CN113502260A - Method for separating and culturing primary myocardial cells of mice suckling mice and application of method - Google Patents
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- 230000002107 myocardial effect Effects 0.000 title claims abstract description 39
- 238000012258 culturing Methods 0.000 title claims abstract description 15
- 241000699670 Mus sp. Species 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims abstract description 12
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 35
- 239000012981 Hank's balanced salt solution Substances 0.000 claims abstract description 26
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 18
- 210000005003 heart tissue Anatomy 0.000 claims abstract description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 12
- 239000006285 cell suspension Substances 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 238000010009 beating Methods 0.000 claims abstract description 9
- 238000004140 cleaning Methods 0.000 claims abstract description 9
- 230000008602 contraction Effects 0.000 claims abstract description 5
- 230000001020 rhythmical effect Effects 0.000 claims abstract description 5
- 210000001519 tissue Anatomy 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 37
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 23
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 14
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 8
- 210000000779 thoracic wall Anatomy 0.000 claims description 8
- 238000012136 culture method Methods 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 241000410367 Clerodendrum thomsoniae Species 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007640 basal medium Substances 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 4
- 230000000747 cardiac effect Effects 0.000 claims description 4
- 230000000249 desinfective effect Effects 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229960005322 streptomycin Drugs 0.000 claims description 4
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 claims description 3
- 229910002091 carbon monoxide Inorganic materials 0.000 claims description 3
- 239000012091 fetal bovine serum Substances 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000009161 Espostoa lanata Nutrition 0.000 description 2
- 240000001624 Espostoa lanata Species 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 230000001237 autorhythmic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008828 contractile function Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002182 neurohumoral effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0657—Cardiomyocytes; Heart cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The invention relates to a method for separating and culturing primary myocardial cells of a mouse suckling mouse and application thereof, which specifically comprises the following steps: anaesthetizing a suckling mouse by using inhaled ether, taking out a beating heart, placing the heart in a precooled D-hank's solution, crushing the heart tissue for 2 times, adding a myocardial cell separating enzyme A solution into a myocardial cell separating enzyme B solution, uniformly mixing, adding the heart tissue, cleaning the heart crushed tissue for 2 times by using the precooled D-hank's solution, adding a culture medium, inoculating the culture medium into a culture dish, transferring a supernatant in the culture dish into a centrifuge tube, uniformly mixing, inoculating the supernatant in the culture dish into a new culture dish, uniformly mixing, inoculating the supernatant in the culture dish into the centrifuge tube, culturing the culture dish into the culture dish, and replacing the culture dish every other day until the cells are contacted to form rhythmical contraction. The experimental operation method for separating the heart of the mouse suckling mouse into the cell suspension containing the single myocardial cell is suitable for separating the myocardial cells of the mouse suckling mice of various strains, and provides basic guarantee for the heart related experiments of the mouse suckling mouse.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for separating and culturing primary myocardial cells of a mouse suckling mouse and application thereof.
Background
Cardiovascular disease is the primary killer of human health. The in vitro cultured myocardial cells eliminate the interference of various in vivo neurohumoral factors, and are ideal for researching the function and mechanism of the myocardial cells. At present, the cardiomyocytes cultured in vitro are mainly divided into commercial cardiomyocyte lines and primary cardiomyocytes isolated and cultured from the heart of suckling mice. Commercial cardiomyocytes retain some of the properties of cardiomyocytes but lose the most important autorhythmic contractile function of cardiomyocytes. The primary myocardial cells separated in vitro well keep the autorhythmicity of the myocardial cells. At present, the separation and culture method of rat primary myocardial cells is relatively mature, and the separation and culture method of mouse primary myocardial cells is obviously insufficient.
Disclosure of Invention
The invention aims to provide a method for separating and culturing primary myocardial cells of mice suckling mice and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: a separation and culture method of primary myocardial cells of mice comprises the culture steps of separating myocardial cells from the heart of a suckling mouse and the separated myocardial cells, and specifically comprises the following steps:
(1) anaesthetizing a suckling mouse by adopting inhalation ether, disinfecting the chest wall of the suckling mouse by using alcohol, cutting the chest wall, and taking out a beating heart;
(2) placing the heart in precooled D-hank's solution, cutting off left and right auricles, flushing the ventricle with precooled D-hank's solution by using an injector until effluent liquid is clear, and placing the ventricle part in new precooled D-hank's solution
(3) Cutting heart into pieces of 1-3mm with ophthalmic scissors3Cleaning the broken heart tissues with precooled D-hank's solution for 2 times, and then discarding the D-hank's solution;
(4) adding 50 μ L of the myocardial cell separating enzyme A solution into 2.5 μ L of the myocardial cell separating enzyme B solution, mixing, adding into the sheared heart tissue, and mixingCO2Culturing at 37 deg.C for 30min in incubator, removing supernatant, and cleaning cardiac crushed tissue with pre-cooled D-hank's solution for 2 times;
(5) adding complete DMEM culture medium, blowing, beating and mixing uniformly the heart tissue to obtain single cell suspension, inoculating said single cell suspension into culture dish, placing it in CO2Attaching the wall in an incubator at a differential speed for 30 min;
(6) transferring the supernatant in the culture dish into a centrifuge tube, uniformly mixing, inoculating the supernatant into a new DMEM culture dish, placing the new DMEM culture dish in a CO (carbon monoxide) culture dish2Attaching the wall in an incubator at a differential speed for 30 min;
(7) transferring the supernatant in the culture dish to a centrifuge tube, mixing uniformly, and adding the supernatant into the centrifuge tube at a speed of 2.5X 105/cm2Inoculating into DMEM culture dish for culturing, changing culture medium for 24h, and changing every other day until cell contact forms rhythmic contraction.
Further, in the step (5), the DMEM medium is: DMEM basal medium, 10% fetal bovine serum, 1% streptomycin.
D-hank's are commercially available products, and the specific manufacturer is Wuhan Punuo race Life technologies, Inc., PB 180322; the myocardial cell separating enzyme A and the myocardial cell separating enzyme B are commercially available products, and the specific manufacturer is Saimer Feishell science and technology (China) Co., Ltd., product number: 88281Y.
The beneficial technical effects of the invention are as follows: the experimental operation method for separating the heart of the mouse suckling mouse into the cell suspension containing the single myocardial cell is suitable for separating the myocardial cells of the mouse suckling mice of various strains, and provides basic guarantee for the heart related experiments of the mouse suckling mouse.
Drawings
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, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a morphological view of a mouse primary cardiomyocyte of the present invention 5 days after culture;
FIG. 2 is an illustration of the identification of mouse primary cardiomyocytes according to the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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 1
A separation and culture method of primary myocardial cells of mice comprises the culture steps of separating myocardial cells from the heart of a suckling mouse and the separated myocardial cells, and specifically comprises the following steps:
(1) dipping a small amount of diethyl ether by using a cotton ball, putting the suckling mouse into a small beaker, anesthetizing the suckling mouse after 5 minutes, disinfecting the chest wall of the suckling mouse by using alcohol, cutting the chest wall, and taking out a beating heart;
(2) placing the heart in a precooled D-hank's solution, cutting off left and right auricles, flushing the ventricle with the precooled D-hank's solution by using an injector until effluent liquid is clear, and placing the ventricle part in a new precooled D-hank's solution;
(3) cutting heart into pieces of 1mm with ophthalmic scissors3Cleaning the broken heart tissues with precooled D-hank's solution for 2 times, and then discarding the D-hank's solution;
(4) adding 50 μ L of the myocardial cell separating enzyme A solution into 2.5 μ L of the myocardial cell separating enzyme B solution, mixing, adding into sheared heart tissue, and adding into CO2Culturing at 37 deg.C for 30min in incubator, removing supernatant, and cleaning cardiac crushed tissue with pre-cooled D-hank's solution for 2 times;
(5) adding complete DMEM culture medium, blowing, beating and mixing uniformly the heart tissue to obtain single cell suspension, inoculating said single cell suspension into culture dish, placing it in CO2Attaching the wall in an incubator at a differential speed for 30 min; the DMEM medium is: DMEM basal medium, 10% fetal calf serum and 1% streptomycin;
(6) will be at the topTransferring the supernatant in the culture dish into a centrifuge tube, uniformly mixing, inoculating into a new DMEM culture dish, and placing in CO2Attaching the wall in an incubator at a differential speed for 30 min;
(7) transferring the supernatant in the culture dish to a centrifuge tube, mixing uniformly, and adding the supernatant into the centrifuge tube at a speed of 2.5X 105/cm2Inoculating into DMEM culture dish for culturing, changing culture medium for 24h, and changing every other day until cell contact forms rhythmic contraction.
Example 2
A separation and culture method of primary myocardial cells of mice comprises the culture steps of separating myocardial cells from the heart of a suckling mouse and the separated myocardial cells, and specifically comprises the following steps:
(1) dipping a small amount of diethyl ether by using a cotton ball, putting the suckling mouse into a small beaker, anesthetizing the suckling mouse after 3 minutes, disinfecting the chest wall of the suckling mouse by using alcohol, cutting the chest wall, and taking out a beating heart;
(2) placing the heart in a precooled D-hank's solution, cutting off left and right auricles, flushing the ventricle with the precooled D-hank's solution by using an injector until effluent liquid is clear, and placing the ventricle part in a new precooled D-hank's solution;
(3) cutting heart into pieces of 2mm with ophthalmic scissors3Cleaning the broken heart tissues with precooled D-hank's solution for 2 times, and then discarding the D-hank's solution;
(4) adding 50 μ L of the myocardial cell separating enzyme A solution into 2.5 μ L of the myocardial cell separating enzyme B solution, mixing, adding into sheared heart tissue, and adding into CO2Culturing at 37 deg.C for 30min in incubator, removing supernatant, and cleaning cardiac crushed tissue with pre-cooled D-hank's solution for 2 times;
(5) adding complete DMEM culture medium, blowing, beating and mixing uniformly the heart tissue to obtain single cell suspension, inoculating said single cell suspension into culture dish, placing it in CO2Attaching the wall in an incubator at a differential speed for 30 min; the DMEM medium is: DMEM basal medium, 10% fetal calf serum and 1% streptomycin;
(6) transferring the supernatant in the culture dish into a centrifuge tube, uniformly mixing, inoculating the supernatant into a new DMEM culture dish, placing the new DMEM culture dish in a CO (carbon monoxide) culture dish2Attaching the wall in an incubator at a differential speed for 30 min;
(7) placing the above culture dishTransferring the supernatant into a centrifuge tube, mixing uniformly at 2.5X 105/cm2Inoculating into DMEM culture dish for culturing, changing culture medium for 24h, and changing every other day until cell contact forms rhythmic contraction.
The identification view of the isolated and cultured mouse primary cardiomyocytes is shown in fig. 2, and the morphological view of the isolated and cultured mouse primary cardiomyocytes 5 days after the isolation of the cardiomyocytes by the method of the present embodiment is shown in fig. 1, since a few cell suspensions are absorbed and dripped on a glass slide to be observed under an optical microscope, a large number of spindle-shaped cardiomyocytes can be seen.
Claims (4)
1. A separation and culture method of primary myocardial cells of mice is characterized by comprising the steps of separating myocardial cells from the hearts of suckling mice and culturing the separated myocardial cells, and specifically comprises the following steps:
(1) anaesthetizing a suckling mouse by adopting inhalation ether, disinfecting the chest wall of the suckling mouse by using alcohol, cutting the chest wall, and taking out a beating heart;
(2) placing the heart in a precooled D-hank's solution, cutting off left and right auricles, flushing the ventricle with the precooled D-hank's solution by using an injector until effluent liquid is clear, and placing the ventricle part in a new precooled D-hank's solution;
(3) cutting heart into pieces of 1-3mm with ophthalmic scissors3Cleaning the broken heart tissues with precooled D-hank's solution for 2 times, and then discarding the D-hank's solution;
(4) adding 50 μ L of the myocardial cell separating enzyme A solution into 2.5 μ L of the myocardial cell separating enzyme B solution, mixing, adding into sheared heart tissue, and adding into CO2Culturing at 37 deg.C for 30min in incubator, removing supernatant, and cleaning cardiac crushed tissue with pre-cooled D-hank's solution for 2 times;
(5) adding complete DMEM culture medium, blowing, beating and mixing uniformly the heart tissue to obtain single cell suspension, inoculating said single cell suspension into culture dish, placing it in CO2Attaching the wall in an incubator at a differential speed for 30 min;
(6) transferring the supernatant in the culture dish into a centrifuge tube, uniformly mixing, inoculating the supernatant into a new DMEM culture dish, placing the new DMEM culture dish in a CO (carbon monoxide) culture dish2Attaching the wall in an incubator at a differential speed for 30 min;
(7) transferring the supernatant in the culture dish toMixing in centrifuge tube at 2.5 × 105/cm2Inoculating into DMEM culture dish for culturing, changing culture medium for 24h, and changing every other day until cell contact forms rhythmic contraction.
2. The method according to claim 1, wherein in step (5), the DMEM medium is: DMEM basal medium, 10% fetal bovine serum, 1% streptomycin.
3. Mouse primary cardiomyocytes obtained according to the method of claim 1 or 2.
4. Use of mouse primary cardiomyocytes according to claim 3.
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| WO2008054819A2 (en) * | 2006-11-02 | 2008-05-08 | The General Hospital Corporation | Cardiovascular stem cells, methods for stem cell isolation, and uses thereof |
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| US20190183971A1 (en) * | 2016-08-11 | 2019-06-20 | Temple University - Of The Commonwealth System Of Higher Education | Ischemia/reperfusion injury |
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- 2021-07-16 CN CN202110806884.XA patent/CN113502260A/en active Pending
Patent Citations (6)
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| CN105505863A (en) * | 2016-01-05 | 2016-04-20 | 中国人民解放军第二军医大学 | Culture method for heterocephalus glaber cardiac muscle cell |
| US20190183971A1 (en) * | 2016-08-11 | 2019-06-20 | Temple University - Of The Commonwealth System Of Higher Education | Ischemia/reperfusion injury |
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