CN105385652A - High-purity cardiac muscle cell primary culture method - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
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Abstract
The invention provides a high-purity cardiac muscle cell primary culture method and is suitable for the field of biotechnology and medical science. The high-purity cardiac muscle cell primary culture method particularly comprises the steps that an isolated heart of a suckling mouse is sheared into tissue fragments; digestion is conducted on the tissue fragments through improved prepared enzyme digestive juice for multiple times, cardiac muscle cell suspension is prepared, resuspension is conducted, and then cardiac muscle cells are inoculated in a culture dish A for differential-time wall sticking; cardiac muscle cell suspension in the culture dish A is sucked into a culture dish B, cardiac muscle cells which sink to the bottom of the culture dish A and do not stick to the wall in the culture dish A are cleaned and resuspended, and then the resuspended cardiac muscle cells are transferred into the culture dish B to be cultured. Compared with the prior art, the high-purity cardiac muscle cell primary culture method has the advantages that the method is simple and effective, the operability is strong, the number of obtained cardiac muscle cells is large, the purity is high, the wall sticking rate is good, and the survival rate is high.
Description
Technical field
The invention provides a kind of high purity Cardiac myocytes method, be applicable to carry out related cardiovascular disease research at cell levels, illustrate mechanism problem from cellular and molecular level, belong to biotechnology and medical field.
Background technology
Along with heart disease rate raises year by year, about the research of cardiomyopathy more and more comes into one's own.Neonatal rat myocardial cell vitro culture can retain the feature on the more original structure and functions of body, there is pulsation, and not by the interference of the factors such as nerve, body fluid, Myocyte growth growth can be carried out, many-sided experimental study such as pathologic, physiologic and pharmacology, therefore the original cuiture of neonatal rat myocardial cell is widely used in the study on prevention field of cardiovascular disorder.
But due to neonatal rat myocardial cell primary culture method of a great variety, and myocardial cell easily sustains damage in original cuiture process, causes survival rate low.And, heart about 80% is made up of myocardial cell, about 20% primarily of non-myocardial infarction compositions such as inoblasts in addition, and myocardial cell is not easily adherent, inoblast energy quick wall attaching, therefore, in the original cuiture process of myocardial cell, easily be formed into fibroblast growth advantage, thus cause the not high defect of myocardial cell's purity.How to obtain the myocardial cell in a large number with normal physiological function, the purity of raising cell is the key issue in myocardial cells culture.
Cardiac myocytes mainly contains 3 kinds of methods: Ex vivo heart perfusion method, tissue block method and enzyme digestion.Ex vivo heart perfusion method is concerning suckling mouse, and operation easier is larger.Tissue block method is simple to operate, can obtain the myocardial cell of higher degree.There is strict control the time of enzyme digestion to the concentration of enzyme and enzyme effect, but can obtain a large amount of unicellular.Therefore, the original cuiture of myocardial cell, multiplex tissue block method and enzyme digestion.Two kinds of methods respectively have relative merits.
1. tissue block method:
Advantage: (1) is simple to operate, does not need enzymic digestion, only need be cut into small pieces heart, evenly be paved with at culture dish.(2) be separated the myocardial ultramicrostructure obtained complete, there is no the digestion of enzyme, so the myocyte survival rate obtained is high, and morphological structure is complete.(3) do not need loaded down with trivial details experimental procedure, require lower to reagent consumptive material, economy.
Shortcoming: the number of myocardial cells of acquisition is few, is difficult to meet needed for experiment.If need a large amount of myocardial cell, then need repeatedly to be separated, required experimental period is long in batches, is difficult to reach experiment purpose.
2. enzyme digestion refers to and adopts trypsinase and II-Collagenase Type mixture slaking heart tissue block, thus separation obtains single myocardial cell.
Advantage: enzyme can obtain the myocardial cell of a large amount of single separation to the digestion of tissue block, can meet and require higher experiment to number of myocardial cells.
Shortcoming: comparatively large to cell injury, because enzyme is to the effect of myocardial cell, and centrifugal, the step such as resuspended, can have chemistry in various degree or physical damnification to the myocardial cell be separated.Further, the concentration of enzyme and enzyme action time difference are also different to the degree of injury of myocardial cell.Inoblast in heart tissue due to adherent ability strong, and myocardial cell has damage in various degree under the digestion of enzyme liquid, so be easy to be formed into fibrocellular growth vigor, thus dilute myocardial cell gradually, cause being separated the myocardial cell's purity obtained very low.
Two kinds of methods cut both ways, and therefore, want to obtain at short notice that large number of viable rate is high and the myocardial cell that purity is high is a technical barrier to meet requirement of experiment.
Summary of the invention
The invention solves deficiency of the prior art, provide and a kind ofly can obtain the primary culture method of the myocardial cell that a large amount of purity is high, vigor is good by sharp separation.
Realizing the technical scheme that above-mentioned purpose of the present invention adopts is:
A kind of high purity Cardiac myocytes method, comprises the following steps:
(1), by vitro suckling mouse heart proceed in the culture dish filling DMEM/F12 basic medium, cardiac scissors is become fragment of tissue;
(2), fragment of tissue is proceeded in EP pipe, add enzymic digestion liquid, described enzymic digestion liquid is made up of II-Collagenase Type, trypsinase and PBS damping fluid, in enzymic digestion liquid, the mass percent concentration of II-Collagenase Type is 0.04%, and tryptic mass percent concentration is 0.09%;
(3), by EP pipe be placed in 35 ~ 40 DEG C of water bath condition and carry out first time digestion, digestion time is 6 ~ 10 minutes, discards after digestion after Aspirate supernatant;
(4), EP pipe is continued be placed in 35 ~ 40 DEG C of water bath condition, continue to add enzymic digestion liquid and carry out follow-up digestion, digestion time is 10 ~ 12 minutes, after Aspirate supernatant supernatant liquor is positioned in centrifuge tube after digestion, placed the DMEM/F12 perfect medium containing foetal calf serum in described centrifuge tube in advance, centrifuge tube is placed on ice;
(5), repeating step (4), supernatant liquor is extracted in circulation, until the fragment of tissue in EP pipe bleaches and after diminishing, completes the collection of supernatant liquor, supernatant liquor collected in centrifuge tube is myocardial cell suspensions;
(6), by centrifugal for the myocardial cell suspensions gathered and remove supernatant, then add DMEM/F12 perfect medium, it is resuspended to carry out myocardial cell, obtained myocardial cell's re-suspension liquid;
(7), then myocardial cell is seeded in adherent 1 ~ 1.5h when differing from culture dish A;
(8), draw myocardial cell suspensions in culture dish A in culture dish B, to be sunken in cleaning culture dish A at the bottom of ware but not adherent myocardial cell resuspended, to be then transferred in culture dish B and to cultivate.
Rock EP pipe when first time digestion in step (3), and take out EP pipe bullet 2-10 time, heating in water bath at once after having played.
Rocking EP pipe when digesting in step (4), taking out EP every 3min and manage and play 2-10 time, after having played, putting into heating in water bath at once.
With the centrifugal 8min of 1000rpm rotating speed in step (6).
Before adherent when differing from step (7) first by myocardial cell's re-suspension liquid through cell 40 μm of strainer filterings, elimination cell mass, rinses centrifuge tube and filter screen with DMEM/F12 perfect medium.
Culture dish B described in step (8) with 0.1% gelatin bag quilt, and opens ultra violet lamp 30min before inoculation.
In the culturing process of step (8), each culture dish B is all rocked according to all around direction with cell dispersion.
In the art, trypsinase can make intercellular protein be hydrolyzed, thus makes cell dispersal, and effect is strong, but larger to cell injury.The effective object of II-Collagenase Type is collagenous tissue, only has digestion to intercellular substance, can collegen filament in peptic cell interstitial thus release cells, action temperature and, less to the injury of cell.In order to reduce the damage to myocardial cell, simultaneously unlikely digestion is too weak, usually by Trypsin pancreas and II-Collagenase Type used in combination.Neonatal rat myocardial cell is very responsive to enzymic digestion, and therefore, the concentration of enzymic digestion liquid and digestion time seem particularly important in the original cuiture of myocardial cell.
Tryptic digestion ability is very strong, and typical concentrations is 0.05 ~ 0.5%, 37 DEG C, act under pH8.0 condition the strongest, easily cause myocardial cell injury, in document, have the method for multiple concentration tryptic digestion.Trypsinase concentration is too low, although it is better to digest the cardiomyocyte viability obtained, digestion time is corresponding elongated, and the number of myocardial cells obtained is few, is difficult to meet requirement of experiment.Trypsinase concentration is too high, then can damaged cardiomyocytes, causes myocardial cell's adherent rate low, does not even paste.
The present invention is on the basis of using for reference forefathers' experimental technique, after many experiments is groped, the concentration of enzymic digestion liquid and proportioning are improved, adopt the trypsinase of low concentration, carry out the short period of time and repeatedly repeat digestion, thus obtain myocardial cell single in a large number, and cardiomyocyte viability is high, myocardial cell's phychology clear-cut, can see comparatively many cells spontaneous beating, and myocardial cell's purity of immunofluorescence dyeing display original cuiture is very high.
Accompanying drawing explanation
The primary cardiomyocytes aspect graph that Fig. 1 cultivates for the present invention;
Fig. 2 is myocardial cell's Purity colored graph.
Embodiment
In order to the technical barrier making the present invention solve clearly is understood, below in conjunction with accompanying drawing, the present invention is further elaborated.
Technology used in the present invention, unless stated otherwise, is routine techniques known to those skilled in the art; Plant and instrument used, reagent etc., unless stated otherwise, the research and the technician that are this area can be obtained by public approach.
The selection in mouse mouse age:
When selecting suckling mouse, select the suckling mouse that date of birth is short, be separated the cardiomyocyte viability obtained so good, adherent rate is high, so preferably select the suckling mouse of newborn 1-3 days as far as possible.
The use of experiment reagent and digestive ferment liquid:
1. foetal calf serum: packing after fire extinguishing, often pipe 50mL, is stored in-20 DEG C.
2.0.1% gelatin: deionized water is prepared, 4 DEG C of preservations after sterilizing.
3. disappear substratum completely: DMEM/F12 basic medium, wherein containing 20%FBS foetal calf serum and 1% dual anti-.
4. enzymic digestion liquid: II-Collagenase Type 0.04%, pancreatin 0.09%, with PBS dilution, general digestion 4 suckling mouse hearts need 5mL Digestive system, and 8 suckling mouse hearts need 10mL enzymic digestion liquid, the like.
The high purity Cardiac myocytes method provided in the present embodiment is as follows:
1. of the right age suckling mouse and the enzymic digestion liquid of respective numbers are provided.
2. heart takes out
(1) 10mLDMEM/F12 basic medium is added in 1 100mm glass dish.Separately add 10mLDMEM/F12 basic medium in the glass dish of other 1 100mm, the precooling of ice basin put into by two plates.
(2) mouse baby is put into 100mm glass dish, tweezer mouse is put into 75% alcohol and soaks the several seconds, sterilize to below its neck, capture mouse, fix upper limbs and lower limb, head is cut from neck, scissors stretches into thoracic cavity by neck and cuts off on the left of breastbone, extrudes thorax gently, allows heart jump out, rapid tweezers take off heart, put into the glass dish filling 10mLDMEM/F12 basic medium.
(3) proceeding to another after softly cleaning the blood of heart fills in the 100mm culture dish of 10mLDMEM/F12 basic medium, by cardiac scissors into about 1mm
3fragment.
(4) above process should complete within 30min on ice.
3. peptic cell
(1) tissue shredded is proceeded in the EP pipe of 1.5mL, discard unnecessary PBS, add enzymic digestion liquid, a 1mL.
Jog in (2) 37 DEG C of water-baths, digestion 8min, middle taking-up is flicked several times, has digested rear Aspirate supernatant, has discarded.The cell now digested is unwanted red corpuscle and cell debris.
Jog in (3) 37 DEG C of water-baths, digestion 10min, take out every 3min and flick several times, dynamics is not too big.Digest rear Aspirate supernatant, put into the 15mL centrifuge tube termination digestion that the 7mL put well in advance contains the DMEM/F12 perfect medium of 20% foetal calf serum, and be placed on ice.
(4) 3 steps are repeated, circulation 2-3 time.Should exhaust when getting supernatant as far as possible, when tissue block bleaches and obviously diminishes, stop digestion.
(5) myocardial cell suspensions will gathered, with the centrifugal 8min of 1000rpm rotating speed, careful supernatant of drawing discards, and adds DMEM/F12 perfect medium, softly blows and beats re-suspended cell.
4. adherent during difference
(1) by cell suspension through cell 40 μm of strainer filterings, with elimination cell mass, rinse centrifuge tube and filter screen with DMEM/F12 perfect medium.
(2) cell is seeded in adherent 60min when differing from the culture dish A of 60mm.
5. inoculating cell
(1) with 0.1% gelatin bag by the culture dish B that will inoculate, open ultraviolet lamp, irradiate 30min.This step should be fulfiled ahead of schedule.
(2) draw myocardial cell suspensions in culture dish A in culture dish B, to be sunken in cleaning culture dish A at the bottom of ware but not adherent myocardial cell resuspended, to be then transferred in culture dish B.
(3) each culture dish B all according to fore-and-aft direction, left and right directions jog with cell dispersion, whirlpool does not rock.
(4) at 37 DEG C, 5%CO
2hatch under condition, clean once with PBS after 48h, replaced medium.
After cultivating 24h, can be observed myocardial cell and start adherent growth under inverted microscope, be just circular, be fusiformis afterwards, accidental individual cells is beaten, and cell forms irregular star subsequently, and it is more obvious that cell sprawls situation, and stretch out pseudopodium.After cultivating 48h, myocardial cell starts to beat in flakes, and vigor is good, and survival rate is high.Fig. 1 is myocardial cell's aspect graph.
In order to identify the purity of cultured myocyte, be reduced to fibrocellular impact, pass through immunofluorescent staining, Fig. 2 is myocardial cell's Purity colored graph, left figure first α-Actinin fluorescent dye display is dyed to myocardial cell's (being shown as light gray in figure) of green fluorescence, middle PI colored graph showed cell core is dyed to red cell (being shown as Dark grey in figure), rightmost figure is the composite diagram on two, the left side, can find out that the cell that nucleus is colored is all myocardial cell substantially, illustrate that cultured myocyte purity is very high, close to 100%.
Above result shows that the present invention's method used can from heart tissue separating myocardium cell, and the method is simple and easy to do, workable, for traditional method, to obtain myocardial cell's purity high, quantity is many, can meet the requirement of general experiment.Be applicable to biological technical field and medical field, at cellular and molecular level research related cardiovascular disease.
Claims (7)
1. a high purity Cardiac myocytes method, is characterized in that comprising the following steps:
(1), by vitro suckling mouse heart proceed in the culture dish filling DMEM/F12 basic medium, cardiac scissors is become fragment of tissue;
(2), fragment of tissue is proceeded in EP pipe, add enzymic digestion liquid, described enzymic digestion liquid is made up of II-Collagenase Type, trypsinase and PBS damping fluid, in enzymic digestion liquid, the mass percent concentration of II-Collagenase Type is 0.04%, and tryptic mass percent concentration is 0.09%;
(3), by EP pipe be placed in 35 ~ 40 DEG C of water bath condition and carry out first time digestion, digestion time is 6 ~ 10 minutes, discards after digestion after Aspirate supernatant;
(4), EP pipe is continued be placed in 35 ~ 40 DEG C of water bath condition, continue to add enzymic digestion liquid and carry out follow-up digestion, digestion time is 10 ~ 12 minutes, after Aspirate supernatant supernatant liquor is positioned in centrifuge tube after digestion, placed the DMEM/F12 perfect medium containing foetal calf serum in described centrifuge tube in advance, centrifuge tube is placed on ice;
(5), repeating step (4), supernatant liquor is extracted in circulation, until the fragment of tissue in EP pipe bleaches and after diminishing, completes the collection of supernatant liquor, supernatant liquor collected in centrifuge tube is myocardial cell suspensions;
(6), by centrifugal for the myocardial cell suspensions gathered and remove supernatant, then add DMEM/F12 perfect medium, it is resuspended to carry out myocardial cell, obtained myocardial cell's re-suspension liquid;
(7), then myocardial cell is seeded in adherent 1 ~ 1.5h when differing from culture dish A;
(8), draw myocardial cell suspensions in culture dish A in culture dish B, to be sunken in cleaning culture dish A at the bottom of ware but not adherent myocardial cell resuspended, to be then transferred in culture dish B and to cultivate.
2. high purity Cardiac myocytes method according to claim 1, is characterized in that: rock EP pipe when first time digestion in step (3), and take out EP pipe bullet 2-10 time, heating in water bath at once after having played.
3. high purity Cardiac myocytes method according to claim 1, is characterized in that: rock EP pipe when digesting in step (4), takes out EP and manages and play 2-10 time, put into heating in water bath at once after having played every 3min.
4. high purity Cardiac myocytes method according to claim 1, is characterized in that: with the centrifugal 8min of 1000rpm rotating speed in step (6).
5. high purity Cardiac myocytes method according to claim 1, it is characterized in that: before adherent when differing from step (7) first by myocardial cell's re-suspension liquid through cell 40 μm of strainer filterings, elimination cell mass, rinses centrifuge tube and filter screen with DMEM/F12 perfect medium.
6. high purity Cardiac myocytes method according to claim 1, is characterized in that: culture dish B described in step (8) with 0.1% gelatin bag quilt, and opens ultra violet lamp 30min before inoculation.
7. high purity Cardiac myocytes method according to claim 1, is characterized in that: in the culturing process of step (8), is all rocked according to all around direction with cell dispersion by each culture dish B.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907708A (en) * | 2016-04-08 | 2016-08-31 | 王晓冰 | Isolation and culture method for primary mice or rat cardiac muscle cells |
| CN106676061A (en) * | 2016-11-19 | 2017-05-17 | 河南医学高等专科学校 | Myocardial cell separation method |
| 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 |
| CN111826342A (en) * | 2019-04-15 | 2020-10-27 | 广西医科大学 | Method for isolating primary neonatal rat cardiomyocytes |
| CN113502260A (en) * | 2021-07-16 | 2021-10-15 | 新疆医科大学第一附属医院 | Method for separating and culturing primary myocardial cells of mice suckling mice and application of method |
| CN115322961A (en) * | 2022-08-19 | 2022-11-11 | 北京全式金生物技术股份有限公司 | Dissociation liquid and dissociation method for in vitro culture of myocardial cells |
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| CN105907708A (en) * | 2016-04-08 | 2016-08-31 | 王晓冰 | Isolation and culture method for primary mice or rat cardiac muscle cells |
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| CN111826342A (en) * | 2019-04-15 | 2020-10-27 | 广西医科大学 | Method for isolating primary neonatal rat cardiomyocytes |
| 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 |
| CN113502260A (en) * | 2021-07-16 | 2021-10-15 | 新疆医科大学第一附属医院 | Method for separating and culturing primary myocardial cells of mice suckling mice and application of method |
| CN115322961A (en) * | 2022-08-19 | 2022-11-11 | 北京全式金生物技术股份有限公司 | Dissociation liquid and dissociation method for in vitro culture of myocardial cells |
| CN115322961B (en) * | 2022-08-19 | 2023-10-20 | 北京全式金生物技术股份有限公司 | Dissociation liquid and dissociation method for in vitro culture of myocardial cells |
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