Yak tissue pretreatment method

Abstract

The invention discloses a yak tissue pretreatment method, and relates to the technical field of yak tissue culture. The method comprises the following steps: contacting liquid containing vitamin E with disinfected yak tissues; and then loading the yak tissues into a container or a packaging bag, and carrying out vacuum-pumping treatment. According to the method provided by the invention, the liquid containing the vitamin E is in contact with the disinfected yak tissue, so that the vitamin E is attached to the surface of the yak tissue, the vitamin E plays a role in resisting oxidation, oxidation of the yak tissue is delayed in a non-toxic and harmless manner, and the yak tissue is protected. Through a vacuumizing mode, air in the container or the packaging bag can be quickly exhausted, so that the high cell survival rate in a long-time transportation or storage environment is maintained. The fibroblasts harvested based on the method are high in survival rate, good in adherence effect and high in reproductive capacity, and protection and utilization of yak germplasm resources are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of yak tissue culture, in particular to a method for pretreating yak tissue. Background Art

[0002] At present, yaks are an important economic pillar for herders, and the protection and research of yak species resources are of great significance. In scientific research, yak somatic cells must be cultured in primary culture. Somatic cells need to be quickly separated and cultured after being removed from the body. Since the environmental conditions required for cell separation and culture are relatively high, the sampling site (plateau area) generally cannot meet the requirements. Tissue samples need to be transported to the laboratory for operation. If samples are collected over long distances, the activity of tissue cells will inevitably be affected, resulting in cell culture failure.

[0003] Currently, commercial tissue preservation fluid is used to transport tissue samples over long distances, but the disadvantage is that it is expensive and has a limited shelf life of up to 48 hours. This preservation method has strict operational requirements and requires the collected tissue to be cleaned and placed in pre-cooled tissue preservation fluid (2-8°C). The sample must be covered with preservation fluid and cannot contain bubbles to prevent prolonged contact with air. During transportation, the sample must be kept at 2-8°C and can be stored for up to 48 hours. After 48 hours, the activity of the sample deteriorates and cells cannot be cultured.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for pretreatment of yak tissue to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] A method for pretreatment of yak tissue, comprising the following steps:

[0008] The liquid containing vitamin E is brought into contact with the sterilized yak tissue; the yak tissue is then placed in a container or a packaging bag and vacuumed.

[0009] The present invention has the following beneficial effects:

[0010] The present invention provides a method for pretreating yak tissue, which contacts a liquid containing vitamin E with the disinfected yak tissue, so that vitamin E is attached to the surface of the yak tissue, and vitamin E plays an antioxidant role, delays the oxidation of yak tissue in a non-toxic and harmless manner, and protects the yak tissue. By vacuuming, the air in the container or packaging bag can be quickly discharged, thereby maintaining a high cell survival rate in a long-term transportation or storage environment. The fibroblasts harvested based on this method have a high survival rate, good wall adhesion effect, and strong reproductive capacity, which is beneficial to the protection and utilization of yak germplasm resources. The present invention provides a solution for long-distance transportation of sample tissues. The method does not require expensive reagents, is simple and easy to operate, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0012] Figure 1 This is a photo of tissue blocks being individually packed into disposable sample bags that have been sterilized and separated in advance and then evacuated;

[0013] Figure 2 Another physical picture of tissue blocks being individually packed into disposable sample bags that have been sterilized and separated in advance and then evacuated;

[0014] Figure 3 This is a micrograph of primary cell culture after 60 hours of vacuum low-temperature transportation;

[0015] Figure 4 This is a micrograph of the culture of subcultured cells;

[0016] Figure 5 The figure is a growth curve of cell proliferation measured by CCK-8 method during 1-7 days of primary cell culture;

[0017] Figure 6 This is the result of identifying vimentin in yak fibroblasts by cell immunofluorescence;

[0018] Figure 7 This is a diagram showing the results of primary cell culture after 60 hours of transportation using the tissue preservation solution in Comparative Example 1. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The present invention provides a method for pretreating yak tissue, which comprises the following steps:

[0021] The liquid containing vitamin E is brought into contact with the sterilized yak tissue; the yak tissue is then placed in a container or a packaging bag and vacuumed.

[0022] The above-mentioned "contact" includes but is not limited to: dipping, soaking, immersing, spraying, fumigation. As long as the liquid containing vitamin E can contact the disinfected yak tissue, it is feasible.

[0023] Disinfection includes but is not limited to surface disinfection methods such as alcohol disinfection, disinfectant (such as pasteurized solution) disinfection, etc.

[0024] The present invention contacts the liquid containing vitamin E with the disinfected yak tissue, so that the surface of the yak tissue is attached with vitamin E, and the vitamin E plays an antioxidant role, delays the oxidation of the yak tissue in a non-toxic and harmless manner, and protects the yak tissue. By vacuuming, the air in the container or packaging bag can be quickly discharged, thereby maintaining a high cell survival rate in a long-term transportation (more than 60 days) or storage environment. The fibroblasts harvested based on this method have a high survival rate, good wall adhesion effect, and strong reproductive capacity, which is beneficial to the protection and utilization of yak germplasm resources.

[0025] Therefore, the present invention provides a solution for long-distance transportation of sample tissues. The method does not require expensive reagents, is simple and easy to operate, and has broad application prospects.

[0026] In a preferred embodiment of the present invention, the vacuuming process is performed as follows: vacuuming is performed 3 to 4 times for 25 to 30 seconds each time. Under the above vacuuming process conditions, the air in the container or bag can be quickly extracted.

[0027] In a preferred embodiment of the present invention, the liquid containing vitamin E is contacted with the disinfected yak tissue for 3-10 minutes. Under the above contact time conditions, a good tissue antioxidant protection effect is achieved. For example, the contact time is 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0028] In a preferred embodiment of the present invention, the concentration of vitamin E in the liquid containing vitamin E is 20 g / L-30 g / L. For example, the concentration of vitamin E in the liquid containing vitamin E is 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L or 30 g / L.

[0029] In a preferred embodiment of the present invention, the container or packaging bag is a container or packaging bag that has been aseptically treated; the aseptic treatment includes the following steps: first soaking the container or packaging bag in 75% alcohol, and then performing ultraviolet sterilization; after drying, adding a solution containing antibiotics into the container or packaging bag.

[0030] Through the combination of chemical sterilization and physical sterilization, the aseptic processing of containers or packaging bags can be achieved, reducing the risk of tissue contamination due to incomplete sterilization.

[0031] In a preferred embodiment of the present invention, the soaking time in 75% alcohol is 1 to 1.5 hours.

[0032] In a preferred embodiment of the present invention, ultraviolet sterilization is to irradiate the container or packaging bag to be sterilized under a 11-15W ultraviolet lamp for 1 to 1.5 hours.

[0033] In a preferred embodiment of the present invention, the solution containing antibiotics refers to physiological saline containing penicillin-streptomycin-amphotericin B. The combination of three antibiotics has a broader spectrum of bactericidal effects.

[0034] In a preferred embodiment of the present invention, the physiological saline contains 200-300 U / ml penicillin, 0.2-0.3 mg / ml streptomycin, and 0.5-0.75 ug / ml amphotericin B.

[0035] In a preferred embodiment of the present invention, after the vacuum treatment, the container or packaging bag is sealed, for example, by heat sealing.

[0036] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0037] Example 1

[0038] This embodiment provides a method for pre-processing yak tissue, which also includes long-distance transportation of yak tissue, and specifically includes the following steps:

[0039] (1) Fix the individual to be collected and measure the ear tissue (size 0.5 cm) 2) parts were disinfected with alcohol. Ear tissue was quickly collected with ear forceps, and surface hair was quickly processed with a skin preparation knife, so that sampling could be done quickly to reduce contamination. After disinfection with 75% alcohol, the processed tissue blocks were placed in physiological saline (salt concentration of 0.9%) containing 0.02g / ml vitamin E and soaked for 5 minutes.

[0040] (2) Soak the disposable sample vacuum bag in 75% alcohol, then irradiate it under a 15W ultraviolet lamp on a clean bench for 1 to 1.5 hours. Dry it in an oven and then soak it in physiological saline containing 250U / ml penicillin, 0.25mg / ml streptomycin, and 0.6ug / ml amphotericin B.

[0041] (3) Without rinsing the soaked tissue blocks in step (1), use tweezers to place them individually into the disposable sample vacuum bags that have been aseptically separated in step (2). Place the opening of the vacuum bag flatly on the sealing strip of the vacuum sealer to ensure that the sealing strip can completely cover the opening of the bag. Turn on the power, preheat the sealing area in advance, and press the vacuum button twice in succession until the air inside is completely extracted.

[0042] The vacuum conditions are as follows:

[0043] Vacuum evacuate for 30 seconds each time for 4 times to completely evacuate the air. Seal and mark. See the actual picture after sealing. Figure 1 and Figure 2 shown.

[0044] (4) All samples are bagged and placed in a constant temperature sample transport box (maintained at 2-8°C) and transported back to the laboratory after 60 hours for the next step of separation and culture.

[0045] Example 2

[0046] This embodiment provides a method for culturing primary cells and passaged cells, which specifically includes the following steps:

[0047] The vacuum low-temperature transport cells were cultured until the 8th day, and the cells were subcultured when the confluence of the primary cells reached about 80%. The culture medium in the culture dish was removed and washed twice with PBS. An appropriate amount of 0.25% trypsin-EDTA solution was added and placed at 37°C and 5% CO. 2 Digest in the incubator for 1 min. When cells become round and a small amount of cells fall off under the microscope, add an equal amount of complete cell culture medium to terminate digestion. Blow the cells to make them fall off completely. Divide them into culture dishes at a ratio of 1:2 for passage and place them in 37°C, 5% CO 2 Continue culturing in the incubator.

[0048] In this example, cell morphology and cell growth were observed under an inverted microscope. Figure 3As shown, when the primary cells were cultured to day 8, the cells were mainly spindle-shaped and polygonal; Figure 4 As shown, on the 8th day of subculture, the cells were mainly spindle-shaped.

[0049] Example 3

[0050] In this example, the cell growth curve of cells cultured from primary cells was measured.

[0051] The growth curve was measured by CCK-8 method. 3000 fibroblasts were inoculated into 96-well plates, with 100 μL culture medium in each well. There were 9 groups in total, with 3 wells in each group. A group was selected every 24 hours, and 90 μL serum-free medium plus 10 μL CCK8 were added to each well. The plates were placed in a 37°C incubator and incubated in the dark for 2 hours. The OD value of each well was measured at a wavelength of 450 nm. The data was read 3 times each time and the average value was taken. The measurement was continued for 7 days. The growth curve was drawn with time as the horizontal axis and OD value as the vertical axis.

[0052] Results reference Figure 5 As shown, the results showed that the cells were in the latent period from 1d to 3d, entered the exponential growth period from 3d to 5d, and entered the plateau period after 5d to 6d, indicating that the fibroblast proliferation activity was good. Therefore, the method of using vitamin E and vacuum low-temperature treatment for long-distance transportation of tissues can still culture fibroblasts 60h after the tissue is separated from the body. The harvested fibroblasts have a high survival rate and strong reproductive capacity.

[0053] Example 4

[0054] In this example, the cell purity of fibroblasts cultured from primary cells was identified.

[0055] The third generation cells were used for the experiment. 2 × 10 3 The fibroblast suspension was placed at a concentration of 100 mL and incubated at 37°C with 5% CO 2 Incubator, remove the culture medium after the cells have reached the appropriate morphology; add 4% paraformaldehyde to fix the cells at room temperature for 30 minutes; add 0.5% Triton-X-100 to permeabilize the membrane for 5 minutes at room temperature; wash 3 times with PBS and place on a shaker, block with blocking solution for 20 minutes, and pour off the liquid; add the primary antibody rabbit anti-vimentin (Anti-vimentin) (1:200) diluted in the primary antibody diluent, and incubate in a 4°C refrigerator overnight; add PBS to wash 3 times, shaking and washing for 5 minutes each time; add the goat anti-mouse IgG fluorescent secondary antibody (1:50) diluted in the secondary antibody diluent, and incubate at room temperature in the dark for 2 hours; add PBS to wash 3 times, 5 minutes each time. Add DAPI to counterstain the nucleus, incubate at room temperature for 5 minutes, wash 3 times with PBS, 5 minutes each time. Observe under a fluorescence microscope.

[0056] Results reference Figure 6 As shown, the results show that the yak fibroblasts cultured from primary cells can express vimentin, and the positive rate of cells expressing vimentin is 100%, indicating that the isolated fibroblasts have high purity, indicating that the method for pretreatment of yak tissue provided by the present invention has better effect.

[0057] Comparative Example 1

[0058] The pretreatment of yak tissue was performed as follows:

[0059] (1) Place the processed ear tissue block into tissue preservation solution ( The samples must be covered with preservation solution, free of bubbles, and prevented from prolonged contact with air. The samples must be kept at 2-8°C during transportation.

[0060] (2) Return to the laboratory for isolation and culture after nearly 60 hours (the steps are the same as those in Example 2).

[0061] The cell migration and proliferation were observed and recorded under a microscope every day using the tissue explant adhesion method.

[0062] Figure 7 The results showed that no cells crawled out of the tissue block transported by the tissue preservation solution after 10 days. That is, in this comparative example, commercial tissue preservation solution was used for long-distance tissue transportation, and no cells were cultured after 60 hours of ex vivo tissue removal.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for pretreatment of yak tissue, characterized in that: It includes the following steps: The liquid containing vitamin E is brought into contact with the sterilized yak tissue; the yak tissue is then placed in a container or a packaging bag and vacuumed.

2. The method for pretreatment of yak tissue according to claim 1, characterized in that: The procedure of the vacuuming treatment is: vacuuming for 25 to 30 seconds each time, and continuously extracting 3 to 4 times.

3. The method for pretreatment of yak tissue according to claim 1, characterized in that: The vitamin E-containing liquid was placed in contact with the sterilized yak tissue for 3-10 minutes.

4. The method for pretreatment of yak tissue according to claim 3, characterized in that: The concentration of vitamin E in the liquid containing vitamin E is 20 g / L-30 g / L.

5. The method for pretreatment of yak tissue according to claim 1, characterized in that: The container or packaging bag is a container or packaging bag that has been aseptically treated; the aseptic treatment includes the following steps: first soaking the container or packaging bag in 75% alcohol, and then performing ultraviolet sterilization; after drying, adding a solution containing antibiotics into the container or packaging bag.

6. The method for pretreatment of yak tissue according to claim 5, characterized in that: The time for the 75% alcohol soaking treatment is 1 to 1.5 hours.

7. The method for pretreatment of yak tissue according to claim 5, characterized in that: The ultraviolet sterilization is to irradiate the container or packaging bag to be sterilized under an ultraviolet lamp with a power of 11W-15W for 1 to 1.5 hours.

8. The method for pretreatment of yak tissue according to claim 5, characterized in that: The solution containing antibiotics refers to physiological saline containing penicillin-streptomycin-amphotericin B.

9. The method for pretreatment of yak tissue according to claim 8, characterized in that: The physiological saline contains 200-300 U / ml penicillin, 0.2-0.3 mg / ml streptomycin, and 0.5-0.75 ug / ml amphotericin B.

10. The method for pretreatment of yak tissue according to claim 1, characterized in that: After the vacuum treatment, the container or packaging bag is sealed.

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