 μg / ml, and ROCK inhibitor 8-12 μM.

2. The tissue preservation solution according to claim 1, characterized in that The tissue preservation solution includes components with the following concentrations: 55 μM 2-mercaptoethanol, 10 g / L bovine serum albumin, 100 μg / ml primary cell antibiotics, and 10 μM ROCK inhibitor.

3. The tissue preservation solution according to claim 1 or 2, characterized in that: The primary cell antibiotics include primocin.

4. The tissue preservation solution according to claim 1 or 2, characterized in that: The ROCK inhibitors include Y-27632.

5. The method for preparing the tissue preservation solution according to any one of claims 1 to 4, characterized in that: The following steps are involved: 2-mercaptoethanol, bovine serum albumin, primary cell antibiotics, ROCK inhibitor and Advanced DMEM / F12 culture medium were mixed in proportion, and filtered through a 0.22 μm filter membrane to obtain a filtrate as the tissue preservation solution.

6. Use of the tissue preservation solution according to any one of claims 1 to 4 or the tissue preservation solution prepared by the preparation method according to claim 5 in the preservation and / or transportation of tissue samples for organoid culture.

7. The use according to claim 6, characterized in that: The tissue sample includes one or more of a lung cancer tissue sample, a breast cancer tissue sample and a colon cancer tissue sample.

8. A method for preserving and / or transporting tissue for organoid culture, characterized in that: The following steps are involved: The tissue sample for organoid culture is placed in a tissue preservation solution and transported or stored at 4°C; the tissue preservation solution is the tissue preservation solution described in any one of claims 1 to 4 or the tissue preservation solution prepared by the preparation method described in claim 5.

9. The storage and / or transportation method according to claim 8, characterized in that: The transportation or storage time is ≤ 72 hours.

10. The storage and / or transportation method according to claim 8, characterized in that: The tissue sample includes one or more of a lung cancer tissue sample, a breast cancer tissue sample and a colon cancer tissue sample.

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