CN105238738A - Isolated culture method of piglet myocardial fibroblasts - Google Patents
Isolated culture method of piglet myocardial fibroblasts Download PDFInfo
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- CN105238738A CN105238738A CN201510634932.6A CN201510634932A CN105238738A CN 105238738 A CN105238738 A CN 105238738A CN 201510634932 A CN201510634932 A CN 201510634932A CN 105238738 A CN105238738 A CN 105238738A
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Abstract
The invention relates to an isolated culture method of piglet myocardial fibroblasts and belongs to the technical field of cell culture in modern biotechnology. The method includes: 1, collecting cardiac tissues from a piglet 1 to 3 days old, shearing ventricular tissues, performing PBS (phosphate buffer saline) pre-cooling, and rinsing several times; 2, digesting the myocardial tissues, namely performing multiple digestion at 37 DEG C with a mixture of trypsin 0.25% and collagenase II 0.1% having a ratio of 1:1, adding DNA deoxyribonuclease (0.02 mg/mL) to reduce cell suspension viscosity and increase cell yield, and adding red blood cell lysis buffer to reduce the amount of red cells; 3, culturing piglet myocardial fibroblasts, namely re-suspending cells with DMEM (Dulbecco modified Eagle medium) high-glucose medium containing fetal calf serum 10-15%, and performing differential attachment to obtain the myocardial fibroblasts. The method is simple and easy to master, time efficient and high in success rate, the obtained piglet myocardial fibroblasts are good in form, good in activity and abundant. Certain basis is provided for establishing a cell model of piglet myocardial fibroblasts to study related diseases such as cardiac hypertrophy in future.
Description
Technical field
The present invention relates to a kind of pig myocardium inoblast isolation cultivation method, belong to the technical field of cell culture of modern biotechnology.
Background technology
The application of cell culture technology in biology, medical scientific is more and more extensive.Original cuiture refers to and directly grows monolayer cell by tissue block or become individual cells to start to cultivate tissue dispersion with enzyme or mechanical means, and the cultivation before going down to posterity first can think original cuiture.Primary cell can be widely used in molecule, cytobiology and biomedical fundamental research, as proteomics, genomics, cell strain research etc., also can be applicable to drug screening, drug metabolism and toxicological study etc.In addition, original cuiture be also set up various clone (strain) must through stage.
Cardiac Fibroblasts accounts for 60 ~ 70% of cardiac bistiocyte's sum, is the chief component of non-myocardial infarction in heart.This cell, by producing extracellular matrix, as collagen protein and fibronectin, constitutes the mechanical framework of myocardial cell; On the other hand, Cardiac Fibroblasts can also produce metalloprotease, various somatomedin and cytokine required in remodeling ventricle process.Therefore, myocardial function normal operation is played an important role.Clinically, the propagation of Cardiac Fibroblasts take part in the pathologic process of the multiple cardiovascular disordeies such as hypertension left ventricular hypertrophy, ischemic heart disease, dilated cardiomyopathy and congestive heart failure, and research Cardiac Fibroblasts is significant for the research of modern cardiovascular disease.
Up to now, studying human heart hypertrophy waits the cell model of heart disease use to be mainly Cardiac Fibroblasts and the neonatal rat myocardial cell of suckling mouse.But, because the affinity of pig and people is nearer, and the heart of pig is very similar with human heart in anatomical structure, cardiovascular distribution, H/BW ratio etc., therefore, if having more advantage using pig myocardium inoblast as the cell model of the diseases such as research human heart is loose.But up to the present, there is no the research about piglet Cardiac Fibroblasts separation and Culture.
Summary of the invention
The invention provides a kind of piglet inoblast isolation cultivation method, this cultural method is simple to operation, saves time, and success ratio is high, and the primary cell form stable of acquisition, active good, quantity foot, ability of cell proliferation is strong.
Technical scheme of the present invention is as follows:
A kind of piglet Cardiac Fibroblasts separation method, is characterized in that, comprise the following steps:
(1) by the piglet in birth 1 ~ 3 day, with 0.1% bromogeramine whole body sterilization, precaval vein bloodletting is put to death rapidly, fixing piglet, 75% alcohol disinfecting skin of chest abdomen.
(2) piglet skin of chest cuts by aseptic operation cutter.
(3) sterile scissors along thoracic cavity last root bone cut off forward, expose heart, put into immediately and fill 50mL precooling PBS beaker, wash away hemocyte and blood clot.
(4) heart is transferred in aseptic plate by aseptic nipper, and Sterile ophthalmic cuts intercepting ventricle, divests pericardium, adds appropriate precooling PBS and rinses, and rejects blood vessel, fat and reticular tissue, precooling PBS rinsing 2 ~ 3 times.
(5) 1 ~ 2mm is cut into
3tissue block, transfers in 50mL centrifuge tube, with precooling PBS rinsing tissue pieces 2 ~ 3 times, to remove hemocyte, discards washings and leaves tissue block in order to digestion.
(6) 0.25% trypsinase and 0.1% II Collagenase Type 1:1 mixed enzyme solution is added, consumption is 3 ~ 5 times of tissue block volume, put in 37 DEG C of water-baths after vibration digestion 15min, abandoning supernatant (being mainly red corpuscle, cell debris and dead cell).
(7) add the mixed enzyme solution containing DNA enzymatic (final concentration is 0.02mg/mL) again, put in 37 DEG C of water-baths the digestion 8 ~ 10min that vibrates.
(8) the careful Aspirate supernatant of transfer pipet moves in another sterile centrifugation tube, and adds the DMEM high sugared nutrient solution termination digestion of 10 ~ 15mL containing 10 ~ 15% foetal calf serums.So repeat digestion 3 ~ 8 times.
(9) cell suspension collects centrifuge tube after filtering respectively with 100 μm and 40 μm of screens, centrifugal 5 ~ the 6min of 1000RPM/min, abandon supernatant, add that erythrocyte cracked liquid 3 ~ 5mL is centrifugal abandons supernatant, with the high sugared nutrient solution re-suspended cell of DMEM containing 10% ~ 15% foetal calf serum, cell suspension is added 25cm
2in 37 DEG C, 5%CO in culturing bottle
2cultivate in incubator.
(10) differential velocity adherent.Timing from incubator put into by culturing bottle, after 60 ~ 90min, discard non-attached cell, attached cell is the Cardiac Fibroblasts of separation and purification.
Compared to existing technology, beneficial effect of the present invention is:
(1) heart of pig is very similar with human heart in anatomical structure, cardiovascular distribution, H/BW ratio etc., is that the heart of the laboratory animal such as mouse can not be compared.For providing stable, reproducible, with clinical more close animal model, we select the pig close with human heart as research object.Up to the present, there is no and be separated the fibroblastic relevant report of pig myocardium, more accurate as the research model of the diseases such as human heart is loose with pig myocardium inoblast.
(2) operation is simple in the present invention, without the need to specific installation, the advantage such as save time, and obtains piglet Cardiac Fibroblasts form stable, activity is good, quantity is many, ability of cell proliferation is strong, can be used for the researchs such as subsequent cell drug sensitivity test, the sorting of cell and qualification.
(3) the present invention uses 0.25% trypsinase and 0.1% II Collagenase Type 1:1 mixed enzyme solution, puts in 37 DEG C of water-baths and vibrates, repeatedly digest.Digestion obtains cell suspension and processes on ice, to improve cytoactive.Adopt low concentration trypsinase and higher concentration II Collagenase Type mixture slaking enzymic digestion cardiac muscular tissue simultaneously, can well Cardiac Fibroblasts be separated.
(4) the present invention uses DNA enzymatic, when cardiac muscular tissue digests, can effectively reduce cell suspension viscosity, improves cell pick-up rate.
(5) the present invention uses erythrocyte cracked liquid splitting erythrocyte, reduces red corpuscle to Cardiac Fibroblasts growth effect.
Accompanying drawing explanation
Fig. 1 embodiment 1 hides pig (piglet) Cardiac Fibroblasts growth regulation 2 days cells.
Fig. 2 embodiment 1 hides pig (piglet) Cardiac Fibroblasts growth regulation 3 days cells.
Fig. 3 embodiment 1 hides pig (piglet) Cardiac Fibroblasts growth regulation 4 days cells.
Fig. 4 embodiment 2 landrace (piglet) Cardiac Fibroblasts growth regulation 2 days cells.
Fig. 5 embodiment 2 landrace (piglet) Cardiac Fibroblasts growth regulation 3 days cells.
Fig. 6 embodiment 2 landrace (piglet) Cardiac Fibroblasts growth regulation 4 days cells.
Fig. 7 embodiment 3 Large White (piglet) Cardiac Fibroblasts growth regulation 2 days cells.
Fig. 8 embodiment 3 Large White (piglet) Cardiac Fibroblasts growth regulation 3 days cells.
Fig. 9 embodiment 3 Large White (piglet) Cardiac Fibroblasts growth regulation 4 days cells.
Figure 10 ~ 13Hoechst stained myocardium inoblast.
Embodiment
Embodiment 1:
A kind of Tibetan pig myocardium inoblast isolation cultivation method, comprises the steps:
(1) by birth 2 age in days piglet, after the bromogeramine whole body sterilization of 0.1%, precaval vein bloodletting is put to death rapidly, fixing piglet, 75% alcohol disinfecting skin of chest abdomen.
(2) piglet skin of chest cuts by aseptic operation cutter.
(3) sterile scissors cuts off thoracic cavity forward along last root bone of piglet, takes off heart, puts into immediately and fills 50mL precooling PBS beaker, wash away hemocyte and blood clot.
(4) heart is transferred in sterilized petri dishes by aseptic nipper, intercepts ventricular organization and divests pericardium, adding appropriate precooling PBS and rinse, and rejects blood vessel, fat and reticular tissue, rinsing 3 times.
(5) tissue is cut into 1mm by eye scissors
3left and right fragment is transferred in 50mL Erlenmeyer flask, adds PBS rinsing 3 times, discards washings and leaves tissue block in order to digestion.
(6) add 0.25% trypsinase and 0.1% II Collagenase Type 1:1 mixed enzyme solution, consumption is tissue volume 3 times, puts in 37 DEG C of water-baths after vibration digestion 15min, suction pipe abandoning supernatant.
(7) add mixed enzyme solution again, put vibration digestion 10min in 37 DEG C of water-baths.。
(8) the careful Aspirate supernatant of transfer pipet moves in another sterile centrifugation tube, and adds the DMEM high sugared nutrient solution termination digestion of 10mL containing 10% foetal calf serum.So repeat digestion 5 times.
(9) cell suspension collects centrifuge tube, the centrifugal 5min of 1000RPM/min after filtering respectively with 100 μm and 70 μm of screens, with the high sugared nutrient solution re-suspended cell of DMEM containing 10% foetal calf serum.
(10) differential velocity adherent 1 90min, separation and purification Cardiac Fibroblasts.
(11) cell is obtained as Fig. 1 ~ 3.
Embodiment 2:
A kind of landrace Cardiac Fibroblasts isolation cultivation method, comprises the steps:
(1) by birth 3 age in days piglet, after 0.1% bromogeramine whole body sterilization, rapid precaval vein sacrificed by exsanguination, fixing piglet, 75% alcohol disinfecting skin of chest abdomen.
(2) piglet skin of chest cuts by aseptic operation cutter.
(3) sterile scissors cuts off forward thoracic cavity exposure heart along last root bone, and sterile scissors cuts off pericardium, takes off heart, puts into immediately and fills 50mL precooling PBS beaker, wash away hemocyte and blood clot.
(4) heart is transferred in plate by aseptic nipper, and intercept ventricular organization and divest myocardium, appropriate precooling PBS rinses, and rejects blood vessel, fat and reticular tissue, rinsing 3 times.
(5) heart is cut into 1mm by Sterile ophthalmic scissors
3left and right fragment, transfers in 50mL Erlenmeyer flask, and PBS rinsing 3 times, to wash away hemocyte, discards washings and leaves tissue block in order to digestion.
(6) add 0.25% trypsinase and 0.1% II Collagenase Type 1:1 mixed enzyme solution, consumption is tissue volume 3 times, and put in 37 DEG C of water-baths after vibration digestion 15min, natural sedimentation, abandoning supernatant drawn by suction pipe.
(7) add the mixed enzyme solution containing DNA enzymatic (final concentration is 0.02mg/mL) again, put vibration digestion 8min in 37 DEG C of water-baths.
(8) the careful Aspirate supernatant of transfer pipet moves in another sterile centrifugation tube, adds 10mL10% foetal calf serum DMEM in high glucose nutrient solution and stops digestion.So repeat digestion 5 times.
(9) cell suspension collects centrifuge tube after filtering respectively with 100 μm and 40 μm of screens, the centrifugal 5min of 1000RPM/min, add erythrocyte cracked liquid 5mL, 1000RPM/min is centrifugal, and 5min abandons supernatant, with the high sugared nutrient solution re-suspended cell of DMEM containing 10% foetal calf serum.
(10) obtain total cell suspension and be all placed on freezen protective in ice bucket.
(11) differential velocity adherent 1 90min, separation and purification Cardiac Fibroblasts.
(12) cell obtained is as Fig. 4 ~ 6.
Embodiment 3:
A kind of Large White Cardiac Fibroblasts cultural method, comprises the steps:
(1) by birth 2 age in days piglet, 0.1% bromogeramine whole body sterilization, the rapid sacrificed by exsanguination of precaval vein, fixing piglet, 75% alcohol disinfecting skin of chest abdomen.
(2) piglet skin of chest cuts by aseptic scalpel.
(3) sterile scissors cuts off thoracic cavity forward along last root bone, takes off heart, puts people immediately and fills in 50mL precooling PBS beaker, wash away hemocyte and blood clot.
(4) heart is transferred in sterilized petri dishes by aseptic nipper, and intercept ventricular organization and divest myocardium, appropriate precooling PBS rinses, and rejects blood vessel, fat and reticular tissue, rinsing 3 times.
(5) tissue is cut into 1mm by Sterile ophthalmic scissors
3left and right fragment, transfers in 50mL Erlenmeyer flask, PBS rinsing 3 times, discards washings and leaves tissue block in order to digestion.
(6) add 0.25% trypsinase and 0.1% II Collagenase Type 1:1 mixed enzyme solution, consumption is tissue volume 3 times, after putting in 37 DEG C of water-baths the digestion 15min that vibrates, uses suction pipe Aspirate supernatant.
(7) mixed enzyme solution containing DNA enzymatic (final concentration is 0.02mg/mL) is added again, after putting in 37 DEG C of water-baths the digestion 8min that vibrates.
(8) the careful Aspirate supernatant of transfer pipet moves in another sterile centrifugation tube, and adds the DMEM high sugared nutrient solution of 10mL containing 10% foetal calf serum to stop digestion.
(9) collect centrifuge tube after filtering respectively with 100 μm and 40 μm of screens, the centrifugal 5min of 1000RPM/min, adds erythrocyte cracked liquid 5mL, and 1000RPM/min is centrifugal, and 5min abandons supernatant, with the high sugared nutrient solution re-suspended cell of DMEM containing 10% foetal calf serum.
(10) collecting cell suspension is all placed on freezen protective in ice bucket.
(11) adherent 2 times of continuous differential, each 60min, separation and purification Cardiac Fibroblasts.
(12) cell obtained is as Fig. 7 ~ 9.
(13) as Fig. 7 ~ 9, Cardiac Fibroblasts is irregular shape or ellipse, becomes spindle shape subsequently, and 2 ~ 3d can be Fusion Strain, and cell arrangement is tight, some juxtaposition growths.Cell is smooth, and kytoplasm is transparent, light, and nucleus is comparatively large, often containing 2 ~ 3 cores.
(14) by Fig. 1 ~ 3, Fig. 4 ~ 6 and Fig. 7 ~ 9 are compared, and can find out, according to Cardiac Fibroblasts form stable, active good, the quantity foot of the inventive method separation and Culture, ability of cell proliferation is strong.
Claims (10)
1. a pig myocardium inoblast isolation cultivation method, is characterized in that, comprises the steps:
1) cardiac muscular tissue's sampling: take out piglet heart under aseptic condition, put into and fill 50mL precooling PBS beaker;
At aseptic operating platform, choose piggy ventricle, reject heart Thin Film Tissue, with precooling PBS rinsing repeatedly, tissue is cut into apple puree shape;
2) cardiac muscular tissue's digestion: will shred tissue and transfer in Erlenmeyer flask, the mixed enzyme solution added containing DNA enzymatic digests;
3) pipette supernatant liquid, the DMEM in high glucose nutrient solution added containing 10% ~ 15% foetal calf serum stops digestion, filters respectively, so repeat, until tissue digestion is complete with 100 μm and 40 μm of screens;
4) centrifugally after collecting cell suspension abandon supernatant, add erythrocyte cracked liquid, mix centrifugal, with containing the high sugared nutrient solution re-suspended cell of 10% ~ 15% foetal calf serum DMEM;
5) Cardiac Fibroblasts is cultivated: will obtain cell and be transferred in Tissue Culture Flask, and be placed in 37 DEG C, 5%CO
2difference is carried out adherent in cell culture incubator;
6) discard supernatant liquid, the substratum more renewed continues to cultivate in incubator.
2. before separation Cardiac Fibroblasts, also comprise following pre-treatment step:
1) by organized processing apparatus and glassware cleaning, drying, wrap with kraft paper and put into high-pressure sterilizing pot, 121 DEG C of sterilizing 20 ~ 30min;
2) prepare thimerosal: get bromogeramine, be after 0.1% with distilled water diluting to final concentration, piglet skin surface is carried out disinfection.
3. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, it is characterized in that: during digestion, use 0.25% trypsinase and 0.1% II Collagenase Type 1:1 mixed enzyme solution, consumption is cardiac muscular tissue's volume 3 ~ 5 times.
4. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, is characterized in that: add the Digestive system digestion tissue containing DNA enzymatic (0.02mg/mL) 3 ~ 5mL.
5. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, is characterized in that: in step 2) in, stop digestion with containing the high sugared nutrient solution of 10% ~ 15% foetal calf serum DMEM, dripping quantity is 5 ~ 15mL.
6. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, it is characterized in that: in 37 DEG C of water-baths, digest about 15min, discard clear liquid last time, continue to add mixture slaking liquid digestion 8 ~ 10min, pipette supernatant liquid and add termination digestion in centrifuge tube, repetition like this 3 ~ 8 times, until tissue block complete digestion.
7. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, is characterized in that: erythrocyte cracked liquid consumption is 3 ~ 5mL.
8. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, is characterized in that: collecting cell suspension is as cryopreservation on ice.
9. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, is characterized in that: difference adherent twice, each 60 ~ 90min, does not also have adherent myocardial cell, purifying Cardiac Fibroblasts to remove.
10. piglet Cardiac Fibroblasts isolation cultivation method according to claim 1, is characterized in that: culturing process uses containing the high sugared nutrient solution of 10% ~ 15% foetal calf serum DMEM, changes nutrient solution every 2 ~ 3 days.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN109666633A (en) * | 2019-01-24 | 2019-04-23 | 中国人民解放军军事科学院军事医学研究院 | A method of improving brown fat stem cell myocardiac differentiation efficiency |
| CN110438067A (en) * | 2019-08-22 | 2019-11-12 | 广东唯泰生物科技有限公司 | Human skin fibroblasts and preparation method thereof |
| CN111647550A (en) * | 2020-05-08 | 2020-09-11 | 嘉禾弘生(深圳)健康产业集团有限公司 | Method for culturing human skin fibroblasts by tissue mass enzyme combination method |
| CN111876378A (en) * | 2020-07-15 | 2020-11-03 | 四川大学华西医院 | Method for efficiently separating and culturing myocardial cells and myocardial fibroblasts of suckling mice |
| CN114149964A (en) * | 2021-12-29 | 2022-03-08 | 中国人民解放军陆军军医大学 | Method for separating and extracting fiber cells from mouse heart |
| CN114921402A (en) * | 2022-06-22 | 2022-08-19 | 北京大学第三医院（北京大学第三临床医学院） | Method for extracting adult rat and mouse heart fibroblast |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109666633A (en) * | 2019-01-24 | 2019-04-23 | 中国人民解放军军事科学院军事医学研究院 | A method of improving brown fat stem cell myocardiac differentiation efficiency |
| CN110438067A (en) * | 2019-08-22 | 2019-11-12 | 广东唯泰生物科技有限公司 | Human skin fibroblasts and preparation method thereof |
| CN111647550A (en) * | 2020-05-08 | 2020-09-11 | 嘉禾弘生(深圳)健康产业集团有限公司 | Method for culturing human skin fibroblasts by tissue mass enzyme combination method |
| CN111876378A (en) * | 2020-07-15 | 2020-11-03 | 四川大学华西医院 | Method for efficiently separating and culturing myocardial cells and myocardial fibroblasts of suckling mice |
| CN114149964A (en) * | 2021-12-29 | 2022-03-08 | 中国人民解放军陆军军医大学 | Method for separating and extracting fiber cells from mouse heart |
| CN114921402A (en) * | 2022-06-22 | 2022-08-19 | 北京大学第三医院（北京大学第三临床医学院） | Method for extracting adult rat and mouse heart fibroblast |
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Application publication date: 20160113 |