CN104073468A - Method for obtaining human OPCs and OPCs medium - Google Patents
Method for obtaining human OPCs and OPCs medium Download PDFInfo
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Abstract
The invention relates to a method for obtaining human OPCs (Oligodendrocyte Progenitor Cells) and an OPCs medium. According to the method, neural stem cells are used as a sorting source, the human OPCs can be obtained by separation through an immunomagnetic bead method, and a more economical medium suitable for OPCs in vitro culture and passage, so that a great amount of high-purity human OPCs can be obtained in a short time, and a new choice is provided for clinical application.
Description
Technical field
The present invention relates to obtain method and the OPCs substratum of people source OPCs.
Background technology
The myelin relative diseases such as Infant Injury in White Matter, Spinal injury, multiple sclerosis are due to neuronic myelinization obstacle or demyelination, have caused the neurone can not normal delivery electrostimulation, thereby cause the function anergies such as body movement.Owing to also can effectively treating these myelin relative diseases without any a kind of medicine at present, this has brought great injury to patient and the family of these diseases, and this makes effectively to treat myelin relative disease becomes current society and difficult medical problem anxious to be resolved.
No matter be myelinization obstacle or Demyelination, its root is all that neuron axon myelin degree reduces or loses, if therefore can recover the myelinization degree of neuron axon, make neurone electric current be stablized conduction rapidly, these diseases just can be cured in theory.Research is found, oligodendrocyte precursor cells (oligodendrocyte progenitor cells, OPCs) be the key cells of being responsible for central nervous system myelinization, in central nervous system, OPCs can be divided into oligodendrocyte (oligodendrocyte, OL), OL further holds neuron axon and forms complete myelin structure, thereby ensures the stable conduction fast of neurone electric current.The disease that myelin is relevant is because disease has been destroyed the myelin structure having formed on the one hand; Especially because OPCs in neural system has been subject to damage, make OL lose source on the other hand.Oligodendrocyte reduces, and causes alba myelinization delay or destroyed, thereby makes electroneurographic signal conduction abnormalities or axonal loss, causes nervous function damage.
Concerning Demyelination patient, due to endogenous, OPCs loses in a large number, promote the regeneration of myelin in brain, and implanting external source pulpefaction sheath cell is a methods for the treatment of likely.Because OPCs is the natural origin of pulpefaction sheath cell in central nervous system, OPCs transplants and may play good curative effect to treatment Demyelination.Experiment is found, the OPCs of rodent is transplanted in newborn rodent brain, implant cell and can in host's brain, extensively move, breed and be divided into ripe OL, formation holds the myelin structure of aixs cylinder and recovers neural function, show that external source implantation OPCs can bring into play the function same with endogenous OPCs, it may be a new direction for the treatment of myelin damage disease that OPCs transplants, and to Demyelination, treatment has positive meaning for this.
Although the OPCs of rodent is easy to obtain, people source OPCs obtains quite difficulty.Depend on immunophenotype, by flow cytometry or immunomagnetic bead technique, sorting from fetal central nervous system tissue or Adult Human Brain white matter tissue can obtain OPCs.Windrem etc. have isolated A from fetus or adult brain tissue
2b
5 +pSA-NCAM
-the cell of phenotype, thinks that this cell is OPCs, this Transplanted cells is entered in the mouse of congenital demyelination, can form myelin.But because the donor tissue in people source is less, separate the OPCs quantity that obtains quite limited, and there is ethnics Problem in it, and therefore also very remote apart from clinical application.Another kind can obtain people source OPCs by embryonic stem cell or nerve stem cell directional induction differentiation.The people such as the Hans of the U.S. study discovery, and embryonic stem cell is external can be divided into OPCs, and OPCs transplants and contribute to the neurological functional recovery of Spinal injury, and the method has entered the treatment research of clinical I phase in the U.S..But the totipotency of embryonic stem cell is double-edged sword, can be divided into various cells, also there is the risk of tumorigenesis, in clinical application, also there is certain tumorigenesis risk in the OPCs that therefore the method obtains.Neural stem cell is a kind of adult stem cell, because adult stem cell differentiation capability is restricted, has avoided to a certain extent the risk of tumorigenesis, safer in clinical application.But the Induction Process of these two kinds of cells all needs the acting in conjunction of long period and cytokine profiles, this must cause the expense of cultivating to increase, the output of restrictive cell.
Therefore, clinical treatment research needs the method for simple acquisition and economic cultivator source OPCs a kind of.Neural stem cell is external the ability that is divided into neurone, astroglia cell and oligodendrocyte, and approximately has 16% cell expressing A
2b
5 +pSA-NCAM
-phenotype, simultaneously owing to being the risk that adult stem cell has been avoided again tumorigenesis to a certain extent.Neural stem cell can also increase in vitro in a large number, can say that neural stem cell is the source of good sorting OPCs.
Summary of the invention
The object of the invention is to address the above problem, a kind of people of acquisition is provided the novel method of source OPCs.For this reason, applicant is through further investigation, neural stem cell (no matter being low algebraically or higher generation number) is originated as sorting, separate and obtain people source OPCs by the method for immunomagnetic beads, and find the substratum of applicable OPCs vitro culture and the less expensive type that goes down to posterity, can obtain at short notice a large amount of highly purified people source OPCs, for clinical application provides new selection.
The method of acquisition provided by the invention people source OPCs is, neural stem cell is originated as sorting, separated and obtained people source OPCs by the method for immunomagnetic beads, and the magnetic bead of employing is Anti-PSA-NCAM magnetic bead.
Preferably, comprise the following steps:
1. neural stem cell is digested individual cells in trypsin solution, crosses screen cloth;
2. centrifugal collecting cell, removes supernatant;
3. cell is resuspended in magnetic bead sorting damping fluid, after mixing in 2-8 DEG C incubation;
4. add Anti-PSA-NCAM magnetic bead, after mixing in 2-8 DEG C incubation;
5. add magnetic bead sorting damping fluid, centrifugal;
6. in centrifugal process, sorting post is put into magnetic bead separator, add magnetic bead sorting damping fluid balance sorting post;
7. after centrifugal, abandoning supernatant, is resuspended in magnetic bead sorting damping fluid completely, and cell suspension is joined in sorting post, collects flowing liquid, and this is unlabelled cell;
8. add magnetic bead sorting damping fluid to wash separator column, centrifugal collecting cell;
9. add Anti-A2B5 magnetic bead, 5. incubation, repeat-8. step, and this time collecting cell should be the cell in set in post, and damping fluid is washed after post, and sorting post is placed in centrifuge tube from taking out in magnetic bead separator;
10. add after magnetic bead sorting damping fluid, pushing piston washes away in conjunction with upper cell immediately, centrifugal, and the cell of collecting is A
2b
5 +pSA-NCAM
-the ubcellular group of phenotype, is OPCs.
More preferably, the concrete operation method of each step is as follows:
1. neural stem cell is digested individual cells in 0.025% trypsin solution, crosses 40 μ m nylon screens with filtering cell mass, the blue total cellular score of calculating of platform phenol; " % " is the usual simple expression mode of " g/100ml " above, and 0.025% is 0.025g/100ml.
2. get 10
7cell, centrifugal 10 minutes collecting cells of 300g, remove supernatant;
3. 10
7cell is resuspended in the magnetic bead sorting damping fluid of 60 μ l, after mixing in 2-8 DEG C incubation 10 minutes;
4. add the Anti-PSA-NCAM magnetic bead of 20 μ l, after mixing in 2-8 DEG C incubation 15 minutes;
5. add 1ml magnetic bead sorting damping fluid, centrifugal 10 minutes of 300g;
6. in centrifugal process, MS sorting post is put into magnetic bead separator, add the magnetic bead sorting damping fluid balance sorting post of 0.5ml;
7. after centrifugal, abandoning supernatant completely, is resuspended in the magnetic bead sorting damping fluid of 0.5ml, and cell suspension is joined in sorting post, collects flowing liquid, and this is unlabelled cell;
8. add the magnetic bead sorting damping fluid of 0.5ml to wash separator column, totally three times, centrifugal collecting cell;
9. add the Anti-A2B5 magnetic bead of 20 μ l, 5. incubation, repeat above-step 8., and this time collecting cell should be the cell in set in post, and damping fluid is washed after post three times, and sorting post is placed in the centrifuge tube of 1.5ml from taking out in magnetic bead separator;
10. add after 1ml magnetic bead sorting damping fluid, pushing piston washes away in conjunction with upper cell immediately, centrifugal 10 minutes of 300g, and the cell of collecting is A
2b
5 +pSA-NCAM
-the ubcellular group of phenotype, is OPCs.
Preferably, utilize OPCs substratum to carry out amplification in vitro cultivation to separating the people source OPCs obtaining, described substratum is made up of Neurobasal A medium, B27, bFGF, heparin and L-glutamin, the volume ratio of Neurobasal A medium, B27 and L-glutamin is 97: 2: 1, the content of bFGF is 20-40ng/ml, and the content of heparin is 5 μ g/ml.
More preferably, the concrete operation method that amplification in vitro is cultivated is: the cell obtaining after magnetic bead sorting is according to 4 × 10
4/ cm
2density is seeded in culture vessel, in OPCs substratum, cultivates, and is placed in 37 DEG C, 5%CO
2in incubator, every 3-4 days, changing 2/3 substratum is fresh OPCs substratum, when cell reaches the degrees of fusion of 80%-90%, the cultivation of going down to posterity, the number of times of cultivating that goes down to posterity is 2-4 time.
Preferably, described neural stem cell adopts nerve stem cell culture medium carry out amplification in vitro and obtain, described nerve stem cell culture medium is made up of DMED/F12, N2, bFGF, EGF, heparin and L-glutamin, the volume ratio of DMED/F12, N2 and L-glutamin is 98: 1: 1, the concentration of bFGF is 10-30ng/ml, and the concentration of EGF is 10-30ng/ml.
More preferably, the operation steps of neural stem cell amplification in vitro is as follows:
1. 100g collects neural stem cell for centrifugal 5 minutes;
2. supernatant being transferred in centrifuge tube, is old substratum;
3. in the neural stem cell of having collected, add 1ml to be diluted 0.025% trypsinase obtaining by 0.01M dPBS, mix, putting 37 DEG C of incubators, to hatch 5-7min loose to cell ball;
4. add 1.2mg/ml pancreatin inhibitor 100 μ l, 200 μ l pipettor suckers are blown and beaten into single cell suspension gently;
5. add 15ml dPBS, cell counting, the centrifugal 5min of 100g;
6. abandon supernatant, add 2/3 fresh nerve stem cell culture medium and 1/3 old substratum, with 2 × 10
6/ mL density is inoculated in T25ml culturing bottle, puts in cell culture incubator and cultivates;
7. to change 2/3 substratum be fresh culture to every 3-4 days, within every 10 days, goes down to posterity once.
The method of acquisition people source OPCs provided by the invention also comprises that the people source OPCs that utilizes OPCs substratum to obtain alternate manner carries out amplification in vitro cultivation, described substratum is made up of Neurobasal A medium, B27, bFGF, heparin and L-glutamin, the volume ratio of Neurobasal A medium, B27 and L-glutamin is 97: 2: 1, the content of bFGF is 20-40ng/ml, and the content of heparin is 5 μ g/ml.
Preferably, the concrete operation method that the people source OPCs amplification in vitro that alternate manner obtains is cultivated is: people source OPCs is according to 4 × 10
4/ cm
2density is seeded in culture vessel, in OPCs substratum, cultivates, and is placed in 37 DEG C, 5%CO
2in incubator, every 3-4 days, changing 2/3 substratum is fresh OPCS substratum, when cell reaches the degrees of fusion of 80%-90%, the cultivation of going down to posterity, the number of times of cultivating that goes down to posterity is 2-4 time.
OPCs substratum is also protection content of the present invention; it is made up of Neurobasal A medium, B27, bFGF, heparin and L-glutamin; the volume ratio of Neurobasal A medium, B27 and L-glutamin is 97: 2: 1; the content of bFGF is 20-40ng/ml, and the content of heparin is 5 μ g/ml.
The method easy handling of acquisition people source OPCs provided by the invention, can obtain a large amount of highly purified people source OPCs, at short notice for clinical application provides new selection.
Brief description of the drawings
Fig. 1 is shown as the cell purity that flow cytometer showed sorting obtains and arrives more than 85%.
Fig. 2 is shown as sorting cells, and to have expressed multiple special OPCs mark: A be sorting after three days, and cell be typical bipolar or three utmost point forms; B-D is respectively immunofluorescence dyeing and shows the special mark of sorting cells expression OPCs O4, PDGF α R and Sox10; E is astroglia cell mark GFAP positive cell; F is neurone mark Tuj-1 positive cell.Scale: 50 μ m (A-E), 100 μ m (F).
Fig. 3 is shown as the OPCs that sorting obtains and can externally at least cultivates for four generations: the microscopic examination s-generation to the four generations OPCs, is typical bipolar or three utmost point forms; Immunofluorescence dyeing shows the equal high expression level oligodendrocyte precursor cells of each algebraically OPCs mark O4.Scale: 50 μ m.
Fig. 4 is shown as the 4th generation OPCs immunofluorescence dyeing result that sorting obtains: OPCs high expression level is prominent precursor cell mark P DGF α R and Sox10, extremely low expression astroglia cell mark GFAP and neurone mark Tuj-1 less.Scale: 50 μ m (PDGF α R, Sox10 and GFAP), 100 μ m (Tuj-1).
Fig. 5 is shown as the first-generation to the four generations OPCs growth curve: through the amplification in vitro in four generations, cell proliferation is more than 14 times.
Fig. 6 is shown as OPCs and is divided into oligodendrocyte.A: the cellular form after micro-Microscopic observation differentiation, cell is complicated multiple-branching construction; B: immunofluorescence dyeing result shows that noble cells can express the O4 of multi-branched; C: immunofluorescence dyeing result shows noble cells expression Galc.Scale: 25 μ m.
Embodiment
Be concrete embodiment of the present invention below.
1 from neural stem cell sorting obtain OPCs
1.1 human nerve stem cell amplification in vitros
1. 100g collects neural stem cell for centrifugal 5 minutes;
2. supernatant being transferred in centrifuge tube, is old substratum;
3. to adding 1ml0.025% trypsin 0.01M dPBS dilution in the neural stem cell of having collected), mix, putting 37 DEG C of incubators, to hatch 5-7min loose to cell ball;
4. add 1.2mg/ml pancreatin inhibitor 100 μ l, 200 μ l pipettor suckers are blown and beaten into single cell suspension gently;
5. add 15ml dPBS, cell counting, the centrifugal 5min of 100g;
6. abandon supernatant, add 2/3 fresh nerve stem cell culture medium and 1/3 old substratum, with 2 × 10
6/ mL density is inoculated in T25ml culturing bottle, puts in cell culture incubator and cultivates.
7. to change 2/3 substratum be fresh culture to every 3-4 days, within every 10 days, goes down to posterity once.
Nerve stem cell culture medium:
1.2 use magnetic bead sorting from neural stem cell to obtain the OPCs in people source
1. neural stem cell is digested individual cells in 0.025% trypsin solution, crosses 40 μ m nylon screens with filtering cell mass, the blue total cellular score of calculating of platform phenol;
2. get 10
7cell, centrifugal 10 minutes collecting cells of 300g, remove supernatant;
3. 10
7cell is resuspended in the magnetic bead sorting damping fluid of 60 μ l, after mixing in 2-8 DEG C incubation 10 minutes;
4. add the Anti-PSA-NCAM magnetic bead of 20 μ l, after mixing in 2-8 DEG C incubation 15 minutes;
5. add 1ml magnetic bead sorting damping fluid, centrifugal 10 minutes of 300g;
6. in centrifugal process, MS sorting post is put into magnetic bead separator, add the magnetic bead sorting damping fluid balance sorting post of 0.5ml;
7., after centrifugal, abandoning supernatant completely, is resuspended in the magnetic bead sorting damping fluid of 0.5ml.Cell suspension is joined in sorting post, collect flowing liquid, this is unlabelled cell;
8. add the magnetic bead sorting damping fluid of 0.5ml to wash separator column, totally three times, centrifugal collecting cell;
9. add Anti-A2B5 magnetic bead, after mixing in 2-8 DEG C incubation, repeat 5.-8. step, after incubation, cross post, this time collecting cell should be the cell in set in post, and damping fluid is washed after post three times, and MS sorting post is placed in the centrifuge tube of 1.5ml from taking out in magnetic bead separator;
10. add after 1ml magnetic bead sorting damping fluid, pushing piston washes away in conjunction with upper cell immediately, centrifugal 10 minutes of 300g, and the cell of collecting is A
2b
5 +pSA-NCAM
-the ubcellular group of phenotype, is OPCs.
Magnetic bead sorting damping fluid: PBS (pH7.2)+0.5%FBS+2mM EDTA.
By the method for immunomagnetic beads, sub-elect A
2b
5 +pSA-NCAM
-phenotype ubcellular group, separating purity is up to more than 85%.This cell subsets high expression level O
4, PDGF α R and Sox10 (mark of OPCs), only have only a few cell expressing GFAP (astroglia cell mark) and Tuj-1 (neurone mark), show can obtain highly purified more early stage OPCs from neural stem cell.And from the neural stem cell of different algebraically, as low algebraically P2, P5, P8 etc., higher generation is counted P25, P30 etc., can use the method for magnetic bead sorting to obtain the OPCs of higher degree.
The amplification in vitro of 2OPCs is cultivated
Clinical application needs a large amount of cells, if the cell amplification cultivation in vitro that therefore sorting obtains will make process simpler, expense is cheaper.The cell obtaining after magnetic bead sorting is according to 4 × 10
4/ cm
2density is seeded in culture vessel, in OPCs substratum, cultivates, and is placed in 37 DEG C, 5%CO
2in incubator.Every 3-4 days, changing 2/3 substratum is fresh OPCs substratum.When cell reaches the degrees of fusion of 80%-90%, the cultivation of going down to posterity.
Every 7-10 days goes down to posterity once, while going down to posterity, cell is blown afloat gently with suction pipe, adds appropriate fresh culture, with 4 × 10
4/ cm
2density is inoculated in Tissue Culture Plate, puts continuation amplification cultivation in cell culture incubator.Get part cell simultaneously and be inoculated in pretreated 48 orifice plates of PDL/laminin, after 4 days, carry out immunofluorescence dyeing.
The highly purified OPCs that sorting obtains can at least go down to posterity constant in the situation that and cultivate four times in vitro maintaining initial proterties.The cell in this four generation all has forms bipolar or three utmost points, continues high expression level O4, the 4th generation cell high expression level Sox10 and PDGF α R, and low expression GFAP and Tuj-1, and the overwhelming majority is still in early days.
After OPCs sorting, count, get 1.5 × 10
5cell is inoculated in 24 orifice plates, is P0.After 10 days, blow afloat gently cell with 200 μ l pipettors, counting, is first-generation P1.In 4 generations of continuous detecting, draw out the growth curve of OPCs.Can find out from growth curve, cell, in logarithmic phase, has stronger multiplication capacity, and through the cultivation in four generations, OPCs quantitatively can breed to more than 14 times.
OPCs substratum:
Conventionally the external long-term cultivation of people source OPCs all needs three kinds of combined utilization to four kinds of cytokines, and this OPCs substratum that inventor uses only contains a kind of cytokine---bFGF, reduce greatly the cost of cultivating, for extensive clinical application provides possibility.
The Differentiation Induction in vitro ability of 3OPCs
In order to determine whether the cell of sorting has differentiation capability, be inoculated in the orifice plate that PDL-Laminin was coated with, in OPCs division culture medium, cultivate.Through the cultivation of 14 days, part synapse cell formed the netted form of multi-branched complexity that grow, dendritic, most of in the non-ripe oligodendrocyte stage, and can be identified by the O4 of multi-branched and Galc.Show that the OPCs obtaining by sorting has the ability that is divided into oligodendrocyte.
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, carries out according to the condition of normal condition or manufacturers's suggestion.Production company and the article No. of following examples agents useful for same or instrument are as shown in the table, the unreceipted person of production firm, and being can be by the conventional products of commercial acquisition.
| Name of product | Company | Article No. |
| DMED/F12 | Gibco | C11330500BT |
| Neurobasal-A?medium | Gibco | 10888-022 |
| N2?supplement | Gibco | 17502-048 |
| B27?supplement | Gibco | 17504-044 |
| Recombinant?human?FGF-basic | PeproTech | AF-100-18B |
| Recombinant?human?EGF | PeproTech | AF-100-15 |
| MS-Column | Miltenyi?Biotec | 130-091-506 |
| Cell?strainer | BD?Falcon | 352340 |
| laminin | Invitrogen | 23017-015 |
| OPCDM | ScienCell | 1631 |
| Poly-D-lysine?hydrobromide | Sigma | p6407 |
| EDTA?disodium?salt?dehydrate | Amresco | 6381-92-6 |
| L-glutamine | Gibco | 35050-061 |
| Pancreatin inhibitor | Sigma | T6522 |
| 0.25% pancreatin | Gibco | 15050-065 |
| Heparin | Sigma | H3149 |
| mouse?anti-Sox10 | R&D | MAB2864 |
| mouse?anti-O4?antibody | R&D | MAB1326 |
| Anti-A2B5?antibodies | Miltenyi?Biotec | 130-093-581 |
| Anti-A2B5?MicroBeads | Miltenyi?Biotec | 130-093-388 |
| Anti-PSA-NCAM?antibodies | Miltenyi?Biotec | 130-093-273 |
| Anti-PSA-NCAM?MicroBeads | Miltenyi?Biotec | 130-092-966 |
| rabbit?anti-PDGFαR?antibody | Cell?Signaling | 5241 |
| rabbit?anti-Galc | Millipore | AB142 |
| mouse?anti-Tuj-1 | Abcam | ab7751 |
| rabbit?anti-GFAP | Invitrogen | 18-0063 |
| Alexa?Fluor?488-AffiniPure | Jackson | 111-545-042 |
| Alexa?Fluor?594-AffiniPure | Jackson | 111-585-144 |
Embodiment 1: divide the source OPCs that chooses from third generation human nerve stem cell
1 third generation neural stem cell is digested to unicellular
1. 100g collects neural stem cell in centrifugal 5 minutes, abandons supernatant;
2. add 1ml0.025% trypsin 0.01M dPBS dilution), mix, putting 37 DEG C of incubators, to hatch 5-7min loose to cell ball;
3. add 1.2mg/ml pancreatin inhibitor 100 μ l, 200 μ l pipettor suckers are blown and beaten into single cell suspension gently;
Nerve stem cell culture medium:
2 use magnetic bead sorting OPCs from human nerve stem cell
1. neural stem cell single cell suspension, crosses 40 μ m nylon screens with filtering cell mass, the blue total cellular score of calculating of platform phenol;
2. get 10
7cell, centrifugal 10 minutes collecting cells of 300g, remove supernatant;
3. be resuspended in the magnetic bead sorting damping fluid of 60 μ l, after mixing in 4 DEG C incubation 10 minutes;
4. add the Anti-PSA-NCAM magnetic bead of 20 μ l, after mixing in 4 DEG C incubation 15 minutes;
5. add 1ml magnetic bead sorting damping fluid, centrifugal 10 minutes of 300g;
6. in centrifugal process, MS sorting post is put into magnetic bead separator, add the magnetic bead sorting damping fluid balance sorting post of 0.5ml;
7., after centrifugal, abandoning supernatant completely, is resuspended in the magnetic bead sorting damping fluid of 0.5ml.Cell suspension is joined in sorting post, collect flowing liquid, this is unlabelled cell;
8. add the magnetic bead sorting damping fluid of 0.5ml to wash separator column, totally three times, centrifugal collecting cell;
9. add Anti-A2B5 magnetic bead, after mixing in 2-8 DEG C incubation, repeat 5.-8. step, this time collecting cell should be the cell in set in post, damping fluid is washed after post three times, and MS sorting post is placed in the centrifuge tube of 1.5ml from taking out in magnetic bead separator;
10. add after 1ml magnetic bead sorting damping fluid, pushing piston washes away in conjunction with upper cell immediately, centrifugal 10 minutes of 300g, and the cell of collecting is A
2b
5 +pSA-NCAM
-the ubcellular group of phenotype, is OPCs.
Magnetic bead sorting damping fluid: PBS (pH7.2)+0.5%FBS+2mM EDTA.
The cell detection A sub-electing
2b
5 +pSA-NCAM
-phenotype ubcellular group purity, streaming result shows that separating purity is up to more than 85% (Fig. 1).
Get part cell simultaneously and be inoculated in pretreated 48 orifice plates of PDL/laminin, after 4 days, carry out immunofluorescence dyeing.
1. by the fixing 15min of 4% paraformaldehyde room temperature for the cell in orifice plate, PBS rinsing 3 × 10min;
2. confining liquid room temperature sealing 60min, sucks confining liquid, PBS rinsing 3 × 10min;
3. add the primary antibodie of diluent preparing, overnight incubation in 4 DEG C of wet boxes; PBS rinsing 3 × 10min;
4. add prepare two anti-, lucifuge, incubated at room 45min, PBS rinsing 3 × 10min;
5. fluorescence microscopy Microscopic observation is taken pictures.
This cell subsets high expression level O
4, PDGF α R and Sox10 (mark of OPCs), only have only a few cell expressing GFAP (astroglia cell mark) and Tuj-1 (neurone mark), show can obtain highly purified more early stage OPCs (Fig. 2) from neural stem cell.
The amplification in vitro ability of 3OPCs and differentiation capability detect
1. the vitro culture of OPCs and the amplification of going down to posterity
The cell obtaining after magnetic bead sorting is according to 4 × 10
4/ cm
2density is seeded in culture vessel, in OPCs substratum, cultivates, and is placed in 37 DEG C, 5%CO2 incubator.Every 3-4 days, changing 2/3 substratum is fresh OPCs substratum.When cell reaches the degrees of fusion of 80%-90%, the cultivation of going down to posterity.
Every 7-10 days goes down to posterity once, while going down to posterity, cell is blown afloat gently with suction pipe, adds appropriate fresh culture, with 4 × 10
4/ cm
2density is inoculated in Tissue Culture Plate, puts continuation amplification cultivation in cell culture incubator.Get part cell simultaneously and be inoculated in pretreated 48 orifice plates of PDL/laminin, after 4 days, carry out immunofluorescence dyeing.
The highly purified OPCs that sorting obtains can at least go down to posterity constant in the situation that and cultivate four times in vitro maintaining initial proterties.The cell in this four generation all has forms bipolar or three utmost points, continues high expression level O4 (Fig. 3) the 4th generation cell high expression level Sox10 and PDGF α R, low expression GFAP and Tuj-1, and the overwhelming majority is still in early stage (Fig. 4).
After OPCs sorting, count, get 1.5 × 10
5cell is inoculated in 24 orifice plates, is P0.After 10 days, blow afloat gently cell with 200 μ l pipettors, counting, is first-generation P1.In 4 generations of continuous detecting, draw out the growth curve of OPCs.Can find out from growth curve, cell, in logarithmic phase, has stronger multiplication capacity, and through the cultivation in four generations, OPCs quantitatively can breed to more than 14 times (Fig. 5).
OPCs substratum:
2. the induction of OPCs differentiation
When OPCs goes down to posterity, get part cell, counting, with 1 × 10
5/ hole is inoculated in pretreated 24 orifice plates of PDL/laminin.After cell attachment, add OPCs division culture medium, every 2-3 days, changing 2/3 substratum is fresh OPC division culture medium.After 14 days, collecting cell carries out immunofluorescence dyeing observation.Part synapse cell has formed the netted form of multi-branched complexity that grow, dendritic, most of in the non-ripe oligodendrocyte stage, and can be identified (Fig. 6) by the O4 of multi-branched and Galc.Show that the OPCs obtaining by sorting has the ability that is divided into oligodendrocyte.
Embodiment 2: source OPCs point chooses from the 30 generation human nerve stem cell
1 the 30 generation neural stem cell be digested to unicellular (method is with embodiment 1)
2 use magnetic bead sorting OPCs (method is with embodiment 1) from human nerve stem cell
The amplification in vitro ability of 3OPCs and differentiation capability detect (method is with embodiment 1)
Micro-Microscopic observation finds that the OPCs of sorting from the neural stem cell of P30 also has typical bipolar or three utmost point forms, and the special mark of high expression level OPCs O4, PDGF α R and Sox10, only have only a few cell expressing astroglia cell mark GFAP and neurone mark Tuj-1, induction rear section synapse cell has formed the netted form of multi-branched complexity that grow, dendritic, and can be identified by the O4 of multi-branched and Galc.
Claims (10)
1. the method that obtains people source OPCs, is characterized in that, neural stem cell is originated as sorting, is separated and is obtained people source OPCs by the method for immunomagnetic beads, and the magnetic bead of employing is Anti-PSA-NCAM and Anti-A2B5 magnetic bead.
2. method according to claim 1, is characterized in that, comprises the following steps:
1. neural stem cell is digested individual cells in trypsin solution, crosses screen cloth;
2. centrifugal collecting cell, removes supernatant;
3. cell is resuspended in magnetic bead sorting damping fluid, after mixing in 2-8 DEG C incubation;
4. add Anti-PSA-NCAM magnetic bead, after mixing in 2-8 DEG C incubation;
5. add magnetic bead sorting damping fluid, centrifugal;
6. in centrifugal process, sorting post is put into magnetic bead separator, add magnetic bead sorting damping fluid balance sorting post;
7. after centrifugal, abandoning supernatant, is resuspended in magnetic bead sorting damping fluid completely, and cell suspension is joined in sorting post, collects flowing liquid, and this is unlabelled cell;
8. add magnetic bead sorting damping fluid to wash separator column, centrifugal collecting cell;
9. add Anti-A2B5 magnetic bead, 5. incubation, repeat-8. step, and this time collecting cell should be the cell in set in post, and damping fluid is washed after post, and sorting post is placed in centrifuge tube from taking out in magnetic bead separator;
10. add after magnetic bead sorting damping fluid, pushing piston washes away in conjunction with upper cell immediately, centrifugal, and the cell of collecting is A
2b
5 +pSA-NCAM
-the ubcellular group of phenotype, is OPCs.
3. method according to claim 2, is characterized in that, the concrete operation method of each step is as follows:
1. neural stem cell is digested individual cells in 0.025% trypsin solution, crosses 40 μ m nylon screens with filtering cell mass, the blue total cellular score of calculating of platform phenol;
2. get 10
7cell, centrifugal 10 minutes collecting cells of 300g, remove supernatant;
3. 10
7cell is resuspended in the magnetic bead sorting damping fluid of 60 μ l, after mixing in 2-8 DEG C incubation 10 minutes;
4. add the Anti-PSA-NCAM magnetic bead of 20 μ l, after mixing in 2-8 DEG C incubation 15 minutes;
5. add 1ml magnetic bead sorting damping fluid, centrifugal 10 minutes of 300g;
6. in centrifugal process, sorting post is put into magnetic bead separator, add the magnetic bead sorting damping fluid balance sorting post of 0.5ml;
7. after centrifugal, abandoning supernatant completely, is resuspended in the magnetic bead sorting damping fluid of 0.5ml, and cell suspension is joined in sorting post, collects flowing liquid, and this is unlabelled cell;
8. add the magnetic bead sorting damping fluid of 0.5ml to wash separator column, totally three times, centrifugal collecting cell;
9. add the Anti-A2B5 magnetic bead of 20 μ l, 5. incubation, repeat above-step 8., and this time collecting cell should be the cell in set in post, and damping fluid is washed after post three times, and sorting post is placed in the centrifuge tube of 1.5ml from taking out in magnetic bead separator;
10. add after 1ml magnetic bead sorting damping fluid, pushing piston washes away in conjunction with upper cell immediately, centrifugal 10 minutes of 300g, and the cell of collecting is A
2b
5 +pSA-NCAM
-the ubcellular group of phenotype, is OPCs.
4. method according to claim 1, it is characterized in that, utilize OPCs substratum to carry out amplification in vitro cultivation to separating the people source OPCs obtaining, described substratum is made up of Neurobasal A medium, B27, bFGF, heparin and L-glutamin, the volume ratio of Neurobasal A medium, B27 and L-glutamin is 97: 2: 1, the content of bFGF is 20-40ng/ml, and the content of heparin is 5 μ g/ml.
5. method according to claim 4, is characterized in that, the concrete operation method that amplification in vitro is cultivated is: the cell obtaining after magnetic bead sorting is according to 4 × 10
4/ cm
2density is seeded in culture vessel, in OPCs substratum, cultivates, and is placed in 37 DEG C, 5%CO
2in incubator, every 3-4 days, changing 2/3 substratum is fresh OPCs substratum, when cell reaches the degrees of fusion of 80%-90%, the cultivation of going down to posterity, the number of times of cultivating that goes down to posterity is 2-4 time.
6. method according to claim 1, it is characterized in that, described neural stem cell adopts nerve stem cell culture medium carry out amplification in vitro and obtain, described nerve stem cell culture medium is made up of DMED/F12, N2, bFGF, EGF, heparin and L-glutamin, the volume ratio of DMED/F12, N2 and L-glutamin is 98: 1: 1, the concentration of bFGF is 10-30ng/ml, and the concentration of EGF is 10-30ng/ml.
7. method according to claim 6, is characterized in that, the operation steps of neural stem cell amplification in vitro is as follows:
1. 100g collects neural stem cell for centrifugal 5 minutes;
2. supernatant being transferred in centrifuge tube, is old substratum;
3. to adding in the neural stem cell of having collected 1ml to be diluted 0.025% the trypsinase obtaining by 0.01M dPBS, mix, putting 37 DEG C of incubators, to hatch 5-7min loose to cell ball;
4. add 1.2mg/ml pancreatin inhibitor 100 μ l, 200 μ l pipettor suckers are blown and beaten into single cell suspension gently;
5. add 15ml dPBS, cell counting, the centrifugal 5min of 100g;
6. abandon supernatant, add 2/3 fresh nerve stem cell culture medium and 1/3 old substratum, with 2 × 10
6/ mL density is inoculated in T25ml culturing bottle, puts in cell culture incubator and cultivates;
7. to change 2/3 substratum be fresh culture to every 3-4 days, within every 10 days, goes down to posterity once.
8. obtain the method for people source OPCs, it is characterized in that, utilize OPCs substratum to carry out amplification in vitro cultivation to people source OPCs, described substratum is made up of Neurobasal A medium, B27, bFGF, heparin and L-glutamin, the volume ratio of Neurobasal A medium, B27 and L-glutamin is 97: 2: 1, the content of bFGF is 20-40ng/ml, and the content of heparin is 5 μ g/ml.
9. method according to claim 8, is characterized in that, the concrete operation method that amplification in vitro is cultivated is: people source OPCs is according to 4 × 10
4/ cm
2density is seeded in culture vessel, in OPCs substratum, cultivates, and is placed in 37 DEG C, 5%CO
2in incubator, every 3-4 days, changing 2/3 substratum is fresh OPCS substratum, when cell reaches the degrees of fusion of 80%-90%, the cultivation of going down to posterity, the number of times of cultivating that goes down to posterity is 2-4 time.
10.OPCS substratum, it is characterized in that, be made up of Neurobasal A medium, B27, bFGF, heparin and L-glutamin, the volume ratio of Neurobasal A medium, B27 and L-glutamin is 97: 2: 1, the content of bFGF is 20-40ng/ml, and the content of heparin is 5 μ g/ml.
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