Tissue sample preserving fluid and tissue preserving method

Abstract

The invention belongs to the field of biology, and discloses a tissue sample preservation solution which comprises a basic culture medium, the basic culture medium contains fetal calf serum and MitoQ, and FBS and MitoQ are added and used on the basis of the basic culture medium, so that the activity of tissue cells can be remarkably improved; in the research process, we have the following specificity that: 1, in the presence of FBS, only MitoQ in the mitochondrial targeting antioxidant can show a relatively strong tissue activity maintaining effect; 2, the effect of MitoQ in a basic culture medium is not fully played, and the MitoQ must be played in the presence of FBS; meanwhile, the invention further provides a tissue preservation method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to a kind of tissue sample preservative and tissue preservation method. BACKGROUND

[0002] MitoQ is a mitochondria-targeted antioxidant, which is used as tissue preservation can be seen as follows:

[0003] Publication No.CA3132533A1, subject to composition comprising antioxidant for preserving corneal tissue; It is described therein: the amount of antioxidant can effectively reduce corneal endothelial cell death, reduce reactive oxygen species (ROS), reduce apoptosis or necrosis, increase mitochondrial function, increase mitochondrial or non-mitochondrial cell respiration, or any combination thereof.

[0004] Through the preliminary experiment, we found the following phenomenon:

[0005] 1. It does not significantly improve the improvement of the culture medium on the activity of the tissue;

[0006] 2. Its performance must be under certain conditions to show.

[0007] Therefore, based on the above findings, the present application is proposed. SUMMARY

[0008] The purpose of the present application is to provide a kind of tissue sample preservative, which is based on the basis of basic culture medium, add FBS and MitoQ, can significantly improve the activity of tissue cells; In the research process, we have the following specific findings: 1. In the presence of FBS, only MitoQ in the mitochondria-targeted antioxidant can show strong tissue viability maintenance effect; 2. MitoQ in the basic culture medium is not sufficient, must be in the presence of FBS to play.

[0009] Meanwhile, the present application also provides a kind of tissue preservation method.

[0010] To achieve the above object, the present application discloses a kind of tissue sample preservative, including basic culture medium;The basic culture medium contains fetal bovine serum and MitoQ.

[0011] In the above-mentioned tissue sample preservative, the basic culture medium contains 5-15wt% FBS fetal bovine serum, 1-10 μM MitoQ.

[0012] Preferably, the basic culture medium contains 5-15wt% FBS fetal bovine serum, 1-2 μM MitoQ.

[0013] In the above-mentioned tissue sample preservative, the basic culture medium also includes double antibody, and the double antibody is penicillin and streptomycin.

[0014] The concentration of penicillin in the culture medium is 50-200 U / mL; the concentration of streptomycin is 50-200 U / mL.

[0015] In the above-mentioned tissue sample storage solution, the basic culture medium is one of DMEM, RPMI-1640 and MEM.

[0016] Meanwhile, the application also discloses a tissue sample storage method, which uses the above-mentioned tissue sample storage solution to store the tissue sample.

[0017] In the above-mentioned storage method, the storage environment is 2-20 DEG C.

[0018] In the above-mentioned storage method, the storage duration is not less than 72 h.

[0019] In the above-mentioned storage method, the tissue sample is a tumor tissue sample or a normal tissue sample.

[0020] The application has the following advantages and effects compared with the prior art:

[0021] 1. The application verifies that MitoQ can improve the tissue viability maintaining ability of the basic culture medium;

[0022] 2. The application verifies that the combination of MitoQ and FBS can more obviously improve the tissue viability maintaining ability of the basic culture medium;

[0023] 3. The application verifies that after the combination of MitoQ and FBS is applied to the basic culture medium, the preservation and transportation of fresh liver tissue samples and fresh spleen tissue samples have stronger advantages than fresh tumor tissue samples; that is, the advantages of the formula of the application in the preservation and transportation of normal tissues are significantly better than those of tumor tissues. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a column chart of the tissue viability maintaining ability of the preservation of fresh tumor tissues by formula 1, formula 2 and formula 3;

[0025] Figure 2 It is a column chart of the 48h tissue viability maintaining ability of the preservation of fresh tumor tissues by formula 1, formula 7, formula 5, formula 6;

[0026] Figure 3 It is a column chart of the 72h tissue viability maintaining ability of the preservation of fresh tumor tissues by formula 1, formula 2, formula 3, formula 4, formula 7 and formula 8;

[0027] Figure 4Bar chart of 72h tissue viability maintenance capability of fresh tumor tissue preserved by Formula 4, Formula 9, Formula 10;

[0028] Figure 5 Bar chart of 72h tissue viability maintenance capability of fresh liver tissue preserved by Formula 1, Formula 2, Formula 4;

[0029] Figure 6 Bar chart of 72h tissue viability maintenance capability of fresh spleen tissue preserved by Formula 2, Formula 4. DETAILED DESCRIPTION

[0030] The application will be described in greater detail with reference to embodiments thereof, in the description of which, it is to be understood that, unless otherwise indicated, the examples are not intended to limit the application. The examples are intended to illustrate the application and to enable one skilled in the art to practice the application without undue experimentation. The reagents or instruments used are not specified by the manufacturer unless otherwise indicated, and are all conventional products that can be purchased on the market.

[0031] MitoQ: selleckchem, Catalog No. S8978

[0032] Trolox: water-soluble vitamin E, selleckchem, Catalog No. S3665

[0033] NAC: N-acetyl-L-cysteine, selleckchem, Catalog No. S1623.

[0034] EMBODIMENT

[0035] Experimental scheme: Fresh breast cancer tumor tissue / fresh liver tissue / fresh spleen tissue was cut, with a diameter of 2-8 mm, and washed 3 times with PBS buffer containing 200 U / mL penicillin / streptomycin. The fresh tissue was immersed in 5 mL of the tissue preservation solution described in Table 1 (the base medium is DMEM). Store at 2-8°C for 24-72 hours, which can be used for subsequent tissue viability detection or experimental requirements.

[0036] The formula of the preservation solution used in this embodiment is shown in Table 1.

[0037] Table 1 Formula Table

[0038] Fetal bovine serum / wt% Superoxide dismutase / catalase MitoQ / μM Trolox / μM NAC / μM Penicillin U / mL Streptomycin U / mL Formula 1 0 0 0 0 0 100 100 Formula 2 5 0 0 0 0 100 100 Formula 3 0 50 mg + 200 mg 0 0 0 100 100 Formula 4 5 0 1 0 0 100 100 Formula 5 0 0 0 1 0 100 100 Formula 6 0 0 0 0 1 100 100 Formula 7 0 0 1 0 0 100 100 Formula 8 0 50 mg + 200 mg 1 0 0 100 100 Formula 9 5 0 2 0 0 100 100 Formula 10 5 0 4 0 0 100 100

[0039] Note: The concentrations of superoxide dismutase and catalase are 50 mg / L and 200 mg / L, respectively.

[0040] Experimental detection method:

[0041] Tumor tissue viability: Place tumor tissue in a well of appropriate size, add an appropriate volume of 5 mg / mL MTT solution, continue to incubate in a cell incubator for 2-4 hours, aspirate the MTT solution, add an appropriate volume of DMSO to immerse the tumor tissue, gently shake to dissolve the purple crystals until the color is uniform.

[0042] Liver tissue viability: Place liver tissue in a well of appropriate size, add an appropriate volume of 5 mg / mL MTT solution, continue to incubate in a cell incubator for 2-4 hours, aspirate the MTT solution, add an appropriate volume of DMSO to immerse the tumor tissue, gently shake to dissolve the purple crystals until the color is uniform.

[0043] Spleen tissue viability: Place spleen tissue in a well of appropriate size, add an appropriate volume of 5 mg / mL MTT solution, continue to incubate in a cell incubator for 2-4 hours, aspirate the MTT solution, add an appropriate volume of DMSO to immerse the tumor tissue, gently shake to dissolve the purple crystals until the color is uniform.

[0044] Experimental results:

[0045] 1. Reference Figure 1 , Figure 1 shows the tissue viability of three different culture media (Formula 1, Formula 2 and Formula 3) preserving fresh tumor tissue samples after 72 hours of transportation at 4°C;

[0046] In Figure 1 , the tissue viability retention ability of Formula 1 is taken as 100%, and the tissue viability retention ability of Formula 2 and Formula 3 is similar to about 110%.

[0047] 2. Reference Figure 2 , Figure 2 shows the tissue viability of Formula 1, Formula 7, Formula 5, Formula 6 preserving fresh tumor tissue samples after 48 hours of transportation at 4°C;

[0048] In Figure 2 , the tissue viability retention ability of Formula 1 is taken as 100%, and only MitoQ can improve the tissue viability ability. In the art, it is believed that MitoQ, water-soluble vitamin E, and N-acetyl-L-cysteine are relatively common antioxidants, and there is no consensus in the art that the three have antioxidant strength. We believe that the reason why the present case can show the superiority of MitoQ in the formula is that MitoQ is an antioxidant targeting mitochondria, which in turn leads to effective maintenance of sample viability. Subsequent experiments selected MitoQ as the target substance for research.

[0049] 3. Reference Figure 3 , Figure 3The formula 1, the formula 2, the formula 3, the formula 4, the formula 7, the formula 8 preserve fresh tumor tissue samples, and the tissue viability after 72 hours of transportation at 4 DEG C is shown;

[0050] In Figure 3 , MitoQ is applied in the formula 1, and the efficiency is increased by about 15%, is applied in the formula 3, and the efficiency is increased by about 20%, is applied in the formula 2, and the efficiency is increased by about 30%, which indicates that the performance of MitoQ is more easily exerted in the formula 2.

[0051] 4. Reference Figure 4 , Figure 4 In , different MitoQs are tested, and the formula 4, the formula 9 and the formula 10 are used for preserving fresh tumor tissue samples, and the tissue viability after 72 hours of transportation at 4 DEG C is shown;

[0052] Figure 4 It can be seen that the optimal MitoQ concentration is 2 micromoles.

[0053] 5. Reference Figure 5 and Figure 6 , Figure 5 The formula 1, the formula 2 and the formula 4 are used for preserving fresh liver tissue samples, and the tissue viability after 72 hours of transportation at 4 DEG C is shown; Figure 6 The formula 1, the formula 2 and the formula 4 are used for preserving fresh spleen tissue samples, and the tissue viability after 72 hours of transportation at 4 DEG C is shown;

[0054] Through Figure 5 , 6 and in combination with Figure 3 , two conclusions can be obtained:

[0055] Conclusion 1: and Figure 3 similarly, after MitoQ is applied in the formula 2, it also has a stronger advantage in the preservation and transportation of fresh liver tissue samples and fresh spleen tissue samples;

[0056] Conclusion 2: unlike Figure 3 , after MitoQ is applied in the formula 2, it has a stronger advantage in the preservation and transportation of fresh liver tissue samples and fresh spleen tissue samples than in fresh tumor tissue samples;

[0057] that is, the advantage of the formula of the application in the transportation and preservation of normal tissues is significantly better than that of tumor tissues.

Claims

1. A tissue sample preservative solution, characterized by, The base culture medium contains fetal bovine serum and MitoQ.

2. The tissue sample preservative solution of claim 1, wherein The base culture medium contains 5-15 wt% FBS fetal bovine serum and 1-10 μM MitoQ.

3. The tissue sample preservative fluid of claim 2, wherein, The base culture medium contains 5-15 wt% FBS fetal bovine serum and 1-2 μM MitoQ.

4. The tissue sample preservative solution of claim 1, wherein The base culture medium further contains double antibiotics, and the double antibiotics are penicillin and streptomycin. The concentration of the penicillin is 50-200 U / mL, and the concentration of the streptomycin is 50-200 U / mL.

5. The tissue sample preserving fluid according to any one of claims 1 to 4, characterized by, The base culture medium is one of DMEM, RPMI-1640 and MEM.

6. A method for preserving a tissue sample, characterized by, The tissue sample is preserved by using the tissue sample preservative liquid as claimed in any one of claims 1 to 5.

7. The preservation method according to claim 6, characterized in that, The preservation environment is 2-20 °C.

8. The preservation method according to claim 6, characterized in that, The preservation time is not less than 72 h.

9. The preservation method according to claim 6, characterized in that, The tissue sample is a tumor tissue sample or a normal tissue sample.

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