CN110172446A - A method of separation mesodermal stroma cell - Google Patents
A method of separation mesodermal stroma cell Download PDFInfo
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- CN110172446A CN110172446A CN201910311628.6A CN201910311628A CN110172446A CN 110172446 A CN110172446 A CN 110172446A CN 201910311628 A CN201910311628 A CN 201910311628A CN 110172446 A CN110172446 A CN 110172446A
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- 108010015865 Transferrins Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000002695 general anesthesia Methods 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000001665 muscle stem cell Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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Abstract
The invention discloses a kind of method for separating mesodermal stroma cell, specifically comprise the following steps: to acquire peripheral blood, gradient centrifugation draws mononuclearcell layer, adhere-wall culture, CXCR4 magnetic bead sorting etc..Method of the invention can effectively improve the separation rate of mesodermal stroma cell, have many advantages, such as easy to operate, quick, efficient, and it is more to obtain cell, efficiently separate method using providing one kind for clinically mesodermal stroma cell.
Description
Technical field
The present invention relates to a kind of methods for separating mesodermal stroma cell.
Background technique
Currently, mesoderm is that organism evolves to one of primary condition of terrestrial, and the stem cell of mesoderma origin, such as make
Hemocytoblast, muscle stem cell etc. are seed cells important in current regenerative medicine and organizational project, in basic research and are faced
It is had broad application prospects in bed treatment.
The treatment of heart failure is that medically a great problem, the only qualified patient of only a few can receive heart shifting
Treatment is planted, and is a kind of potential replacement therapy method to the stem-cell therapy of heart failure.In clinical application, marrow is stem cell
The most common source, and have been demonstrated that cardiac function can be improved.However, marrow acquisition modes (i.e. bone marrow aspiration) are also brought
Many problems: for patient, this is the behavior of a kind of property invaded and pain；Bone marrow aspiration also has the risk of infection；At certain
In a little situations, patient also needs general anesthesia.These factors make this process become complicated and expensive.
Mesodermal stroma cell is the fibroblast-like cells separated from adult peripheral blood, their form, surface
Label and its differentiation potential are similar to mesenchymal stem cell (BM-MSC).Existing research shows to move in mesodermal stroma cell
Planting impaired myocardial region is a kind of effective therapeutic modality, it can substitute or repair necrosis, have scar or function not just
Normal cardiac muscle cell.In clinical studies, a large amount of clinical test achieves good progress, including myocardial infarction, it is chronic
Ischemic left ventricular dysfunction and heart failure patient.In terms of curative effect, is all detected in most of tests and penetrate blood system
Several increases.
In addition to this, existing research person is tested using the monoclonal strain of dog, and the mesodermal stroma for showing survival is thin
Cellular expression osteocalcin, by the way that bone lining cell * can be formed after its autogenous vein graft.Separately there is test to confirm, by the mesoderm of rabbit
Stroma cell carries out autotransplantation after combining with loose calcium phosphate soluble substrate, as a result, it has been found that in rabbit ulnar critical size
Bone defect model on osteanagenesis can be enhanced, this show the circulation skeletonization that mesodermal stroma cell can be used as osteanagenesis
A kind of new resources of cell.In addition to this, it has been also reported that people, rabbit, cavy, mouse and rat mesodermal stroma cell can
To be divided into osteoblast, cartilage cell, fat cell, desmacyte, myotube and fibroblast.Because blood is more than marrow
It is easy to get, uses peripheral blood fairly obvious as the advantage of MSC potential resource.
In view of mesodermal stroma cell in the good prospect of clinical research, the invention discloses a kind of mesodermal stroma cells
Efficiently separate method, cooperate its dedicated serum free medium, provided for clinical research and effective obtain cell and carry out body
The method of outer culture amplification.
Summary of the invention
It is a kind of for separating the side of mesodermal stroma cell object of the present invention is to provide in view of the defects existing in the prior art
Method.
The present invention to achieve the above object, adopts the following technical scheme that a kind of for separating the side of mesodermal stroma cell
Method acquires peripheral blood, carries out gradient centrifugation, draws mononuclearcell layer, and carry out adhere-wall culture, finally uses CXCR4 magnetic bead
Cell after culture is sorted, it is final to obtain mesodermal stroma cell and carry out in vitro culture.
In further embodiment, before gradient centrifugation, peripheral blood need to be subjected to 1:2 dilution with dilution, dilution
It is formulated as follows:
Wherein, the molecular weight of hydroxyethyl starch is 100K-400K.
In further embodiment, the peripheral blood after dilution carries out ladder using Ficoll Hypaque human lymphocyte separating liquid
Degree centrifugation, relative density 1.074-1.079g/L.
In further embodiment, centrifugal force when gradient centrifugation is 300~400g, 25~35min, using without brake nature
Reduction of speed.
In further embodiment, supernatant is removed, draws intermediate nebulous tunica albuginea confluent monolayer cells.
In further embodiment, cleaning and culture cell use the dedicated serum free medium of mesoderm, count according to 1000ml,
It is formulated as follows:
L-Alanine (sigma) 0.3mmol, L-arginine (sigma) 1mmol, L-Aspartic acid (sigma) 0.4mmol/
L, l-cysteine (sigma) 0.2mmol, L-cysteine (sigma) 0.6mmol, l-Isoleucine (sigma) 0.4mmol, L-
Leucine (sigma) 0.4mmol, L-lysine (sigma) 0.2mmol, l-methionine (sigma) 0.1mmol, L-PROLINE
(sigma) 0.3mmol, Serine (sigma) 0.6mmol, L-threonine (sigma) 0.6mmol, L-Trp (sigma)
0.1mmol, l-tyrosine (sigma) 0.2mmol, Valine (sigma) 0.8mmol, biotin (sigma) 8 × 10- 4mmol、
Calciferol (sigma) 3 × 10-4Mmol, folic acid (sigma) 6 × 10-2Mmol, Vitamin K3 (sigma) 6 × 10-5Mmol, niacinamide (sigma) 2 × 10-4Mmol, D- pantothen calcium (sigma) 1 × 10-3Mmol, pyridoxal (sigma) 2 ×
10-4Mmol, riboflavin (sigma) 3 × 10-4Mmol, thiamine (sigma) 3 × 10-3Mmol, vitamin E (sigma) 2 × 10- 5Mmol, vitamin B12 (sigma) 1 × 10-3Mmol, sodium selenite (sigma) 3 × 10-3mmol、CaCl2(sigma)
0.3mmol, KCl content are 3mmol, MgSO4(sigma)0.6mmol、NaCl(sigma)128mmol、NaHCO3(sigma)
14mmol、Na2HPO4(sigma)1mmol、CuSO4·5H2O(sigma)1.6×10-5mmol、FeSO4·7H2O(sigma)3×
10-3mmol、ZnSO4·7H2O(sigma)3×10-3Mmol, adenosine (sigma) 3 × 10-2Mmol, desoxyadenossine (sigma) 4 ×
10-2Mmol, deoxycytidine (sigma) 4 × 10-2Mmol, deoxyguanosine (sigma) 4 × 10-2Mmol, hypoxanthine (sigma)
1.5×10-2Mmol, uridine (sigma) 4 × 10-2Mmol, D-Glucose (sigma) 10mmol, Sodium Pyruvate (sigma)
1mmol, unsaturated fatty acid (sigma) 5 × 10-4Mmol, Tween-80 (sigma) 2 × 10-2Mmol, astragalus polyose (sigma)
2g, 120 μ g of neurotrophic factor -3 (NT-3) (sigma), 60 μ g of platelet derived growth factor (PDGF) (sigma), turn iron
Albumen (sigma) 20mg, 100 μ g of vitronectin (sigma), laminin (sigma) 2.73g, phenol red 12mg.
In further embodiment, after mesoderm stroma cell is expanded to 80%~90% fusion, using cell dissociation buffer
It digests the mesodermal stroma cell and the mesodermal stroma cell is sorted with CXCR4 magnetic bead.
In further embodiment, the pH value of the dilution is 7.15~7.35.
In further embodiment, the molecular weight of the hydroxyethyl starch is 200K, 20g/L.
In further embodiment, the peripheral blood after dilution carries out ladder using Ficoll Hypaque human lymphocyte separating liquid
Degree centrifugation, relative density 1.077g/L.
Beneficial effects of the present invention: method of the invention can effectively improve the separation rate of mesodermal stroma cell, have
It is easy to operate, quick, efficient, the advantages that cell is more are obtained, are had for clinically mesodermal stroma cell using providing one kind
The separation method of effect.
Detailed description of the invention
Fig. 1 is the first aspect graph of cell of the invention；
Fig. 2 is the second aspect graph of cell of the invention；
Fig. 3-Fig. 7 is cell first stage detection schematic diagram of the invention；
Fig. 8-Fig. 9 is cell second stage detection schematic diagram of the invention；
Figure 10 is cell of the invention using form schematic diagram after Alizarin red staining；
Figure 11 is cell of the invention using form schematic diagram after A Erxinlan dyeing；
Figure 12 is cell of the invention using form schematic diagram after oil red O stain.
Specific embodiment
Below with reference to example, invention is further described in detail:
The preparation of embodiment 1:1000ml mesodermal stroma cell culture medium:
Weigh l-Alanine (sigma) 0.3mmol, L-arginine (sigma) 1mmol, L-Aspartic acid (sigma)
0.4mmol/L, l-cysteine (sigma) 0.2mmol, L-cysteine (sigma) 0.6mmol, l-Isoleucine (sigma)
0.4mmol, L-Leu (sigma) 0.4mmol, L-lysine (sigma) 0.2mmol, l-methionine (sigma)
0.1mmol, L-PROLINE (sigma) 0.3mmol, Serine (sigma) 0.6mmol, L-threonine (sigma) 0.6mmol,
L-Trp (sigma) 0.1mmol, l-tyrosine (sigma) 0.2mmol, Valine (sigma) 0.8mmol, biotin
(sigma)8×10-4Mmol, calciferol (sigma) 3 × 10-4Mmol, folic acid (sigma) 6 × 10-2Mmol, Vitamin K3
(sigma)6×10-5Mmol, niacinamide (sigma) 2 × 10-4Mmol, D- pantothen calcium (sigma) 1 × 10-3Mmol, pyridoxal
(sigma)2×10-4Mmol, riboflavin (sigma) 3 × 10-4Mmol, thiamine (sigma) 3 × 10-3Mmol, vitamin E
(sigma)2×10-5Mmol, vitamin B12 (sigma) 1 × 10-3Mmol, sodium selenite (sigma) 3 × 10-3mmol、CaCl2
(sigma) 0.3mmol, KCl content are 3mmol, MgSO4(sigma)0.6mmol、NaCl(sigma)128mmol、NaHCO3
(sigma)14mmol、Na2HPO4(sigma)1mmol、CuSO4·5H2O(sigma)1.6×10-5mmol、FeSO4·7H2O
(sigma)3×10-3mmol、ZnSO4·7H2O(sigma)3×10-3Mmol, adenosine (sigma) 3 × 10-2Mmol, desoxyadenossine
(sigma)4×10-2Mmol, deoxycytidine (sigma) 4 × 10-2Mmol, deoxyguanosine (sigma) 4 × 10-2Mmol, secondary Huang are fast
Purine (sigma) 1.5 × 10-2Mmol, uridine (sigma) 4 × 10-2Mmol, D-Glucose (sigma) 10mmol, Sodium Pyruvate
(sigma) 1mmol, unsaturated fatty acid (sigma) 5 × 10-4Mmol, Tween-80 (sigma) 2 × 10-2Mmol, astragalus polyose
(sigma) 2g, 120 μ g of neurotrophic factor -3 (NT-3) (sigma), 60 μ of platelet derived growth factor (PDGF) (sigma)
G, transferrins (sigma) 20mg, 100 μ g of vitronectin (sigma), laminin (sigma) 2.73g, phenol red 12mg are mixed
It closes, after addition 800ml pure water stirs and evenly mixs, is settled to 1000ml.After mixing well, pH value is adjusted to 7.2,0.22 μm of filter membrane mistakes
Filter out bacterium, 4 DEG C of preservations.
Embodiment 2: the isolation and culture (1 used medium of embodiment) of mesodermal stroma cell
1, Healthy Volunteers peripheral blood (~18ml) is taken under aseptic condition；
2, peripheral blood is subjected to 1:2 dilution with HBSS；
3, the Ficoll Hypaque human lymphocyte separating liquid of 15ml is added in each 50ml centrifuge tube, it is relatively close
Degree is 1.077g/L；
4, the peripheral blood diluted is slowly added to the human lymphocyte separating liquid of Hypaque containing Ficoll upper layer along tube wall
(the two ratio is 1:1)；
5, it is centrifuged at room temperature, 400g, 35min, natural reduction of speed；
6, under the premise of not destroying tunica albuginea layer, the supernatant as much as possible (HBSS and blood of top layer is removed with pipette
Slurry)；
7, intermediate nebulous tunica albuginea confluent monolayer cells (i.e. mononuclearcell) are drawn with pipette, shifts and is incorporated into new
(volume for drawing mononuclearcell is recorded with graduated pipette) in 50ml centrifuge tube；
8, it is diluted with the HBSS of at least 3 times mononuclearcell volumes；
9, it is centrifuged at room temperature, 500g, 15min；
10, supernatant is abandoned；
11, cell, adjustment cell density to 1*10 is resuspended with culture medium matched in embodiment 16/ml；
12, it is seeded to culture bottle, at 37 DEG C, 5%CO2It is cultivated under the conditions of incubator；
13, liquid is changed after adherent 5-7 days, pours out the cell conditioned medium in culture bottle, added 5ml culture medium and continue to cultivate；
14, it when culture to 90% fusion, is digested with the digestive juice containing 0.25% trypsase；
15, cell is sorted with CXCR4 magnetic bead；
16, for cell inoculation to culture bottle culture, cellular morphology is as shown in Figure 1 after sorting.
Embodiment 3: the isolation and culture (culture medium containing FBS) of mesodermal stroma cell
Experimental procedure is with embodiment 2, and used medium changes DMEM/F12 (containing 10%FBS) into, and the cellular morphology of culture is such as
Shown in Fig. 2.
Embodiment 4: the identification of mesodermal stroma cell
The mesodermal stroma cell culture sorted in above-described embodiment 2 carries out streaming identification, concrete operations step to the 3rd generation
It is rapid as follows:
The 3rd generation cell is taken, is digested when reaching 80%~90% fusion with trypsase/EDTA digestive juice, is made 1 × 106/
Ml cell suspension.It is each to be separately added into mouse anti-human monoclonal's antibody CD45-PE, CD73-PE, CD90-PE, CD105-PE, HLA-DR
10 μ l, mix well, and react 30min at room temperature, and 1.5mlPBS is added in every pipe, and 1200r/min is centrifuged 5min, abandon supernatant, every pipe
Be added 100 μ lPBS, flow cytomery, process detection such as Fig. 3-Fig. 7, detection the result is as follows:
Table 1: cell surface molecule Mark Detection result table
| Surface molecular label | Positive rate |
| HLA-DR | 2.75% |
| CD45 | 1.85% |
| CD73 | 98.0% |
| CD90 | 98.6% |
| CD105 | 98.1% |
Embodiment 5: the identification of mesodermal stroma cell surface early stage stem cell molecular marker
The mesodermal stroma cell culture sorted in above-described embodiment 2 carries out streaming identification, concrete operations step to the 3rd generation
It is rapid as follows:
The 3rd generation cell is taken, is digested when reaching 80%~90% fusion with trypsase/EDTA digestive juice, is made 1 × 106/
Ml cell suspension.It is separately added into mouse anti-human monoclonal's antibody SSEA-4PE, CXCR4PE10 μ l, mixes well, reacts at room temperature
1.5mlPBS is added in 30min, every pipe, and 1200r/min is centrifuged 5min, abandons supernatant, and 100 μ lPBS, flow cytometer inspection is added in every pipe
It surveys, detection process is shown in Fig. 8 and Fig. 9, the result is as follows:
Table 2: cell surface placenta molecular marker testing result table
| Surface molecular label | Positive rate |
| SSEA-4 | 84.42% |
| CXCR4 | 91.97% |
The differentiation of 6 mesodermal stroma cell of embodiment
Osteogenic induction and detection:
Take P4 for mesodermal stroma cell, respectively with 6 × 10 after cell count4The density in a/hole is inoculated in 6 well culture plates
In, use osteogenic induction liquid instead afterwards for 24 hours, every 3d changes liquid, induces 2 Zhou Houyong Alizarin red stainings, testing result such as Figure 10.
At chondrocyte induction and detection:
Take P4 for mesodermal stroma cell, respectively with 3 × 10 after cell count5A cell is set in 15ml centrifuge tube, 220g
Low-speed centrifugal 8min makes cell form micelle.It is cultivated in centrifuge tube at chondrocyte induction liquid+TGF-β 3, changes liquid at interval of 3d,
Induction 5 weeks, 4% paraformaldehyde is fixed, conventional to make paraffin section.Slice dewaxes through dimethylbenzene, graded ethanol dehydration, distilled water
It is dyed after washing with A Erxinlan, as a result as shown in figure 11.
Adipogenic induction and detection:
Take P4 for mesodermal stroma cell, respectively with 1 × 10 after cell count5/ hole is inoculated in 6 well culture plates, conventional
Culture medium culture be changed to afterwards for 24 hours fat induction liquid A start to induce, after 3d replace adipogenic induction liquid B maintain, for 24 hours after replace again
It is induced at adipogenic induction liquid A, 3 Zhou Houyong oil red O stain of circulation-induced, as a result as shown in figure 12.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method for separating mesodermal stroma cell, which is characterized in that acquisition peripheral blood carries out gradient centrifugation, inhales
Mononuclearcell layer is taken, and carries out adhere-wall culture, finally the cell after culture is sorted using CXCR4 magnetic bead, is finally obtained
It takes mesodermal stroma cell and carries out in vitro culture.
2. the method for separation mesodermal stroma cell according to claim 1, which is characterized in that before gradient centrifugation,
Peripheral blood need to be subjected to 1:2 dilution with dilution, the formula of dilution is as follows:
Wherein, the molecular weight of the hydroxyethyl starch is 100K-400K.
3. the method for separation mesodermal stroma cell according to claim 1, which is characterized in that the peripheral blood after dilution is adopted
Gradient centrifugation, relative density 1.074-1.079g/L are carried out with Ficoll Hypaque human lymphocyte separating liquid.
4. the method for separation mesodermal stroma cell according to claim 1, which is characterized in that centrifugation when gradient centrifugation
Power is 300~400g, 25~35min, using without brake nature reduction of speed.
5. the method for separation mesodermal stroma cell according to claim 1, which is characterized in that remove supernatant, draw
Intermediate nebulous tunica albuginea confluent monolayer cells.
6. it is according to any one of claims 1 to 5 separation mesodermal stroma cell method, which is characterized in that cleaning and
It cultivates cell and uses the dedicated serum free medium of mesoderm, counted according to 1000ml, is formulated as follows:
L-Alanine (sigma) 0.3mmol, L-arginine (sigma) 1mmol, L-Aspartic acid (sigma) 0.4mmol/L, L-
Cystine (sigma) 0.2mmol, L-cysteine (sigma) 0.6mmol, the bright ammonia of l-Isoleucine (sigma) 0.4mmol, L-
Acid (sigma) 0.4mmol, L-lysine (sigma) 0.2mmol, l-methionine (sigma) 0.1mmol, L-PROLINE
(sigma) 0.3mmol, Serine (sigma) 0.6mmol, L-threonine (sigma) 0.6mmol, L-Trp (sigma)
0.1mmol, l-tyrosine (sigma) 0.2mmol, Valine (sigma) 0.8mmol, biotin (sigma) 8 × 10- 4Mmol, calciferol (sigma) 3 × 10-4Mmol, folic acid (sigma) 6 × 10-2Mmol, Vitamin K3 (sigma) 6 × 10- 5Mmol, niacinamide (sigma) 2 × 10-4Mmol, D- pantothen calcium (sigma) 1 × 10-3Mmol, pyridoxal (sigma) 2 × 10-4Mmol, riboflavin (sigma) 3 × 10-4Mmol, thiamine (sigma) 3 × 10-3Mmol, vitamin E (sigma) 2 × 10- 5Mmol, vitamin B12 (sigma) 1 × 10-3Mmol, sodium selenite (sigma) 3 × 10-3mmol、CaCl2(sigma)
0.3mmol, KCl content are 3mmol, MgSO4 (sigma) 0.6mmol, NaCl (sigma) 128mmol, NaHCO3(sigma)
14mmol、Na2HPO4(sigma)1mmol、CuSO4·5H2O(sigma)1.6×10-5mmol、FeSO4·7H2O(sigma)3×
10-3mmol、ZnSO4·7H2O(sigma)3×10-3Mmol, adenosine (sigma) 3 × 10-2Mmol, desoxyadenossine (sigma) 4 ×
10-2Mmol, deoxycytidine (sigma) 4 × 10-2Mmol, deoxyguanosine (sigma) 4 × 10-2Mmol, hypoxanthine (sigma)
1.5×10-2Mmol, uridine (sigma) 4 × 10-2Mmol, D-Glucose (sigma) 10mmol, Sodium Pyruvate (sigma)
1mmol, unsaturated fatty acid (sigma) 5 × 10-4Mmol, Tween-80 (sigma) 2 × 10-2Mmol, astragalus polyose (sigma)
2g, 120 μ g of neurotrophic factor -3 (NT-3) (sigma), 60 μ g of platelet derived growth factor (PDGF) (sigma), turn iron
Albumen (sigma) 20mg, 100 μ g of vitronectin (sigma), laminin (sigma) 2.73g, phenol red 12mg.
7. the method for separation mesodermal stroma cell according to claim 1, which is characterized in that when mesoderm stroma cell
After being expanded to 80%~90% fusion, which is digested and with CXCR4 magnetic bead in this using cell dissociation buffer
Germinal layer stroma cell is sorted.
8. the method for separation mesodermal stroma cell according to claim 2, which is characterized in that the pH value of the dilution
It is 7.15~7.35.
9. the method for separation mesodermal stroma cell according to claim 2, which is characterized in that the hydroxyethyl starch
Molecular weight is 200K, 20g/L.
10. the method for separation mesodermal stroma cell according to claim 3, which is characterized in that the peripheral blood after dilution
Gradient centrifugation, relative density 1.077g/L are carried out using Ficoll Hypaque human lymphocyte separating liquid.
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