CN117660305A - Method for remotely collecting animal tissues and method for establishing cell line - Google Patents

Abstract

The invention provides a method for long-distance collection of animal tissue and a method for establishing cell lines, and belongs to the technical field of cell culture. The invention provides a method for long-distance collection of animal tissue, which includes the following steps: soaking the animal tissue in an ethanol aqueous solution to obtain a first-processed tissue; soaking the first-processed tissue in a PBS solution containing penicillin and streptomycin. , obtain the second-processed tissue; preserve the second-processed tissue in a PBS solution containing penicillin and streptomycin. The method for long-distance collection of animal tissues provided by the present invention is suitable for the preservation of various animal tissues. Through this collection method, animal tissues can be preserved for 3 to 5 days, which is suitable for long-distance sampling in the wild, and the animal tissues are preserved and cells are established. Growth is almost unaffected and ideal growth conditions can be achieved.

Description

A method for long-distance collection of animal tissue and a method for establishing cell lines

Technical field

The invention belongs to the technical field of cell culture, and specifically relates to a method of collecting animal tissue from a long distance and a method of establishing a cell line.

Background technique

Animal genetic resources are an important part of natural resources and an important biological resource on which human society depends for the survival and development. It is also a major national strategic basic resource. The Inner Mongolia Autonomous Region has a vast territory and is home to animals unique to the Mongolian plateau, such as wild horses, wild donkeys, wild camels, goose-throated antelopes, gazelles, Mongolian horses, Mongolian cattle, Mongolian sheep and rodents unique to the plateau. They are the source of my country's animal genetic resources. An important part of. Maintaining the diversity of these animal genetic resources, strengthening the protection of characteristic animal breed resources, and achieving reasonable, effective, and sustainable utilization and development are not only of great significance to the protection of earth resources, but also of great significance to the actual social and economic development and life science research. Practical value.

At present, there are no genetic resource libraries and corresponding academic research institutions at home and abroad specifically focusing on the unique animal resources in the Mongolian Plateau region. Establish a unique animal genetic resource library and research information platform in northern my country, and use preserved cells, embryos, and semen resources to carry out basic research in life sciences. At the same time, modern biotechnological means such as genome testing, somatic cell cloning, and gender control can be used to analyze, Research representative life science topics related to these animals, protect these unique genetic resources, and develop and utilize these genetic resources.

At present, there are three main ways to preserve animal germplasm resources in my country: in situ conservation, ex situ conservation and in vitro conservation. The essence of the preservation of genetic resources is the protection of living species and sperm, eggs, embryos, cells, tissues, organs and genomes that carry all the information of life. Since all genetic information is carried in animal body cells, any animal body cell can be grown into a complete animal body. Therefore, establishing an animal somatic cell bank, especially establishing a somatic cell bank for important and endangered livestock and poultry genetic resources, is the best way to collect, organize, preserve and utilize livestock and poultry germplasm resources. How to obtain animal tissues to establish cell lines and establish a cell resource bank without harming animals has always been a problem that animal resource collection and conservation workers strive to solve. However, when collecting animal tissues in the wild, there are often problems such as improper tissue processing or preservation, which affects the establishment of cell lines and results in a low success rate of cell line establishment. Therefore, it is of great significance in the establishment of cell lines to provide a method suitable for long-distance collection of animal tissues to keep the animal tissues in the state of fresh tissues.

Contents of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for collecting animal tissue from a long distance. The method for collecting animal tissue is suitable for the preservation of various animal tissues, and the animal tissue is established after preservation. Cell growth is almost unaffected and ideal growth conditions can be achieved.

The purpose of the present invention is achieved through the following technical solutions:

The invention provides a method for long-distance collection of animal tissue, which includes the following steps:

Soak the animal tissue in the ethanol aqueous solution to obtain the first treated tissue;

Soak the first-processed tissue in a PBS solution containing penicillin and streptomycin to obtain the second-processed tissue;

The second treated tissue was stored in PBS solution containing penicillin and streptomycin.

Preferably, the soaking time of the animal tissue in the ethanol aqueous solution is 10 to 15 seconds; the volume percentage of ethanol in the ethanol aqueous solution is 70% to 80%.

Preferably, the first treated tissue is soaked 3 to 5 times in a PBS solution containing penicillin and streptomycin, and the soaking time for each time is 10 to 15 seconds; in the PBS solution containing penicillin and streptomycin, penicillin and streptomycin are The volume percentage of element is 1% to 3%.

Preferably, the animal tissue includes ear tissue; the area of the animal tissue is 1 to 2 cm ² .

Preferably, the storage temperature is 4°C; the storage time is 3 to 5 days.

The present invention provides a method for establishing a cell line of animal tissue collected remotely using the method described in the above technical solution, which includes the following steps:

After sterilizing the animal tissue, rinse it with DPBS solution containing penicillin and streptomycin to obtain pretreated tissue;

Rinse the pretreated tissue and cut it into finely divided tissue pieces for primary culture in culture medium;

Primary culture is performed until the confluence of the primary cells reaches 80-90%, and then subculture is performed to obtain a cell line.

Preferably, the rinsing is performed using PBS solution; the number of rinsing is 5 to 6 times; the size of the finely divided tissue pieces is 0.5 to 1 mm ³ .

Preferably, the disinfection is carried out using a 70% to 80% ethanol aqueous solution; the volume percentage of penicillin and streptomycin in the DPBS solution containing penicillin and streptomycin is 1% to 3%.

Preferably, the culture medium used in the process of establishing the cell line includes 10% FBS by volume and 1% penicillin-streptomycin by volume; the culture medium includes α-MEM basic culture medium; The temperature for primary culture and subculture is 37°C to 38.5°C; the carbon dioxide concentration is 5%.

The present invention provides a cell line established by the establishment method described in the above technical solution.

Beneficial effects of the present invention:

The invention provides a method for long-distance collection of animal tissue, which includes the following steps: soaking the animal tissue in an ethanol aqueous solution to obtain a first-processed tissue; soaking the first-processed tissue in a PBS solution containing penicillin and streptomycin. , obtain the second-processed tissue; preserve the second-processed tissue in a PBS solution containing penicillin and streptomycin. The long-distance collection method of animal tissue provided by the present invention is suitable for long-distance (long-distance) collection of animal tissue samples in the wild. This collection method can preserve animal tissues for 3 to 5 days, and is suitable for the preservation of a variety of animal tissues, and the animals After the tissue is preserved and established, cell growth is almost unaffected and the ideal growth state can be achieved.

Furthermore, the present invention provides a method for establishing a cell line based on animal tissue collected remotely. The method for establishing a cell line is a culture method for obtaining primary cells from animal tissues. It can be generally used for primary cell culture and establishment of tissues of different species. It is suitable for cultivating primary cells of multiple species. The success rate is high and it can be continuously It can be passed down through generations without mutation, and the state of somatic cells can be better maintained in terms of morphological structure and biological characteristics.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the culture results of primary reindeer cells;

Figure 2 shows the culture results of reindeer cell passage cells;

Figure 3 shows the chromosome metaphase division phase and karyotype diagram of reindeer fibroblast cells;

Figure 4 shows the chromosome metaphase division phase and karyotype diagram of reindeer fibroblast cells;

Figure 5 shows the culture results of primary cells of Sahu cells;

Figure 6 shows the culture results of sand fox cells passaged cells;

Figure 7 is a phase diagram of chromosome metaphase division in Shahu cells;

Figure 8 shows the karyotype diagram of sand fox cells;

Figure 9 shows the culture results of primary wild boar cells;

Figure 10 shows the culture results of wild boar cell passage cells;

Figure 11 is a phase diagram of chromosome metaphase division in wild boar fibroblast cells;

Figure 12 shows the karyotype of wild boar fibroblasts;

Figure 13 shows the culture results of primary roe deer cells;

Figure 14 is a diagram of the culture results of roe deer cell passage cells;

Figure 15 shows the chromosome metaphase division phase and karyotype diagram of roe deer fibroblasts;

Figure 16 shows the chromosome metaphase division phase and karyotype diagram of roe deer fibroblasts.

Detailed ways

The invention provides a method for long-distance collection of animal tissue, which includes the following steps:

Soak the animal tissue in the ethanol aqueous solution to obtain the first treated tissue;

Soak the first-processed tissue in a PBS solution containing penicillin and streptomycin to obtain the second-processed tissue;

The second treated tissue was stored in PBS solution containing penicillin and streptomycin.

In the present invention, animal tissue is soaked in ethanol aqueous solution to obtain the first treated tissue. In the present invention, the animal tissue is preferably a freshly collected animal tissue, and more preferably is an animal ear tissue. In the present invention, the area of the animal tissue is preferably 1 to 2 cm ² , and more preferably 1 cm ² . The present invention has no special limitations on the original collection method of animal tissue, and any conventional animal tissue collection method in the field can be used. In the present invention, the animal tissue collection is preferably performed after the animal is fixed and the sampling site is sterilized, and then animal tissue of appropriate size is clamped. In the present invention, the method for disinfecting the sampling site preferably includes disinfection with iodophor. After obtaining the animal tissue, the present invention preferably performs shaving treatment on the animal tissue to obtain depilated animal tissue. After obtaining the depilated animal tissue, the present invention preferably soaks the depilated animal tissue in an ethanol aqueous solution. In the present invention, the ethanol aqueous solution preferably includes an ethanol aqueous solution with an ethanol volume percentage content of 70% to 80%, and more preferably an ethanol aqueous solution with an ethanol volume percentage content of 75%. In the present invention, the soaking time of the depilated animal tissue in the ethanol aqueous solution is preferably 10 to 15 s, and more preferably 12 s. In the present invention, the depilated animal tissue is soaked in an ethanol aqueous solution to achieve a sterilization effect.

After obtaining the first treated tissue, the present invention soaks the first treated tissue in a PBS solution containing penicillin and streptomycin to obtain the second treated tissue.

In the present invention, the volume percentage of penicillin and streptomycin in the PBS solution containing penicillin and streptomycin is preferably 1% to 3%, and more preferably 2%. The number of soakings in the present invention is preferably 3 to 5 times, more preferably 5 times; the time of each soaking is preferably 10 to 15 s, further preferably 12 to 15 s, and more preferably 15 s. In the present invention, the soaking function is to remove impurities on the tissue surface and ensure the cleanliness of the sample tissue. In the present invention, the PBS solution containing penicillin and streptomycin is preferably obtained by self-preparation. In the present invention, the penicillin and streptomycin are preferably derived from a double antigen solution; the double antigen solution preferably includes Gibco15140-122.

After obtaining the second-processed tissue, the present invention preserves the second-processed tissue in a PBS solution containing penicillin and streptomycin.

In the present invention, the storage temperature is preferably 4°C; the storage time is preferably 3 to 5 days, and more preferably 4 days. In the present invention, the volume percentage of penicillin and streptomycin in the PBS solution containing penicillin and streptomycin used for preservation is preferably 1% to 3%, and more preferably 2%. In the present invention, the purpose of preserving the second-processed tissue in a PBS solution containing penicillin and streptomycin is to maintain the preservation conditions of the animal tissue close to the in vivo environmental conditions.

The method for long-distance collection of animal tissue provided by the present invention is suitable for long-distance sampling in the field, and the collected fresh sample tissue can maintain the best state within 1 to 5 days, and more preferably, the best state can be maintained within 3 to 5 days. , and thus does not affect the subsequent use of the sample tissue to establish cell lines. At the same time, the method for long-distance collection of animal tissues provided by the present invention is suitable for collecting a variety of animal tissues, has no species specificity, and has a wide range of applications.

The present invention provides a method for establishing a cell line of animal tissue collected remotely using the method described in the above technical solution, which includes the following steps:

After disinfecting the animal tissue, rinse it with a DPBS solution containing penicillin and streptomycin to obtain pretreated tissue;

Rinse the pretreated tissue and cut it into finely divided tissue pieces for primary culture in culture medium;

Primary culture is performed until the confluence of the primary cells reaches 80% to 90%, and then subculture is performed to obtain a cell line.

After disinfecting the animal tissue, the present invention rinses it with a DPBS solution containing penicillin and streptomycin to obtain pretreated tissue.

The disinfection in the present invention is preferably performed using an ethanol aqueous solution; the volume percentage of the ethanol aqueous solution is preferably 70% to 80%, and more preferably 75%. After sterilizing animal tissues, the present invention obtains preliminary processed tissues. After obtaining the preliminarily treated tissue, the present invention rinses the preliminarily treated tissue with a DPBS solution containing penicillin and streptomycin to obtain the pretreated tissue. In the present invention, the volume percentage of penicillin and streptomycin in the DPBS solution containing penicillin and streptomycin is preferably 1% to 3%, and more preferably 2%. The number of flushings in the present invention is preferably 4 to 5 times, and more preferably 5 times. The advantage of using the DPBS solution containing penicillin and streptomycin for flushing is that it can perform disinfection and sterilization and achieve sterile treatment of tissue blocks. In the present invention, the disinfection and flushing are preferably performed under aseptic conditions.

After obtaining the pretreated tissue, the present invention rinses the pretreated tissue and cuts it into finely divided tissue pieces for primary culture in the culture medium.

The rinsing in the present invention is preferably carried out using PBS solution; the number of rinsing is preferably 5 to 6 times, and more preferably 6 times. After rinsing is completed, the present invention preferably cuts the sample tissue into fine tissue pieces. In the present invention, ophthalmic scissors are preferably used for cutting. In the present invention, the size of the finely divided tissue pieces is preferably 0.5 to 1 mm ³ , and more preferably 1 mm ³ .

After obtaining the finely divided tissue pieces, the present invention performs primary culture on the finely divided tissue pieces in the culture medium. After obtaining the finely divided tissue pieces, the present invention preferably spreads the finely divided tissue pieces evenly on the bottom of the culture bottle; the culture bottle preferably includes a T25 culture bottle. After the finely divided tissue pieces are spread on the bottom of the culture bottle, the present invention preferably inverts the culture bottle and adds culture medium to the tissue-free surface; the culture medium is preferably based on α-MEM culture medium and also includes a volume percentage of 10%. FBS and 1% penicillin-streptomycin by volume. In the present invention, the added amount of the culture medium is preferably 5 to 8 mL, more preferably 6 to 8 mL, and most preferably 8 mL.

After adding the culture medium, the present invention preferably places the culture bottle upside down in a CO ₂ incubator for inverted culture. In the present invention, the culture temperature is preferably 37°C to 38.5°C, more preferably 37°C to 38°C, and more preferably 37°C. In the present invention, the inversion culture time is preferably 5 to 6 hours, and more preferably 6 hours. After the inversion culture is completed, the present invention preferably turns the culture bottle over, allowing the culture solution to slowly infiltrate the tissue block, and then gently places it in the incubator for primary culture; the temperature of the primary culture is preferably 37 to 38.5°C, and more preferably It is further preferably 37°C to 38°C, more preferably 37°C; the carbon dioxide concentration in the culture environment is preferably 5%. During the primary culture process of the present invention, it is preferable to change the medium when the color of the culture medium turns yellow. In the present invention, the medium change is preferably performed once after 5 to 7 days of culture, and more preferably once every 6 days. The culture medium used when changing the medium in the present invention is based on α-MEM culture medium and also includes FBS with a volume percentage of 10% and penicillin and streptomycin with a volume percentage of 1%.

In the present invention, the rinsing, shearing of finely divided tissue pieces, and operations during cell culture are preferably performed in a clean workbench.

In the present invention, primary culture is performed until the confluence of the primary cells reaches 80-90%, and subculture is performed to obtain a cell line.

In the present invention, the culture medium used for the subculture is preferably based on α-MEM culture medium and also includes 10% FBS by volume and 1% penicillin and streptomycin by volume. The present invention has no special limitations on the subculture method, and any conventional subculture method in this field can be used. In the present invention, the subculture is preferably performed once every 2 to 3 days, more preferably once when the cell confluence reaches 80% to 90%; the subculture is preferably performed for more than 15 generations, more preferably 15 generations. In the present invention, the subculture temperature is preferably 37°C to 38.5°C, more preferably 37°C to 38°C, and more preferably 37°C; the carbon dioxide concentration in the culture environment is preferably 5%.

The cell line establishment method provided by the present invention is a culture method for obtaining primary cells from animal tissues. It can be universally used for primary cell culture and line establishment in tissues of different species. It is suitable for cultivating primary cells of multiple species and has a high success rate. , can be continuously passaged without mutation, and better maintains the state of somatic cells in terms of morphological structure and biological characteristics.

The present invention provides a cell line established by the establishment method described in the above technical solution. In the present invention, the cell line better maintains the state of somatic cells in terms of morphological structure and biological characteristics.

In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present invention.

Reagents and culture media: 0.25% Trypsin-EDTA (1X) (Gibco 25200-056), MEM-Alpha culture medium (Gibco 12561-056), special grade fetal calf serum (BI 04-001-1ACS), hydrochloric acid phosphate buffer ( DPBS: Gibco14287-080), double antibody (Penicillin-Streptomycin, Liquid: Gibco15140-122), DPBS (BI 02-023-1ACS), 0.9% NaCl injection (Jingxin Pharmaceutical), 75% ethanol (Lierkang ).

Experimental equipment: upright phase contrast microscope (Nikon), CO ₂ incubator (RS Biotech), T25 cell culture flask (Corning), ordinary stereo microscope (Nikon), programmed cooling box (Nalgene).

Example 1

Methods for collecting reindeer ear tissue cell resources

Preparation for the experiment: Dry heat sterilize the sampling equipment for later use; in a sterile environment, pack the sampling reagents and prepare sampling consumables.

1. Sample collection

Bind the animal, disinfect the sampling site with iodophor, use sampling forceps to take ^1cm2 of animal ear tissue, shave the hair clean with a razor, and put it into an ethanol aqueous solution with an ethanol volume percentage of 75% for 12 seconds. , pass 5 times in PBS solution containing 2% penicillin and streptomycin, 15 seconds each time, put into PBS solution containing 2% penicillin and streptomycin, and store at 4°C for 1 to 5 days;

2. Sample pre-processing

Under sterile conditions, remove the ear tissue stored for 5 days in step 1 above, disinfect it again with an aqueous ethanol solution with an ethanol volume percentage of 75%, and rinse it 5 times with a DPBS solution containing 2% double antibody;

3. Sample processing

Place the taken tissue pieces in a small petri dish, move them to a clean workbench, and rinse them 6 times with PBS;

Use ophthalmic scissors to cut the tissue block into fine pieces of ^1mm3 ;

Spread the finely chopped tissue pieces evenly on the bottom of the T25 culture bottle;

4. Sample establishment

Invert the culture bottle, add 8 mL of culture medium to the tissue-free surface, cover the bottle and place it in a 37°C CO ₂ incubator; the composition of the culture medium is α-MEM culture medium as the base culture medium, and the base culture medium contains 10 % FBS and 1% double antibody;

5. Sample cell culture

The culture bottle in step 4 is placed upside down for 6 hours, and then the culture bottle is gently turned over to allow the culture solution to slowly infiltrate the tissue block, and then gently placed in the incubator for culture; the culture temperature is 37°C; the culture is in a carbon dioxide concentration of 5%. carried out under ambient conditions. After culturing for 6 days, when the color of the culture medium turns yellow, change the medium; the medium when changing the medium is the same as above, specifically α-MEM culture medium is used as the base culture medium, and the base culture medium contains 10% FBS and 1% of double resistance;

After the primary cells crawl out of the tissue block and cover the bottom of the culture bottle, they are subcultured. The subculture temperature is 37°C and the carbon dioxide concentration is 5%. During the subculture, cells are cultured every 2 to 3 times. Within days, the cell confluence reaches 80-90%, and the cells are passaged once for 15 generations to obtain a cell line.

6. After obtaining the cell line sample, freeze the cell line sample.

7. Observation of experimental results.

The culture results of primary reindeer cells are shown in Figure 1, and the culture results of reindeer cell passage cells are shown in Figure 2.

It can be seen from Figures 1 and 2 that the line establishment method provided by the present invention can maintain the characteristics of cells and enable stable subculture.

Karyotype analysis was performed on cells cultured for 5 generations to detect whether mutations or deletions occurred during cell passage.

The chromosome metaphase division phase of cells cultured for 5 generations is shown in the left picture in Figure 3; the karyotype analysis results are shown in the right picture in Figure 3. The chromosome metaphase division phase of cells cultured for 5 generations is shown in the left picture in Figure 4; the karyotype analysis results are shown in the right picture in Figure 4.

It can be seen from Figures 3 and 4 that after the present invention obtains cell tissue through the method of collecting cell tissue from a distance, the cell line cultured using the method for establishing the cell line of the present invention does not have chromosomal mutations or deletions.

Example 2

Methods for collecting sand fox cell resources

Preparation for the experiment: Dry heat sterilize the sampling equipment for later use; in a sterile environment, pack the sampling reagents and prepare sampling consumables.

1. Sample collection

Bind the animal, disinfect the sampling site with iodophor, use sampling forceps to take ^1cm2 of animal ear tissue, shave the hair clean with a razor, and put it into an ethanol aqueous solution with an ethanol volume percentage of 75% for 15 seconds. , pass it through the PBS solution containing 2% penicillin and streptomycin 5 times, 15 seconds each time, and put it into the PBS solution containing 2% penicillin and streptomycin. It can be stored at 4°C for 1 to 5 days;

2. Sample pre-processing

Under sterile conditions, remove the ear tissue stored for 5 days in step 1 above, disinfect it again with an ethanol aqueous solution with an ethanol volume percentage of 75%, and rinse 4 times with DPBS containing 2% double antibody;

3. Sample processing

Place the tissue block in a small petri dish, move it to a clean workbench, and rinse it 5 times with PBS;

Use ophthalmic scissors to cut the tissue block into fine pieces of ^1mm3 ;

Spread the finely chopped tissue pieces evenly on the bottom of the T25 culture bottle;

4. Sample establishment

Invert the culture bottle, add 8 mL of culture medium to the tissue-free surface, cover the bottle and place it in a 37°C CO ₂ incubator; the composition of the culture medium is α-MEM culture medium as the base culture medium, and the base culture medium contains 10 % FBS and 1% double antibody;

5. Sample cell culture

The culture bottle in step 4 is placed upside down for 5 hours, and then the culture bottle is gently turned over to allow the culture solution to slowly infiltrate the tissue block, and then gently placed in the incubator for culture; the culture temperature is 37°C; the culture is cultured in a carbon dioxide concentration of 5%. carried out under ambient conditions. After culturing for 7 days, when the color of the culture medium turns yellow, change the medium; the medium when changing the medium is the same as above, specifically α-MEM culture medium is used as the base culture medium, and the base culture medium contains 10% FBS and 1% of double resistance;

After the primary cells crawl out of the tissue block and cover the bottom of the culture bottle, they are subcultured. The subculture temperature is 37°C and the carbon dioxide concentration is 5%. During the subculture, the cells are cultured every 2 to After 3 days, the cell confluence reached 80-90%, and the cells were passaged once for 15 generations to obtain a cell line.

6. After obtaining the cell line sample, freeze the cell line sample.

7. Observation of experimental results.

Figure 5 shows the culture results of the primary cells of Sahu cells, and Figure 6 shows the culture results of the passage cells of Sahu cells.

It can be seen from Figures 5 and 6 that the line establishment method provided by the present invention can maintain the characteristics of cells and enable stable subculture.

Karyotype analysis was performed on the cells cultured for 5 generations to detect whether mutations occurred during cell passage.

The chromosome metaphase division phase of cells cultured for 5 generations is shown in Figure 7; the karyotype analysis results are shown in Figure 8.

It can be seen from Figures 7 and 8 that after the present invention obtains cell tissue through the method of collecting cell tissue from a distance, the cell line cultured using the cell line establishment method of the present invention does not have chromosomal mutations or deletions.

Example 3

Methods for collecting wild boar cell resources

Preparation for the experiment: Dry heat sterilize the sampling equipment for later use; in a sterile environment, pack the sampling reagents and prepare sampling consumables.

1. Sample collection

Bind the animal, disinfect the sampling site with iodophor, use sampling forceps to take ^1cm2 of animal ear tissue, shave the hair clean with a razor, and put it into an ethanol aqueous solution with an ethanol volume percentage of 75% for 10 seconds. , pass it through a PBS solution containing 2% penicillin and streptomycin three times, 10 seconds each time, and then put it into a PBS solution containing 2% penicillin and streptomycin. It can be stored at 4°C for 1 to 5 days;

2. Sample pre-processing

Under sterile conditions, remove the ear tissue that has been maintained for 5 days in step 1 above, disinfect it again with an aqueous ethanol solution with an ethanol volume percentage of 75%, and rinse 4 times with a DPBS solution containing 2% double antibody;

3. Sample processing

Place the tissue block in a small petri dish, move it to a clean workbench, and rinse it 5 times with PBS;

Use ophthalmic scissors to cut the tissue block into fine pieces of ^1mm3 ;

Spread the tissue quickly and evenly on the bottom of the T25 culture bottle;

4. Sample establishment

Invert the culture bottle, add 8 mL of culture medium to the tissue-free surface, cover the bottle and place it in a 37°C CO ₂ incubator; the composition of the culture medium is α-MEM culture medium as the base culture medium, and the base culture medium contains 10 % FBS and 1% double antibody;

5. Sample cell culture

The culture bottle in step 4 is placed upside down for 5 hours, and then the culture bottle is gently turned over to allow the culture solution to slowly infiltrate the tissue block, and then gently placed in the incubator for culture; the culture temperature is 37°C; the culture is cultured in a carbon dioxide concentration of 5%. carried out under environmental conditions. After culturing for 7 days, when the color of the culture medium turns yellow, change the medium; the medium when changing the medium is the same as above, specifically α-MEM culture medium is used as the base culture medium, and the base culture medium contains 10% FBS and 1% of double resistance;

After the primary cells crawl out of the tissue block and cover the bottom of the culture bottle, they are subcultured. The subculture temperature is 37°C and the carbon dioxide concentration is 5%. During the subculture, the cells are cultured every 2 to After 3 days, the cell confluence reached 80-90%, and the cells were passaged once for 15 generations to obtain a cell line.

6. After obtaining the cell line sample, freeze the cell line sample.

7. Observation of experimental results.

The culture results of wild boar primary cells are shown in Figure 9, and the culture results of wild boar cells are shown in Figure 10. The scale bars in Figures 9 and 10 are 100 μm.

It can be seen from Figures 9 and 10 that the line establishment method provided by the present invention can maintain the characteristics of cells and enable stable subculture.

Karyotype analysis was performed on cells cultured for 5 generations to detect whether mutations or deletions occurred during cell passage.

The chromosome metaphase division phase of cells cultured for 5 generations is shown in Figure 11; the karyotype analysis results are shown in Figure 12.

It can be seen from Figures 11 and 12 that after the present invention obtains cell tissue through the method of collecting cell tissue from a distance, the cell line cultured using the method of establishing the cell line of the present invention does not have chromosomal mutations or deletions.

Example 4

Methods for collecting roe deer cell resources

Preparation for the experiment: Dry heat sterilize the sampling equipment for later use; in a sterile environment, pack the sampling reagents and prepare sampling consumables.

1. Sample collection

Bind the animal, disinfect the sampling site with iodophor, use sampling forceps to take an animal ear tissue with an area of ^1cm2 , shave the hair clean with a razor, and place it in an ethanol aqueous solution with an ethanol volume percentage of 75%. It acts for 10 to 15 seconds, and is placed in a PBS solution containing 2% penicillin and streptomycin five times for 15 seconds each time, and then placed in a PBS solution containing 2% penicillin and streptomycin. It can be stored at 4°C for 1 to 5 days. ;

2. Sample pre-processing

Under sterile conditions, remove the ear tissue that has been maintained for 5 days in step 1 above, disinfect it again with an ethanol aqueous solution with an ethanol volume percentage of 75%, and rinse it 5 times with DPBS containing 2% double antibody;

3. Sample processing

Place the tissue block in a small petri dish, move it to a clean workbench, and rinse it 6 times with PBS;

Use ophthalmic scissors to cut the tissue block into fine pieces of ^1mm3 ;

Spread the tissue pieces evenly on the bottom of the T25 culture bottle;

4. Sample establishment

Invert the culture bottle, add 8 mL of culture medium to the tissue-free surface, cover the bottle and place it in a 37°C CO ₂ incubator; the composition of the culture medium is α-MEM culture medium as the base culture medium, and the base culture medium contains 10 % FBS and 1% double antibody;

5. Sample cell culture

The culture bottle in step 4 is placed upside down for 6 hours, and then the culture bottle is gently turned over to allow the culture solution to slowly infiltrate the tissue block, and then gently placed in the incubator for culture; the culture temperature is 37°C; the culture is in a carbon dioxide concentration of 5%. carried out under ambient conditions. When culturing for 7 days and until the color of the culture medium turns yellow, change the medium; the medium when changing the medium is the same as above, specifically α-MEM culture medium is used as the base culture medium, and the base culture medium contains 10% FBS and 1% of double resistance;

After the primary cells crawl out of the tissue block and cover the bottom of the culture bottle, they are subcultured. The subculture temperature is 37°C and the carbon dioxide concentration is 5%. During the subculture, the cells are cultured every 2 to After 3 days, the cell confluence reached 80-90%, and the cells were passaged once for 15 generations to obtain a cell line.

6. After obtaining the cell line sample, freeze the cell line sample.

7. Observation of experimental results.

Cells were observed through an inverted microscope.

The culture results of the primary roe deer cells are shown in Figure 13, and the culture results of the roe deer secondary cells are shown in Figure 14.

It can be seen from Figures 13 and 14 that the line establishment method provided by the present invention can maintain the characteristics of cells and enable stable subculture.

Karyotype analysis was performed on cells cultured for 5 generations to detect whether mutations or deletions occurred during cell passage.

The chromosome metaphase division phase of cells cultured for 5 generations is shown in the left picture in Figure 15; the karyotype analysis results are shown in the right picture in Figure 15. The chromosome metaphase division phase of cells cultured for 5 generations is shown in the left picture in Figure 16; the karyotype analysis results are shown in the right picture in Figure 16.

It can be seen from Figures 15 and 16 that after the present invention obtains cell tissue through the method of collecting cell tissue from a distance, the cell line cultured using the method for establishing the cell line of the present invention does not have chromosomal mutations or deletions.

In summary, the method for long-distance collection of animal tissues provided by the present invention is suitable for the preservation of a variety of animal tissues. This collection method can preserve animal tissues for 3 to 5 days, which is suitable for long-distance sampling in the field, and the animal tissues are constructed after preservation. system, cell growth is almost unaffected and the ideal growth state can be achieved. The method for establishing cell lines based on long-distance collected animal tissues provided by the present invention can be universally used for primary cell culture and establishment of tissues of different species, is suitable for cultivating primary cells of multiple species, has a high success rate, and can be continuously passaged. There will be no mutation, and the state of somatic cells can be better maintained in terms of morphological structure and biological characteristics.

Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without any inventive step. These embodiments All belong to the protection scope of the present invention.

Claims (10)

1. A method for remotely harvesting animal tissue, comprising the steps of:

soaking animal tissue in ethanol water solution to obtain first treated tissue;

soaking the first treated tissue in PBS solution containing the green streptomycin to obtain a second treated tissue;

the second treated tissue was preserved in PBS solution containing green streptomycin.

2. The method of claim 1, wherein the animal tissue is immersed in the aqueous ethanol for a period of time ranging from 10 to 15 seconds; the volume percentage of the ethanol in the ethanol water solution is 70-80%.

3. The method of claim 1, wherein the first treated tissue is soaked in a PBS solution containing green streptomycin for 3 to 5 times, each soaking time being 10 to 15 seconds; the volume percentage of the blue-chain mycin in the PBS solution containing the blue-chain mycin is 1-3%.

4. The method of claim 1, wherein the animal tissue comprises ear tissue; the area of the animal tissue is 1-2 cm ² 。

5. The method of claim 1, wherein the stored temperature is 4 ℃; the preservation time is 3-5 d.

6. A method of establishing a cell line of animal tissue harvested remotely using the method of any one of claims 1 to 5, comprising the steps of:

after animal tissues are disinfected, washing is carried out by DPBS solution containing the green streptomycin, and pretreated tissues are obtained;

rinsing the pretreated tissue, shearing the pretreated tissue into finely divided tissue blocks, and carrying out primary culture in a culture medium;

primary culture is carried out until the confluence of primary cells reaches 80% -90%, and subculture is carried out to obtain a cell line.

7. The method of claim 6, wherein the rinsing is performed with a PBS solution; the rinsing times are 5-6 times; the size of the finely divided tissue blocks is 0.5-1 mm ³ 。

8. The method according to claim 6, wherein the sterilization is performed with 70% -80% ethanol aqueous solution; the DPBS solution containing the blue-chain mycin contains the blue-chain mycin with the volume percentage of 1-3 percent.

9. The method according to claim 6, wherein the medium used in the establishment of the cell line comprises 10% FBS and 1% penicillin; the culture medium comprises alpha-MEM basal medium; the temperature of primary culture and subculture is 37-38.5 ℃; the carbon dioxide concentration was 5%.

10. A cell line established by the establishment method of any one of claims 6 to 9.