CN101525595B - Method for largely expanding late endothelial progenitor cells from peripheral blood - Google Patents
Method for largely expanding late endothelial progenitor cells from peripheral blood Download PDFInfo
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- CN101525595B CN101525595B CN2009100685116A CN200910068511A CN101525595B CN 101525595 B CN101525595 B CN 101525595B CN 2009100685116 A CN2009100685116 A CN 2009100685116A CN 200910068511 A CN200910068511 A CN 200910068511A CN 101525595 B CN101525595 B CN 101525595B
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Abstract
The invention discloses a method for largely expanding endothelial outgrowth cells from peripheral blood. Mononuclear cells separated from the peripheral blood are planted to a pretreated culture dish spread with endothelial outgrowth cells or umbilical endothelial cells as nutritive layers to obtain more endothelial outgrowth cell colonies, and then the endothelial outgrowth cells are largely expanded. By previously spreading the nutritive layer cells containing endothelial cells in the culture dish before planting the mononuclear cells of the peripheral blood, and then planting the mononuclear cells in the culture dish, the method can obtain colony numbers of the endothelial outgrowth cells 20 times more than the traditional method about two weeks in advance, and can obtain the endothelial outgrowth cells with high quality, high quantity and treatment value from the limited peripheral blood.
Description
Technical field
The present invention relates to biological technical field, thus but especially a kind of cultural method that from human peripheral, obtains more late endothelial progenitor cells colony largely expanding late endothelial progenitor cells.
Background technology
People such as Asahara were in report adult peripheral blood CD34 in 1997
+Cell mass is divided into endothelioid cells external can inducing, the a series of endotheliocyte antigens of this class cell expressing, have and participate in angiopoietic function in vivo, be named as endothelial progenitor cells (Endothelial progenitor cells, EPCs), thereby upgraded the notion of angiogenesis, promptly in becoming human body, not only existed the angiogenesis phenomenon (angiogenesis) that it has been recognized that, also existed vasculogenesis phenomenon (vasculogenesis).Further investigation along with endothelial progenitor cells, reports such as Hur J EPCs can be divided into early stage EPCs and late period EPCs, describing as them is the culture plate of the mononuclearcell kind from the peripheral blood source of certain density having been gone into to spread fibronectin or collagen, then with the commercial culture medium culturing that contains VEGF. cultivating had adherent cell to occur in 4-7 days, cellular form is fusiformis, more vigorous in 2-3 week growth, 4 weeks began death later, present limited multiplication capacity, passage number is limited, and this class cell occurs early claiming early stage endothelial progenitor cells, real be the EPCs that relatively breaks up, and this also is our EPCs of indication usually.Cultivating the EPCs that occurs late period is to continue to cultivate at above mononuclearcell 2-3 week to begin to grow, be the growth of colony shape, cellular form is cobblestone-appearance, and is vigorous in 4-8 week growth, can grow to for 12 weeks, multiplication capacity is stronger, can pass about 30 generations, this class cell of proofs such as Lin derives from marrow, may be the offspring of blood blood vessel stem cell (hemangioblast), its growth characteristics shows that it belongs to the EPCs in differentiation stage morning, is called late endothelial progenitor cells.Function ratio is proof: the early stage EPCs of EPCs in late period has the ability that forms blood vessel in stronger ability that is integrated into endothelium and the body.Because the EPCs in bleeding of the umbilicus source exists the immunogenicity problem in clinical application, thus at present more can near clinical be EPCs in the autologous peripheral blood.But the problem that exists is into the content rareness of endothelial progenitor cells in the human peripheral at present, cardiovascular risk factors is particularly arranged, in patient's body that old-age group and heart failure need EPC to treat few relatively EPCs quantity is arranged, and the function of learning EPCs formation blood vessel in some diseases patient body from existing analysis of clinical reduces, quantity EPCs rare and that function lowers has limited it in clinical effective application in a word, so obtain high quantity and high-quality EPCs is the difficult point that present EPCs research field is badly in need of solution.
The method of cultivation EPCs in late period commonly used is as follows at present: separate the mononuclearcell layer with known Ficoll density gradient method from peripheral blood; The mononuclearcell kind of certain density has been gone into to spread in the culture dish of fibronectin or collagen, then use the commercial culture medium culturing of EGM2, every other day change substratum once, cultivation begins to have the growth of the colony of being shape 2-3 week, and cellular form is the late endothelial progenitor cells of cobblestone-appearance.The traditional method disadvantage is that acquisition late endothelial progenitor cells colony is less, every 100ml normal people's peripheral blood obtains two late endothelial progenitor cells colonies only, this like cell pick-up rate is low and use duration, laboratory stage is the quantity of the required peripheral blood of waste, be because of cell acquisition amount has influenced its clinical efficacy less aspect clinical application, hindered its clinical use prospect.
Summary of the invention
Technical problem to be solved by this invention is, thereby but provides a kind of cultural method that obtains more late endothelial progenitor cells colony largely expanding late endothelial progenitor cells from human peripheral.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of from peripheral blood the method for largely expanding late endothelial progenitor cells, it is characterized in that, will be from peripheral blood isolating mononuclearcell plant through pretreated that to be covered with late endothelial progenitor cells or umbilical cord endotheliocyte be in the trophoblastic culture dish, obtain more late endothelial progenitor cells colony, again largely expanding late endothelial progenitor cells.
Described from peripheral blood the method for largely expanding late endothelial progenitor cells, follow these steps to carry out:
(1) separation of human peripheral blood mononuclear cell: it is an amount of to get peripheral blood, separates mononuclearcell with the Ficoll density gradient method;
(2) vitro culture mononuclearcell: it is to cultivate in the culture dish of trophoblastic glue primordial covering that mononuclearcell is planted pretreated being covered with late endothelial progenitor cells or umbilical cord endotheliocyte, and nutrient solution is EGM2;
(3) evaluation of late endothelial progenitor cells colony number and cell function: carry out the expression of cellular form, function and cell surface molecule.
The nurse cell that overlays is through 10Gy-20Gy dosage radiotreatment.
The described peripheral blood of getting separates mononuclearcell, and the amount of peripheral blood is 100ml-400ml.
The cell concn that has overlay nurse cell in the culture dish is 1 * 10
3Individual/ml-1 * 10
4Individual/ml.
The concentration of the peripheral blood mononuclear cell of plantation is: 1 * 10
7Individual/ml-8 * 10
7Individual/ml.
Described bag is 1% by the collagen mass percent concentration of culture dish.
Specifically, follow these steps to carry out:
(1) separation of human peripheral blood mononuclear cell: get people's vein anticoagulation 100ml, separate mononuclearcell with the Ficoll density gradient centrifugation;
(2) trophocyte's preparation in the culture dish: late endothelial progenitor cells or the umbilical cord endotheliocyte 1 * 10 that will before cultivate acquisition
3Individual cell seeding is a nutrient solution with EGM2 in 6 orifice plates of 1% glue primordial covering, at 37 ℃, and 5%CO
2Cultivated in the incubator 6 hours, treat the cell attachment growth after, after the 10Gy dose irradiation, suppress its growth;
(3) vitro culture mononuclearcell: the mononuclearcell of separator well is pressed 2.5 * 10
7Individual cell/ml kind is planted in the 6 above-mentioned orifice plates, is nutrient solution with EGM2, at 37 ℃, and 5%CO
2Cultivate in the incubator, every other day change liquid once; Cultivate and began to have colony to occur on the 6th day, promptly can digest colony at the 9th to 12 day that grows into cultivation cell is continued to go down to posterity;
(4) evaluation of late endothelial progenitor cells characteristic.
The invention has the beneficial effects as follows: the cultural method that a kind of new peripheral blood late endothelial progenitor cells colony is provided.By before the plantation peripheral blood mononuclear cell, in culture dish, overlay the trophocyte who contains late endothelial progenitor cells or umbilical cord endotheliocyte in advance, then mononuclearcell is planted into, obtained about first two weeks to Duo clone's number of late endothelial progenitor cells of 20 times thereby will carry, can obtain the late endothelial progenitor cells with therapeutic value of the high quantity of high quality with limited peripheral blood than traditional method.
Description of drawings
Fig. 1 is the peculiar form explanation of growth of late endothelial progenitor cells colony and cell:
This colony of A is that the peculiar colony form and the distinctive cell of late endothelial progenitor cells growth is pebbles sample growthhabit;
B has the growth conditions of mononuclearcell cultivation in the time of 6 days only for not spreading the trophocyte;
C plants the growth conditions of mononuclearcell cultivation in the time of 6 days after the shop trophocyte is arranged, and as seen has little, many colonies to exist;
D plants the growth conditions of mononuclearcell cultivation in the time of 9 days after the shop trophocyte is arranged, and as seen has big, many colony to exist;
Fig. 2 is late endothelial progenitor cells Function Identification (late endothelial progenitor cells that increases into the growth of colony shape mixes visible tubular structure formation after 12 hours with Matrigel);
Fig. 3 is the evaluation of the biological nature of late endothelial progenitor cells.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail:
The present invention be a kind of from peripheral blood mononuclear cell the method for largely expanding late endothelial progenitor cells, realize by following steps:
(1) separation of human peripheral blood mononuclear cell: get people's vein anticoagulation 100ml, separate mononuclearcell with the Ficoll density gradient centrifugation.
(2) trophocyte's preparation in the culture dish: the late endothelial progenitor cells or the HUVECs (umbilical cord endotheliocyte) 1 * 10 that will before cultivate acquisition
3Individual cell seeding is a nutrient solution with EGM2 in 6 orifice plates of 1% glue primordial covering, at 37 ℃, and 5%CO
2Cultivated in the incubator 6 hours, treat the cell attachment growth after, after the 10Gy dose irradiation, suppress its growth.
(3) vitro culture mononuclearcell: the mononuclearcell that is about to separator well is planted in 6 orifice plates that prepare, and is nutrient solution with EGM2, at 37 ℃, and 5%CO
2Cultivate in the incubator, every other day change liquid once.
Above-mentioned vitro culture liquid is the nutrient solution of unified sale on the market; Mononuclearcell is by 5 * 10
7Individual cell seeding is in 6 orifice plates of 1% glue primordial covering; The bag of collagen is 5-10ul/cm by concentration
2Trophoderm radiation dose 10Gy.
(4) evaluation of late endothelial progenitor cells characteristic:
Morphocytology is identified: every ml peripheral blood can obtain 1-2 * 10 through the Ficoll density gradient centrifugation
6Individual mononuclearcell through planting on the trophocyte, every other day changes liquid once, the growth of colony shape promptly occurred being on the 6th day after plantation, cellular form is the colony of cobblestone-appearance, and growth rapidly, every 100ml can obtain the colony of the late endothelial progenitor cells about 40, as Fig. 1.
Cell function is identified: cultivating the 9th day, and after the late endothelial progenitor cells colony is digested separately, after EGM2 cultivates 2-3 days, getting 2 * 10 after the digestion once more
4Individual cell mixes back kind implantation to be had in the culture dish of the slide glass that overlays the Matrigel matrix membrane with the 500ulEGM2 nutrient solution, through 37 ℃, and 5%CO
2Cultivated 12 hours in the incubator, have tubular structure to form, as Fig. 2.
Characteristics of cell biology is identified: after the granulocyte colony digestion of cultivation, be passaged to 75cm
2Culturing bottle in continue to cultivate, after reaching 70% fusion, digest, use the corresponding antibodies mark, the detection of capable flow cytometer, detected result such as Fig. 3: CD31 99.9%, CD34 12.4%, and eNOS 62.9%, Flt-137.1%, PIH12 100%, Sendo 43.4%.
Present method agents useful for same is on the market can purchase reagent, and used all experimental techniques of present method are laboratory normal experiment method.
The present invention is a trophoderm with the late endothelial progenitor cells or the HUVECs of radiotreatment, then implant again from the isolating mononuclearcell of peripheral blood, observe its cultivation state, promptly begin to grow cell colony after 6 days, the quantity that its colony generates is 20 times of traditional cultural method output, and the growth of this cell colony has the distinctive characteristics of late endothelial progenitor cells, promptly is the growth of colony shape, cellular form is cobblestone-appearance, and this can obviously make a distinction on form with early stage endothelial progenitor cells.These cells have the distinctive characteristic of endothelial progenitor cells simultaneously: function aspects is the ability of external formation blood vessel; Cell biological characteristic aspect: have the distinctive cell sign of the endothelial progenitor cells of expression.The present invention grows the theory that can not leave microenvironment having used for reference hemopoietic stem cell, by simply providing trophoderm for mononuclearcell, it is microenvironment, colony than the late endothelial ancestral of many 20 times of traditional method, obviously improved the output capacity of the late endothelial progenitor cells of unit week blood, and the time of occurrence of colony is than traditional method early about two weeks, a large amount of all blood requirements and time have not only been saved, also saved Financial cost, and can obtain a large amount of competent late endothelial progenitor cells that clinical treatment is worth that have, for amplification in vitro and clinical application thereof provide necessary experimental data.The present invention cultivates the late endothelial progenitor cells with clinical value for effective high speed experimental technique is provided.
Embodiment 1:
1, separation of human peripheral blood mononuclear cell: get people's vein anticoagulation 100ml, after the dilution in 1: 1 of PBS balanced salt solution, the 32ml diluent slowly is taped against on the Ficoll parting liquid that contains 18ml along tube wall, and with 2000r/m, 30 minutes centrifugal, after the density gradient centrifugation with the sucking-off of white rings cloud and mist layer, use the PBS balanced salt solution again, with 1000r/m, 10 minutes washed twice, use the nutrient solution re-suspended cell at last, and with the blue numeration of placenta cell.
2, trophocyte's preparation in the culture dish: the late endothelial progenitor cells or the HUVECs 1 * 10 that will before cultivate acquisition
3Individual cell seeding is a nutrient solution with EGM2 in 6 orifice plates of 1% glue primordial covering, at 37 ℃, and 5%CO
2Cultivated in the incubator 6 hours, treat the cell attachment growth after, after the 10Gy dose irradiation, suppress its growth.
3, vitro culture mononuclearcell: the mononuclearcell that is about to separator well is with 5 * 10
7Individual mononuclearcell is planted in 6 orifice plates that prepare, and is nutrient solution with EGM2, at 37 ℃, and 5%CO
2Cultivate in the incubator, every other day change liquid once.
4, cell is identified and is carried out with method described above.
The application of EPCs has in recent years moved towards clinical from experimentation on animals, mainly concentrates on: 1. Atherosclerotic Obliteration of Lower Extremity ASO or BurgerShi disease, can obviously increase the neovascularization at sufferer place after the application, and increase ankle end finger number, alleviate rest pain.2. be not suitable for the PCI percutaneous catheter behind the acute myocardial infarction and interfere the patient of CABG coronary bypass, EPCs can obviously improve blood perfusion coronarius and improve the function of left ventricle.3. a following carrier that can be used as gene therapy, as do EPCs treatment behind the genetic modification etc.Experiment is reported in and exists EPCs in the bleeding of the umbilicus equally in succession, lays a good foundation for the widespread use of EPCs, makes the research of EPCs become present focus, for a fine prospect is opened up in the treatment of cardiovascular and cerebrovascular diseases.It is the cell quantity of applied EPCs etap of living in and use that EPCs participates in vascularization onset key.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (2)
1. the method for a largely expanding late endothelial progenitor cells from peripheral blood is characterized in that, follows these steps to carry out:
(1) separation of human peripheral blood mononuclear cell: it is an amount of to get peripheral blood, separates mononuclearcell with the Ficoll density gradient method;
(2) vitro culture mononuclearcell: it is to cultivate in the culture dish of trophoblastic glue primordial covering that mononuclearcell is planted pretreated being covered with late endothelial progenitor cells, and nutrient solution is EGM2;
(3) evaluation of late endothelial progenitor cells colony number and cell function: the expression of identification of cell form, function and cell surface molecule;
The described nurse cell that overlays is through 10Gy-20Gy dosage radiotreatment;
The described peripheral blood of getting separates mononuclearcell, and the amount of peripheral blood is 100ml-400ml;
The cell concn that has overlay nurse cell in the described culture dish is 1 * 10
3Individual/ml-1 * 10
4Individual/ml;
The concentration of the peripheral blood mononuclear cell of described plantation is: 1 * 10
7Individual/ml-8 * 10
7Individual/ml;
Described bag is 1% by the collagen mass percent concentration of culture dish.
2. according to claim 1 from peripheral blood the method for largely expanding late endothelial progenitor cells, it is characterized in that, follow these steps to carry out:
(1) separation of human peripheral blood mononuclear cell: get people's vein anticoagulation 100ml, separate mononuclearcell with the Ficoll density gradient centrifugation;
(2) trophocyte's preparation in the culture dish: the late endothelial progenitor cells 1 * 10 that will before cultivate acquisition
3Individual cell seeding is a nutrient solution with EGM2 in 6 orifice plates of 1% glue primordial covering, at 37 ℃, and 5%CO
2Cultivated in the incubator 6 hours, treat the cell attachment growth after, after the 10Gy dose irradiation, suppress its growth;
(3) vitro culture mononuclearcell: the mononuclearcell of separator well is pressed 2.5 * 10
7Individual cell/ml kind is planted in the 6 above-mentioned orifice plates, is nutrient solution with EGM2, at 37 ℃, and 5%CO
2Cultivate in the incubator, every other day change liquid once, cultivate and began to have colony to occur on the 6th day, promptly can digest colony at the 9th to 12 day that grows into cultivation cell is continued to go down to posterity;
(4) evaluation of late endothelial progenitor cells characteristic.
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| CN103484423A (en) * | 2013-07-18 | 2014-01-01 | 浙江大学 | Method used for separation and culture of poultry endothelial progenitor cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103484423A (en) * | 2013-07-18 | 2014-01-01 | 浙江大学 | Method used for separation and culture of poultry endothelial progenitor cells |
| CN103484423B (en) * | 2013-07-18 | 2015-06-24 | 浙江大学 | Method used for separation and culture of poultry endothelial progenitor cells |
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