CN111088218A - Three-dimensional continuous culture method for porcine mammary epithelial cells - Google Patents
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Abstract
The invention relates to a three-dimensional continuous culture method of porcine mammary epithelial cells, which belongs to the field of cell culture methods and specifically comprises the following steps: (1) separating and extracting primary epithelial cells of the porcine mammary gland, and selecting cells before 10 generations; (2) digesting into single cells with trypsin, centrifuging, removing supernatant, resuspending with common culture medium, adjusting cell concentration to 1 × 106~5×106Taking 1mL of cells/mL, centrifuging again, discarding supernatant, resuspending by using experimental Matrigel, and uniformly mixing on ice for later use; (3) sucking a proper amount of the suspension, dripping 7-8 drops of the suspension into each hole of a six-hole plate, and after dripping, reversely buckling the six-hole plate and incubating the six-hole plate in an incubator at 37 ℃ for 30-40 min to solidify the six-hole plate; (4) adding 2mL of three-dimensional culture medium into each hole, continuously culturing for 12-16 days, and replacing the three-dimensional culture medium every 2-3 days according to the color of the culture medium; (5) removing the culture medium, washing for three times by using D-PBS, adding 2mL of trypsin into each hole, blowing by using a gun head, culturing at 37 ℃, blowing and uniformly mixing every 2-4 min until the cells become single cells, neutralizing, digesting, centrifuging at 4 ℃, removing the supernatant, and carrying out passage according to the steps (2) - (4).
Description
Technical Field
The invention relates to the field of cell culture methods, in particular to a three-dimensional continuous culture method for porcine mammary epithelial cells.
Background
Mammary gland epithelial cells are the main cells constituting mammary glands and have a special function of milk secretion, and many components of milk can be synthesized only by mammary gland epithelial cells. The study of mammary gland cells began in the 70 s of the 20 th century, and it has become a hot spot to culture primary mammary gland epithelial cells at the cellular level by an effective cell culture method and to study the growth, development and lactation cells of mammary glands or molecular biological mechanisms by passage. At present, the study using mammary gland epithelial cells of human, mouse and cattle as models is quite abundant, but the study on the mammary gland epithelial cells of pigs has few reports.
Cell technology, the most core and basic technology in medical biotechnology, has become the necessary skills in medical scientific research at present, and is widely applied to many fields of life science. The traditional cell culture usually means that cells grow on a single-layer glass or plastic plane, has better extensibility, but cannot sufficiently and truly simulate in vivo growth mode, and finally influences cell processes such as cell proliferation, differentiation, apoptosis, gene and protein expression and the like. Three-dimensional cell culture systems more accurately reflect the actual microenvironment of cells in a tissue than monolayer cell culture systems. The three-dimensional cell culture method is that cells are placed in a three-dimensional culture carrier imitating the in-vivo cell environment for in-vitro culture, and the cells grow in the three-dimensional space structure of the three-dimensional carrier to form a three-dimensional cell carrier compound. Compared with the traditional monolayer cell culture, the three-dimensional cell culture method can better simulate the in-vivo conditions, can more closely simulate the complex interaction between cell tissues and the microenvironment in vivo, provides a proper microenvironment for the optimal growth and differentiation of cells, and has the capability of creating tissue-like structures in vitro. By allowing individual cells to maintain their normal three-dimensional shape, it facilitates complex interactions and signal reception and transmission between cells and adjacent cells, creating a more natural growth environment for different types of cell cultures.
At present, the research combining the porcine mammary gland epithelial cells and the three-dimensional cell culture method is rarely reported, the three-dimensional continuous culture method of the porcine mammary gland epithelial cells is provided for improving the defects of the traditional monolayer cell culture method in the culture of the mammary gland epithelial cells, and the method has important significance for the research of the porcine mammary gland epithelial cells.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a three-dimensional continuous culture method for porcine mammary epithelial cells, the method enables the cells to be differentiated to generate a certain three-dimensional tissue specific structure, and the created cell growth environment simulates the in-vivo environment to the maximum extent and can be continuously cultured.
In order to achieve the purpose, the invention adopts the technical scheme that:
a three-dimensional continuous culture method of porcine mammary epithelial cells comprises the following steps:
(1) separating and extracting cells before 10 generations of primary epithelial cells of the pig mammary gland for experiment;
(2) digesting porcine mammary gland epithelial cells into single cells by trypsin, centrifuging at 1000rpm for 4-6 min, discarding supernatant, adding 1mL of common culture medium for resuspension, and adjusting cell concentration to 1 × 106~5×106centrifuging the cells/mL at 600-800 rpm for 4-6 min, removing supernatant, adding 1mL of experimental Matrigel for heavy suspension, and uniformly mixing on ice for later use;
(3) sucking the suspension obtained in the step (2) by using a 200-microliter gun head, dripping 7-8 drops of the suspension into a six-hole plate by using the amount of 20-40 microliter per drop, and after dripping, inversely buckling and placing the six-hole plate in a carbon dioxide incubator at 37 ℃ for incubation for 30-40 min to solidify the suspension;
(4) adding 2mL of three-dimensional culture medium into each hole of the six-hole plate, continuously culturing for 12-16 days, and replacing the three-dimensional culture medium every 2-3 days according to the color of the culture medium;
(5) removing the culture medium, washing for three times by using a D-PBS buffer solution, adding 2mL of trypsin into each hole, blowing off by using a 1mL gun head, culturing at 37 ℃, blowing off and uniformly mixing every 2-4 min until the cells become single cells, neutralizing and digesting the three-dimensional culture medium, centrifuging at the temperature of 4 ℃ and at the speed of 600-1000 rpm for 4-6 min, removing the supernatant, carrying out passage according to the steps (2) - (4), and extracting and detecting RNA and protein of the three-dimensional cultured cells;
the three-dimensional cell RNA extraction method comprises the following steps:
removing the culture medium, washing for 3 times by using D-PBS, directly adding Trizol for cracking and collecting, and storing at-100 to-60 ℃; extracting RNA by using a Trizol method;
the extraction method of the three-dimensional culture cell protein comprises the following steps:
removing the culture medium, washing for 3 times by using precooled D-PBS, adding 2mL of trypsin into each hole of a six-hole plate, blowing off by using a 1mL gun head, putting the six-hole plate into a carbon dioxide incubator at 37 ℃ for complete digestion, neutralizing and digesting the three-dimensional culture medium, centrifuging at the temperature of 4 ℃ to 1200rpm for 4 to 6min, removing the supernatant, adding 1mL of D-PBS for re-suspension, centrifuging at the temperature of 4 ℃ to 1200rpm for 4 to 6min, removing the supernatant, adding a protein lysate, and cracking the protein to collect a sample, and storing at the temperature of-100 ℃ to-60 ℃;
the preparation of the three-dimensional culture medium comprises the following steps:
1mL of Advanced DMEM/F12 culture medium, 40-60% of L-WRN cells culture supernatant (a conditioned medium rich in Wnt3 α, R-spondins and Noggin protein factors), 8-12% of fetal bovine serum, 1-2% of penicillin streptomycin, 8-12 μ M Y27632 (ROCK inhibitor), 8-12 μ M SB202190 (P38 inhibitor), 8-12 ng/mL of epithelial growth factor, 0.4-0.6 μ g/mL of hydrocortisone, 1B-27, 1 glutamine, 0.01-0.02% of ITS (Insulin-Insulin, HumanTransfern-human transferrin, Selenous Acid-selenic Acid), 1 gentamycin B & damycin, 25 μ M mycoplasma remover, 1.00-1.50 mM of N-acetylcysteine, and 0.20 μ M of culture medium filter.
Preparation of Matrigel: 50-60% of Matrigel stock solution and 40-50% of three-dimensional culture medium, preserving for one month at-25 to-15 ℃, and putting the mixture into water or ice at 3-5 ℃ for preheating 2-3 hours before treatment.
The invention has the beneficial effects that: the technology provided by the invention can better provide a proper microenvironment for the optimal growth and differentiation of cells, so that the matrix and the cells are in full contact, the microenvironment in vivo is better met, the operation is more time-saving and simple, and the continuous culture can be realized.
Drawings
FIG. 1 is a graph comparing the effect of porcine mammary epithelial cells grown for 5 days in monolayer and three-dimensional culture methods;
FIG. 2 is a graph showing the effect of growth of porcine mammary epithelial cells for 1, 5, 9 and 13 days in a three-dimensional culture method.
Detailed Description
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention is described in further detail below with reference to the figures and the specific examples, which are provided only for illustrating the invention and do not limit the scope of protection of the invention.
Preparation of Matrigel: 50-60% of Matrigel stock solution and 40-50% of three-dimensional culture medium, preserving for one month at-25 to-15 ℃, and putting the mixture into water or ice at 3-5 ℃ for preheating 2-3 hours before treatment.
Preparation of three-dimensional culture medium
1mL of Advanced DMEM/F12 medium, 40-60% of L-WRN cells culture supernatant (Wnt 3 α, R-spondins, Noggin protein factor-rich conditioned medium), 8-12% of fetal bovine serum, 1-2% of penicillin streptomycin, 8-12 μ MY27632 (ROCK inhibitor), 8-12 μ M SB202190 (P38 inhibitor), 8-12 ng/mL of epithelial growth factor, 0.4-0.6 μ g/mL of hydrocortisone, 1B-27, 1 glutamine, 0.01-0.02% of ITS (Insulin-Insulin, HumanTransferrin-human transferrin, Selenous Acid-selenic Acid), 1 gentamycin B & damycin, 25 μ M mycoplasma remover, 1.00-1.50 mM of N-acetylcysteine, and the percentages of the culture medium, the filter volume and the filter volume are 20 μ M.
Example 1
A three-dimensional continuous culture method of porcine mammary epithelial cells comprises the following steps:
(1) separating and extracting cells before 10 generations of primary epithelial cells of the pig mammary gland;
(2) digesting porcine mammary gland epithelial cells with trypsin to form single cells, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1mL commonResuspending the medium and adjusting the cell concentration to 1X 106centrifuging at 800rpm for 5min for cells/mL, removing supernatant, adding 1mL of experimental Matrigel for heavy suspension, and uniformly mixing on ice for later use;
(3) sucking the suspension by using a 200-mu-L gun head, dripping into a six-hole plate with the amount of 30 mu L per drop, dripping 8 drops into each hole, and reversely turning over the six-hole plate to be placed in a carbon dioxide incubator at 37 ℃ for incubation for 30min after dripping to solidify the suspension;
(4) adding 2mL of three-dimensional culture medium into each hole of a six-hole plate, continuously culturing for 14 days, and replacing the three-dimensional culture medium every 3 days according to the color of the culture medium;
(5) on the 14 th day, removing the culture medium, washing for three times by using a D-PBS buffer solution, adding 2mL of trypsin into each hole, blowing off by using a 1mL gun head, culturing at 37 ℃, blowing off and uniformly mixing every 3min until the cells become single cells, neutralizing and digesting, centrifuging at the temperature of 4 ℃ and the rpm of 600 for 6min, removing the supernatant, carrying out passage according to the steps (2) to (4), and extracting and detecting RNA and protein of the three-dimensional cultured cells;
the three-dimensional cultured cell RNA extraction method in the step (5) is a Trizol method, namely, a culture medium is removed, and after washing for 3 times by using precooled D-PBS, the culture medium is directly added into Trizol for cracking and collection and is preserved at-80 ℃;
the method for extracting the three-dimensional culture cell protein in the step (5) comprises the following steps: removing the culture medium, washing for 3 times by using precooled D-PBS, adding 2mL of trypsin into each hole of a six-hole plate, blowing off by using a 1mL gun head, putting the six-hole plate into a 37 ℃ carbon dioxide incubator to completely digest the six-hole plate, neutralizing and digesting the three-dimensional culture medium, centrifuging at 4 ℃ and 800rpm for 6min, removing the supernatant, adding 1mL of D-PBS for heavy suspension, centrifuging at 4 ℃ and 800rpm for 6min, removing the supernatant, adding a proper protein lysate, cracking the protein, collecting a sample, and preserving at-80 ℃.
In this example, a comparison of the effect of growth of porcine mammary epithelial cells in a monolayer and three-dimensional culture method in the light field for 5 days is taken using an inverted biomicroscope, as shown in FIG. 1.
Example 2
A three-dimensional continuous culture method of porcine mammary epithelial cells comprises the following steps:
(1) separating and extracting cells before 10 generations of primary epithelium of the porcine mammary gland;
(2) digesting porcine mammary gland epithelial cells with trypsin to form single cells, centrifuging at 1000rpm for 4min, discarding supernatant, adding 1mL common culture medium, resuspending, and adjusting cell concentration to 5 × 106centrifuging at 800rpm for 6min in cells/mL, removing supernatant, adding 1mL of experimental Matrigel for heavy suspension, and uniformly mixing on ice for later use;
(3) sucking the suspension obtained in the step (2) by using a 200-mu-L gun head, dripping the suspension into a six-hole plate with 8 drops per hole in an amount of 20 mu L per drop, and after dripping, reversing the suspension and incubating the suspension in a carbon dioxide incubator at 37 ℃ for 40min to solidify the suspension;
(4) adding 2mL of three-dimensional culture medium into each hole of the six-hole plate, continuously culturing for 12 days, and replacing the three-dimensional culture medium every 2 days according to the color of the culture medium;
(5) on the 12 th day, removing the culture medium, washing for three times by using a D-PBS buffer solution, adding 2mL of trypsin into each hole, blowing off by using a 1mL gun head, culturing at 37 ℃, blowing off and uniformly mixing every 2min until the cells become single cells, neutralizing and digesting the three-dimensional culture medium, separating at 4 ℃ and 800rpm for 5min, removing the supernatant, carrying out passage according to the steps (2) to (4), and extracting and detecting RNA and protein of the three-dimensional cultured cells;
the three-dimensional cultured cell RNA extraction method in the step (5) is a Trizol method, namely, a culture medium is removed, the cell is washed for 3 times by using D-PBS, and then the cell is directly added with Trizol for cracking and collection and is preserved at the temperature of minus 80 ℃;
the three-dimensional culture cell protein extraction method in the step (5) comprises the following steps: removing the culture medium, washing for 3 times by using D-PBS, adding 2mL of trypsin into each hole of a six-hole plate, blowing off by using a 1mL gun head, putting the six-hole plate into a carbon dioxide incubator at 37 ℃ to completely digest the mixture, neutralizing and digesting the three-dimensional culture medium, centrifuging at 4 ℃ and 1000rpm for 5min, removing the supernatant, adding 1mLD-PBS for heavy suspension, centrifuging at 4 ℃ and 1000rpm for 5min to remove the supernatant, adding a proper protein lysate, collecting a sample of the cleaved protein, and storing at-60 ℃.
In this example, the effect of the growth of the porcine mammary epithelial cells in the three-dimensional culture method in the bright field is shown in FIG. 2 in a graph of the effect of the growth for 1, 5, 9 and 13 days by using an inverted biomicroscope.
Example 3
A three-dimensional continuous culture method of porcine mammary epithelial cells comprises the following steps:
(1) separating and extracting cells before 10 generations of primary epithelial cells of the pig mammary gland;
(2) digesting porcine mammary gland epithelial cells with trypsin to form single cells, centrifuging at 1000rpm for 6min, discarding supernatant, adding 1mL common culture medium, resuspending, and adjusting cell concentration to 5 × 106centrifuging at 800rpm for 4min in cells/mL, removing supernatant, adding 1mL experimental Matrigel for heavy suspension, and uniformly mixing on ice for later use;
(3) sucking the suspension obtained in the step (2) by using a 200-mu-L gun head, dripping the suspension into a six-hole plate with the amount of 40 mu L per drop, dripping 7 drops of the suspension into each hole, and reversely turning the plate into a carbon dioxide incubator at 37 ℃ to incubate for 35min after dripping to solidify the suspension;
(4) adding 2mL of three-dimensional culture medium into each hole of the six-hole plate, continuously culturing for 16 days, and replacing the three-dimensional culture medium every 2 days according to the color of the culture medium;
(5) on the 16 th day, removing the culture medium, washing for three times by using a D-PBS buffer solution, adding 2mL of trypsin into each hole, blowing off by using a 1mL gun head, culturing at 37 ℃, blowing off and uniformly mixing every 4min until the cells become single cells, neutralizing and digesting the three-dimensional culture medium, centrifuging at 4 ℃ and 1000rpm for 4min, removing the supernatant, carrying out passage according to the steps (2) to (4), and extracting and detecting RNA and protein of the three-dimensional cultured cells;
the three-dimensional cultured cell RNA extraction method in the step (5) is a Trizol method, namely, the culture medium is removed, and after washing for 3 times by using precooled D-PBS, the culture medium is directly added into Trizol for cracking and collection and is preserved at-80 ℃;
the three-dimensional cell protein extraction method in the step (5) comprises the following steps: removing the culture medium, washing for 3 times by using D-PBS, adding 2mL of trypsin into each hole of a six-hole plate, blowing off by using a 1mL gun head, putting the six-hole plate into a carbon dioxide incubator at 37 ℃ to completely digest the trypsin, centrifuging for 4min at 4 ℃ and 1200rpm after the digestion is neutralized, removing the supernatant, adding 1mL of D-PBS for resuspension, centrifuging for 4min at 4 ℃ and 1200rpm to remove the supernatant, adding a proper protein lysate, collecting the cleaved protein and storing at-60 ℃.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (5)
1. A three-dimensional continuous culture method of porcine mammary epithelial cells is characterized by comprising the following steps: the method comprises the following steps:
(1) separating and extracting cells before 10 generations of primary epithelial cells of the pig mammary gland for experiment;
(2) digesting porcine mammary gland epithelial cells into single cells by trypsin, centrifuging at 1000rpm for 4-6 min, discarding supernatant, adding 1mL of common culture medium for resuspension, and adjusting cell concentration to 1 × 106~5×106centrifuging the cells/mL at 600-800 rpm for 4-6 min, removing supernatant, adding 1mL of experimental Matrigel for heavy suspension, and uniformly mixing for later use;
(3) sucking the suspension obtained in the step (2) by using a 200-microliter gun head, dripping 7-8 drops of the suspension into a six-hole plate by using the amount of 20-40 microliter per drop, and after dripping, inversely buckling and placing the six-hole plate in a carbon dioxide incubator at 37 ℃ for incubation for 30-40 min to solidify the suspension;
(4) adding 2mL of three-dimensional culture medium into each hole of the six-hole plate, continuously culturing for 12-16 days, and replacing the three-dimensional culture medium every 2-3 days according to the color of the culture medium;
(5) removing the culture medium, washing for three times by using a D-PBS buffer solution, adding 2mL of trypsin into each hole, blowing off by using a 1mL gun head, culturing at 37 ℃, blowing off and uniformly mixing every 2-4 min until the cells become single cells, neutralizing and digesting the three-dimensional culture medium, centrifuging at the temperature of 4 ℃ and at the speed of 600-1000 rpm for 4-6 min, removing the supernatant, carrying out passage according to the steps (2) - (4), and extracting and detecting RNA and protein of the three-dimensional cultured cells.
2. The three-dimensional continuous culture method of the porcine mammary epithelial cells according to claim 1, which is characterized in that: preparing the Matrigel in the step (2): 50-60% of Matrigel stock solution and 40-50% of three-dimensional culture medium, preserving for one month at-25 to-15 ℃, and putting the mixture into water at 3-5 ℃ or ice for preheating 2-3 hours before treatment.
3. The three-dimensional continuous culture method of the porcine mammary epithelial cells as claimed in claim 1, wherein the three-dimensional culture medium of step (4) is prepared from 1mL of Advanced DMEM/F12 culture medium, 40-60% of L-WRN cells culture supernatant, wherein the supernatant is a conditioned medium rich in Wnt3 α, R-spondins and Noggin protein factor, 8-12% of fetal bovine serum, 1-2% of penicillin streptomycin, 8-12 μ M of ROCK inhibitor Y27632, 8-12 μ M P38 inhibitor SB202190, 8-12 ng/mL of epidermal growth factor, 0.4-0.6 μ g/mL of hydrocortisone, 1. about.B-27, 1. about.glutamine, 0.01-0.02% of ITS, 1. about.gentamicin B & gentamicin, 25 μ M of mycoplasma remover, 1.00-1.50 mM of N-acetylcysteine, and a filter of culture medium with 0.20 μ M filter.
4. The three-dimensional continuous culture method of the porcine mammary epithelial cells according to claim 1, which is characterized in that: the extraction method of the three-dimensional cultured cell RNA in the step (5) is a Trizol method, the culture medium is removed, the cell RNA is washed for 3 times by precooled D-PBS and then directly added with Trizol for cracking and collection, and the cell RNA is stored at the temperature of minus 100 to minus 60 ℃.
5. The three-dimensional continuous culture method of the porcine mammary epithelial cells according to claim 1, which is characterized in that: the extraction method of the three-dimensional culture cell protein in the step (5) comprises the following steps: removing the culture medium, washing for 3 times by using precooled D-PBS, adding 2mL of trypsin into each hole of a six-hole plate, blowing off by using a 1mL gun head, putting the six-hole plate into a carbon dioxide incubator at 37 ℃ for complete digestion, neutralizing and digesting the three-dimensional culture medium, centrifuging at the temperature of 800-1200 rpm for 4-6 min, removing the supernatant, adding 1mL of D-PBS for re-suspension, centrifuging at the temperature of 800-1200 rpm for 4-6 min, removing the supernatant, adding a protein lysate, and cracking the protein to collect a sample, and storing at the temperature of-100 ℃ to-60 ℃.
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| CN114196620A (en) * | 2022-02-14 | 2022-03-18 | 广东省农业科学院动物科学研究所 | In-vitro culture method for breast tissue of sow |
| CN117025516A (en) * | 2023-10-08 | 2023-11-10 | 广东省农业科学院动物科学研究所 | Separation digestion and culture method for pig mammary gland epithelial cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113564114A (en) * | 2021-01-16 | 2021-10-29 | 浙江工商大学 | Construction method and application of three-dimensional cell model of CMT93-DC-T cell |
| CN113564114B (en) * | 2021-01-16 | 2023-04-07 | 浙江工商大学 | Construction method and application of three-dimensional cell model of CMT93-DC-T cell |
| CN114196620A (en) * | 2022-02-14 | 2022-03-18 | 广东省农业科学院动物科学研究所 | In-vitro culture method for breast tissue of sow |
| CN117025516A (en) * | 2023-10-08 | 2023-11-10 | 广东省农业科学院动物科学研究所 | Separation digestion and culture method for pig mammary gland epithelial cells |
| CN117025516B (en) * | 2023-10-08 | 2023-12-15 | 广东省农业科学院动物科学研究所 | Separation digestion and culture method for pig mammary gland epithelial cells |
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