CN104830759A - Rat myocardial cell separation culture method - Google Patents
Rat myocardial cell separation culture method Download PDFInfo
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Abstract
The present invention discloses a rat myocardial cell separation culture method. According to the present invention, the myocardial cells being subjected to separation culture through the method have characteristics of high survival rate, complete myocardial cell and non-myocardial cell separation, rapid cell adherence, high purity, and early rhythmicity synchronized contraction time, and the method has characteristics of simple operation and good repeatability, is an ideal myocardial cell primary culture method, and can meet requirements of cardiovascular disease generation and development mechanisms, cardiovascular drug and cardiac tissue engineering researches, and a variety of physiological and biochemical experiments.
Description
Technical field
The invention belongs to technical field of cell culture, be specifically related to a kind of isolation cultivation method of rat myocardial cell.
Background technology
In the researchs such as Myocyte growth growth, physiology, metabolism, pathology, there is vital role.Therefore, Cardiac myocytes technology has been widely used in the research of cardiovascular disease mechanisms and cardiovascular agent and heart tissue engineering.The common myocardial cells culture method in current this area has: 1. adopt suckling mouse four limbs to be fixed on operating panel, dirty such words operation opening of coring is large, hemorrhage many, is easily mixed into a large amount of hemocytes in myocardial cell.2. adopt 10%FCS/DMEM (high sugar) cultured myocardial, the suitable equally Cardiac Fibroblasts growth of this substratum, so the myocardial cell's purity obtained is low.3. to reach time of synchronous longer for traditional method cultured myocyte, generally at least 3 days, is not well positioned to meet requirement of experiment.
Summary of the invention
According to above-mentioned cited problem, the invention provides a kind of isolation cultivation method of rat myocardial cell, the neonatal rat myocardial cell survival rate with separation and Culture is high, myocardial cell is separated more thorough with non-myocardial infarction, cell attachment is fast, purity is high, the synchronous time of rhythmicity early and easy and simple to handle, reproducible, be a kind of ideal Cardiac myocytes method; Cardiovascular disorder generation, development mechanism and the research of cardiovascular agent and heart tissue engineering and the requirement of multiple bio-chemical characteristics can be met.
The invention provides following technical proposals to solve the problems of the technologies described above.
Technical scheme provided by the invention is: a kind of isolation cultivation method of rat myocardial cell, is characterized in that: comprise the steps:
(1) sterilization wiped in the spray of newborn Sprague-Dawlcy (SD) rat (hereinafter referred to as suckling mouse) alcohol and being placed on surrounding and lower floor is all covered with in the aseptic square box of gauze, to wipe the unnecessary alcohol of dry mouse with it;
(2) left hand holds suckling mouse, suckling mouse is lain on the back after fixing, iodophor disinfection thorax abdomen also takes off allusion quotation, afterwards with right hand ophthalmic tweezers between upper limbs slightly under place carry out cutting " V " mouth with ophthalmologic operation and cut off, left hand refers to that hand top mouse backbone makes heart expose, and the apex of the heart jumps out at once, gets another sterile scissors clip apex, and the apex of the heart is loaded in the culture dish of PBS liquid and washs, fully remove hemocyte;
(3) apex of the heart tissue is moved in microbiotic bottle, and add the DMEM substratum of 3ml, with eye scissors, rat heart muscle tissue is cut into 1X1mm
2tissue block, inhale abandon substratum; Add the trypsinase of 3ml 0.125%, left hand holds antibiotic bottle bottle wall, to keep 37 DEG C of pancreatin the best digestion temperature; The right hand takes elbow straw piping and druming 1min, and Digestive system shifts out Digestive system after becoming muddiness and with 10%FCS/DMEM neutralization, do not digest and be organized in antibiotic bottle, rejoin pancreatin, and so upper method digests 3 times and neutralizes;
(4) after cell suspension being crossed 200 mesh sieves, the centrifugal 5min of 1200rpm/min, abandons supernatant, and cell precipitation 5%FCS/CMM substratum is resuspended, and cell suspension inoculation 6 orifice plate 1 piece, cultivates in incubator;
(5) after cell attachment 90min, draw not adherent myocardial cell suspensions, microscopic examination, under mirror, visible myocardial cell 80-90% merges, and has stronger automaticity and shrinkability feature.
Wherein, Sprague-Dawlcy (SD) rat described in step (1) takes from new born 1-3 days, and male and female regardless of.
Wherein, being loaded in by the apex of the heart in the culture dish of PBS liquid described in step (2) is washed, and washing times is 3 times, each 1min.
Wherein, the PBS liquid described in step (2) is 4 DEG C of precoolings; Culture dish is placed on ice.
Wherein, step (5) draws not adherent myocardial cell suspensions needs inoculation 6 orifice plate 1 piece, every hole 5X10
5individual/mL cell, 24h replaced medium.
The beneficial effect that the present invention realizes: the present invention draws materials conveniently, rat regardless of male and female, simple, the strong operability of method in culturing process, external myocardial cell is separated, low, consuming time short, the program simplification of method cost cultivated; Opening operation is young, hemorrhage few, avoids a large amount of blood cells contamination; Use myocardial cell's special culture media (Cardiac MyocyteMedium, sciencell) CMM, obtain the myocardial cell that a large amount of purity is higher, thus ensure the use of subsequent experimental, and the myocardial cell that separation and Culture obtains has automaticity and shrinkability feature, its original many structure and function in vivo can be kept.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.Agents useful for same of the present invention and raw material are known technology, the technology known by those skilled in that art, and are commercially easy to buy.
The invention provides a kind of isolation cultivation method of rat myocardial cell, the neonatal rat myocardial cell survival rate with separation and Culture is high, myocardial cell is separated more thorough with non-myocardial infarction, cell attachment is fast, purity is high, the synchronous time of rhythmicity early and easy and simple to handle, reproducible, be a kind of ideal Cardiac myocytes method: can meet that cardiovascular disorder occurs, development mechanism and the research of cardiovascular agent and heart tissue engineering and the requirement of multiple bio-chemical characteristics.
One of technical scheme provided by the invention is: a kind of isolation cultivation method of rat myocardial cell, comprises the steps:
(1) sterilization wiped in the spray of newborn Sprague-Dawlcy (SD) rat (hereinafter referred to as suckling mouse) alcohol and being placed on surrounding and lower floor is all covered with in the aseptic square box of gauze, to wipe the unnecessary alcohol of dry mouse with it;
(2) left hand holds suckling mouse, suckling mouse is lain on the back after fixing, iodophor disinfection thorax abdomen also takes off allusion quotation, afterwards with right hand ophthalmic tweezers between upper limbs slightly under place carry out cutting " V " mouth with ophthalmologic operation and cut off, left hand refers to that hand top mouse backbone makes heart expose, and the apex of the heart jumps out at once, gets another sterile scissors clip apex, and the apex of the heart is loaded in the culture dish of PBS liquid and washs, fully remove hemocyte;
(3) apex of the heart tissue is moved in microbiotic bottle, and add the DMEM substratum of 3ml, with eye scissors, rat heart muscle tissue is cut into 1X1mm
2tissue block, inhale abandon substratum; Add the trypsinase of 3ml 0.125%, left hand holds antibiotic bottle bottle wall, to keep 37 DEG C of pancreatin the best digestion temperature; The right hand takes elbow straw piping and druming 1min, and Digestive system shifts out Digestive system after becoming muddiness and with 10%FCS/DMEM neutralization, do not digest and be organized in antibiotic bottle, rejoin pancreatin, and so upper method digests 3 times and neutralizes;
(4) after cell suspension being crossed 200 mesh sieves, the centrifugal 5min of 1200rpm/min, abandons supernatant, and cell precipitation 5%FCS/CMM substratum is resuspended, and cell suspension inoculation 6 orifice plate 1 piece, cultivates in incubator;
(5) after cell attachment 90min, draw not adherent myocardial cell suspensions, microscopic examination, under mirror, visible myocardial cell 80-90% merges, and has stronger automaticity and shrinkability feature.
Wherein, Sprague-Dawlcy (SD) rat described in step (1) takes from new born 1-3 days, and male and female regardless of.
Wherein, being loaded in by the apex of the heart in the culture dish of PBS liquid described in step (2) is washed, and washing times is 3 times, each 1min.
Wherein, the PBS liquid described in step (2) is 4 DEG C of precoolings; Culture dish is placed on ice.
Wherein, step (5) draws not adherent myocardial cell suspensions needs inoculation 6 orifice plate 1 piece, every hole 5X10
5individual/mL cell, 24h replaced medium.
The beneficial effect that the present invention realizes: the present invention draws materials conveniently, rat regardless of male and female, simple, the strong operability of method in culturing process, external myocardial cell is separated, low, consuming time short, the program simplification of method cost cultivated; Opening operation is young, hemorrhage few, avoids a large amount of blood cells contamination; Use myocardial cell's special culture media (Cardiac MyocyteMedium, sciencell) CMM, obtain the myocardial cell that a large amount of purity is higher, thus ensure the use of subsequent experimental, and the myocardial cell that separation and Culture obtains has automaticity and shrinkability feature, its original many structure and function in vivo can be kept.
Above, be only the embodiment utilizing this origination techniques content, the modification that any those skilled in the art use this creation to do, change, all belong to the scope of the claims that this creation is advocated, and be not limited to those disclosed embodiments.
Claims (5)
1. an isolation cultivation method for rat myocardial cell, is characterized in that: comprise the steps:
(1) sterilization wiped in the spray of newborn Sprague-Dawlcy (SD) rat (hereinafter referred to as suckling mouse) alcohol and being placed on surrounding and lower floor is all covered with in the aseptic square box of gauze, to wipe the unnecessary alcohol of dry mouse with it;
(2) left hand holds suckling mouse, suckling mouse is lain on the back after fixing, iodophor disinfection thorax abdomen also takes off allusion quotation, afterwards with right hand ophthalmic tweezers between upper limbs slightly under place carry out cutting " V " mouth with ophthalmologic operation and cut off, left hand refers to that hand top mouse backbone makes heart expose, and the apex of the heart jumps out at once, gets another sterile scissors clip apex, and the apex of the heart is loaded in the culture dish of PBS liquid and washs, fully remove hemocyte;
(3) apex of the heart tissue is moved in microbiotic bottle, and add the DMEM substratum of 3ml, with eye scissors, Neonatal myocardial tissue is cut into 1X1mm
2tissue block, inhale abandon substratum; Add the trypsinase of 3ml 0.125%, left hand holds antibiotic bottle bottle wall, to keep 37 DEG C of pancreatin the best digestion temperature; The right hand takes elbow straw piping and druming 1min, and Digestive system shifts out Digestive system after becoming muddiness and with 10%FCS/DMEM neutralization, do not digest and be organized in antibiotic bottle, rejoin pancreatin, and so upper method digests 3 times and neutralizes;
(4) after cell suspension being crossed 200 mesh sieves, the centrifugal 5min of 1200rpm/min, abandons supernatant, and cell precipitation 5%FCS/CMM substratum is resuspended, and cell suspension inoculation 6 orifice plate 1 piece, cultivates in incubator;
(5) after cell attachment 90min, draw not adherent myocardial cell suspensions, microscopic examination, under mirror, visible myocardial cell 80-90% merges, and has stronger automaticity and shrinkability feature.
2. the isolation cultivation method of a kind of rat myocardial cell according to claim 1, is characterized in that: Sprague-Dawlcy (SD) rat described in step (1) takes from new born 1-3 days, and male and female regardless of.
3. the isolation cultivation method of a kind of rat myocardial cell according to claim 1, is characterized in that: being loaded in by the apex of the heart in the culture dish of PBS liquid described in step (2) is washed, and washing times is 3 times, each 1min.
4. the isolation cultivation method of a kind of rat myocardial cell according to claim 1 or 3, is characterized in that: the PBS liquid described in step (2) is 4 DEG C of precoolings; Culture dish is placed on ice.
5. the isolation cultivation method of a kind of rat myocardial cell according to claim 1, is characterized in that: step (5) draws not adherent myocardial cell suspensions needs inoculation 6 orifice plate 1 piece, every hole 5X10
5individual/mL cell, 24h replaced medium.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385652A (en) * | 2015-12-24 | 2016-03-09 | 中南民族大学 | High-purity cardiac muscle cell primary culture method |
| 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 |
| CN105907708A (en) * | 2016-04-08 | 2016-08-31 | 王晓冰 | Isolation and culture method for primary mice or rat cardiac muscle cells |
| CN106244525A (en) * | 2016-08-16 | 2016-12-21 | 中国农业科学院兰州兽医研究所 | A kind of myocardial cell and the Combined culture method of sustentacular cell of testis |
| CN108949673A (en) * | 2018-08-13 | 2018-12-07 | 武汉华联科生物技术有限公司 | A kind of primary separation method of Fetal Rat rat cardiomyocyte |
| CN111154715A (en) * | 2019-12-27 | 2020-05-15 | 广东博溪生物科技有限公司 | Myocardial cell separation culture method |
| CN111808794A (en) * | 2020-06-24 | 2020-10-23 | 江西博领生物技术有限公司 | Method for efficiently obtaining primary organ cells of mice |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385652A (en) * | 2015-12-24 | 2016-03-09 | 中南民族大学 | High-purity cardiac muscle cell primary culture method |
| 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 |
| CN105505863B (en) * | 2016-01-05 | 2019-03-22 | 中国人民解放军第二军医大学 | A kind of cultural method of naked mole cardiac muscle cell |
| CN105907708A (en) * | 2016-04-08 | 2016-08-31 | 王晓冰 | Isolation and culture method for primary mice or rat cardiac muscle cells |
| CN106244525A (en) * | 2016-08-16 | 2016-12-21 | 中国农业科学院兰州兽医研究所 | A kind of myocardial cell and the Combined culture method of sustentacular cell of testis |
| CN106244525B (en) * | 2016-08-16 | 2019-06-18 | 中国农业科学院兰州兽医研究所 | A kind of Combined culture method of cardiac muscle cell and sustentacular cell of testis |
| CN108949673A (en) * | 2018-08-13 | 2018-12-07 | 武汉华联科生物技术有限公司 | A kind of primary separation method of Fetal Rat rat cardiomyocyte |
| CN111154715A (en) * | 2019-12-27 | 2020-05-15 | 广东博溪生物科技有限公司 | Myocardial cell separation culture method |
| CN111808794A (en) * | 2020-06-24 | 2020-10-23 | 江西博领生物技术有限公司 | Method for efficiently obtaining primary organ cells of mice |
| CN111808794B (en) * | 2020-06-24 | 2022-04-22 | 江西博领生物技术有限公司 | Method for efficiently obtaining primary organ cells of mice |
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