CN104673744B - A kind of vascular endothelial cells extracorporeal culturing method - Google Patents
A kind of vascular endothelial cells extracorporeal culturing method Download PDFInfo
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Abstract
The present invention provides a kind of vascular endothelial cells extracorporeal culturing method, including taking rat aorta, is rinsed, is immersed in the DMEM/F12 culture solutions containing heparin with sterile PBS liquid；Aortic tunica intima is flipped out using sterile needlework, and is knotted at both ends；Sustainer after overturning is placed in clostridiopetidase A and is digested；After being cultivated in 37 DEG C of incubators, the sustainer being digested is put into the DMEM/F12 culture solutions of serum-free, and is divided into the sustainer thin slice of fritter；Sustainer thin slice is subjected to adhere-wall culture, the DMEM/F12 culture solutions containing heparin and serum are immersed in the aortic tunica intima part of sustainer thin slice, and a culture solution is replaced every 48h；3rd 5 day, sustainer thin slice is removed, culture solution is replaced daily afterwards, substantial amounts of vascular endothelial cell can be obtained within the 9th day, after the 14th day cell can be used for pass on.
Description
Technical field
The invention belongs to pharmaceutical technical field more particularly to a kind of methods of vascular endothelial cells in vitro culture.
Background technology
Angiocardiopathy is that a kind of disease of great threat is formed to human health.The formation of atherosclerotic lesion and
Revascularization after injury of blood vessel is directed to the activation of vascular endothelial cell, and vascular endothelial cell is the reason of vascular pathological research
Think vehicles cells, therefore how easily, steadily the vascular endothelial cell for obtaining purifying is always a popular research direction.Mesh
Before have the cultural method of substantial amounts of documents and materials report human umbilical vein endothelial cell and its widely apply to vascular pathological
Research, but since human umbilical vein endothelial cell is from vein rather than artery, so limiting its researching value；The same period
Also documents and materials are reported from muroid, eyeball and the vascular endothelial cell of lung primary culture method, but most of originals
Special instrument, such as magnetic bead, specific antibody, FACS selection etc. are required for cultural method.
For a long time, since mouse aorta pectoralis caliber is tiny, intercostal arteries branch is more, although having using enzymic digestion and inner membrance
The methods of overturning, but primary be separately cultured of thoracic aorta endothelium cell is asked there are still cell pick-up rate and purity are more low
Topic is restricted its application.Overturning blood vessel is also needed in digestion process, not only increases the risk of pollution, is also directly affected interior
The vigor of chrotoplast.Inner membrance inversion method is the method more used in recent years, and the blood vessel endothelium that can obtain higher degree is thin
The primary isolated culture method of born of the same parents, but the key point of this method is to need to rout up endangium.It is difficult to avoid that during routing up thin
The friction of intercellular adds the probability of cellular damage and pollution.160-220g rat aortas caliber only about 2mm, in addition blood vessel
Wall its thickness, thus inner membrance overturning operation success rate it is relatively low and to materials blood vessel also underuse.
The content of the invention
In view of this, the present invention is directed to propose a kind of vascular endothelial cells extracorporeal culturing method, to overcome existing skill
The shortcomings that art.
In order to achieve the above objectives, the technical solution of the invention is realized in：A kind of vascular endothelial cells
Extracorporeal culturing method includes the following steps：
(1) rat aorta is taken, is rinsed with sterile PBS liquid, is then immersed in the DMEM/F12 trainings containing heparin
With spare in nutrient solution；
(2) aortic tunica intima is flipped out using sterile needlework, and knotted at both ends；
(3) sustainer after overturning is placed in II Collagenase Types and digested；
(4) after being cultivated in 37 DEG C of incubators, the sustainer being digested is put into the DMEM/F12 culture solutions of serum-free, by master
Two leaf of artery, which is cut off, becomes hollow tubular sample, and the sustainer thin slice cut off along longitudinal direction and be divided into fritter is standby
With；
(5) sustainer thin slice is subjected to adhere-wall culture, the aortic tunica intima part of the sustainer thin slice is immersed containing heparin
With the DMEM/F12 culture solutions of serum, a culture solution is replaced every 48h；
(6) the 3-5 days, sustainer thin slice is removed, replaces culture solution daily afterwards, the culture solution is identical with step (5),
Substantial amounts of vascular endothelial cell can be obtained within 9th day, after the 14th day cell can be used for pass on.
Further, the DMEM/F12 culture solutions heparin containing 1000U/ml in the step (1).
Further, clostridiopetidase A is II Collagenase Types or type i collagen enzyme in the step (3), and the concentration of the clostridiopetidase A is
2g/L。
Preferably, cultivated 60 minutes in 37 DEG C of incubators in the step (4).
Preferably, the sustainer of the step (4) may be partitioned into 4-5 block sustainer thin slices, the sustainer thin slice it is big
Small is 2cm*4cm, which obtains sustainer thin slice and be highly suitable for primary adhere-wall culture.
Further, in the DMEM/F12 culture solutions of the step (5) serum content be 20%, heparin content 50ug/ml.
Preferably, aortic tunica intima is flipped out using sterile needlework in the step (2), and the tool to knot at both ends
Body method is：4-0 nylon wires are fastened with a sterile fine needle, by sterile needle thread passing sustainer inner cavity, by sterile needle after passing through
It cuts, is knotted and fixed in one end of sustainer using nylon wire；The one end not knotted using ophthalmic tweezers fixation nylon wire, Ran Houli
Separated sustainer is overturn along knotted end with another ophthalmic tweezers, gently pull aortic tunica adventitia until entire sustainer is
Inwardly, one end that the sustainer after overturning is not knotted knots aortic tunica adventitia aortic tunica intima outwardly.
Preferably, the mode of adhere-wall culture is that sustainer thin slice is laid on six orifice plates in the step (5), six holes
DMEM/F12 culture solutions of the 800ul containing heparin and serum is added in only aortic tunica intima to be made to be immersed in culture solution per hole in plate
In rather than the entire sustainer including aortic tunica adventitia immerse wherein.In whole process, sustainer thin slice need to be kept to paste always
Wall is also required to prevent mobile sustainer thin slice when replacing culture solution.
Preferably, after removing sustainer thin slice in the step (6), culture solution is replaced daily, and addition is 3ml/ holes.
Compared with the prior art, vascular endothelial cells extracorporeal culturing method of the present invention has the advantage that：
1. simple and quick, repeatability is high, short the time required to experiment, greatly reduces pollution and the influence of Human Umbilical Vein Endothelial Cells activity；
2. blood vessel amount is few needed for, takes full advantage of materials blood vessel, is separately cultured a rat chest aorta is taken to may be partitioned into 4-5 every time
Fritter, a fritter tissues, which can be stablized, readily obtains a large amount of vascular endothelial cells；3. it is grown at first using vascular endothelial cell
The characteristics of, tissue block is discarded in due course, reduces the opportunities for contamination of fibroblast and smooth muscle cell；4. add in quantitative culture
Liquid only makes inner membrance be contacted with culture solution, and outer membrane is avoided to contact culture solution, can reduce the opportunities for contamination of pericyte etc.；5. training
Somatomedin class substance need not be added in nutrient solution, makes this method more economical and practical；6. sterile needlework are utilized by endangium
It flips out, intercellular friction and damage can be avoided to the greatest extent, simultaneously as needlework are all sterile, so cell
The probability of pollution is very low, and the success rate of the method turning operation is close to 100%.
Description of the drawings：
After Fig. 1 is primary endothelial cell culture the 3rd day, photo under * 100 mirrors
Fig. 2 is photo under * 200 mirrors after primary endothelial cell culture the 3rd day
Fig. 3 is photo under * 400 mirrors after primary endothelial cell culture the 3rd day
Fig. 4 is photo under * 100 mirrors after primary endothelial cell culture the 4th day
Fig. 5 is photo under * 200 mirrors after primary endothelial cell culture the 4th day
Fig. 6 is photo under * 400 mirrors after primary endothelial cell culture the 4th day
Fig. 7 is photo under * 100 mirrors after primary endothelial cell culture the 5th day
Fig. 8 is photo under * 200 mirrors after primary endothelial cell culture the 5th day
Fig. 9 is photo under * 400 mirrors after primary endothelial cell culture the 5th day
Figure 10 is photo under * 100 mirrors after primary endothelial cell culture the 7th day
Figure 11 is photo under * 200 mirrors after primary endothelial cell culture the 7th day
Figure 12 is photo under * 100 mirrors after primary endothelial cell culture the 7th day
Figure 13 is photo under * 100 mirrors after primary endothelial cell culture the 9th day
Figure 14 is photo under * 200 mirrors after primary endothelial cell culture the 9th day
Figure 15 is photo under * 400 mirrors after primary endothelial cell culture the 9th day
Figure 16 is photo under * 100 mirrors after primary endothelial cell culture fortnight
Figure 17 is CD31 antibody mediated immunity cytochemistries SABC colour developing photos
Figure 18 is two analysis resistant photo of CD31 antibody mediated immunity cytochemistry FITC fluorescence
Figure 19 is from the separated primary endothelial cell of rat chest aorta and cultured cell line, in Matrigel matrigels
Photo after middle culture 2h
Figure 20 is from the separated primary endothelial cell of rat chest aorta and cultured cell line, in Matrigel matrigels
Photo after middle culture 6h
Figure 21 be implementation result example 4 in 1-3 for cell growth curve, wherein series 1 be the vascular endothelial cell first generation；
Series 2 is the vascular endothelial cell second generation；Series 3 is the vascular endothelial cell third generation
Specific embodiment
Embodiment 1
Vascular endothelial cells extracorporeal culturing method includes the following steps：
1st, rat aorta is taken, separated sustainer is rinsed twice with sterile PBS liquid, is then immersed in DMEM/F12
With spare in the culture solution of heparin containing 1000U/ml.
2nd, it is spare to fasten 4-0 nylon wires for a sterile fine needle, will be sterile after passing through by sterile needle thread passing sustainer inner cavity
Pin is cut, and is knotted and fixed in one end of sustainer using nylon wire.The one end not knotted using ophthalmic tweezers fixation nylon wire, then
Separated sustainer is overturn along knotted end using another ophthalmic tweezers, gently pull aortic tunica adventitia until entire sustainer
It is that inwardly, one end that the sustainer after overturning is not knotted knots aortic tunica adventitia aortic tunica intima outwardly.
3rd, the sustainer after overturning that will knot, which is placed in II Collagenase Types, to be digested so that aortic tunica intima completely contacts glue
Protoenzyme, the concentration of II Collagenase Types is 2g/L.Since sustainer is overturn and is knotted, aortic tunica adventitia can not be by collagenase digesting.
4th, cultivated in 37 DEG C of incubators after sixty minutes, the sustainer being digested is put into and is cultivated in ware, wherein filling containing nothing
Then the DMEM/F12 culture solutions of serum are cut two leaf of sustainer so that it becomes hollow tubular sample.It cuts off along longitudinal direction
Hollow sustainer opens tubulose sample sustainer and is divided into that 4-5 fritter sustainer thin slices are spare, and sustainer thin slice size is usual
It is 2cm*4cm.These sustainer thin slices being digested are highly suitable for primary adhere-wall culture.
5th, one piece of sustainer thin slice is laid on six orifice plates, in six orifice plates per hole add in 800ul containing 20% serum,
The DMEM/F12 culture solutions of 50ug/ml heparin are only to make aortic tunica intima be immersed in culture solution rather than including outer membrane
Entire sustainer immerse wherein.It keeps sustainer thin slice adherent always, and culture solution is replaced once when 48 is small, simultaneously
Mobile sustainer thin slice need to be prevented when replacing culture solution for the first time.
6th, at the 3rd day or the 4th day, visible minority vascular endothelial cell around sustainer thin slice.If blood vessel endothelium
For cell, it is apparent that removable sustainer thin slice, the time for removing sustainer thin slice is not later than 120 hours.Then daily more
Change culture solution, 3ml/ holes.At the 9th day, we will obtain substantial amounts of vascular endothelial cell, and fortnight can be used for cell biography
Generation.The sustainer being generally separated can be divided into 4-5 block sustainer thin slices, and every piece of sustainer thin slice will obtain a large amount of at the 9th day
Vascular endothelial cell.
1 growing state of implementation result example and morphological observation：
* 100 Microscopic observations after primary endothelial cell culture the 3rd day, sustainer chip edge is visible a little to be sporadicly distributed
Flat, fusiformis or polygon cell, are shown in Fig. 1.* 200 Microscopic observations after primary endothelial cell culture the 3rd day, are shown in Fig. 2.In primary
* 400 Microscopic observations after chrotoplast culture the 3rd day, are shown in Fig. 3.* 100 Microscopic observations after primary endothelial cell culture the 4th day, it is bright
Showing flat visible sustainer chip edge, fusiformis or polygon cell quantity increases, and part cell is migrated to outside, sees Fig. 4.
* 200 Microscopic observations after primary endothelial cell culture the 4th day, are shown in Fig. 5.It is seen after primary endothelial cell culture the 4th day under * 400 mirrors
It examines, sees Fig. 6.* 100 Microscopic observations after primary endothelial cell culture the 5th day, remove sustainer thin slice, visible cell quantity under mirror
It is multiplied, form is uniform in size mostly into fusiformis or polygon, sees Fig. 7.After primary endothelial cell culture the 5th day under * 200 mirrors
Observation, is shown in Fig. 8.* 400 Microscopic observations after primary endothelial cell culture the 5th day, are shown in Fig. 9.Primary endothelial cell culture the 7th day
* 100 Microscopic observation afterwards, it is seen that cell quantity increases severely, and illustrates that ability of cell proliferation is strong, sees Figure 10.Primary endothelial cell culture
* 200 Microscopic observations, are shown in Figure 11 after seven days.* 100 Microscopic observations after primary endothelial cell culture the 7th day, are shown in Figure 12.In primary
* 100 Microscopic observations after chrotoplast culture the 9th day, cell quantity further increase, and multiplication rate is accelerated, and sees Figure 13.In primary
* 400 Microscopic observations after chrotoplast culture the 9th day, are shown in Figure 15.* 100 Microscopic observations after primary endothelial cell culture fortnight,
After visible cell growth fusion forms individual layer, in typical cobblestone or paving stone zyklopisch aligned growth, and there is contact to press down
Phenomenon processed, at this time can secondary culture see Figure 16.
Implementation result example 2CD31 immunocytes are analyzed：
CD31 antibody mediated immunities cytochemistry (1) SABC develops the color, it is seen that fallow positive cell in endochylema (see Figure 17)；
(2) two analysis resistant of FITC fluorescence, it is seen that endochylema Green fluorescence strong positive cell (see Figure 18).
3 vascular endothelial cell of implementation result example is tested into pipe：
From the separated primary endothelial cell of rat chest aorta and cultured cell line, cultivated in Matrigel matrigels
After 2h, visible formation part tubular structure (see Figure 19)；Complete blood vessel sample tubule is formed to culture 6h is visible (see Figure 20).
4 vascular endothelial cell secondary culture growth curve of implementation result example
Primary rat chest aorta vascular endothelial cell, the second generation and the third generation are taken, adjusts cell density to 2*105/ml。
24 orifice plates add in cell suspension 100ul, the DMEM/F12 culture solution 900ul containing 20%FBS, in 37 DEG C, 5%CO per hole2Culture.
Start within 2nd day, daily 4 hole continuous counter 7d, draw growth curves of the 1-3 for cell respectively.Rat chest aorta blood vessel endothelium
Cell has carried out three generations's secondary culture, and unchanged in different generation cellular morphologies, cell growth curve is shown in Figure 21.Cell is in sub-bottle
2 days to exponential phase afterwards, in identical incubation time difference generation cell number no significant difference (P ＞ 0.05).
Presently preferred embodiments of the present invention is described in detail above, but the content is only the preferable implementation of the present invention
Example, it is impossible to be construed as limiting the practical range of the present invention.All all the changes and improvements made according to the present patent application scope
Deng, should all still belong to the present invention patent covering scope within.
Claims (10)
1. a kind of vascular endothelial cells extracorporeal culturing method, which is characterized in that include the following steps：
(1) rat aorta is taken, is rinsed with sterile PBS liquid, is then immersed in the DMEM/F12 culture solutions containing heparin
In with spare；
(2) sterile needle is cut behind the sterile needle thread passing sustainer inner cavity that nylon wire will be fastened, using nylon wire in sustainer
One end, which knots, to be fixed；One end that fixed nylon wire does not knot, pulls aortic tunica adventitia and turns over separated sustainer along knotted end
Turn, until entire sustainer is that aortic tunica adventitia is inwardly outwardly for aortic tunica intima；
(3) sustainer after overturning is placed in clostridiopetidase A and digested；
(4) after being cultivated in 37 DEG C of incubators, the sustainer being digested is put into the DMEM/F12 culture solutions of serum-free, by sustainer
Two leafs, which are cut off, becomes hollow tubular sample, and the sustainer thin slice cut off along longitudinal direction and be divided into fritter is spare；
(5) sustainer thin slice is subjected to adhere-wall culture, the aortic tunica intima part of the sustainer thin slice is immersed containing heparin and blood
Clear DMEM/F12 culture solutions replace a culture solution every 48h；
(6) the 3-5 days, sustainer thin slice is removed, replaces culture solution daily afterwards, the culture solution is identical with step (5), and the 9th
It can obtain substantial amounts of vascular endothelial cell, after the 14th day cell can be used for pass on.
2. vascular endothelial cells extracorporeal culturing method according to claim 1, it is characterised in that：The step (1)
In DMEM/F12 culture solutions heparin containing 1000U/ml.
3. vascular endothelial cells extracorporeal culturing method according to claim 1, it is characterised in that：The step (3)
Middle clostridiopetidase A is II Collagenase Types or type i collagen enzyme.
4. vascular endothelial cells extracorporeal culturing method according to claim 3, it is characterised in that：The II Collagen Type VIs
The concentration of enzyme or type i collagen enzyme is 2g/L.
5. vascular endothelial cells extracorporeal culturing method according to claim 1, it is characterised in that：The step (4)
In cultivate 60 minutes in 37 DEG C of incubators.
6. vascular endothelial cells extracorporeal culturing method according to claim 1, it is characterised in that：The step (4)
Sustainer may be partitioned into 4-5 block sustainer thin slices, the size of the sustainer thin slice is 2cm*4cm.
7. vascular endothelial cells extracorporeal culturing method according to claim 1, it is characterised in that：The step (5)
DMEM/F12 culture solutions in serum content be 20%, heparin content 50ug/ml.
8. according to claim 1-7 any one of them vascular endothelial cells extracorporeal culturing methods, which is characterized in that institute
It states in step (2) and is flipped out aortic tunica intima using sterile needlework, and be in the specific method that both ends knot：With a nothing
Bacterium fine needle fastens 4-0 nylon wires, by sterile needle thread passing sustainer inner cavity, cuts sterile needle after passing through, is existed using nylon wire
One end of sustainer, which knots, to be fixed；The one end not knotted using ophthalmic tweezers fixation nylon wire, then will using another ophthalmic tweezers
Separated sustainer is overturn along knotted end, gently pull aortic tunica adventitia until entire sustainer is that aortic tunica intima is led outwardly
Inwardly, one end that the sustainer after overturning is not knotted knots tunica adventitia of artery.
9. according to claim 1-7 any one of them vascular endothelial cells extracorporeal culturing methods, which is characterized in that institute
The mode for stating adhere-wall culture in step (5) is that sustainer thin slice is laid on six orifice plates, adds in 800ul in six orifice plates per hole
DMEM/F12 culture solutions containing heparin and serum are only to make aortic tunica intima be immersed in culture solution.
10. vascular endothelial cells extracorporeal culturing method according to claim 9, which is characterized in that the step (6)
After middle removal sustainer thin slice, culture solution is replaced daily, and addition is 3ml/ holes.
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| CN104988113B (en) * | 2015-07-22 | 2018-06-01 | 南阳师范学院 | A kind of separation of cavy arteria carotis communis endothelial cell and cultural method |
| CN109055298A (en) * | 2018-08-10 | 2018-12-21 | 佛山科学技术学院 | A kind of isolated culture method of primary dog vascular endothelial cell |
| CN109679894A (en) * | 2019-02-28 | 2019-04-26 | 深圳市龙华区中心医院 | A kind of method of separating small pig endothelial cell |
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| CN104031879A (en) * | 2014-05-21 | 2014-09-10 | 北京农学院 | Method for in-vitro separation and culture of rat brain microvessel endothelial cells |
| CN104371968A (en) * | 2014-06-25 | 2015-02-25 | 北京农学院 | Method for separating and purifying mouse lung microvascular endothelial cell |
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Patent Citations (7)
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| CN101463341A (en) * | 2008-01-31 | 2009-06-24 | 中国人民解放军第四军医大学 | Isolation in vitro and amplification method for adult rat heart microvascular endothelial cell |
| CN102475913A (en) * | 2010-11-22 | 2012-05-30 | 大连创达技术交易市场有限公司 | Valve tissue built through tissue engineering |
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