CN113774013A - Separation and culture method for chick yellow follicle adventitial layer cells - Google Patents
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Abstract
The invention discloses a method for separating and culturing cells of an adventitial layer of a chicken small yellow follicle, which comprises the steps of collecting the small yellow follicle; obtaining an outer membrane layer of the small yellow follicle; cleaning, shearing, digesting and filtering the outer membrane layer; then carrying out primary culture on the adventitia layer cells for 36-48 h; continuously culturing for 36-48 h after liquid is changed; and finally, subculturing the cultured cells for 24-48 h. The invention provides a pollution-free method for collecting a small yellow follicle, optimizes the acquisition of an adventitia layer of the small yellow follicle and a membrane cell culture method, and also provides a primary membrane cell subculture method. The method can be used for separating the membrane cells with higher purity, and has the advantages of high consistency of growth and development periods, high cell survival rate, high success rate of cell proliferation test, high result consistency, strong practicability, strong stability and high feasibility. The method of the invention has the advantages of collecting a large number of cells of the small yellow follicles, continuously supplying the cells to the subsequently developed experiment, reducing the cost of purchasing poultry and improving the experiment efficiency.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a method for separating, primary culturing and subculturing chicken small yellow follicle adventitia layer cells.
Background
The egg laying performance of poultry is mainly dependent on the developmental status of the follicles in the ovaries. One follicle of the high-yield laying hens is recruited out every day to enter a grade follicle, and the follicle cannot be locked once entering the grade follicle; a follicle (Small yellow follicle, SYF) with the size of 5-10 mm is just a selection key point for judging whether the follicle can enter the priority level or not in the follicle screening, and therefore the development condition of the Small yellow follicle is indistinguishable from the egg laying performance of chickens. Unlike mammals, chicken oocyst cells can synthesize estrogen in addition to providing structural support, synthesizing androgen, mediating the interaction between oocytes and granulosa cells. The proliferation of theca cell is an important guarantee for the completion of the above physiological functions, and is also a necessary condition for the selection of the small yellow follicle into a grade follicle. However, at present, the poultry yellow blastocyst cell line is not available on the market. Therefore, collecting the poultry yellow follicle membrane cells in vivo for culture is an important technical basis for researching the egg laying performance of the poultry.
However, in the prior art, pure buffy coat follicle adventitia cells are difficult to separate, the phenomena of cell pollution, cell non-adherence and the like easily occur when membrane cells are subjected to primary culture, and subculture cannot be performed for subsequent experiments.
Disclosure of Invention
Therefore, the invention provides a method for separating and culturing chick small yellow follicle adventitia layer cells, which aims to solve the problems that pure small yellow follicle adventitia layer cells are difficult to separate in the prior art, and primary culture of the membrane cells is easy to cause cell pollution, cell non-adherence and the like.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to the invention, the method for separating and culturing the cells of the adventitia layer of the chicken small yellow follicle comprises the following steps:
step one, collecting small yellow follicles
Collecting small yellow follicles on hen ovaries with regular egg laying period in an aseptic laboratory, flushing the surface grease and blood stains of the follicles with PBS buffer solution, cleaning, placing the follicles in a beaker containing the PBS buffer solution, sealing the beaker, and transporting the follicles back to a cell room;
step two, obtaining the adventitial layer of the small yellow follicle
Washing the small yellow follicle by using PBS buffer solution again, taking the small yellow follicle to be placed in a sterile culture dish containing the PBS buffer solution after washing until no blood exists, cutting off connective tissues connected with the ovary on the small yellow follicle to expose a small opening on the outer membrane layer, extruding blood in the blood vessel along the blood vessel on the surface of the outer membrane layer by using an elbow forceps, taking down the outer membrane layer along the small opening, and placing the taken down outer membrane layer in another clean sterile culture dish containing the PBS buffer solution;
step three, cutting the outer film layer
Gently washing the taken outer membrane layer in PBS buffer solution for 5-6 times, washing off residual blood on the membrane, transferring the membrane layer into a culture dish containing type II collagenase after cleaning, and shearing the outer membrane layer into a thickness of 0.5-1 mm in the culture dish3A size tissue mass;
step four, digesting the adventitia tissue block
Covering a culture dish cover, transferring the culture dish into an incubator for digestion culture, gently blowing and beating the culture dish for a plurality of times by using a pipettor every 5-7 min, and finally adding an equal amount of all-contained culture medium into the culture dish to terminate the digestion reaction to obtain an outer membrane tissue block suspension;
step five, obtaining the adventitia layer cells
Filtering the adventitia tissue mass suspension by using a cell filter screen, removing the undigested and complete adventitia tissue mass, preparing a crude adventitia cell suspension, putting the cell suspension in a centrifuge tube, centrifuging, and removing supernatant; adding PBS buffer solution, blowing, beating, mixing, centrifuging, and removing supernatant; adding PBS buffer solution again, blowing, beating, mixing uniformly, centrifuging, and removing supernatant; finally adding all culture media, blowing, beating and uniformly mixing to obtain refined membrane cell suspension;
step six, primary culture
Diluting 10 mu L of refined membrane cell suspension by 100 times, counting cells under a microscope, inoculating the cells into a T25 culture bottle, adding all culture media into the culture bottle, blowing and uniformly mixing, and culturing in an incubator to obtain primary culture cells;
step seven, replacing the primary culture solution
After culturing for 36-48 h, removing the culture solution, cleaning adherent cells by using a PBS buffer solution, removing the cleaning solution, adding all culture media, and culturing in an incubator;
step eight, subculturing
Continuously culturing for 36-48 h in the incubator until the anchorage rate reaches 70-80%, removing the culture solution, adding a PBS (phosphate buffer solution) to clean anchorage-dependent cells, and removing the cleaning solution; adding pancreatin, and digesting and culturing in an incubator at constant temperature; adding a culture medium which is equal to pancreatin in quantity to terminate the digestion reaction, and blowing and beating the adherent cells to completely fall off; transferring the cell suspension in the culture bottle to a centrifuge tube, centrifuging, and removing supernatant; adding PBS buffer solution into the cell sediment, blowing, uniformly mixing, centrifuging and removing supernatant; finally adding all culture media, uniformly blowing and mixing, and counting cells under a microscope; then inoculating the mixture into a 6-hole plate or a 96-hole plate, adding a complete culture medium, and continuously culturing in an incubator; obtaining subculture cells.
Further, in the step one, small yellow follicles are collected to be pre-grade follicles with the diameter of 5-10 mm.
Further, the PBS buffer is a buffer mixed by 99mL of PBS buffer and 1mL of SK.
Further, the beaker sealing method adopts 4 layers of sterile newspaper and 8 layers of sterile gauze for sealing.
Further, in the third step, the culture dish for type II collagenase contains 8mL of type II collagenase with the concentration of 1 mg/mL; the 1mg/mL collagenase II is prepared by dissolving 100mg collagenase II powder in 100mL DMEM/F12 culture medium and mixing uniformly.
Further, in the fourth step, the digestion culture conditions are 38 ℃, and digestion is carried out in a 5% CO2 incubator for 30 min.
Further, the total culture medium comprises 84mL of DMEM/F12 culture medium, 15mL of fetal bovine serum and 1mL of streptomycin for cells.
Furthermore, the refined membrane cell suspension is diluted 100 times by taking 10 μ L of the cell suspension to a 1.5mL centrifuge tube containing 990 μ L of PBS buffer solution, and uniformly mixing by blowing.
Further, the centrifugation condition is 1500r centrifugation for 5 min.
Further, the number of the cells inoculated in the sixth step is 5X 107One/bottle; in the eighth step, the number of cells to be seeded into the 6-well plate is 1X 106Number of cells seeded in 96-well plate per well was 1.2X 104Per well.
Further, in the seventh step, the volume of the PBS buffer solution is 2 mL; the total amount of the culture medium added was 4 mL.
The invention has the following advantages:
the invention provides a pollution-free method for collecting a small yellow follicle, optimizes the acquisition of an adventitia layer of the small yellow follicle and a membrane cell culture method, and also provides a primary membrane cell subculture method. The method can be used for separating the membrane cells with higher purity, almost has no pollution, has high consistency in the growth and development period, high cell survival rate, high success rate of cell proliferation test, high result consistency, strong practicability, strong stability and high feasibility. In addition, the method of the invention has the advantages of collecting a large number of cells of the small yellow follicles, continuously supplying the cells to the subsequently developed experiments, reducing the cost for purchasing poultry, improving the experiment efficiency and reducing the experiment cost.
The method is more specific, and people in the field of research all adopt the whole membrane cells and do not further separate the outer membrane cells, but the method can separate the outer membrane cells and is obviously different from related researches of other researchers, thereby laying a solid foundation for the subsequent research of molecular mechanisms of related functions of the outer membrane cells.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a diagram of ovulation-period-graded follicles (F1-F6) of a chicken provided by the invention;
FIG. 2 is a diagram of a chicken yellow follicle provided by the present invention;
FIG. 3 is a diagram illustrating the method of taking the adventitia layer of a small yellow follicle according to the present invention;
FIG. 4 is a diagram illustrating the manner in which the invention provides for cutting the tissue mass of the adventitia layer;
FIG. 5 is a diagram illustrating a manner of digesting adventitia tissue mass according to the present invention;
FIG. 6 illustrates a manner of filtering the cells of the adventitia layer according to the present invention;
FIG. 7 is a microscopic image of cells after being inoculated with a T25 flask and mixed in a primary culture according to the present invention;
FIG. 8 is a cell map under microscope of 48h for primary culture according to the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example a method for separating and culturing cells in the adventitial layer of a chicken yellow follicle
Collagenase type ii, BioFroxx, batch No.: 2275MG 100;
DMEM/F12 medium, WISENT inc., lot No.: 319085029, respectively;
0.25% pancreatin (0.1% EDTA), WISENT inc, lot No.: 325043027, respectively;
cells were purified using Streptomycin (SK), Penicillin-Streptomycin Solution, HyClone INC., batch No.: j190007;
fetal Bovine serum fbs (total Bovine serum): gibco, lot number: 2166446, respectively;
PBS buffer, WISENT inc, lot number: 311010050, respectively;
microscope: olympus CX31, photograph 10 × 10, with 10 x objective and eyepiece.
The follicle of the adventitial layer is a small yellow follicle of 5-10 mm, and is not selected for preovulatory follicles (>10mm), large white follicles (2-4 mm) and small white follicles (<1 mm).
First, preparation before experiment
1. High-pressure sterilization experimental article
Gun heads (1 box 10 muL, 4 box 1mL, 1 box 200 muL, 1 box 5mL), 3 beakers (250mL), 3250 mL glass bottles, 15mL centrifuge tubes (unscrewing covers and 20), 50mL centrifuge tubes (unscrewing covers and 4), 1.5mL centrifuge tubes, 3 small scissors, 6 long tweezers, 6 elbow tweezers, 8 culture dishes, 4 pieces of newspaper and distilled water, and autoclaving for 30min at 121 ℃ and 147 KPa;
2. putting other articles except distilled water in an oven, and drying for 6h at 70 ℃;
3. solution preparation:
preparing a culture medium: 84mL of DMEM/F12 culture solution, 15mL of Fetal Bovine Serum (FBS) and 1mL of streptomycin for cells (SK) are uniformly mixed;
preparation of 1% SK-containing PBS buffer: 99mL of PBS buffer solution and 1mL of SK are mixed uniformly; the PBS buffer mentioned in the present invention is PBS buffer containing 1% SK.
4. The cell chamber was UV-sterilized for 30min in advance.
Second, separating the cells of the adventitial layer
1) Preparing an autoclaved 250mL beaker, adding 200mL of PBS buffer solution preheated at 38 ℃, sealing 4 layers of newspaper and 8 layers of gauze, and taking the sealed newspaper and the sealed gauze to a sterile room for later use;
2) the method comprises the steps of purchasing hens with regular egg laying periods from a vegetable market, cutting the throats, bleeding, dying, carefully cutting off the lower abdomen of the hens by using sterile scissors, pulling the internal organs open to see the ovaries of the hens, taking down the small yellow follicles with the length of 5-10 mm, washing the small yellow follicles once by using PBS (phosphate buffer solution) as shown in figure 2, placing the washed small yellow follicles in a beaker containing the PBS, sealing, and transporting to a cell room;
3) washing the small yellow follicles in the biological safety cabinet again by using PBS buffer solution, putting the small yellow follicles in a beaker containing the PBS buffer solution after washing till no bloody water for standby, and putting the small yellow follicles in a sterile culture dish containing the PBS buffer solution, wherein the process is shown in figure 2; cutting off connective tissue connected with ovary on the small yellow follicle to expose a small opening on the adventitia layer, and squeezing out blood in the blood vessel along blood vessel on the surface of the adventitia layer with elbow forceps, as shown in fig. 3; the adventitia layer is then carefully removed along the small opening, as shown in fig. 4; placing the removed outer membrane layer in another clean sterile culture dish containing PBS buffer solution;
4) clamping a small adventitia layer, slightly washing in PBS buffer solution for 5-6 times, washing residual blood on the membrane as much as possible to separate membrane cells with higher purity, transferring the membrane cells into another culture dish containing 8mL of 1mg/mL type II collagenase after cleaning, and shearing the adventitia layer into 1-1.5 mm by using an ophthalmic scissors in the culture dish3A size tissue mass;
5) cover the dish lid and transfer the dish to 38 ℃ with 5% CO2Digesting in the incubator for 30min, as shown in FIG. 5; lightly beating the tissue blocks for several times by a pipette every 5-7 min so as to facilitate the full digestion of the tissue blocks, and then adding an equal amount of all-contained culture medium into a culture dish to terminate the digestion reaction;
6) filtering the liquid with a cell filter mesh with a pore size of 70 μm, as shown in FIG. 6; removing undigested adventitia tissue mass to prepare a crude adventitia cell suspension, putting 10-11 mL of cell suspension in a 15mL centrifuge tube, centrifuging at 1500r for 5min, and removing supernatant; adding 4mL of PBS buffer solution, blowing, beating and mixing uniformly, centrifuging at 1500r for 5min, and removing supernatant; adding 4mL PBS buffer solution again, blowing, beating and mixing uniformly, centrifuging at 1500r for 5min, and removing supernatant; finally adding 4mL of all-contained culture medium, blowing, beating and uniformly mixing to obtain refined membrane cell suspension;
7) 10. mu.L of the purified membrane cell suspension was diluted 100-fold, and then the cells were counted under a microscope at 5X 107One/bottle was inoculated into a T25 flask, as shown in fig. 7; adding 4mL of all-contained culture medium into a culture bottle, blowing, beating and uniformly mixing, and culturing in an incubator to obtain primary culture cells, wherein the primary cells cultured for 48 hours are shown in figure 8;
8) after culturing for 36-48 h, removing the culture solution, cleaning adherent cells by using 2mL of PBS buffer solution, removing the cleaning solution, adding 4mL of all-contained culture medium, and culturing in an incubator;
thirdly, subculturing membrane cells
Culturing for 36-48 h in an incubator, removing the culture solution after the anchorage rate reaches 70% -80%, adding 3mL of PBS buffer solution to clean anchorage-dependent cells for 1 time, and removing the cleaning solution; adding 2mL of pancreatin, and digesting for 1.5min in an incubator at constant temperature; adding a culture medium which is equal to pancreatin in quantity to terminate the digestion reaction, and blowing and beating the adherent cells to completely fall off; transferring the cell suspension in the culture bottle to a 15mL centrifuge tube, centrifuging at 1500r for 5min, and removing the supernatant; adding 4mL of PBS buffer solution into the cell sediment, blowing, uniformly mixing, centrifuging at 1500r for 5min, and removing supernatant; finally, 4mL of all-contained culture medium is added, the mixture is uniformly blown and beaten, and the cells are counted under a microscope; at 1.2X 104One/well inoculation into 96-well plates or 1X 106Inoculating each cell/well into a 6-well plate, adding a corresponding culture medium, and culturing in an incubator; obtaining subculture cells.
The method for separating and culturing the cells of the adventitia layer of the small yellow follicle of the chicken can separate the cells of the adventitia layer of the small yellow follicle for primary culture and can also perform subculture under the condition of a laboratory. The method can be used for separating the membrane cells with higher purity, almost has no pollution, has high consistency in the growth and development period, high cell survival rate, high success rate of cell proliferation test, high result consistency, strong practicability, strong stability and high feasibility.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for separating and culturing chicken yellow follicle adventitial layer cells is characterized by comprising the following steps:
step one, collecting small yellow follicles
Collecting small yellow follicles on hen ovaries with regular egg laying period in an aseptic laboratory, flushing the surface grease and blood stains of the follicles with PBS buffer solution, cleaning, placing the follicles in a beaker containing the PBS buffer solution, sealing the beaker, and transporting the follicles back to a cell room;
step two, obtaining the adventitial layer of the small yellow follicle
Washing the small yellow follicle by using PBS buffer solution again, taking the small yellow follicle to be placed in a sterile culture dish containing the PBS buffer solution after washing until no blood exists, cutting off connective tissues connected with the ovary on the small yellow follicle to expose a small opening on the outer membrane layer, extruding blood in the blood vessel along the blood vessel on the surface of the outer membrane layer by using an elbow forceps, taking down the outer membrane layer along the small opening, and placing the taken down outer membrane layer in another clean sterile culture dish containing the PBS buffer solution;
step three, cutting the outer film layer
Gently washing the taken outer membrane layer in PBS buffer solution for 5-6 times, washing off residual blood on the membrane, transferring the membrane layer into a culture dish containing type II collagenase after cleaning, and shearing the outer membrane layer into a thickness of 0.5-1.5 mm in the culture dish3A size tissue mass;
step four, digesting the adventitia tissue block
Covering a culture dish cover, transferring the culture dish into an incubator for digestion culture, gently blowing and beating the culture dish for a plurality of times by using a pipettor every 5-7 min, and finally adding an equal amount of all-contained culture medium into the culture dish to terminate the digestion reaction to obtain an outer membrane tissue block suspension;
step five, obtaining the adventitia layer cells
Filtering the adventitia tissue mass suspension by using a cell filter screen, removing the undigested and complete adventitia tissue mass, preparing a crude adventitia cell suspension, putting the cell suspension in a centrifuge tube, centrifuging, and removing supernatant; adding PBS buffer solution, blowing, beating, mixing, centrifuging, and removing supernatant; adding PBS buffer solution again, blowing, beating, mixing uniformly, centrifuging, and removing supernatant; finally adding all culture media, blowing, beating and uniformly mixing to obtain refined membrane cell suspension;
step six, primary culture
Diluting 10 mu L of refined membrane cell suspension by 100 times, counting cells under a microscope, inoculating the cells into a T25 culture bottle, adding all culture media into the culture bottle, blowing and uniformly mixing, and culturing in an incubator to obtain primary culture cells;
step seven, replacing the primary culture solution
After culturing for 36-48 h, removing the culture solution, cleaning adherent cells by using a PBS buffer solution, removing the cleaning solution, adding all culture media, and culturing in an incubator;
step eight, subculturing
Continuously culturing for 36-48 h in the incubator until the anchorage rate reaches 70-80%, removing the culture solution, adding a PBS (phosphate buffer solution) to clean anchorage-dependent cells, and removing the cleaning solution; adding pancreatin, and digesting and culturing in an incubator at constant temperature; adding a culture medium which is equal to pancreatin in quantity to terminate the digestion reaction, and blowing and beating the adherent cells to completely fall off; transferring the cell suspension in the culture bottle to a centrifuge tube, centrifuging, and removing supernatant; adding PBS buffer solution into the cell sediment, blowing, uniformly mixing, centrifuging and removing supernatant; finally adding all culture media, uniformly blowing and mixing, and counting cells under a microscope; then inoculating the mixture into a 6-hole plate or a 96-hole plate, adding a complete culture medium, and continuously culturing in an incubator; obtaining subculture cells.
2. The method for separating and culturing the adventitial layer cells of the small yellow follicles according to claim 1, wherein, in the first step, the small yellow follicles are collected to pre-grade follicles with a diameter of 5 to 10 mm.
3. The method for separating and culturing the cells of the adventitia layer of the small yellow follicle of the chicken as claimed in claim 1, wherein the PBS buffer is a mixture of 99mL of PBS buffer and 1mL of SK.
4. The method for separating and culturing the cells of the adventitial layer of the small yellow follicle of the chicken as claimed in claim 1, wherein the sealing beaker method is sealing by using 4 layers of sterile newspaper and 8 layers of sterile gauze.
5. The method for separating and culturing the adventitia layer cells of chicken small yellow follicles according to claim 1, wherein in the third step, the culture dish for type II collagenase contains 8mL of type II collagenase of 1 mg/mL; the 1mg/mL collagenase II is prepared by dissolving 100mg collagenase II powder in 100mL DMEM/F12 culture medium and mixing uniformly.
6. The method for separating and culturing the cells in the adventitial layer of the small yellow follicle of the chicken as claimed in claim 1, wherein the digestion culture conditions in the fourth step are 38 ℃ and 5% CO2Digesting in the incubator for 30 min.
7. The method for separating and culturing the cells in the adventitial layer of the chicken small yellow follicle according to claim 1, wherein the total culture medium comprises 84mL of DMEM/F12 culture medium, 15mL of fetal bovine serum and 1mL of streptomycin for cells.
8. The method for separating and culturing the cells in the adventitial layer of the chicken yellow follicle according to claim 1, wherein the refined membranous cell suspension is diluted by 100 times by taking 10 μ L of the cell suspension into a 1.5mL centrifuge tube containing 990 μ L of PBS buffer solution and blowing and stirring the cell suspension uniformly.
9. The method for separating and culturing the cells of the adventitial layer of a chicken yellow follicle according to claim 1, wherein the centrifugation condition is 1500r centrifugation for 5 min.
10. The method for separating and culturing the cells in the adventitial layer of the small yellow follicle of the chicken as claimed in claim 1, wherein the number of the cells inoculated in the sixth step is 5X 107One/bottle; in the eighth step, the number of cells to be seeded into the 6-well plate is 1X 106Number of cells seeded in 96-well plate per well was 1.2X 104Per well.
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| CN108588005A (en) * | 2018-04-27 | 2018-09-28 | 四川农业大学 | A kind of isolated culture method of duck folliculus ovarii film layer cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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