CN108728405A - A kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes - Google Patents
A kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes Download PDFInfo
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- CN108728405A CN108728405A CN201710264517.5A CN201710264517A CN108728405A CN 108728405 A CN108728405 A CN 108728405A CN 201710264517 A CN201710264517 A CN 201710264517A CN 108728405 A CN108728405 A CN 108728405A
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- ventricular cardiomyocytes
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The present invention provides a kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes, including step：（a）Perfusion system is preheated, and fully cleans perfusion pipeline；（b）Rat abdominal cavity priority injecting heparin sodium and yellow Jackets；（c）Rapid opening rat thoracic cavity exposes heart, and taking out lower heart from aortic root is placed in primer solution, and after appropriate removing surrounding tissue, aortic knob is tied on catheter needle；（d）Heart after intubation is connected into perfusion system, no Ca2+Primer solution perfused hearts；（e）Heart continues that mixture slaking enzyme solution is perfused；（f）It removes the heart after perfusion to be placed in transfer liquid, removes atrium and big blood vessel removal atrium and big blood vessel, ventricle is cut into small pieces, suction pipe piping and druming makes to be organized into monodisperse cell, and screen filtration cell suspension is stood；（g）Gradient answers Ca2+；（h）Multiple Ca is resuspended in complete medium2+Cell precipitation afterwards is inoculated in the Tissue Culture Flask after coating and cultivates.Isolation Methods of Adult Rat Ventricular Cardiomyocytes provided by the invention can get the Adult Rat Ventricular Cardiomyocytes of high quality, high yield, time-to-live length.
Description
Technical field
The invention belongs to cell biologies, and in particular to a kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes.
Background technology
Cardiac muscle cell (Cardiomyocytes) is cannot to be proliferated, and it is cardiac electrophysiology to obtain normal Cardiomyocytes
The basis of research.And the cell of most of researchs both is from the newborn rat heart of culture, the cardiac muscle cell of newborn rat and adult
Rat cardiomyocyte in form, structure, function there are prodigious difference, as the biological experiment tool of cardiovascular research, at
The functional characteristic of year cardiac muscle cell is that other stages of development cardiac muscle cell is irreplaceable, the in vitro culture mould of adult cardiomyocytes
Type is necessary, but the culture for rat cardiomyocyte of growing up is relatively difficult.Point provided by the present invention for obtaining Adult Rat Ventricular Cardiomyocytes
From the Adult Rat Ventricular Cardiomyocytes that method can obtain high quality, high yield, time-to-live length.
Invention content
The purpose of the present invention is establish a kind of Adult Rat Ventricular Cardiomyocytes obtaining high quality, high yield, time-to-live length
The method of separation.
Above-mentioned involved in order to solve the problems, such as, the present invention takes following method to obtain Adult Rat Ventricular Cardiomyocytes：
The step of Isolation Methods of Adult Rat Ventricular Cardiomyocytes includes：(a) perfusion system is preheated, and fully cleans perfusion pipeline；(b)
Rat abdominal cavity priority injecting heparin sodium and yellow Jackets；(c) rat thoracic cavity is opened rapidly and expose heart, taken from aortic root
Go out lower heart to be placed in primer solution, after appropriate removing surrounding tissue, aortic knob is tied on catheter needle；(d) after being intubated
Heart connect perfusion system, no Ca2+Primer solution perfused hearts；(e) heart continues that mixture slaking enzyme solution is perfused；(f) it takes
Heart after lower perfusion is placed in transfer liquid, removes atrium and big blood vessel removal atrium and big blood vessel, ventricle is cut into small pieces, is inhaled
Pipe piping and druming makes to be organized into monodisperse cell, and screen filtration cell suspension is stood；(g) gradient answers Ca2+；(h) complete medium is resuspended
Multiple Ca2+Cell precipitation afterwards is inoculated in the Tissue Culture Flask after coating and cultivates.
Optional rat is weight 150-300g, the SD rats of raising 6-8 weeks.
Contain 137 mM NaCl after optionally primer solution is pre-chilled for 4 DEG C2、4 mM KCl、1 mM MgCl、10 mM
HEPES、0.33 mM NaH2PO4, 10 mM D-Glucoses, 5 mM taurines and 10 mM BDM buffer solutions.
Optionally without Ca2+The specific methods of primer solution perfused hearts be perfusion rate be 3-5 mL/min, when perfusion
Between be 2-3 min.
Clostridiopetidase A II, 0.05%-0.1% clostridiopetidase A IV and 0.05%- that optional mixture slaking enzyme solution is 0.05%-0.1%
0.1% protease mixed enzyme.
The perfusion rate of optional perfusion mixture slaking enzyme solution is 5-10mL/min, Hemoperfusion time 25-40min.
Optional transfer liquid is the primer solution containing 0.05-0.1%BSA.
The sieve of optional filtration cell suspension is the sieve in 250 μm of aperture.
Optional gradient answers Ca2+Ca2+Final concentration is followed successively by 0.06mM, 0.24mM, 0.6mM and 1.0mM；
Optional gradient answers Ca2+Method be with contain different Ca2+Cell precipitation is resuspended in the solution of concentration, and 10min is settled in 37 DEG C
Afterwards, it inhales and abandons supernatant.
Optional complete medium is that carnitine containing 5mM, 5mM creatines, 5mM taurines, the 5%FBS and 1% of preheating are dual anti-
M199 complete mediums.
Optional coated cell culture bottle is the coated Tissue Culture Flask of 0.01-0.015mg/mL Laminin lens.
The present invention solves the problems, such as that Adult Rat Ventricular Cardiomyocytes separation process survival rate is low and yield is few, make acquisition at
Year rat myocardial cell yield is high, activity is high and the time-to-live is long.
Description of the drawings
Fig. 1 answers Ca2+Preceding cell picture 100 ×
Fig. 2 cultures cell picture 100 for 24 hours ×
The cell picture 100 of Fig. 3 cultures 48h ×
The cell picture 100 of Fig. 4 cultures 72h ×
Specific implementation mode
In order to make the purpose of the present invention and advantage it is more pure and fresh show, now specific implementation mode is expanded on further.Herein
The specific implementation mode illustrated is explained only for the present invention, is not intended to limit the present invention.
This experiment laboratory apparatus used is as follows：
Inverted microscope（XDS-1A, Shanghai）, superclean bench（Model：SW-CJ-1FD, Suzhou are ultra-clean）；Cell incubator（Type
Number：240i Thermo）, thermostatic water-circulator bath pot（Model：SeaBird grand science and education equipment Co., Ltd in HH-501 types）, constant flow pump
（Longer Pump, BT 100-1L）, langendorff perfusion systems（The many real enlightening wound development in science and technology Limited Liabilities in Beijing are public
Department）.
It is the SD rats of 150-300 g that the present invention, which selects raising 6-8 weeks, weight, detaches Adult Rat Ventricular Cardiomyocytes.Specifically
Operation is as follows：
1,42 DEG C of preheating perfusion systems are first perfused cleaning 10-15 min with 75% second, then clean 10-15 with aseptic deionized water
Min fully cleans perfusion pipeline；
2, the heparin of 0.2 mL is injected intraperitoneally in experimental rat 20-30 min before anesthesia（1000 U/mL）, then it is injected intraperitoneally 1%
Amobarbital is with anesthetized rat；
3, after anesthesia fully, rapid rat thoracic cavity of opening exposes heart, and the filling that lower heart is placed in 4 DEG C is taken out from aortic root
It notes solution and aorta is isolated, with ready No. 12 perfusion needles for being connected in 10 ml syringes after suitably removing surrounding tissue
It is gently inserted into aorta, injects the primer solution of about 10 mL, so that part blood stream goes out in heart, then will be filled with surgical thread
Stream needle is fixed on aorta；
4, No. 12 perfusion needles of fixed heart are connected on Langendorff pipes, with the primer solution perfused hearts of no Ca2+,
Perfusion rate is 3-5 mL/min, and Hemoperfusion time is 2-3 min；
5, no Ca is drained2+Primer solution, with mixed enzyme solution perfused hearts to heart soften, color is thin out, and mixed enzyme solution is
Clostridiopetidase A II, 0.05%-0.1% clostridiopetidase A IV and 0.05%-0.1% protease of 0.05%-0.1%, perfusion rate are 5-10 mL/
Min, Hemoperfusion time are 25-40 min；
6, in the heart to transfer liquid after transfer perfusion, atrium and big blood vessel removal atrium and big blood vessel, the heart that will be obtained are removed
Room is cut into small pieces, and suction pipe piping and druming makes to be organized into monodisperse cell；
7, monodispersed cell suspension is after 250 μm of screen filtrations, microscopy, in being stored at room temperature 10 min；
8, it inhales and abandons supernatant, be added and contain 0.06 mM, 0.24 mM, 0.6 mM and 1.0 mM Ca2+Solution be resuspended cell precipitation,
10 min are settled in 37 DEG C；
9, it fully inhales and abandons supernatant, the dual anti-containing 5 mM carnitines, 5 mM creatines, 5 mM taurines 5%FBS and 1% of preheating is added
Cell precipitation is resuspended in M199 complete mediums, and the cell bottle being inoculated in after 0.01-0.015 mg/mL Laminin lens coating is set
In 37 DEG C, 5%（m/v）CO2, saturated humidity incubator in cultivate up to five days.
The preferred embodiment of the invention is described in detail above, but the invention be not limited to it is above-mentioned
Embodiment, makes any modification all within the spirits and principles of the present invention, and equivalent replacement and improvement etc. should be included in this hair
In bright protection domain.
Claims (10)
1. a kind of Adult Rat Ventricular Cardiomyocytes isolation and culture method, which is characterized in that including step：（a）Perfusion system is preheated,
And fully clean perfusion pipeline；（b）Rat abdominal cavity priority injecting heparin sodium and yellow Jackets；（c）It is rapid to open rat thoracic cavity
Exposure heart takes out lower heart from aortic root and is placed in primer solution, after appropriate removing surrounding tissue, aorta is ligatured
In on catheter needle；（d）Heart after intubation is connected into perfusion system, no Ca2+Primer solution perfused hearts；（e）Heart continues
Mixture slaking enzyme solution is perfused；（f）It removes the heart after perfusion to be placed in transfer liquid, removes atrium and big blood vessel, ventricle is cut into
Fritter, suction pipe piping and druming make to be organized into monodisperse cell, and screen filtration cell suspension is stood；（g）Gradient answers Ca2+；（h）Training completely
Support the outstanding multiple Ca of base weight2+Cell precipitation afterwards is inoculated in the Tissue Culture Flask after coating and cultivates.
2. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that the rat is weight
150-300g, the SD rats of raising 6-8 weeks.
3. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that be perfused in the step c
Solution is the NaCl containing 137mM after 4 DEG C of precoolings2、4mM KCl、1mM MgCl、10mM HEPES、0.33mM NaH2PO4、10mM
D-Glucose, 5mM taurines and 10mM BDM buffer solutions.
4. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that without Ca in the step d2+
The specific methods of primer solution perfused hearts be perfusion rate be 3-5mL/min, Hemoperfusion time 2-3min.
5. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that mixed in the step e
Digest the clostridiopetidase A that enzyme solution is 0.05%-0.1%, 0.05%-0.1% clostridiopetidase AsWith 0.05%-0.1% protease mixed enzymes.
6. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that be perfused in the step e
The perfusion rate of mixture slaking enzyme solution is 5-10mL/min, Hemoperfusion time 25-40min.
7. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that shifted in the step f
Liquid is the primer solution containing 0.05-0.1%BSA.
8. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that sieve in the step f
The sieve that filtration cell suspension uses is the sieve in 250 μm of aperture.
9. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that the ladder in the step g
The multiple Ca of degree2+Ca2+Final concentration is followed successively by 0.06mM, 0.24mM, 0.6mM and 1.0mM, and gradient answers Ca2+Method be containing difference
Ca2+Cell precipitation is resuspended in the solution of concentration, and after 37 DEG C settle 10min, supernatant is abandoned in suction.
10. Isolation Methods of Adult Rat Ventricular Cardiomyocytes according to claim 1, which is characterized in that complete in the step h
Full culture medium is the dual anti-M199 complete mediums of the carnitine containing 5mM, 5mM creatines, 5mM taurines 5%FBS and 1% of preheating, coating
Tissue Culture Flask is the coated Tissue Culture Flask of 0.01-0.015mg/mL Laminin lens.
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| CN201710264517.5A CN108728405A (en) | 2017-04-21 | 2017-04-21 | A kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes |
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| CN201710264517.5A CN108728405A (en) | 2017-04-21 | 2017-04-21 | A kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576214A (en) * | 2018-12-21 | 2019-04-05 | 武汉大学人民医院（湖北省人民医院） | A kind of method of acute isolation neonatal rat cardiomyocytes exposed |
| CN113234670A (en) * | 2021-05-26 | 2021-08-10 | 西安交通大学 | Separation and culture method of adult mouse atrial myocytes |
Citations (4)
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| CN102911909A (en) * | 2012-03-05 | 2013-02-06 | 遵义医学院附属医院 | Method for separating and extracting myocardial cells of grown-up rats by adopting one-step enzyme digestion process |
| CN104087550A (en) * | 2014-07-10 | 2014-10-08 | 上海益诺思生物技术有限公司 | Culture method of rat myocardial cell |
| CN105907708A (en) * | 2016-04-08 | 2016-08-31 | 王晓冰 | Isolation and culture method for primary mice or rat cardiac muscle cells |
| CN106497863A (en) * | 2015-09-07 | 2017-03-15 | 江苏齐氏生物科技有限公司 | A kind of separation of cornea of rats endothelial cell, purifying and cultural method |
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2017
- 2017-04-21 CN CN201710264517.5A patent/CN108728405A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911909A (en) * | 2012-03-05 | 2013-02-06 | 遵义医学院附属医院 | Method for separating and extracting myocardial cells of grown-up rats by adopting one-step enzyme digestion process |
| CN104087550A (en) * | 2014-07-10 | 2014-10-08 | 上海益诺思生物技术有限公司 | Culture method of rat myocardial cell |
| CN106497863A (en) * | 2015-09-07 | 2017-03-15 | 江苏齐氏生物科技有限公司 | A kind of separation of cornea of rats endothelial cell, purifying and cultural method |
| CN105907708A (en) * | 2016-04-08 | 2016-08-31 | 王晓冰 | Isolation and culture method for primary mice or rat cardiac muscle cells |
Non-Patent Citations (2)
| Title |
|---|
| JUSTIN JUDD等: "Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| 廖华 等: "成年大鼠心肌细胞分离方法的改良", 《中国组织工程研究与临床康复》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576214A (en) * | 2018-12-21 | 2019-04-05 | 武汉大学人民医院（湖北省人民医院） | A kind of method of acute isolation neonatal rat cardiomyocytes exposed |
| CN113234670A (en) * | 2021-05-26 | 2021-08-10 | 西安交通大学 | Separation and culture method of adult mouse atrial myocytes |
| CN113234670B (en) * | 2021-05-26 | 2022-07-12 | 西安交通大学 | Separation and culture method of adult mouse atrial myocytes |
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Application publication date: 20181102 |