CN103789260A - Method for separating and amplifying blood mesenchymal stem cells - Google Patents
Method for separating and amplifying blood mesenchymal stem cells Download PDFInfo
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- CN103789260A CN103789260A CN201410046445.3A CN201410046445A CN103789260A CN 103789260 A CN103789260 A CN 103789260A CN 201410046445 A CN201410046445 A CN 201410046445A CN 103789260 A CN103789260 A CN 103789260A
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Abstract
The invention relates to the field of the biotechnology, and in particular relates to a method for separating and amplifying blood mesenchymal stem cells. The method comprises the steps of a. collecting a blood sample; b. separating a mononuclear cell component in the blood sample; c. inoculating the mononuclear cell component into a culture dish modified by a high polymer material; d. separating the mesenchymal stem cells; and e. culturing and amplifying the mesenchymal stem cells. According to the method, a cell culture plate is coated by a coating component with definite components, so that the purity and efficiency of separation can be effectively improved. The cell factors such as b-FGF (fibroblast growth factor), EGF (epidermal growth factor) and TGF (transforming growth factor)-beta are added into alpha-MEM (minimum essential medium), so that the growth of the stem cells is stably maintained, and mass application of the blood mesenchymal stem cells can be realized.
Description
Technical field
The present invention relates to biological technical field, especially a kind of method that separates and increase blood mescenchymal stem cell.
Background technology
Stem cell refers in organism can carry out self, and maintains the metabolic a group cell of the each histocyte of body.Meanwhile, when various tissues are subject to endogenous or ectogenic infringement in vivo, the stem cell execution differentiation function that can be activated, thus reach the object of repairing this tissue.Mescenchymal stem cell under suitable condition, has powerful competence for added value and differentiation capability, can repair Various Tissues damage, comprises bone, muscle, and the neural tissue that waits, therefore, mescenchymal stem cell has caused increasing attention in regenerative medicine treatment field.
Prove to contain in blood differentiation capability higher, the mescenchymal stem cell that immunological rejection is less.Being established as of blood banks at different levels, Cord blood bank realized autogenous cell transplantation basis is provided.But, due to mescenchymal stem cell content in blood extremely low (take Cord blood as example, 1 × 10
8in individual monocyte, only contain 0.5-30, and in the blood of growing up stem cell population far below this quantity), therefore how high efficiency separation and these cells of amplification become and hinder the difficult problem of blood mescenchymal stem cell to clinical conversion.At present, utilize the adsorptive power of this cell at culture dish more, mesenchymal stem cells in umbilical cord blood is separated, but success ratio is low, and in amplification procedure, be difficult to keep stem cell characteristic.The present invention includes and utilize macromolecule chemical material to modify culture dish, coordinate and add the substratum of multiple somatomedin, build blood mescenchymal stem cell amplification environment, the efficient amplification that has realized this cell with separate.
In the middle of marrow, contain abundant mescenchymal stem cell, therefore obtained the most extensive research.Studies confirm that, mesenchymal stem cells MSCs is expressed specific surface antigen phenotype, such as, CD45-, CD34-, CD29+, CD90+, CD73+ and CD105+.In vitro and in vivo can be to bone in environment, cartilage, the cytodifferentiation such as nerve and pancreas islet.In addition, mesenchymal stem cells MSCs has immunoregulatory function, secretion panimmunity related signaling molecules, thus in immunological disease therapeutic process, play a role.But, although mesenchymal stem cells MSCs cell is more easily separated and cultivation,, derived from bone marrow is limited, is difficult to meet clinical a large amount of demand.Even if carry out patient's autotransplantation, the misery of patient outside also must commitment.At its hetero-organization, such as muscle, fat, other organizes brain and blood etc. and also can separate acquisition mescenchymal stem cell.These cells have the antigenic phenotype similar to mesenchymal stem cells MSCs and differentiation capability.But, remove outside blood, apply its hetero-organization between fill stem cell and be faced with the problem identical with applying mesenchymal stem cells MSCs, and sample is rare, is not enough to meet clinical demand.
Verified, in blood, contain active very strong population of stem cells, comprise hemopoietic stem cell and mescenchymal stem cell.Mescenchymal stem cell in blood is similar to bone marrow stem cell, has identical surface antigen phenotype, and similar differentiation capability.Document shows, blood stem cell has more original stem cell characteristic and lower immunogenicity.Meanwhile, application haemodialysis diabetes, the diseases such as nerve injury and angionecrosis are also appeared in the newspapers repeatly.
Summary of the invention
Method of the present invention, by obtaining blood, is utilized specific polymer chemistry material rather than is used composition to be difficult to definite extracellular matrix, mescenchymal stem cell is wherein carried out to separation and purification, and coordinate suitable substratum, realizes the efficient amplification to blood.
The technical solution adopted for the present invention to solve the technical problems is: a kind of method that separates and increase blood mescenchymal stem cell, comprises the following steps:
Step a: collect blood sample,
Step b: monocyte composition in separating blood sample,
Step c: monocyte composition is inoculated in to the culture dish that macromolecular material is modified,
Steps d: separating mesenchymal stem cell,
Step e: cultivate and amplification of mesenchymal stem cells.
According to another embodiment of the invention, further comprise that described blood sample gathers from hospital, or obtain in autoblood storehouse.
According to another embodiment of the invention, further comprise that described blood sample is people's blood sample.
According to another embodiment of the invention, further comprise that described blood sample collection is certainly in premature labor or normal labor fetus and grownup's blood.
According to another embodiment of the invention, further comprise that described blood sample all passes through anticoagulant heparin, Walocel MT 20.000PV splitting erythrocyte, density gradient centrifugation is inoculated after obtaining monocyte composition.
According to another embodiment of the invention, further comprise in described step c, macromolecular material refers to that polyoxyethylene glycol (PEG) and arginine (R)-glycine (G)-aspartic acid (D) (RGD) mix by concentration 1ml:0.01g of the same race.The present invention proposes polyoxyethylene glycol (PEG) and (RGD) the ratio mixing of 1ml:0.01g of arginine (R)-glycine (G)-aspartic acid (D), can reasonable separation and purifying blood in mescenchymal stem cell, thereby both improved separation and purification efficiency, also avoided the extracellular matrix that uses chemical composition extremely complicated.
According to another embodiment of the invention, further comprise in described step e, cultivation and amplification of mesenchymal stem cells refer to that applying blood mescenchymal stem cell substratum cultivates and increase.
According to another embodiment of the invention, further comprise that described blood mescenchymal stem cell substratum comprises: α-MEM, Pen-Strep, b-FGF, EGF, TGF-β.The present invention uses and contains α-MEM, Pen-Strep, and b-FGF, EGF, the substratum of TGF-β, can maintain the activity of primary cell in early stage, at the later stage blood mescenchymal stem cell that can efficiently increase.Wherein, α-MEM is the essential minimal medium of α-minimum; Pen-Strep is penicillin-Streptomycin sulphate, and b-FGF is b-fibroblast growth factor, and EGF is epithelical cell growth factor, and TGF-β is transforming growth factor.
The key step of separation of the present invention and amplification blood mescenchymal stem cell is as follows:
Under aseptic condition, obtain blood, 25-200ml, through anticoagulant heparin.The separation and purification process of blood mescenchymal stem cell is carried out in latter 24 hours in sampling.
First, use the blood of same volume α-MEM dilution anti-freezing, mix by 4:1 with the methylcellulose gum of 5g/L, leave standstill 30 minutes, sedimented red cell.Draw supernatant, centrifugal after, make single cell suspension with PBS damping fluid, on the lymphocyte separation medium of the relative density that is added to 1.077, the centrifugal 20min of 2500rpm, gets interfacial layer, adds PBS damping fluid and makes single cell suspension, centrifuge washing.Cell is added on the Tissue Culture Plate of modifying with polyoxyethylene glycol (PEG) and RGD.Cell is cultivated in α-MEM, in this substratum, contains Pen-Strep, b-FGF, EGF, the added ingredientss such as TGF-β.When cell reaches 90% fusion, go down to posterity.While going down to posterity, use common culture dish, and in the above-mentioned substratum that contains added ingredients, carry out the amplification of cell.
The invention has the beneficial effects as follows, the coated composition that the present invention has used composition to determine is coated with Tissue Culture Plate, has effectively improved the purity and the efficiency that separate.In α-MEM, add b-FGF, EGF, what the cytokines such as TGF-β can be stable maintains stem cell growth, can realize a large amount of amplifications of blood mescenchymal stem cell, thereby complete the present invention.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 is that the present invention cultivates the result of mesenchymal stem cells in umbilical cord blood after 20 days.
Embodiment
Now carry out the more detailed description method that separates and increase for Cord blood mescenchymal stem cell of the present invention in conjunction with following instance.Provide the object of example to be only exemplary elaboration name invention, can not be understood as is the restriction to the scope of the invention and essence.
Embodiment 1: modified cells culture plate
Polyoxyethylene glycol (PEG) and RGD material are heavily dissolved in chloroform in the ratio of 0.25g:8ml, get culture vessel, above-mentioned solution is evenly coated with thereon, under low temperature, aspirate 48h, obtain the transparent biomaterial on slide that is covered in.Add Sulfo.LC.SPDP phosphate buffered saline buffer (PBS, the pH:8.0) 0.5mL of 10mg/mL, under room temperature, failure of oscillation is not moving, reaction 12h; PBS damping fluid rinses 3 times, and under room temperature, failure of oscillation is not moving, reaction 24h; PBS damping fluid rinses 3 times, seasoning.For subsequent use after the sterilization of material ethane via epoxyethane.
Embodiment 2: separate and amplification mesenchymal stem cells in umbilical cord blood
By the Cord blood gathering under aseptic condition with volume ratio 1:1 and cell at α-MEM mixed diluting, mix by 4:1 with the methylcellulose gum of 5g/L, leave standstill 30 minutes, sedimented red cell.Draw supernatant, centrifugal after, make single cell suspension with PBS damping fluid, the lymphocyte separation medium Ficoll-Hypaque of the relative density that is added to 1.077 goes up, the centrifugal 20min of 2500rpm, gets interfacial layer, adds PBS damping fluid and makes single cell suspension, centrifuge washing.
Because the proportion of lymphocyte separation medium Ficoll-Hypaque is 1.077g/ml, it is heavier but lighter than red corpuscle than monocyte, therefore monocyte can be separated with residual red corpuscle.Collect interfacial layer and can collect purer monocyte.
By PBS damping fluid dilution for the monocyte obtaining, 2000rpm, centrifugal 10min, removes supernatant liquor, and adds new PBS damping fluid to break up, dilution.Wash once with same condition, visible deposition is in the cell of centrifuge tube bottom again.
Subsequently, use the mesenchymal stem cells in umbilical cord blood substratum that adds 10% foetal calf serum evenly to break up the cell of collecting.Mesenchymal stem cells in umbilical cord blood substratum refers to and is adding 1%Pen-Strep, 50ng/ml b-FGF, 10ng/mlEGF, α-MEM substratum of the somatomedins such as 10ng/ml TGF-β.
After cell is broken up, be inoculated in Tissue Culture Plate prepared by embodiment 1, after 7 days, change substratum.Washing culture plate, removes not attached cell.Continue to use the mesenchymal stem cells in umbilical cord blood culture medium culturing of adding 10% foetal calf serum, within every 7 days, change liquid once, until cell approaches 90% fusion.
90% fused cell is digested with pancreatin, be inoculated in general culture plate, use the mesenchymal stem cells in umbilical cord blood culture medium culturing of 10% foetal calf serum, within 3 days, change liquid once.Until merge, and go down to posterity next time.
Embodiment 3: the mescenchymal stem cell surface antigen of cultivation characterizes
In order to determine that above-mentioned cell has the feature of mescenchymal stem cell surface antigen, use FACs to analyze cell surface.The results are shown in table 1.
Table 1
Table 1 confirms, for the stem cell that the present invention separated and cultivated, CD34, CD45 and CD3 are negative, and CD73, CD105 and CD90 are positive.This result shows that the cell that the present invention separates and increases is mescenchymal stem cell.
The present invention is not limited to described embodiment, and those skilled in the art, in the situation that not departing from main idea of the present invention, design and aim, can change, add and replace the succession of each step in mixture of the present invention and method or method.
Claims (8)
1. a method for separation and amplification blood mescenchymal stem cell, is characterized in that, the method comprises the following steps:
Step a: collect blood sample,
Step b: monocyte composition in separating blood sample,
Step c: monocyte composition is inoculated in to the culture dish that macromolecular material is modified,
Steps d: separating mesenchymal stem cell,
Step e: cultivate and amplification of mesenchymal stem cells.
2. the method for separation according to claim 1 and amplification blood mescenchymal stem cell, is characterized in that, described blood sample gathers from hospital, or obtains in autoblood storehouse.
3. the method for separation according to claim 1 and amplification blood mescenchymal stem cell, is characterized in that, described blood sample is people's blood sample.
4. according to the method for the separation described in claim 1 or 2 or 3 and amplification blood mescenchymal stem cell, it is characterized in that, described blood sample collection is certainly in premature labor or normal labor fetus and grownup's blood.
5. the method for separation according to claim 4 and amplification blood mescenchymal stem cell, is characterized in that, described blood sample all passes through anticoagulant heparin, Walocel MT 20.000PV splitting erythrocyte, and density gradient centrifugation is inoculated after obtaining monocyte composition.
6. the method for separation according to claim 1 and amplification blood mescenchymal stem cell, is characterized in that, in described step c, macromolecular material refers to that polyoxyethylene glycol and arginine-glycine-aspartic acid mix by concentration 1ml:0.01g of the same race.
7. the method for separation according to claim 1 and amplification blood mescenchymal stem cell, is characterized in that, in described step e, cultivation and amplification of mesenchymal stem cells refer to that applying blood mescenchymal stem cell substratum cultivates and increase.
8. the method for separation according to claim 7 and amplification blood mescenchymal stem cell, is characterized in that, described blood mescenchymal stem cell substratum comprises: α-MEM, Pen-Strep, b-FGF, EGF, TGF-β.
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| CN106754673A (en) * | 2016-12-09 | 2017-05-31 | 北京雨泽瑞清生物科技有限公司 | The method of modifying and mescenchymal stem cell at a kind of culture medium bottom separate amplification method |
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| CN101032692A (en) * | 2007-04-17 | 2007-09-12 | 武汉理工大学 | Polyurethane material for improving adhering growing of cells and rombolytic function |
| CN101810559A (en) * | 2009-02-20 | 2010-08-25 | 北京大学 | Hirudin polyion micelle composition of targeted platelet |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106754673A (en) * | 2016-12-09 | 2017-05-31 | 北京雨泽瑞清生物科技有限公司 | The method of modifying and mescenchymal stem cell at a kind of culture medium bottom separate amplification method |
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Application publication date: 20140514 |