CN105695395A - Method for separating and enriching vascular endothelial progenitor cells from peripheral blood - Google Patents
Method for separating and enriching vascular endothelial progenitor cells from peripheral blood Download PDFInfo
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- CN105695395A CN105695395A CN201610257564.2A CN201610257564A CN105695395A CN 105695395 A CN105695395 A CN 105695395A CN 201610257564 A CN201610257564 A CN 201610257564A CN 105695395 A CN105695395 A CN 105695395A
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Abstract
The invention relates to the technical field of vascular cell separation, in particular to a method for separating and enriching vascular endothelial progenitor cells from peripheral blood. The method comprises the steps of obtaining a peripheral blood sample, and using PBS phosphate buffer for dilution; adding diluted blood into a hydroxyethyl starch solution, performing centrifuging and sucking monocular cell sap of a middle albuginea cell layer; using the PBS phosphate buffer for dilution, performing centrifuging, abandoning a supernatant, and performing resuspension on residues through a nutrition solution; performing culture transferring. The method is high in separation efficiency, adopts few parenchyma cells, only needs a small number of experiment instruments, is good in separation effect and has the repeatability.
Description
Technical field
The present invention relates to vascular cell separation technology field, particularly to a kind of method of separation and concentration endothelial progenitor cell from peripheral blood。
Background technology
EPC (endothelialprogenitorcells, endothelial progenitor cell) is that a class can circulate, breed and be divided into vascular endothelial cell, but not yet expresses ripe vascular endothelial cell phenotype, does not also form the precursor of blood vessel。The physiology and appearance of this cell injuring model is cobblestone sample endothelial layer, EPC identify general surface be labeled as: CD45 (-), CD34 (+), KDR (+), CD31 (+)。
The EPC cell mass deriving from peripheral blood identified and describe after, the angiogenesis in adult also begins to be in the news out。Substantial amounts of bibliographical information is all at the importance highlighting EPC maintenance the integrity of endothelium。Wherein EPC promotes angiogenesis by playing paracrine effect and self is incorporated into neovascularity and organizes these two approach to maintain the integrity of endothelium。The blood circulation number of EPC and cardiovascular risk factor also exist an inverse relation。EPC is in numerous vascular conditions (vascular trauma, apoplexy, cardiovascular disease, diabetic complication etc.) vascular repair in play an important role, simultaneously EPC can be used as producing the carrier of engineering blood vessel, artificial heart valve and the treatment of cancer, gene therapy。
Along with scientific research personnel to EPC (endothelial progenitor cell) in numerous vascular conditions (vascular trauma, apoplexy, cardiovascular disease, diabetic complication etc.) vascular repair in the understanding that plays an important role constantly deepen, how obtaining quick obtaining EPC becomes primary solution problem in EPC basic research and clinical research。
From peripheral blood, the research of separation and concentration EPC is a research EPC biological characteristics, reconstructing blood vessel and the extraordinary instrument of angiogenesis, is have very important clinical value simultaneously for studying EPC in the mechanism of adult medium vessels reparation。
Separating in present stage and the step of enrichment EPC is different in different bibliographical informations, there is very big-difference in multiple important indicator such as separation efficiency, proliferation potential, and lacks the result of an effective comparability。Comparatively general method for separating and concentrating is: gather people's fresh peripheral blood, from human peripheral blood separate mononuclearcell, recycling be substantially suitable for EPC growth culture medium culturing；Select the single clone of EPC sample according to cellular morphology, recycling surface marker identifies that fluidic cell sorting, RT-PCR are analyzed, whether endotheliocyte picked-up Ac-LDL functional analysis gained cell is EPC。But different seminars, the separation and Culture scheme of different bibliographical information (or applying for a patent), medium component differ greatly, and also difference is huge for the effect (cell proliferation potential etc.) declared。
In present stage due to separate and enrichment EPC step in each team different, and lack an effective comparability result。Simultaneously as the difference that various separation and Culture conditions select, all influence whether kind and the quantity of the cell of separation and concentration including the factor such as ratio of the volume of blood, process time, blood anticoagulant, coating mechanism and hyclone。For in the middle of the basic research of EPC and clinical research, the method for separating and concentrating that mutually can compare between result that equal neither one is unified。Meanwhile, it is commercially available wide variety of EPC culture medium EBM-2 and actually develops for the separation and Culture of Cord blood endothelial progenitor cells (HUVEC), still lack adult's endothelial progenitor cells of peripheral blood special culture media of mature and reliable at present。
Summary of the invention
In order to solve the separation efficiency that above prior art exists in EPC separates, the problem that proliferation potential difference is big, it is contemplated that set up a standard, the method for separation and concentration endothelial progenitor cell from peripheral blood general, efficient, optimum condition in the separation process found out by experiment, make, between each index of separating resulting, there is comparability, solve to separate the problem that cell results is unstable, improve separation and concentration cell yield, for the clinical practice of the basic research of cell physiological characteristic of EPC and vascular repair, regeneration。
The present invention is achieved by the following measures:
A kind of method of separation and concentration endothelial progenitor cell from peripheral blood, comprises the following steps:
(1) peripheral blood sample (processing in 2 hours) of enrichment to be separated is obtained, in centrifuge tube, reverse mixing is once, dilute the blood obtaining dilution with the PBS phosphate buffer 1:1 of pH7.2, in pipe, first add PBS phosphate buffer during dilution, add peripheral blood sample；
(2) 4 50mL centrifuge tubes are taken, each add the hydroxyethyl starch solution 15mL that mass/volume percent concentration is 3.5-5.0%, hydroxyethyl starch solution is slowly added to the blood of 20mL dilution, guarantee that the blood of dilution stays hydroxyethyl starch solution upper strata, room temperature 375-425r/min, centrifugal 30-35min, the mononuclearcell liquid of tunica albuginea cellular layer in the middle of drawing after terminating, about 20mL, the absorption thing of every two pipes merges into a pipe；
(3) 2 new pipes are taken, often pipe adds the absorption thing obtained in 15-20mL step (2), dilute with the PBS phosphate buffer 1:1 of pH7.2 respectively, room temperature 275-325r/min, centrifugal 15-20min, abandoning supernatant, residue 1mL culture fluid is resuspended, and culture fluid consists of addition FGF20.1-0.2mL, VEGF0.1-0.2mL, IGF-10.1-0.2mL, BMP-40.1-0.2mL, LiF0.3-0.5mL and GA0.1-0.2mL in every 80mLEBM-2 basal medium；
(4) 8mL culture fluid is added a wherein pipe, add another pipe after mixing, altogether 10mL suspension；
(5) take 10 μ L and join in 490 μ LDPBS, mixing, therefrom take 10 μ L countings, wherein 4 big lattice sum sums within the visual field are N, total cell number=104× 50 × 10 × N/4, wherein N is visual field inner cell sum, N >=35, total cell number >=4.375 × 107；
(6) Collagenase I spreads Tissue Culture Flask: the Collagenase I105 μ L of 50 μ g/mL is dissolved in the acetic acid 7.5mL of 0.02M, and 0.22 μm of filter filters, and is laid on T75 bottle, 37 DEG C 1 hour, room temperature 1 hour, 10mLDPBS rinses 2 times, adds 5mL culture fluid；10mL suspension step (4) obtained injects, and 48h is later half changes liquid once, operates as extracting 7.5mL original fluid out, is newly added 7.5mL culture fluid, and hereafter every 72h partly changes liquid once, condition of culture CO237 DEG C, CO in incubator2Concentration 5% is cultivated, first 12 days can not observation of cell；
(7) go down to posterity: when cell proliferation to cell density is 90-100%, reach 3 culture bottles, continue to cultivate。
Described method, it is preferable that go down to posterity 10-15 generation。
Described method, it is preferable that in step (2), centrifugal speed is 390-410r/min。
Described method, it is preferable that in step (3), centrifugal speed is 280-300r/min。
Described method, it is preferable that in step (2), centrifugal speed is 380r/min, centrifugal 30min。
Described method, it is preferable that in step (3), centrifugal speed is 280r/min, centrifugal 20min。
When just separating, overwhelming majority cell is that (EPC only accounts for 1/10 to heteroproteose cell6Left and right), and EPC cell cannot observe to go out respectively by profile (being also not easy to the direct detectable concentration of other technologies means) initial 2-4 week。After separation and Culture 2-4 week, cell appearance that EPC cell just starts to occur having typical characteristic also can be observed division growth colony (" colony " means greatly the cell mass that a cell division repeatedly produces afterwards), and propagation colony grows to about 1 and is multiplied by 105Another culture bottle need to be reached during individual cell so that cell distribution obtain comparatively dispersion (condition of culture is the same---Collagenase I spreads Tissue Culture Flask, 15ml culture fluid, CO237 DEG C, CO in incubator2Concentration 5% is cultivated)。From separating calculating the first day, go down to posterity, be cultured to total 100th day sustainable propagation, the endotheliocyte of whole last current state will not be divided into, also would not observe that obvious apoptosis, necrosis。
Beneficial effects of the present invention:
(1) present invention has carried out standardization the program of separation and concentration human endothelial progenitor cells from peripheral blood；
(2) in operation, user can conveniently be easily separated, it is only necessary to provides a small amount of experiment equipment；
(3) separating effect is better, and separation efficiency is high, and heteroproteose cell is few, has repeatability, is particularly suited for the separation of endothelial progenitor cells of peripheral blood of human body；
(4) present invention can the needs of volume per sample, the reagent solution being proportionally added into different volumes is added, and decreases the waste of reagent, and user can be tried one's best all reagent utilized fully in test kit。
Accompanying drawing explanation
There is EPC colony when being embodiment 1 separation and Culture the 15th day in Fig. 1, and in figure, relatively minicell is heteroproteose cell；
Fig. 2 is that embodiment 1EPC cell colony grows to local shooting in the 21st day (colony central authorities)；
Fig. 3 is that embodiment 1EPC cell colony grows to the 21st day local shooting (colony edge)；
Fig. 4 is that embodiment 1EPC goes down to posterity and grows to density 100% after 1 time, local shooting；
Fig. 5 is that embodiment 1EPC goes down to posterity and grows to density 100% after 5 times, local shooting；
Fig. 6 is that embodiment 1 the 15th generation cell is taken pictures。
Detailed description of the invention
In order to be better understood from the present invention, further illustrate below in conjunction with specific embodiment。
Embodiment 1:
The method of separation and concentration endothelial progenitor cell from peripheral blood, comprises the following steps:
(1) the peripheral blood sample 30mL (processing in 2 hours) of enrichment to be separated is obtained, in centrifuge tube, reverse mixing is once, dilute the blood obtaining dilution with the PBS phosphate buffer 1:1 of pH7.2, in pipe, first add PBS phosphate buffer during dilution, add peripheral blood sample；
(2) 4 50mL centrifuge tubes are taken, each add the hydroxyethyl starch solution 15mL that mass/volume percent concentration is 5.0%, hydroxyethyl starch solution is slowly added to the blood of 20mL dilution, guarantee that the blood of dilution stays hydroxyethyl starch solution upper strata, room temperature 375r/min, centrifugal 35min, the mononuclearcell liquid of tunica albuginea cellular layer in the middle of drawing after terminating, about 20mL, the absorption thing of every two pipes merges into a pipe；
(3) 2 new pipes are taken, often pipe adds the absorption thing obtained in 15-20mL step (2), dilute with the PBS phosphate buffer 1:1 of pH7.2 respectively, room temperature 275r/min, centrifugal 20min, abandoning supernatant, residue 1mL culture fluid is resuspended, and culture fluid consists of addition FGF20.1mL, VEGF0.2mL, IGF-10.1mL, BMP-40.2mL, LiF0.3mL and GA0.2mL in every 80mLEBM-2 basal medium；
(4) 8mL culture fluid is added a wherein pipe, add another pipe after mixing, altogether 10mL suspension；
(5) take 10 μ L and join in 490 μ LDPBS, mixing, therefrom take 10 μ L countings, wherein 4 big lattice sum sums within the visual field are N, N is 35, and total cell number is 4.375 × 107；
(6) Collagenase I spreads Tissue Culture Flask: the Collagenase I105 μ L of 50 μ g/mL is dissolved in the acetic acid 7.5mL of 0.02M, and 0.22 μm of filter filters, and is laid on T75 bottle, 37 DEG C 1 hour, room temperature 1 hour, 10mLDPBS rinses 2 times, adds 5mL culture fluid；10mL suspension step (4) obtained injects, and 48h is later half changes liquid once, operates as extracting 7.5mL original fluid out, is newly added 7.5mL culture fluid, and hereafter every 72h partly changes liquid once, condition of culture CO237 DEG C, CO in incubator2Concentration 5% is cultivated, first 12 days can not observation of cell；
(7) go down to posterity: when cell proliferation to cell density is 90-100%, reach 3 culture bottles, before being passaged to for the 10th generation, would not observe that obvious cell differentiation, apoptosis or necrosis；From separating calculating the first day, going down to posterity, be cultured to total 100th day sustainable propagation, undifferentiated be the endotheliocyte of last current state at end, does not also observe obvious apoptosis, necrosis。Reach 14-15 for time, cell occur differentiation or stop propagation。
Cell is cultivated situation and is seen accompanying drawing 1-6, and amplification is 40 times。Fig. 1. being cultured to the 15th day after separation, it was observed that typical " paving stone " shape EPC cell proliferation colony, each 15-30 cell of colony proliferation number is not etc.；Fig. 2. colony growth was to the 21st day, and cell number is multiplied by 4 powers of 10 more than 1, and colony intermediate cell gap is less；Fig. 3. colony growth was to the 21st day, and cell number is multiplied by 4 powers of 10 more than 1, and colony edge intercellular substance is bigger；Fig. 4. going down to posterity and cultivate more than 7 days after 1 time, cell density reaches close to 100%；After Fig. 5 .EPC is passaged to the 5th, cultivating more than 7 days, grow to density 100%, form, growth rate are unchanged compared with previously；Fig. 6. passage is to the 15th generation, and cell proliferation rate substantially slows down, and from from going down to posterity 20 days, density not yet reached 50%。
Embodiment 2:
The method of separation and concentration endothelial progenitor cell from peripheral blood, comprises the following steps:
(1) the peripheral blood sample 60mL (processing in 2 hours) of enrichment to be separated is obtained, in centrifuge tube, reverse mixing is once, dilute the blood obtaining dilution with the PBS phosphate buffer 1:1 of pH7.2, in pipe, first add PBS phosphate buffer during dilution, add peripheral blood sample；
(2) 4 50mL centrifuge tubes are taken, each add the hydroxyethyl starch solution 15mL that mass/volume percent concentration is 3.5%, hydroxyethyl starch solution is slowly added to the blood of 20mL dilution, guarantee that the blood of dilution stays hydroxyethyl starch solution upper strata, room temperature 425r/min, centrifugal 30min, the mononuclearcell liquid of tunica albuginea cellular layer in the middle of drawing after terminating, about 20mL, the absorption thing of every two pipes merges into a pipe；
(3) 2 new pipes are taken, often pipe adds the absorption thing obtained in 20mL step (2), dilute with the PBS phosphate buffer 1:1 of pH7.2 respectively, room temperature 275r/min, centrifugal 20min, abandoning supernatant, residue 1mL culture fluid is resuspended, and culture fluid consists of addition FGF20.1mL, VEGF0.2mL, IGF-10.1mL, BMP-40.2mL, LiF0.3mL and GA0.2mL in every 80mLEBM-2 basal medium；
(4) 8mL culture fluid is added a wherein pipe, add another pipe after mixing, altogether 10mL suspension；
(5) take 10 μ L and join in 490 μ LDPBS, mixing, therefrom take 10 μ L countings, wherein 4 big lattice sum sums within the visual field are N, N is 37, and total cell number is 4.625 × 107；
(6) Collagenase I spreads Tissue Culture Flask: the Collagenase I105 μ L of 50 μ g/mL is dissolved in the acetic acid 7.5mL of 0.02M, and 0.22 μm of filter filters, and is laid on T75 bottle, 37 DEG C 1 hour, room temperature 1 hour, 10mLDPBS rinses 2 times, adds 5mL culture fluid；10mL suspension step (4) obtained injects, and 48h is later half changes liquid once, operates as extracting 7.5mL original fluid out, is newly added 7.5mL culture fluid, and hereafter every 72h partly changes liquid once, condition of culture CO237 DEG C, CO in incubator2Concentration 5% is cultivated, first 12 days can not observation of cell；
(7) go down to posterity: when cell proliferation to cell density is 90-100%, reach 3 culture bottles, go down to posterity and would not observe that obvious cell differentiation, apoptosis or necrosis；Generally reach 12-13 for time, cell occur differentiation or stop propagation。
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention also should not be limited by the examples; the change made under other any spirit without departing from the present invention and principle, modification, combination, replacement, simplification all should be equivalence substitute mode, are included within protection scope of the present invention。
Claims (8)
1. the method for separation and concentration endothelial progenitor cell from peripheral blood, it is characterised in that comprise the following steps:
(1) obtaining the peripheral blood sample of enrichment to be separated, in centrifuge tube, reverse mixing once, dilutes the blood obtaining dilution with the PBS phosphate buffer 1:1 of pH7.2, first adds PBS phosphate buffer in pipe, add peripheral blood sample during dilution；
(2) 4 50mL centrifuge tubes are taken, each add the hydroxyethyl starch solution 15mL that mass/volume percent concentration is 3.5-5.0%, hydroxyethyl starch solution is slowly added to the blood of 20mL dilution, guarantee that the blood of dilution stays hydroxyethyl starch solution upper strata, room temperature 375-425r/min, centrifugal 30-35min, the mononuclearcell liquid of tunica albuginea cellular layer in the middle of drawing after terminating, the absorption thing of every two pipes merges into a pipe；
(3) 2 new pipes are taken, often pipe adds the absorption thing obtained in 15-20mL step (2), dilute with the PBS phosphate buffer 1:1 of pH7.2 respectively, room temperature 275-325r/min, centrifugal 15-20min, abandoning supernatant, residue 1mL culture fluid is resuspended, and culture fluid consists of addition FGF20.1-0.2mL, VEGF0.1-0.2mL, IGF-10.1-0.2mL, BMP-40.1-0.2mL, LiF0.3-0.5mL and GA0.1-0.2mL in every 80mLEBM-2 basal medium；
(4) 8mL culture fluid is added a wherein pipe, add another pipe after mixing, altogether 10mL suspension；
(5) counting；
(6) Collagenase I spreads Tissue Culture Flask: the Collagenase I105 μ L of 50 μ g/mL is dissolved in the acetic acid 7.5mL of 0.02M, and 0.22 μm of filter filters, and is laid on T75 bottle, 37 DEG C 1 hour, room temperature 1 hour, 10mLDPBS rinses 2 times, adds 5mL culture fluid；10mL suspension step (4) obtained injects, and 48h is later half changes liquid once, operates as extracting 7.5mL original fluid out, is newly added 7.5mL culture fluid, and hereafter every 72h partly changes liquid once, condition of culture CO237 DEG C, CO in incubator2Concentration 5%；
(7) go down to posterity: when cell proliferation to cell density is 90-100%, reach 3 culture bottles。
2. method according to claim 1, it is characterised in that go down to posterity 10-15 generation。
3. method according to claim 1, it is characterised in that in step (2), centrifugal speed is 390-410r/min。
4. method according to claim 1, it is characterised in that in step (3), centrifugal speed is 280-300r/min。
5. method according to claim 1, it is characterised in that in step (2), centrifugal speed is 380r/min, centrifugal 30min。
6. method according to claim 1, it is characterised in that in step (3), centrifugal speed is 280r/min, centrifugal 20min。
7. method according to claim 1, it is characterized in that in step (5), counting operation is that the suspension 10 μ L taken in step (4) joins in 490 μ L Du Shi phosphate buffer DPBS, mixing, therefrom take 10 μ L countings, wherein 4 big lattice total cellular score sums within the visual field are N, total cell number=104×50×10×N/4。
8. method according to claim 7, it is characterised in that N >=35, total cell number >=4.375 × 107。
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| CN109897817A (en) * | 2019-03-21 | 2019-06-18 | 广州沙艾生物科技有限公司 | The method and its application of isolating immune cells |
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