CN116064381A - Method for culturing primary muscle cells of coilia ectenes - Google Patents
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Abstract
The invention provides a method for culturing primary muscle cells of coilia ectenes, and belongs to the technical field of cell culture. The coilia ectenes muscle tissue is sheared and placed in a culture flask treated by rat tail collagen I, and is subjected to collagenase and trypsin digestion treatment and then is cultured in a primary complete culture medium, so that the obtained cells are the primary coilia ectenes muscle cells. The method can obtain high-quality coilia ectenes muscle cells from coilia ectenes muscle tissues and carry out passage, can be applied to molecular mechanism research related to the coilia ectenes muscle cells, and provides theoretical and technical support for the coilia ectenes muscle cells.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a method for culturing primary muscle cells of coilia ectenes.
Background
Muscle growth is primarily dependent on the growth and proliferation of muscle cells. However, research on muscle growth and quality is now in an early stage. There is currently no systematic study of the mechanism of muscle growth regulation.
Coilia ectenes (coilia ectenes)Coilia nasus) Belonging to the order Clupeiformes (Clupeiformes), engraulidae (Engraulidae), and Engraulidae (Coilia) are widely distributed in the sea area around east Asia. Because it has delicious and nutritious tasteHigh value and long-term importance of economic fish. Because of the problems of strong stress response, water output, death and the like of the coilia ectenes, the development of breeding and biological experiments is difficult, and the promotion of scientific research of the coilia ectenes is severely restricted, and the coilia ectenes becomes a bottleneck for expanding breeding. Therefore, it is necessary to establish an in vitro cell culture technology, which lays a technological foundation for advancing the scientific research of coilia ectenes.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for culturing primary muscle cells of coilia ectenes, so as to meet the requirement of researching materials for the muscle growth regulation mechanism of coilia ectenes.
The invention provides a method for culturing primary muscle cells of coilia ectenes, which comprises the following steps:
sterile muscle tissue is obtained from coilia ectenes, sheared into fragments, digested by collagenase and trypsin to obtain muscle granules;
and (3) placing the muscle pellets in a rat tail collagen I-type treated culture flask for culturing until the cells migrate out and grow into a monolayer of cells, so as to obtain primary muscle cells.
Preferably, the mass percentage of the collagenase is 0.1% -0.17% when the collagenase and trypsin are digested;
the mass percentage of the trypsin is 0.1-0.125%.
Preferably, the collagenase is 0.1% by mass;
the mass percentage of the trypsin is 0.125%.
Preferably, the volume ratio of the collagenase solution to the trypsin solution is 1:1;
the collagenase solution has a mass percentage of 0.20%;
the trypsin solution has a mass percentage of 0.25%.
Preferably, the concentration of the rat tail collagen I in the culture flask is 0.025-0.05 mg/ml.
Preferably, the treatment method comprises the steps of coating the tail collagen I type solution on the bottom of a culture flask, removing liquid overnight, and then cleaning to obtain the tail collagen I type treated culture flask.
Preferably, the coating amount of the rat tail collagen I is 3-6 mug/cm 2 。
Preferably, the culture medium is a primary complete medium, which is an L15 medium containing fetal bovine serum with a volume concentration of 20%, 100ng/ml fibroblast growth factor, 100IU/ml penicillin and 100 μg/ml streptomycin.
Preferably, the primary complete medium is changed every 3 days.
Preferably, the temperature of the culture is 27-29 ℃.
The invention provides a method for culturing primary muscle cells of coilia ectenes, which comprises the following steps: sterile muscle tissue is obtained from coilia ectenes, sheared into fragments, digested by collagenase and trypsin to obtain muscle granules; the muscle pellets were plated in rat tail collagen type i treated flasks until the cells migrate and develop into a monolayer of primary muscle cells. According to the invention, rat tail collagen is used for coating the bottom of a culture flask so as to improve the adhesion and migration efficiency of muscle cells, and meanwhile, a method of enzymolysis by collagenase and trypsin is used for preparing the primary muscle cells of coilia ectenes with higher quantity. Therefore, the method provided by the invention can be used for effectively preparing the coilia ectenes primary myocytes with large quantity and mobility, fills the blank of the coilia ectenes primary myocyte separation culture technology, and provides an in vitro model for research on coilia ectenes muscle growth regulation.
Drawings
FIG. 1 shows the number of migratory muscle cells in different volume ratios of 0.2% collagenase solution to 0.25% trypsin solution;
FIG. 2 shows the number of myocytes migrating at different culture times;
FIG. 3 shows muscle cell viability at various culture times;
FIG. 4 shows primary muscle cells of coilia ectenes cultured according to the present invention.
Detailed Description
The invention provides a method for culturing primary muscle cells of coilia ectenes, which comprises the following steps:
sterile muscle tissue is obtained from coilia ectenes, sheared into fragments, digested by collagenase and trypsin to obtain muscle granules;
and (3) placing the muscle pellets in a rat tail collagen I-type treated culture flask for culturing until the cells migrate out and grow into a monolayer of cells, so as to obtain primary muscle cells.
The invention obtains sterile muscle tissue from coilia ectenes, cuts the tissue into fragments, and digests the fragments by collagenase and trypsin to obtain muscle granules.
In the present invention, the coilia ectenes are preferably subjected to aseptic treatment. The coilia ectenes are preferably 20-50g healthy coilia ectenes. The method for aseptic treatment preferably comprises soaking coilia ectenes in 75% alcohol for 1min, washing fish body with sterile DPBS buffer solution for 2 times, and collecting blood from tail vein to bluish white. The method for acquiring the sterile muscle tissue preferably uses sterilized ophthalmic scissors to cut 2-3 cm from the back of the coilia ectenes to the lateral line direction, cuts one part of the dorsal fin at the front and back of the dorsal fin, and cuts the two parts transversely; the skin here was carefully torn open with sterilized forceps; the dorsal white muscle is cut down by another pair of sterilized ophthalmic scissors against the bone. The dorsal white muscle is preferably washed 3 times with highly resistant DPBS and then placed in a petri dish containing highly resistant basal medium. The high DPBS resistance is preferably a DPBS buffer containing 200IU/ml penicillin and 200. Mu.g/ml streptomycin. The high-resistance basal medium is preferably an L15 medium containing 200IU/ml penicillin+200 ug/ml streptomycin. The volume of the fragments is preferably 0.8-1.2 mm 3 . Prior to digestion, the fragments are preferably washed 2 times with high-impedance basal medium, centrifuged at 1000rmp for 5min, and the supernatant discarded.
In the present invention, the collagenase is preferably 0.1 to 0.17% by mass, more preferably 0.1% by mass, of the collagenase when digested with trypsin. The content of trypsin is preferably 0.1-0.125% by mass, more preferably 0.125% by mass. In the embodiment of the invention, the volume ratio of the collagenase solution to the trypsin solution is preferably 1:1 during digestion; the collagenase solution has a mass percentage of 0.20%; the trypsin solution has a mass percentage of 0.25%. According to the invention, by optimizing the volume ratio of the collagenase solution to the trypsin solution, the volume ratio of the collagenase solution to the trypsin solution is preferably in the range of 1-1.4:1, and higher number of primary muscle cells can be obviously obtained compared with the volume ratio of 0.4-0.8:1. The sources of collagenase and trypsin are not particularly limited in the present invention, and the methods for preparing collagenase and trypsin known in the art may be employed. In the examples of the present invention, collagenase was purchased from Shanghai Seiyaka Biotechnology Co., ltd, and trypsin was purchased from Gibco Co. The temperature of the digestion is preferably 27 to 29 ℃, more preferably 28 ℃. The digestion time is preferably 8 to 12min, more preferably 10min. The volume of the mixture of collagenase and trypsin at the time of digestion is 5 times that of the fragments to be digested.
In the present invention, the enzyme is preferably inactivated after digestion. The enzyme deactivation method comprises the steps of adding an equal volume of complete culture medium into digestive juice to stop digestion reaction, centrifuging, discarding supernatant, and washing 3 times with the high-resistance basic culture medium to obtain digested myogranules. The complete medium is L15 medium containing 10% fetal bovine serum, 100IU/ml penicillin and 100 μg/ml streptomycin.
After obtaining the muscle small granules, the invention cultures the muscle small granules in a culture flask treated by rat tail collagen I until the cells migrate out and grow into monolayer cells, thus obtaining primary muscle cells.
In the present invention, the concentration of the rat tail collagen type I treated culture flask is preferably 0.025 to 0.05mg/ml, more preferably 0.04mg/ml. The rat tail collagen type I is preferably diluted with sterile 0.006mol/L acetic acid. The flask is preferably a T25 flask. The treatment method is preferably to coat the rat tail collagen type I solution on the bottom of a culture flask overnight, remove liquid and then clean the flask to obtain the rat tail collagen type I treated culture flask. The coating amount of the rat tail collagen type I is preferably 3 mug/cm 2 -6μg/cm 2 All together, most preferably 4. Mu.g/cm 2 . In the embodiment of the invention, the adding volume of the rat tail collagen I type solution is preferably 4mL/T25 culture flask. The cleaning solution is sterile DPBS. The number of times of the washing is preferably 3.
In the present invention, before placing the muscle pellets in the flask, it is preferable to wet the flask coated with rat tail collagen type I with 1ml of the primary complete medium, and then uniformly spread the treated muscle pellets on the bottom of the flask at a distance of 3-5mm.
In the present invention, the temperature of the culture is preferably 27 to 29℃and more preferably 28 ℃. After 1h of culture, the flask was allowed to fall overnight, and complete medium was added until the myopellets were gone, and forward culture was continued. The primary complete medium is preferably changed every 3 days. The culture medium is preferably a primary complete medium, which is preferably an L15 medium containing fetal bovine serum at a volume concentration of 20%, 100ng/ml fibroblast growth factor, 100IU/ml penicillin and 100. Mu.g/ml streptomycin.
The method for culturing primary muscle cells of coilia ectenes according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
The brands and formulas of the reagents required for the embodiments are as follows:
DPBS, penicillin streptomycin mixed solution, rat tail collagen type I and L15 culture medium are purchased from Beijing Soy Bao technology Co., ltd;
absolute ethanol was purchased from Shanghai Taitan technologies Co., ltd;
fetal bovine serum was purchased from Shanghai Xiao Peng Biotechnology Co., ltd;
collagenase was purchased from Shanghai Seiyaka Biotechnology Co., ltd;
trypsin, fibroblast growth factor were purchased from Gibco;
the CCK-8 cell viability detection kit is purchased from Nanjing's institute of biological engineering.
Example 1
Method for culturing primary muscle cells of coilia ectenes
1. The preparation stage:
(1) Coating of the culture flask: diluting 5mg/ml of rat tail collagen type I with sterile 0.006mol/L acetic acid aqueous solution to 0.025mg/ml on an ultra-clean bench, sucking 4ml of rat tail collagen type I diluent, adding into a T25 culture flask, gently shaking the culture flask to uniformly spread the rat tail collagen type I on the bottom of the culture flask, and standing overnight in the ultra-clean bench; excess liquid was carefully poured off and washed 3 times with sterile DPBS for use.
(2) Preparation of collagenase: adding 1mlL culture medium into a reagent bottle containing collagenase, and lightly inverting the reagent bottle for several times to fully dissolve the collagenase; centrifuging at 3000rpm for 10s, sucking out the mixed solution, and placing the mixed solution into a 50ml centrifuge tube; repeatedly cleaning the reagent bottle for several times, and finally preparing collagenase into 0.2% liquid by using an L15 culture medium, and placing the liquid in a refrigerator for standby.
(3) Preparation of the complete medium: the complete medium is L15 medium containing 10% fetal bovine serum, 100IU/ml penicillin and 100 mug/ml streptomycin; preparation of primary complete medium: l15 medium+20% foetal calf serum+100 ng/ml fibroblast growth factor+100 IU/ml penicillin+100 ug/ml streptomycin.
2. Separation of muscle tissue:
(1) Selecting 20-50g healthy coilia ectenes, soaking in 75% alcohol for 1min, washing fish body with sterile DPBS buffer solution for 2 times, and collecting blood from tail vein until the gill is slightly white.
(2) 2-3 cm of the sterilized ophthalmic scissors are cut from the back of the coilia ectenes to the lateral line direction, one part of the dorsal fin is cut at the front and the back of the dorsal fin, and the two parts are transversely cut; the skin here was carefully torn open with sterilized forceps; the dorsal white muscle was cut off with another sterilized ophthalmic scissors against the bone, washed 3 times with DPBS containing high antibody (200 IU/ml penicillin and 200. Mu.g/ml streptomycin), and subsequently placed in L15 medium containing high antibody.
(3) The obtained muscle tissue is cut into 1mm with sterilized ophthalmic scissors 3 The left and right myopellets were transferred to a 50ml centrifuge tube, washed 2 times with high-resistance basal medium, centrifuged at 1000rmp for 5min, and the supernatant discarded.
(4) The 0.2% collagenase solution and the 0.25% trypsin solution are uniformly mixed into digestive juice according to the volume ratio of 0.4:1, 0.6:1, 0.8:1, 1:1, 1.2:1 and 1.4:1 respectively. Adding 5 times volume of digestive juice into the muscle granules, digesting for 10min at 28 ℃, stopping the reaction with an equal volume of complete culture medium, centrifuging for 5min at 1000rmp, and discarding the supernatant; the cells were washed 3 times with L15 medium containing high antibody.
3. Isolation and culture of muscle cells:
(1) The rat tail collagen type I coated flask was wetted with 1ml of primary complete medium.
(2) And uniformly spreading the cleaned myopellets at the bottom of the culture flask, and uniformly spreading the myopellets at intervals of 3-5mm.
(3) Placing the culture flask with the muscle granules at 28deg.C without CO 2 Culturing for 1h in an incubator; the flask was then placed upside down overnight, primary complete medium was added until the myopellets were gone, and the flask was kept in the incubator, after which the medium was replaced every 3 days.
(4) After 5 days, muscle cells are observed to migrate under a microscope, when the cells grow and fuse into a monolayer cell, 1.5ml of trypsin containing EDTA is added, and when the cells shrink and become round, the same amount of complete culture medium is added; tapping the bottom of the culture bottle to make the cells fall off; the cell mixture was transferred to a 15ml centrifuge tube and centrifuged at 100rpm for 5min, and the supernatant was discarded. The cells were resuspended in complete medium and passaged 1:2, and continued to be placed in a 28℃incubator for subculture.
Cultured muscle cells were analyzed on days 0, 3, 5, 7, 10, and 14, respectively, and the cells were counted by a cytometer.
The different volume ratios of the two enzyme solutions to the amount of myocyte migration are shown in figure 1. As can be seen from the results of FIG. 1, the volume ratio of the 0.2% collagenase solution to the 0.25% trypsin solution is in the range of 1-1.4:1, and the number of the migrating cells is significantly higher than the volume ratio of 0.4-0.8:1. Considering the cost factor of the enzyme, 1:1 is selected as the volume ratio of the optimal digestive juice.
In addition, the CCK-8 method was used to determine the viability of the cultured primary muscle cells. The number of muscle cells during the different culture times is shown in FIG. 2. Cell viability is shown in FIG. 3. Cell number and cell viability results and cost factors, the optimal time to culture the myocytes was 7 days. The morphology of the primary cells of the mobilized coilia ectenes is shown in FIG. 4. As can be seen from fig. 4, the primary cells of coilia ectenes have typical migration characteristics and good growth conditions.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for culturing primary muscle cells of coilia ectenes, which is characterized by comprising the following steps:
sterile muscle tissue is obtained from coilia ectenes, sheared into fragments, digested by collagenase and trypsin to obtain muscle granules;
and (3) placing the muscle pellets in a rat tail collagen I-type treated culture flask for culturing until the cells migrate out and grow into a monolayer of cells, so as to obtain primary muscle cells.
2. The method for culturing coilia ectenes primary myocytes according to claim 1, characterized in that the collagenase is present in an amount of 0.1% to 0.17% by mass when digested with trypsin;
the mass percentage of the trypsin is 0.1-0.125%.
3. The method for culturing coilia ectenes primary myocytes according to claim 1, characterized in that the collagenase is present in an amount of 0.1% by mass;
the mass percentage of the trypsin is 0.125%.
4. The method of claim 1, wherein the volume ratio of collagenase solution to trypsin solution is 1:1;
the collagenase solution has a mass percentage of 0.20%;
the trypsin solution has a mass percentage of 0.25%.
5. The method for culturing primary muscle cells of coilia ectenes according to claim 1, wherein the concentration of rat tail collagen type i in the culture flask is 0.025-0.05 mg/ml.
6. The method for culturing coilia ectenes primary muscle cells according to claim 1 or 5, wherein the treatment method comprises the steps of coating a rat tail collagen type i solution on the bottom of a culture flask, removing liquid overnight, and then washing to obtain the rat tail collagen type i treated culture flask.
7. The method for culturing primary myoblasts of coilia ectenes according to claim 6, wherein the coating amount of the rat tail collagen type i is 3-6 μg/cm 2 。
8. The method of claim 1, wherein the culture medium is a primary complete medium comprising 20% fetal bovine serum, 100ng/ml fibroblast growth factor, 100IU/ml penicillin and 100 μg/ml streptomycin in volume concentration in L15 medium.
9. The method of claim 8, wherein the primary complete medium is changed every 3 days.
10. The method of claim 1 or 8, wherein the temperature of the culture is 27-29 ℃.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002335955A (en) * | 2001-05-23 | 2002-11-26 | Kenji Sofue | Method for vascular smooth muscle cell culture |
| CN104830760A (en) * | 2015-06-01 | 2015-08-12 | 中国海洋大学 | Turbot muscular cell line establishment method |
| CN113388576A (en) * | 2021-07-12 | 2021-09-14 | 新乡医学院 | Rat vascular smooth muscle cell culture medium and application thereof |
| WO2022140847A1 (en) * | 2020-12-30 | 2022-07-07 | Cell Ag Tech Inc | Methods and processes for culturing cells |
| CN114854676A (en) * | 2022-06-01 | 2022-08-05 | 华中农业大学 | Construction method and application of grass carp skeletal muscle myoblast cell line |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002335955A (en) * | 2001-05-23 | 2002-11-26 | Kenji Sofue | Method for vascular smooth muscle cell culture |
| CN104830760A (en) * | 2015-06-01 | 2015-08-12 | 中国海洋大学 | Turbot muscular cell line establishment method |
| WO2022140847A1 (en) * | 2020-12-30 | 2022-07-07 | Cell Ag Tech Inc | Methods and processes for culturing cells |
| CN113388576A (en) * | 2021-07-12 | 2021-09-14 | 新乡医学院 | Rat vascular smooth muscle cell culture medium and application thereof |
| CN114854676A (en) * | 2022-06-01 | 2022-08-05 | 华中农业大学 | Construction method and application of grass carp skeletal muscle myoblast cell line |
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