CN104099291A - Method for improving proliferation ability and performance of umbilical cord blood OECs (outgrowth endothelial cells) - Google Patents
Method for improving proliferation ability and performance of umbilical cord blood OECs (outgrowth endothelial cells) Download PDFInfo
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Abstract
The invention discloses a method for improving proliferation ability and performance of umbilical cord blood OECs (outgrowth endothelial cells). The method comprises the steps as follows: pure umbilical cord blood-derived OECs are obtained by separation from fresh umbilical cord blood and purification, and mixed with umbilical cord-derived mesenchymal stem cells for culture, and a culture medium is a EGM-2BulletKit complete medium containing the fetal bovine serum with the volume fraction of 10%. By the aid of the method, the proliferation efficiency and the vascularization ability of the OECs are improved. After the method is applied, due to the immune down-regulatory effect of the mesenchymal stem cells and the expansion of the total treatment cell amount, the cell loss proportion caused by immune reactions is reduced, the treatment effect is guaranteed, and the application possibility of the umbilical cord blood-derived OECs is increased. The mesenchymal stem cells also have the ability of assisting in maintaining blood vessels, so that the long-term treatment effect is guaranteed. The method is mainly applied to blood vessel repair treatment, tissue engineering and the like.
Description
Technical field
The invention belongs to cell therapy field, particularly a kind of method that improves bleeding of the umbilicus export-oriented growth endothelial cell proliferation ability and performance.
Background technology
Export-oriented growth endotheliocyte (outgrowth endothelial cell, OEC), belong to blood vessel endothelium precursor species cell, during vitro culture, be the growth of paving stone sample, form cell monolayer, consistent with mature endothelial cell form, but its ability that there is rapid amplifying and generate blood vessel, make it to become the new lover in cell therapy field, investigators wish to use it for the treatment of the diseases such as cardiovascular and cerebrovascular diseases, heart trouble, limb ischemia.(the Jumi Kim such as Jumi Kim, Young-Joo Jeon.Comparative proteomic analysis of endothelial cells progenitor cells derived from cord blood-and peripheral blood for cell therapy.Biomaterials.Volume34, Issue6, February2013, Pages1669 – 1685.) compared the difference of bleeding of the umbilicus export-oriented growth endotheliocyte and the export-oriented growth of peripheral blood endotheliocyte, find no matter be the reparative experiment of ischemic injuries in external one-tenth blood vessel ability or the body of animal, the export-oriented growth of bleeding of the umbilicus endotheliocyte is all better than peripheral blood.(the David A.Ingram such as David A, Laura E..Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.blood, 2004,104:2752-2760.) also report when extroversion is grown endotheliocyte vitro culture in bleeding of the umbilicus and show higher amplification ability, can in long-term cultivation, keep precursor cell characteristic.
Although the export-oriented growth of the export-oriented growth of bleeding of the umbilicus endotheliocyte phase human peripheral blood endotheliocyte has considerable benefit, but because its indication mostly is degenerative disease, similar cardiovascular and cerebrovascular diseases, diabetic foot etc., need long-term infusion repeatedly, and cell concentration just becomes the bottleneck of its application.The primary inductivity of bleeding of the umbilicus export-oriented growth endotheliocyte is not high, poor growth especially under low density, and the cell concentration that cell therapy needs is larger, how to increase cell concentration, and strengthen its ability that generates blood vessel is the main direction of preclinical study simultaneously.
Mescenchymal stem cell has proved secretes a large amount of cytokines in breeding, can promote revascularization, increases blood vessel intensity in body, is now widely used in the regenerative medicine assisting therapy fields such as diabetic foot.
Summary of the invention
In order to overcome the shortcoming and deficiency of prior art, the object of the present invention is to provide a kind of method that improves derived from umbilical cord blood export-oriented growth endothelial cell proliferation ability and performance.By mescenchymal stem cell and the export-oriented growth of bleeding of the umbilicus endotheliocyte mixed culture, improve the proliferate efficiency of the export-oriented growth of bleeding of the umbilicus endotheliocyte and become blood vessel ability.The method adopts reliable donor, through separated, cultivation, obtains stem cell product.Preparation process is simple, reliable, reproducible.The export-oriented growth of mescenchymal stem cell and bleeding of the umbilicus endotheliocyte is cultivated altogether, improves the multiplication capacity under the export-oriented growth of bleeding of the umbilicus endotheliocyte low density, and the paracrine action that can make full use of mescenchymal stem cell simultaneously promotes the export-oriented growth of bleeding of the umbilicus endotheliocyte to become blood vessel.Experimental results show that when extracellular matrix is not enough to support blood vessels, mescenchymal stem cell can supplemental support blood vessel, maintains vascular morphology.
Object of the present invention is achieved through the following technical solutions: a kind of method that improves derived from umbilical cord blood export-oriented growth endothelial cell proliferation ability and performance, comprises the steps:
By mescenchymal stem cell and endotheliocyte mixed culture, can improve bleeding of the umbilicus export-oriented growth endothelial cell proliferation ability and performance; Wherein said mescenchymal stem cell is the Mesenchymal Stem Cells from Umbilical Cord of Healthy People, and described endotheliocyte is the export-oriented growth of Healthy People derived from umbilical cord blood endotheliocyte, hereinafter to be referred as endotheliocyte.
Described raising derived from umbilical cord blood export-oriented growth endothelial cell proliferation ability and the method for performance, specifically comprise the steps:
(1) separation of mescenchymal stem cell and former culture: umbilical cord is rejected to blood vessel, remove residual blood, separate jelly of Wharton, be cut into 1～2mm
2tissue block, at the bottom of being laid in T25 culturing bottle, add 1～2mL mescenchymal stem cell substratum (LONZA; Hereinafter to be referred as mescenchymal stem cell substratum), cultivate 7～11 days, isolate primary mescenchymal stem cell;
(2) going down to posterity of mescenchymal stem cell: the primary mescenchymal stem cell that step (1) is obtained goes down to posterity, massfraction 0.25% trysinization, mescenchymal stem cell substratum stops digestion, the centrifugal supernatant that goes, leave and take cell precipitation, resuspended with mescenchymal stem cell substratum, be seeded in T75 culturing bottle and cultivate, ratio in 1:3 goes down to posterity, and within 3～4 days, goes down to posterity once;
(3) separation of endotheliocyte and former culture: fresh bleeding of the umbilicus density gradient centrifugation is isolated mononuclearcell, gained mononuclearcell is resuspended in Endothelial cell culture base, obtains primary endotheliocyte after cultivating 10～20 days in 6 orifice plates of the pre-bed board of collagen;
(4) endotheliocyte P1 goes down to posterity: the primary endotheliocyte granulocyte colony number obtaining in step (3) is greater than at 1000 o'clock, go down to posterity, massfraction 0.25% trysinization of 37 ℃ of preheatings, Endothelial cell culture base stops digestion, collecting cell suspension, the centrifugal supernatant that goes, leaves and takes cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in 6 orifice plates of the pre-bed board of collagen and cultivate;
(5) endotheliocyte P2 goes down to posterity: after P1 cell 70% merges, go down to posterity, massfraction 0.25% trysinization of 37 ℃ of preheatings, Endothelial cell culture base stops digestion, collecting cell suspension, the centrifugal supernatant that goes, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in the T25 culturing bottle of the pre-bed board of collagen and cultivate;
(6) endotheliocyte P3 goes down to posterity: after P2 cell 70% merges, go down to posterity, massfraction 0.25% trysinization of 37 ℃ of preheatings, Endothelial cell culture base stops digestion, collecting cell suspension, the centrifugal supernatant that goes, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in the T75 culturing bottle of the pre-bed board of collagen and cultivate;
(7) endotheliocyte and mescenchymal stem cell mixed culture: endotheliocyte and mescenchymal stem cell are counted respectively, ratio with number of cells 1:1 is mixed, with the amount of every bottle of 10mL, be seeded in the T75 culturing bottle of the pre-bed board of collagen and cultivate, can improve bleeding of the umbilicus export-oriented growth endothelial cell proliferation ability and performance.
The condition optimization of the cultivation described in step (1) is for being placed in 37 ℃, and saturated humidity, cultivates in volume fraction 5% CO2gas incubator;
Centrifugal condition optimization described in step (2) is centrifugation time 5min, centrifugal force 500g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature;
Mescenchymal stem cell medium optimization described in step (1) and (2) is the TheraPEAK of LONZA company
tMmSCGM-CD
tMmedium;
Fresh bleeding of the umbilicus described in step (3) preferably should adopt fresh bleeding of the umbilicus within 24 hours;
The condition optimization that fresh bleeding of the umbilicus density gradient centrifugation described in step (3) is isolated mononuclearcell is after fresh bleeding of the umbilicus and physiological saline volume ratio 1:1 dilute, through the separated mononuclearcell of Ficoll lymphocyte separation medium, centrifugation time 25min, centrifugal force 500g, centrifugal acceleration 1g, retarded velocity 1g, 20 ℃ of temperature; After centrifugal end, get tunica albuginea layer (tunica albuginea layer is between plasma layer and separated liquid layer); The physiological saline that adds 3 times of volumes, washs once centrifugal force 800g, centrifugation time 8min, centrifugal acceleration 7g, retarded velocity 7g, collecting cell precipitation, the physiological saline of 30mL is washed once again, centrifugal force 200g, centrifugation time 10min, centrifugal acceleration 4g, retarded velocity 4g, collecting cell precipitation, is mononuclearcell;
Going down to posterity preferably in 1:(3～5 described in step (4), (5) and (6)) ratio goes down to posterity; 1:3 more preferably;
Centrifugal condition optimization described in step (4), (5) and (6) is centrifugation time 4～8min, centrifugal force 400g～800g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature; Centrifugation time 5min more preferably, centrifugal force 500g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature;
Endothelial cell culture base described in step (3)～(7) is preferably volume fraction 10% foetal calf serum+EGM-2BulletKit perfect medium (LONZA company)
Endotheliocyte described in step (7) and mesenchyme mixed culture, during inoculation, total cell density is preferably 3 * 10
4～5 * 10
4individual/mL; More preferably 4 * 10
4individual/mL;
Endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, endotheliocyte algebraically was preferably for 3～6 generations, more preferably 3 generations;
Endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, mescenchymal stem cell algebraically was preferably in 4 generations, more preferably 2 generations;
Endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, incubation time is preferably no more than 7 days, more preferably 3～4 days;
Endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, cell mixing can obtain the amplification of 5～6 times in a generation, effectively reduces passage number, damaging cells and cause differentiation while avoiding going down to posterity;
Cell after mixed culture described in step (7) shows better one-tenth blood vessel ability in matrigel;
Under the condition of the exogenous factor in removing substratum of the cell after the mixed culture described in step (7), the time keeping at matrigel medium vessels is longer;
Endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, two kinds of cells being characterized as before mixing:
1. endotheliocyte paving stone sample growth, cellular form homogeneous, during high-density culture, propagation is rapidly; CD309, CD34 positive expression; CD105>80%; In matrigel, can become rapidly blood vessel; In cultivation, after 5～6 generations, enter differentiation, CD34 expression amount increases, and multiplication capacity weakens, and becomes blood vessel ability to weaken in matrigel;
2. Mesenchymal Stem Cells from Umbilical Cord becomes fiber-like growth, cellular form homogeneous, propagation rapidly, streaming phenotype is CD105>90%, CD90>90%, CD73>90%, CD45<2%, CD34<2%.
Endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, two kinds of cells are characterized as mixed: propagation rapidly, can become rapidly blood vessel, streaming phenotype CD105>80% in matrigel.
Mechanism of the present invention is: mescenchymal stem cell is secreted a large amount of vasculogenesis relevant cell factors, contribute to the propagation of endotheliocyte and the formation of blood vessel, such as VEGF-A, VEGF-D, bFGF etc., and umbilical cord mesenchymal stem cells promotes to be better than mesenchymal stem cells MSCs aspect vasculogenesis at secrete cytokines.Export-oriented growth endotheliocyte is the cell that a kind of density relies on shape, propagation needs certain cell quantity and growth factor content, the export-oriented growth of bleeding of the umbilicus endotheliocyte can not be effective to clinical due to the restriction of cell concentration, mesenchymal cell just can provide such condition, accelerates the growth of the export-oriented growth of bleeding of the umbilicus endotheliocyte as a kind of stroma cell.
Simultaneously, mescenchymal stem cell is supported endotheliocyte vascularization as a kind of stroma cell, increases angiopoietic quantity, particularly when extracellular matrix can not support formed blood vessel, mesenchymal cell can play the effect of support, the auxiliary vascular morphology that maintains.
The present invention, with respect to prior art, has following advantage and effect:
(1) the present invention by mescenchymal stem cell at short notice paracrine promote the ability of revascularization to use, first promoted the export-oriented growth of bleeding of the umbilicus endotheliocyte to breed rapidly, shorten the needed time of application, increase cell quantity; Next strengthens cell and becomes blood vessel ability, in matrigel, can become rapidly blood vessel, improves result for the treatment of; Moreover mescenchymal stem cell has the auxiliary ability that maintains blood vessel, for the effect of long-term treatment provides assurance; The streaming phenotype CD105>80% of the cell mixing that in addition, the present invention obtains.
(2) in cell therapy, cell total amount is a very important problem, and foreign cell enters people and knows from experience initiation immune response to a certain degree, and these immune responses cause cell concentration to reduce.And after method application of the present invention, expanded the total cell concentration for the treatment of, reduce the ratio by immune response loss cell.Due to the immune down regulation effect of mescenchymal stem cell, further reduce the cell concentration of loss simultaneously, guaranteed result for the treatment of
(3) for patient, the extroversion of autologous peripheral blood growth endothelial cell activity is generally poor, although the abundant immunogenicity in variant cell source is stronger, is not suitable for cell therapy application.The export-oriented growth of bleeding of the umbilicus endotheliocyte confirms through lot of documents, its treatment prospect is better than derived from peripheral blood, but because source is limited, cause cell concentration to be restricted and be difficult to enter application, method of the present invention has reduced the loss in treatment when having expanded total cell concentration, strengthening its ability to function.
Accompanying drawing explanation
Fig. 1 is the aspect graph of observing 2nd generation mescenchymal stem cell under inverted microscope.
Fig. 2 is the expression figure that flow cytometer detects the surface antigen of 2nd generation mescenchymal stem cell.
Fig. 3 be under inverted microscope, observe the 3rd generation endotheliocyte aspect graph.
Fig. 4 is the expression figure of the surface antigen of flow cytometer detection the 3rd generation endotheliocyte.
Fig. 5 be the 3rd generation, 4 generations, 5 generations with 6 generation endotheliocyte become blood vessel lab diagram.
Fig. 6 be under inverted microscope, observe 2nd generation mescenchymal stem cell and the 3rd generation the aspect graph cultivated altogether of endotheliocyte.
Fig. 7 is into blood vessel lab diagram; Wherein, to be 2nd generation mescenchymal stem cell become blood vessel lab diagram with the cell mixing of the 3rd generation endotheliocyte after cultivating altogether to figure A; Figure B is that endotheliocyte becomes blood vessel lab diagram; Figure C is that mescenchymal stem cell becomes blood vessel lab diagram.
Fig. 8 is the CD105 expression figure that flow cytometer detects embodiment 5 mixing group cells.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The experimental technique of unreceipted specific experiment condition in the following example, the experiment condition of conventionally advising according to manufacturer according to normal experiment conditioned disjunction.
Mescenchymal stem cell substratum is the TheraPEAK of LONZA company
tMmSCGM-CD
tMmedium;
Endothelial cell culture base is volume fraction 10% foetal calf serum+EGM-2BulletKit perfect medium, and EGM-2BulletKit is purchased from LONZA company.
Embodiment 1
Bleeding of the umbilicus and umbilical cord sample are that the placenta of being given birth to by healthy puerpera obtains by aseptic technique.Hepatitis B, hepatitis C, syphilis, AIDS, cytomegalovirus, TORCH detection, mycoplasma, chlamydozoan, G-6PD and ground are poor etc. all negative after testing.After collection of specimens, transport blood bank's process back and keep 4～8 ℃ of transport conditions.Process in accordance with the following methods.
1, the preparation of umbilical cord mesenchymal stem cells
From the umbilical cord tissue of Healthy People, extract mescenchymal stem cell, the mescenchymal stem cell obtaining carries out vitro culture, amplification.Primary cell collect after in the cultivation of going down to posterity of the ratio of 1:3, at the bottom of cell is paved with bottle approximately during 80% density, utilize pancreatin to digest, collect, in identical ratio, go down to posterity, go down to posterity at most and be cultured to for the 3rd generation, collect the cell in the 3rd generation, the cytobiology proterties of now collecting is stable, cytoactive is good, and multiplication capacity is strong.Detailed process is as follows:
1) the aseptic umbilical cord of taking Healthy People;
2) remove blood vessel and the outer amnion in umbilical cord, leave and take jelly of Wharton matter;
3) umbilical cord tissue is cut into 1～2cm
2fritter.
4) by the culturing bottle of the fritter inoculation T25 after shearing, add 1～2mL mesenchyme substratum, be placed in 37 ℃, saturated humidity, cultivates in volume fraction 5% CO2gas incubator (Thermo company, the U.S.);
5) after 7 days, cell changes liquid, and after 3～4 days, cell can go down to posterity.
6) utilize the pancreatin (GIBCO company, the U.S.) of massfraction 0.25% to digest, collecting cell, is seeded in the cultivation of going down to posterity in the culturing bottle of T25.
7) at the bottom of being paved with bottle, cell approximately during 80% density, utilize the pancreatin of massfraction 0.25% to digest, collecting cell.
2, the preparation of the export-oriented growth of bleeding of the umbilicus endotheliocyte
Fresh and healthy people's bleeding of the umbilicus in 24 hours, obtain mononuclearcell, be cultured to P1 generation in Endothelial cell culture base, detailed process is as follows.
1) dilution proportion of the fresh bleeding of the umbilicus 1:1 of physiological saline and 50mL, after mixing, is added on lymphocyte separation medium, carries out the separation of mononuclearcell, centrifugation time 25min, centrifugal force 500g, centrifugal acceleration 1g, retarded velocity 1g, 20 ℃ of temperature.
2), after centrifugal end, get tunica albuginea layer (tunica albuginea layer is between plasma layer and separated liquid layer).The physiological saline that adds 3 times of volumes, washs once centrifugal force 800g, centrifugal 8min.
3) collecting cell precipitation, washes once with the physiological saline of 30mL again, centrifugal force 200g, and centrifugation time 10min, centrifugal acceleration 4g, retarded velocity 4g, collecting cell precipitation, obtains mononuclearcell.
4) by Cord Blood Mononuclear Cell with 1 * 10
5individual/mL is seeded in 6 orifice plates of the pre-bed board of collagen, adds 2mL Endothelial cell culture base/hole.
5) within the 5th day, incite somebody to action not attached cell and discard, within 3 days, change liquid once until find paving stone like cell colony.Continue to cultivate and get final product collecting cell in about 4 days.
6) P1 goes down to posterity: utilize the pancreatin of the massfraction 0.25% of 37 ℃ of preheatings to digest, Endothelial cell culture base stops digestion, collecting cell suspension, centrifugal, centrifugation time 5min, centrifugal force 500g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature, remove supernatant, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in 6 orifice plates of the pre-bed board of collagen and cultivate.
7) P2 goes down to posterity: after P1 cell 70% merges, utilize the pancreatin of the massfraction 0.25% of 37 ℃ of preheatings to digest, Endothelial cell culture base stops digestion, collecting cell suspension, centrifugal, centrifugation time 5min, centrifugal force 500g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature, remove supernatant, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in the T25 culturing bottle of the pre-bed board of collagen and cultivate.
8) P3 goes down to posterity: after P2 cell 70% merges, utilize the pancreatin of the massfraction 0.25% of 37 ℃ of preheatings to digest, Endothelial cell culture base stops digestion, collecting cell suspension, centrifugal, centrifugation time 5min, centrifugal force 500g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature, remove supernatant, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in the T75 culturing bottle of the pre-bed board of collagen and cultivate.
3, the evaluation of mescenchymal stem cell and endotheliocyte
1) evaluation of mescenchymal stem cell by the following method
A) morphological analysis of mescenchymal stem cell and cell counting: will be cultured to the cell of 2nd generation, at inverted microscope observation of cell state, and take pictures.
B) phenotype test of flow cytometer: get 2nd generation cell, after massfraction 0.25% trysinization is collected, physiological saline centrifuge washing 2 times.Homotype control tube adds mouse IgG-FITC, IgG-PE (BD company, the U.S.), and detector tube adds respectively mouse-anti people CD105-PE, CD90-FITC, CD73-PE, each 5 μ L of CD45-FITC, CD34-PE (BD company, the U.S.).Room temperature lucifuge is hatched 30min, and flow cytometer (Fascalibur, BD company, the U.S.) detects.
2) evaluation of endotheliocyte by the following method
A) morphological analysis of endotheliocyte and cell counting: by P3 culture to the cell of 2 days, at inverted microscope, observe, and take pictures.In had digestive transfer culture process, get few cells suspension, counting cells quantity.
B) phenotype test of flow cytometer: by the cell of the 3rd culture, the amount of taking fully cell suspension in had digestive transfer culture process, after centrifugal collection, physiological saline centrifuge washing 2 times.Homotype control tube adds mouse IgG-FITC, IgG-PE, and detector tube adds respectively mouse-anti people CD309-PE, each 5 μ L of CD34-PE, CD105-PE (BD company, the U.S.).Room temperature lucifuge is hatched 30min, and flow cytometer (Fascalibur, BD company, the U.S.) detects.
C) become blood vessel experimental identification: 4 ℃ melt matrigel (BD company, the U.S.), with pre-sniper's shot head by the matrigel melting completely add precooling 96 orifice plates, every hole 40 μ L, hatch 30～60min for 37 ℃.Results endotheliocyte, is resuspended in EBM-2 basic medium (LONZA company), cell density 5 * 10
5individual/mL, adds 96 orifice plates, and every hole adds 50 μ L, cultivates 18h for 37 ℃, observes vascularization effect.
3) cell qualification result:
Under inverted microscope, observe 2nd generation mescenchymal stem cell form more single, spindle shape cell is common, a small amount of stellate cell, and round cell is few.During cell low density, grow up to flat monolayer cell, when Growth of Cells density increases, it is elongated that cellular form becomes, and the similar inoblast of form is be arranged in parallel growth or swirl shape growth (seeing Fig. 1).Through flow cytometer (Fascalibur, BD company, the U.S.) detection display, cell-surface antigens CD105, CD90, CD73 positive expression rate reach more than 95%, and CD45, CD34 positive expression rate (are shown in Fig. 2) below 2%.In sum, the mescenchymal stem cell purity of preparing according to patented method of the present invention is high, active good, meets the requirements.
Under inverted microscope, observed for the 3rd generation (P3 generation) endothelial cell morphology more single, the growth of paving stone sample.During cell low density, be colony growth, colony not obvious (seeing Fig. 3) after going down to posterity.Through flow cytometer detection display, cell-surface antigens CD309, CD34 positive expression, CD105 positive expression rate reaches more than 80% (sees Fig. 4).Through becoming blood vessel experiment, endotheliocyte that the application obtains all can be stable into blood vessel (seeing Fig. 5) in 3 generations to six generations.After 6 generations, cell proliferation rate declines, and after 8～9 generations, no longer breeds.In sum, the endothelial cell activity of preparing according to patented method of the present invention is good, meets the requirements.
4, this experiment repeats 3 times, and all the follow-up embodiment 2 that carries out tests at every turn.
Embodiment 2
With reference to the method for embodiment 1, by the endotheliocyte of collecting with whole density to 1 * 10 of Endothelial cell culture keynote
5individual/mL, is designated as cell suspension A; By the mescenchymal stem cell of collecting with whole density to 1 * 10 of Endothelial cell culture keynote
5individual/mL, is designated as cell suspension B, and obtained cell suspension A and each 2mL of cell suspension B join in 6mL Endothelial cell culture base, i.e. mixing group.
Establish endotheliocyte group is OEC group simultaneously, and mescenchymal stem cell group is MSC group, and OEC group obtained cell suspension A2mL joins in 8mL Endothelial cell culture base.MSC group obtained cell suspension B2mL joins in 8mL Endothelial cell culture base.
In the T75 culturing bottle that 3 groups join respectively the pre-bed board of collagen, cultivate.
This experiment repeats 3 times, and each experiment is all carried out interpretation of result according to embodiment 3, embodiment 4 and embodiment 5.
Embodiment 3
With reference to the method for embodiment 2, harvested cell after 4 days, carries out cell counting to the cell of results, contrasts 3 groups of cell concentrations, with following formula, calculates its propagation multiple.
Propagation multiple=harvested cell amount/inoculating cell amount
The results are shown in Table 1: mixing group cell proliferation rate is larger compared with endotheliocyte group and mescenchymal stem cell group, and difference has statistical significance, reaches the requirement of the application's rapid amplifying.
Table 1
| ? | OEC group | Mixing group | MSC group |
| Propagation multiple | 2.37 doubly | 5.35 doubly | 3.22 doubly |
Embodiment 4
With reference to the method for embodiment 2, harvested cell after 4 days, becomes blood vessel experimental analysis:
4 ℃ melt matrigel, with pre-sniper's shot head by the matrigel melting completely add precooling 96 orifice plates, every hole 40 μ L, hatch 30～60min for 37 ℃.
OEC group cell, mixing group cell, the MSC of results are organized to cell, be resuspended in respectively EBM-2 basic medium, total cell density is respectively 5 * 10
5individual/mL, adds respectively (3 holes of every group of cell parallel laboratory test) every hole in 96 orifice plates that contain matrigel to add 50 μ L, cultivates 48h for 37 ℃, continues to observe 18h, 24h, 48h vascularization effect.
Cellular form (seeing Fig. 6) while observing 48h under inverted microscope before results, in Matrigel vascularization experiment, mixing group and OEC group all have vascularization in various degree, and MSC group does not become blood vessel ability completely.
Observe into blood vessel experimental result, shown in Fig. 7 (Fig. 7 A is mixing group, and Fig. 7 B is OEC group, and Fig. 7 C is MSC group);
1) within 18 hours, observe into blood vessel experimental result, mixing group cell becomes blood vessel ability apparently higher than OEC group, and counting mixing group and OEC organize formed blood vessel, and mixed culture group forms 69 sections, and OEC group forms 52 sections.Cell mixing becomes blood vessel ability higher than endotheliocyte group.
2) within 24 hours, observe into blood vessel experimental result, owing to there is no enough cytokine supports, all there is disintegration in various degree in two groups of cells (mixing group and OEC group), simultaneously because anchorage force is inadequate, collagen atrophy, causes cell to be united, and no longer becomes blood vessel structure, in mixing group cell, more visible cell attachment, around blood vessel, do not participate in into blood vessel.
3) within 48 hours, observe into the complete disintegration of the plastidogenetic blood vessel of blood vessel experimental result endotheliocyte group and unite, and in mixed culture group, still have a small amount of blood vessel residual.
Result proof mixing group cell and OEC group cell by comparison, have stronger vascularization ability, and mesenchymal cell can be assisted and be maintained vascular morphology simultaneously, assists support blood vessels when extracellular matrix is not enough to support blood vessels.
Embodiment 5
With reference to the method for embodiment 2, harvested cell after 4 days, detects through flow cytometer, and detection method is with reference to embodiment 1, and detected result is as Fig. 8.
Result demonstration, the cell-surface antigens CD105 positive expression rate of OEC group reaches more than 80%, and the cell-surface antigens CD105 positive expression rate of MSC group reaches more than 90%; The cell-surface antigens CD105 positive expression rate of mixing group cell all reaches more than 80%, proves that cell mixing still has good dryness.
The result of comprehensive embodiment 4, the cell mixing of can reaching a conclusion is still keeping stem cell characteristic, is applicable to scientific research or clinical demand.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. a method that improves bleeding of the umbilicus export-oriented growth endothelial cell proliferation ability and performance, is characterized in that comprising the steps:
By mescenchymal stem cell and endotheliocyte mixed culture, can improve bleeding of the umbilicus export-oriented growth endothelial cell proliferation ability and performance; Wherein said mescenchymal stem cell is the Mesenchymal Stem Cells from Umbilical Cord of Healthy People, and described endotheliocyte is the export-oriented growth of Healthy People bleeding of the umbilicus endotheliocyte.
2. the method for raising bleeding of the umbilicus according to claim 1 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that comprising following concrete steps:
(1) separation of mescenchymal stem cell and former culture: umbilical cord is rejected to blood vessel, remove residual blood, separate jelly of Wharton, be cut into 1～2mm
2tissue block, at the bottom of being laid in T25 culturing bottle, add 1～2mL mescenchymal stem cell substratum, cultivate 7～11 days, isolate primary mescenchymal stem cell;
(2) going down to posterity of mescenchymal stem cell: the primary mescenchymal stem cell that step (1) is obtained goes down to posterity, massfraction 0.25% trysinization, mescenchymal stem cell substratum stops digestion, the centrifugal supernatant that goes, leave and take cell precipitation, resuspended with mescenchymal stem cell substratum, be seeded in T75 culturing bottle and cultivate, ratio in 1:3 goes down to posterity, and within 3～4 days, goes down to posterity once;
(3) separation of endotheliocyte and former culture: fresh bleeding of the umbilicus density gradient centrifugation is isolated mononuclearcell, gained mononuclearcell is resuspended in Endothelial cell culture base, obtains primary endotheliocyte after cultivating 10～20 days in 6 orifice plates of the pre-bed board of collagen;
(4) endotheliocyte P1 goes down to posterity: the primary endotheliocyte granulocyte colony number obtaining in step (3) is greater than at 1000 o'clock, go down to posterity, massfraction 0.25% trysinization of 37 ℃ of preheatings, Endothelial cell culture base stops digestion, collecting cell suspension, the centrifugal supernatant that goes, leaves and takes cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in 6 orifice plates of the pre-bed board of collagen and cultivate;
(5) endotheliocyte P2 goes down to posterity: after P1 cell 70% merges, go down to posterity, massfraction 0.25% trysinization of 37 ℃ of preheatings, Endothelial cell culture base stops digestion, collecting cell suspension, the centrifugal supernatant that goes, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in the T25 culturing bottle of the pre-bed board of collagen and cultivate;
(6) endotheliocyte P3 goes down to posterity: after P2 cell 70% merges, go down to posterity, massfraction 0.25% trysinization of 37 ℃ of preheatings, Endothelial cell culture base stops digestion, collecting cell suspension, the centrifugal supernatant that goes, leave and take cell precipitation, outstanding with Endothelial cell culture basic weight, be seeded in the T75 culturing bottle of the pre-bed board of collagen and cultivate;
(7) endotheliocyte and mescenchymal stem cell mixed culture: endotheliocyte and mescenchymal stem cell are counted respectively, ratio with number of cells 1:1 is mixed, be seeded in the T75 culturing bottle of the pre-bed board of collagen and cultivate, can improve bleeding of the umbilicus export-oriented growth endothelial cell proliferation ability and performance.
3. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that:
Fresh bleeding of the umbilicus described in step (3) should adopt fresh bleeding of the umbilicus within 24 hours;
The condition that fresh bleeding of the umbilicus density gradient centrifugation described in step (3) is isolated mononuclearcell is after fresh bleeding of the umbilicus and physiological saline volume ratio 1:1 dilute, through the separated mononuclearcell of Ficoll lymphocyte separation medium, centrifugation time 25min, centrifugal force 500g, centrifugal acceleration 1g, retarded velocity 1g, 20 ℃ of temperature; After centrifugal end, get tunica albuginea layer; The physiological saline that adds 3 times of volumes, washs once centrifugal force 800g, centrifugation time 8min, centrifugal acceleration 7g, retarded velocity 7g, collecting cell precipitation, the physiological saline of 30mL is washed once again, centrifugal force 200g, centrifugation time 10min, centrifugal acceleration 4g, retarded velocity 4g, collecting cell precipitation, is mononuclearcell.
4. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that: going down to posterity in 1:(3～5 described in step (4), (5) and (6)) ratio goes down to posterity.
5. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that:
Centrifugal condition described in step (2) is centrifugation time 5min, centrifugal force 500g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature;
Centrifugal condition described in step (4), (5) and (6) is centrifugation time 4～8min, centrifugal force 400g～800g, centrifugal acceleration 7g, retarded velocity 7g, 20 ℃ of temperature.
6. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that: the endotheliocyte described in step (7) and mesenchyme mixed culture, during inoculation, total cell density is 3 * 10
4～5 * 10
4individual/mL.
7. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that: the endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, endotheliocyte algebraically was 3～6 generations.
8. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that: the endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, mescenchymal stem cell algebraically is in 4 generations.
9. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that: the endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, incubation time is no more than 7 days.
10. the method for raising bleeding of the umbilicus according to claim 2 export-oriented growth endothelial cell proliferation ability and performance, is characterized in that: the endotheliocyte described in step (7) and mescenchymal stem cell mixed culture, and two kinds of cells being characterized as before mixing:
1. endotheliocyte paving stone sample growth, cellular form homogeneous, during high-density culture, propagation is rapidly; CD309, CD34 positive expression, CD105>80%; In matrigel, can become rapidly blood vessel; In cultivation, after 5～6 generations, enter differentiation, CD34 expression amount increases, and multiplication capacity weakens, and becomes blood vessel ability to weaken in matrigel;
2. Mesenchymal Stem Cells from Umbilical Cord becomes fiber-like growth, cellular form homogeneous, propagation rapidly, streaming phenotype is CD105>90%, CD90>90%, CD73>90%, CD45<2%, CD34<2%.
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| CN112210528A (en) * | 2020-09-18 | 2021-01-12 | 广州市天河诺亚生物工程有限公司 | Method for improving proliferation capacity and performance of endothelial cells of cord blood |
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| CN110982779A (en) * | 2019-12-25 | 2020-04-10 | 广州市天河诺亚生物工程有限公司 | Method for improving utilization rate of umbilical cord blood |
| CN110982779B (en) * | 2019-12-25 | 2020-12-29 | 广州市天河诺亚生物工程有限公司 | Method for improving utilization rate of umbilical cord blood |
| CN112210528A (en) * | 2020-09-18 | 2021-01-12 | 广州市天河诺亚生物工程有限公司 | Method for improving proliferation capacity and performance of endothelial cells of cord blood |
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