A method for pretreatment of yak tissues

Abstract

The application discloses a kind of yaks tissue pretreatment method, it is related to yak tissue culture technical field.It includes the following steps: the liquid containing vitamin E is contacted with the yak tissue after disinfection;Then yak tissue is loaded into container or packaging bag, and vacuum treatment is handled.The method provided by the application contacts the liquid containing vitamin E with the yak tissue after disinfection, so that the surface of yak tissue is attached with vitamin E, vitamin E plays the role of antioxidant, and the oxidation of yak tissue is delayed in a non-toxic and harmless way, which protects the yak tissue.By the way of vacuum, air in the container or packaging bag can be quickly discharged, so that high cell survival rate can be maintained under the transportation or storage environment for a long time.Based on the harvested fibroblasts by the method, the survival rate is high, the adhesion effect is good, and the reproduction ability is strong, which is beneficial to the protection and utilization of yak germplasm resources.

Description

Technical Field

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

[0002] Yaks are currently an important economic support for herders, and research on the conservation of yak resources is of great significance. Primary culture of yak somatic cells is essential for scientific research. Once isolated from the body, somatic cells need to be rapidly isolated and cultured. However, due to the high environmental conditions required for cell isolation and culture, sampling sites (in high-altitude areas) generally cannot meet these requirements. Therefore, tissue samples must be transported to the laboratory for processing. Collecting samples over long distances can negatively impact tissue cell activity, leading to 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. The collected tissue needs to be cleaned and placed in pre-cooled tissue preservation fluid (2-8°C). The sample must be covered with the 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 packaging bag and vacuumed.

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

[0010] The application provides a yak tissue pretreatment method, which comprises the following steps: contacting a liquid containing vitamin E with sterilized yak tissue, so that the yak tissue surface is attached with vitamin E, the vitamin E plays an antioxidant role, and the yak tissue is protected in a non-toxic and harmless way. Through the vacuum extraction mode, the air in the container or packaging bag can be quickly discharged, so that the high cell survival rate under the long-time transportation or storage environment can be maintained. The fibroblasts harvested based on the method have high survival rate, good adhesion effect and strong reproduction capacity, and are beneficial to the protection and utilization of yak germplasm resources. The application provides a solution for long-distance transportation of sample tissues, and the method does not need expensive reagents, is simple and easy to operate, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0012] Figure 1 A physical map of the tissue block independently loaded into a disposable sample bag which is separated by pre-sterile processing and vacuumed;

[0013] Figure 2 Another physical map of the tissue block independently loaded into a disposable sample bag which is separated by pre-sterile processing and vacuumed;

[0014] Figure 3 A microscopic map of the primary cell culture after 60 hours of vacuum low-temperature transportation;

[0015] Figure 4 A microscopic map of the subculture cell culture;

[0016] Figure 5 A growth curve graph of the cell proliferation of the primary cell culture measured by the CCK-8 method for 1-7 days;

[0017] Figure 6 A result graph of the identification of yak fibroblast vimentin by the cell immunofluorescence method;

[0018] Figure 7 A result graph of the primary cell culture after 60 hours of transportation by using the tissue preservation liquid in the comparative example 1. DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are 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 packaging bag and vacuumed.

[0022] The above-mentioned "contact" includes but is not limited to: dipping, soaking, immersing, spraying, and fumigation. Any method that allows the liquid containing vitamin E to come into contact with the disinfected yak tissue is acceptable.

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

[0024] The present invention brings a liquid containing vitamin E into contact with the disinfected yak tissue, so that vitamin E is attached to the surface of the yak tissue. 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 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 transporting sample tissues over long distances. 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 vitamin E-containing liquid is contacted with the disinfected yak tissue for 3-10 minutes. Under these contact time conditions, a good tissue antioxidant protection effect is achieved. For example, 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 sterilized; the sterilization process 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 to the container or packaging bag.

[0030] Through the combination of chemical sterilization and physical sterilization, the aseptic treatment 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, the ultraviolet sterilization is to irradiate the container or packaging bag to be sterilized under an 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 a physiological saline solution containing penicillin, streptomycin, and amphotericin B. The combination of the 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 further sealed, for example, by heat sealing.

[0036] The features and performance of the present invention are further described in detail below with reference to 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, specifically comprising the following steps:

[0039] (1) Fix the individual to be collected and make a sample of the ear tissue (size 0.5 cm) 2Disinfect the ear tissue with alcohol. Quickly collect ear tissue using ear forceps and quickly remove surface hair with a dermatome to minimize contamination. After disinfecting with 75% alcohol, soak the prepared tissue block in 0.9% saline solution containing 0.02g / ml vitamin E for 5 minutes.

[0040] (2) Soak the disposable sample vacuum bag in 75% alcohol and then irradiate it under a 15W UV lamp on a clean bench for 1-1.5 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 all the air inside is completely extracted.

[0042] The vacuum conditions are as follows:

[0043] Vacuum the container for 30 seconds each time for 4 times to remove all the air. Seal the container and mark it. Figure 1 and Figure 2 shown.

[0044] (4) All samples were 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 cells were cultured at low temperature by vacuum transport until day 8. When the confluence of the primary cells reached approximately 80%, the cells were subcultured. 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 the cells were incubated in a 37°C, 5% CO2 incubator for 1 min. When the cells were observed to be rounded and a small amount of cells had detached under a microscope, an equal amount of complete cell culture medium was added to terminate the digestion. The cells were pipetted to completely detach, and the culture dishes were divided into subcultures at a ratio of 1:2. The cells were then placed in a 37°C, 5% CO2 incubator for further culture.

[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 in the figure, when the subcultured cells were cultured to day 8, 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] Growth curves were measured using the CCK-8 method. 3,000 fibroblasts were seeded per well in a 96-well plate, with 100 μL of culture medium added to each well. Nine groups, each containing three wells, were formed. A new group was selected every 24 hours, and 90 μL of serum-free culture medium plus 10 μL of CCK-8 were added to each well. The plates were incubated in a 37°C incubator protected from light for 2 hours. The OD value of each well was measured at a wavelength of 450 nm, with the average value of three readings taken for each reading. Measurements were continued for 7 consecutive days. Growth curves were plotted with time as the horizontal axis and OD value as the vertical axis.

[0052] Results reference Figure 5 As shown in the results, the cells were in the latent phase from 1 to 3 days, entered the exponential growth phase from 3 to 5 days, and entered the plateau phase after 5 to 6 days, indicating that the fibroblasts had good proliferation activity. Therefore, the method of using vitamin E and vacuum low-temperature treatment for long-distance tissue transportation can still culture fibroblasts 60 hours after tissue ex vivo. 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, and 2×10 3 A fibroblast suspension at a concentration of 100 μg / ml was placed in a 37°C, 5% CO2 incubator. After reaching the appropriate morphology, the medium was removed. 4% paraformaldehyde was added to fix the cells for 30 minutes at room temperature. 0.5% Triton-X-100 was added to permeabilize the membrane for 5 minutes at room temperature. The cells were washed three times with PBS and blocked with blocking solution for 20 minutes on a shaker. The solution was then decanted. Rabbit anti-vimentin (1:200) diluted in primary antibody diluent was added and incubated overnight at 4°C. The cells were washed three times with PBS, with shaking for 5 minutes each time. Goat anti-mouse IgG (1:50) fluorescent secondary antibody was added in secondary antibody diluent and incubated at room temperature in the dark for 2 hours. The cells were washed three times with PBS, each for 5 minutes. Nuclei were counterstained with DAPI and incubated for 5 minutes at room temperature. The cells were washed three times with PBS, each for 5 minutes each. The cells were observed under a fluorescence microscope.

[0056] Results referenceFigure 6 As shown, the results showed that the primary cell cultured yak fibroblasts can express vimentin, and the positive rate of cells expressing vimentin is 100%, indicating that the isolated fibroblasts are of high purity. This shows that the yak tissue pretreatment method provided by the present invention is more effective.

[0057] Comparative Example 1

[0058] Yak tissue was pretreated as follows:

[0059] (1) Place the processed ear tissue block into tissue preservation solution ( In the Tissue Storage Solution (Cat. No. 5230905926), samples must be covered with preservation solution, free of air bubbles, and protected from prolonged exposure to air. Keep samples 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 were cultured in the tissue blocks transported in the tissue preservation solution after 10 days. In other words, in this comparative example, no cells were cultured after 60 hours of tissue ex vivo using commercial tissue preservation solution for long-distance tissue transportation.

[0063] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for pretreatment of yak tissue, characterized in that: It includes the following steps: The vitamin E-containing liquid is contacted with the sterilized yak tissue for 3-10 minutes; the yak tissue is then placed in a container or packaging bag and vacuumed; the vacuuming procedure is as follows: vacuuming for 25-30 seconds each time, 3-4 times continuously; the concentration of vitamin E in the vitamin E-containing liquid is 20g / L-30g / L; The container or packaging bag is a container or packaging bag that has been sterilized; the sterilization process includes the following steps: first soaking the container or packaging bag in 75% alcohol for 1 to 1.5 hours, and then performing ultraviolet sterilization; after drying, adding physiological saline containing penicillin-streptomycin-amphotericin B into the container or packaging bag; the physiological saline contains a concentration of 200 to 300 U / ml penicillin, 0.2 to 0.3 mg / ml streptomycin, and 0.5 to 0.75 ug / ml amphotericin B.

2. The method for pretreatment of yak tissue according to claim 1, 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 1-1.5 hours.

3. 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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