CN100587063C - Method for removing mouse embryonic stem cells in differentiation system by immunomagnetic bead sorting - Google Patents
Method for removing mouse embryonic stem cells in differentiation system by immunomagnetic bead sorting Download PDFInfo
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Abstract
The invention discloses a method for removing embryonic stem cells in a differentiation system by sorting immunomagnetic beads, belonging to the technical field of medical biology. The invention comprises the following steps: (1) preparing a single cell suspension; (2) MACS isolation and purification of differentiated cells: adding mouse anti-mouse SSEA-1IgM antibody in a dilution ratio of 1: 100 to make the total volume 500. mu.l, and incubating at 4 ℃ for 10 min; washing the cells with 2ml of MACS buffer for two times of centrifugation to remove excess antibody; resuspending the cells in 80. mu.l of MACS buffer, adding 20. mu.l of immunomagnetic beads coated with goat anti-mouse IgM antibody, and standing at 4 ℃ for 15 min; adding MACS buffer solution for washing and centrifuging, discarding supernatant, and re-suspending cells by 500 ul buffer solution; after pre-washing the LD column with 2ml of buffer solution, 500. mu.l of cell suspension was passed through the column; washing the column twice with 1ml of buffer solution to remove non-labeled negative cells; moving the column out of the magnetic field, washing the column by using buffer solution, and collecting the washing solution, namely the positive cells. The invention has the advantages that: simple and fast operation, no need of expensive experimental equipment, high purity and high survival rate of the screened cells.
Description
Technical field
The present invention relates to a kind of immunological magnetic bead sorting and remove the method for embryonic stem cell in the differentiated system, adopt magnetic activated cell (sorting) (MACS) that embryonic stem cell is separated from its noble cells more precisely and remove, go in the animal body to reduce the method for tumorigenicity in the hope of the noble cells of transplanting purifying, belong to medical biotechnology field.
Background technology
The central nervous system injury reparation is the important difficult problem of medical circle always, and conventional medicament treatment and surgical intervention method all exist the problem that is difficult to overcome.Along with the fast development of biomedical technology, cell therapy (cell therapy) just more and more comes into one's own in 20 years in the past.Cell therapy refer to of the same race or xenogeneic, from viable cell body or allosome through or after processing, transplant in patient's brain or the specific region in the spinal cord, thereby treat certain disease.This method provides possibility for solving nervous system disorderss such as nerve cells transplantation treatment Parkinson's disease.
There are some researches show, original embryo stem cell transplantation is gone in the animal body to cause teratoma, so remove initiating cell in the embryonic stem cell noble cells, the noble cells that obtains large-scale purification become and solve the key of transplanting back tumorigenicity problem.
Traditional has selected by flow cytometry apoptosis, differential centrifugation etc. with the noble cells method of purification, but aforesaid method all exists that purity is not high, plant and instrument is expensive, influence deficiency such as cell viability, therefore is difficult to widespread use in real work.(Magnetic Cell Sorting is a kind of emerging cell separation technology MACS) to magnetic activated cell (sorting), is widely used in numerous areas such as cellular segregation, protein nucleic acid purifying, and MACS has been used for stem-cell research at present.The core of this technology is by the antibody of tool immune response originality at the magnetic bead surfaces bag, directly carry out antigen antibody reaction with the antigen molecule of target cell or antibody indirect and that be combined in the target cell surface in advance, in externally-applied magnetic field, these cells that combine magnetic bead will hive off with other not combined cell, the superpower paramagnetic magnetic bead of tool breaks away from behind the magnetic field disappearing magnetism immediately, so just can extract or remove the cell of institute's mark, thereby reach the purpose that positive or negative is selected cell.In addition, MACS have that incubation time is short, operating process fast and do not influence cell in streaming detects scattering properties and the function and the active characteristics of cell.In addition, magnetic bead can biological degradation in the process of cell cultures, and need not again magnetic bead to be carried out special removal.From these characteristic analysis, magnetic bead sorting can be isolated highly purified cell, and cytoactive remains intact.
The sophisticated methodology report that how to separate the original embryonic stem cell of removing in the embryonic stem cell differentiated system is not arranged at present as yet.In the present invention, we attempt utilizing magnetic activated cell (sorting) (MACS) to reduce the ratio of original embryonic stem cell in the differentiated system cell, and detect its purity through flow cytometer, to inquire into the purifying noble cells from methodology, screen out the wherein concrete approach of initiating cell, lay the foundation with the research that reduces tumorigenicity for further the noble cells behind the purifying being implanted in the mouse body.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of easy and simple to handle, obvious results is removed initiating cell in the embryonic stem cell differentiated system with immunological magnetic bead sorting method.
For achieving the above object, the present invention is by the following technical solutions:
Immunological magnetic bead sorting is removed the method for initiating cell in the embryonic stem cell differentiated system, comprises the steps:
(1) preparation single cell suspension: get the embryonic stem cell differentiation phase cell of cultivation) with 0.25% trypsin Trypsin-and EDTA digest 2-4min, and increase serum stops digesting, and breaks up afterwards centrifugally, abandons supernatant; With MACS damping fluid re-suspended cell, cross 40 μ m filter screens and obtain single cell suspension, adjust cell density to 10
7/ ml;
(2) MACS separation and purification noble cells: add mouse anti mouse SSEA-1IgM antibody by 1: 100 Dilution ratio, making cumulative volume is 500 μ l, hatches 10min for 4 ℃; With 2ml MACS damping fluid washed cell twice, the antibody that centrifugal removal is unnecessary; With 80 μ l MACS damping fluid re-suspended cells, add bag by the immunomagnetic beads 20 μ l of goat-anti mouse IgM antibody, place 15min for 4 ℃; Add 1ml MACS damping fluid washed twice, centrifugal removal magnetic bead antibody is abandoned supernatant, uses 500 μ l MACS damping fluid re-suspended cells again; Behind the 2ml damping fluid prewashing LD magnetic sorting post, 500 μ l cell suspensions are crossed post; The 1ml damping fluid is washed post twice, removes the non-marked negative cells; Move post and go out magnetic field, wash post, collect washing lotion and be positive cell with the MACS damping fluid.
The present invention applies to the indirect negative sorting of MACS the branch that screens out of the noble cells of embryonic stem cell first and chooses, and screens out the antibodies positive cells, the negative cells behind the reservation purifying, and the research that is used for stem cell for MACS provides new field.Embryonic stem cell specific surfaces antibody (Special Stage Embryonic Angent-1, SSEA-1) be the specificity after birth surface marker of internationally recognized mouse embryonic stem cell commonly used, two anti-FITC have very high joint efficiency with fluorescence, it is the basis that sorting is carried out smoothly, but because the direct combination of its no magnetic bead, so can only use indirect separating method.The prerequisite of sorting success is to seek suitable activity, operative temperature and the action time of the specific marker thing SSEA-1 of embryonic stem cell, to guarantee to obtain best sub-sieve result.For validity with keep 2 considerations of cytoactive,, finally can obtain suitable activity by 1: 100 dilution proportion SSEA-1 antibody through experiment screening repeatedly; Through repeatedly differing temps and the SSEA-1 antibody test of different action times, finally selecting 4 ℃ of effect 10min is best screening mode.
As this area routine operation, the time of Trypsin-EDTA digestion is 3min among the present invention.Centrifugal speed and time are 1000rpm, 3min.These data can be adjusted according to concrete experiment content, are not innovative point of the present invention place.
Advantage of the present invention is: under the SSEA-1 of this optimization action condition, the ratio of initiating cell can obviously reduce in the noble cells behind the sub-sieve, and difference has statistical significance, illustrates that this method is easy, effective.Transplant noble cells for further carrying out in the nude mouse, carry out tumorigenicity research and lay the first stone.The MACS method is easy and simple to handle rapidly, need not expensive experimental equipment, and purity is higher, and the cell behind the sub-sieve has survival rate preferably, and separating effect can obtain the affirmation of cell cultures, flow cytometry, fluorescent microscope and PCR equimolecular biological method.Therefore, the present invention will have broad application prospects in stem-cell research.
The present invention will be further described below in conjunction with the drawings and specific embodiments, and do not limit the present invention in any way, and every any this area of carrying out according to the disclosure of invention is equal to replacement, all belongs to protection scope of the present invention.
Description of drawings
Fig. 1 is a SSEA-1 activity gradient map.
Fig. 2 be SSEA-1 in 37 ℃ action time gradient map.
Fig. 3 is that SSEA-1 is in 4 ℃ of activity gradient map.
Fig. 4-A is the negative control figure of cell sorting experiment.
Fig. 4-B is the preceding SSEA-1 negative cells middle-jiao yang, function of the spleen and stomach sex ratio figure of the sub-sieve of cell sorting experiment.
Fig. 4-C is SSEA-1 negative cells middle-jiao yang, function of the spleen and stomach sex ratio figure behind the sub-sieve of cell sorting experiment.
Embodiment
Material that uses among the following embodiment and source
1. (murine embryonic stem cells is the sv129 strain mESC) to the mouse embryonic stem cell, and ATCC is numbered CRL-11379.
2. main agents and instrument: mouse anti mouse SSEA-1IgM antibody (R﹠amp; D company, the U.S.); Bag is by the immunomagnetic beads of goat-anti mouse IgM (Miltenyi Biotech company, Germany); The goat-anti mouse IgM of FITC (fluorescein isothiocyanate) mark (Jackson ImmunoResearch company, the U.S.); Mini MACS magnetic bead sorting system (Miltenyi Biotech company, Germany); Aperture 40 μ m screen clothes (Millipore company, the U.S.), FACS Calibur type flow cytometer (Becton Dickinson company, the U.S.).
One. method (known method)
1. cultivation embryonic stem cell: undifferentiated mESC is containing 1000U/mL human leukemia supressor (hLIF, human Leukemia Inhibition Factor, ESC substratum CHEMICON) (DMEM (Dulbecco ' smodified eagle ' s medium), GIBCO; The 15mL/L foetal calf serum (fetus bovine serum, FBS), GIBCO; 2mM non-essential amino acid (Non-essensial amino acid), Invetrogen; 0.55mM beta-mercaptoethanol (β-mercaptoethanol), GIBCO; 2mM L-glutaminate (L-glutamine), GIBCO; Mycillin (Penicillin-Stroptomyicn liquid) carries out amplification in vitro in Invetrogen), changes liquid every other day, goes down to posterity in 1: 5 ratio after at the bottom of about 5 days cells cover with bottle.
2. the inducing embryo body forms: centrifugal with 0.25%Trypsin-EDTA (Invetrogen) digestion mESC, and 1000rpm, 3min abandons supernatant; Add 1ml ESC substratum re-suspended cell, blow and beat gently to unicellular with the pasteur pipe; By 2.5 * 10
4/ cm
2Cell density be inoculated in the low adhesion culture dish (Petridish) postdigestive ES is unicellular, with the cultivation of the ESC substratum that does not contain hLIF 6 days, cell is by the unicellular round cell group that grows up to suspension in when inoculation, promptly embryoid body (Embryonic body, EBs).
3. screen the nestin positive cell: after EBs forms, embryoid body is inoculated in adds the ES substratum that does not contain LIF in the common culture dish of 0.1% gelatin shop fixtures, behind the 24h ESC substratum is replaced by ITSF screening culture medium (DMEM/F-12, the GIBCO of serum-free; Wherein add 5 μ g/mL Regular Insulin (Insulin), GIBCO; 50 μ g/mL Transferrins,iron complexess (Transferrin), Sigma; 5 μ g/mL fibronectins (Fibronectin), Sigma; 30nmol/L sodium selenate (Sodium selenium) Sigma) was cultivated 6-10 days.
4.Nestin positive cell amplification: centrifugal with the cell after the 0.25% trysinization screening, 1000rpm, 3min; By 2 * 10
5/ cm
2Density inoculating cell in be covered with in advance 15 μ g/mL poly-ornithines (Poly-L-OrnithineSolution, Sigma) (Laminin is in culture dish GIBCO) with 1 μ g/mL mouse laminin; Add proliferated culture medium and (add 1 * N2 among the DMEM/F12,1 * B27, GIBCO; 10 μ g/mL bFGF, R﹠amp; D; 1 μ g/mLLaminin) amplification Nestin positive cell, the next day change liquid, cultured continuously 6 days.
5. directional induction differentiation: after amplification finished, substratum was replaced by division culture medium and (is added 1 * N2 among the DMEM/F12,1 * B27; 1mg/L Laminin; 200 μ M Ascorbic Acid Sigma) induce differentiation 6～15 days.
Two. the result
MESC is the roughly cell mass of the sub-circular of rule of edge at the culture dish bottom growth that is covered with gelatin.The embryonic stem cell of breaing up is suspension culture in petri dish, and removes hLIF, can form spheric EBs.Afterwards EBs is broken up with the screening of ITSF substratum, cell still is inclined to assembles agglomerating growth, much is dispersed in the cell that is fusiformis but have.Noble cells quantity increases after the amplification of mitogen, and the spindle cell that is dispersed in the culturing cell is seen more.The last cell that originally was fusiformis in inductive differentiation medium gradually becomes circular, has abundant projection to connect between the cell.
Embodiment 2. explores optimal SSEA-1 antibody and ES cytosis concentration, temperature and action time
One. method
1. cultivation embryonic stem cell: undifferentiated mESC places 37 ℃ with containing in the ESC substratum of 1000U/mL hLIF, and cultured continuously in the 5%CO2 incubator is changed liquid every other day.Can cover with a ware in about 2-4 days, and after 0.25% trysinization, get cell suspension, but final every ware collecting cell amount be about 2 * 10
6/ ml.
2. mESC is done the mouse SSEA-1 antibodies analysis of different concns, understand the activity of SSEA-1 the best: mESC discards nutrient solution, and 0.01M PBS (pH7.4) washes one time, and 0.25% trysinization is centrifugal, 300g, 3min; 4% Paraformaldehyde 96 re-suspended cell is in 4 ℃ of 20min; 0.01M PBS solution is washed one time; Drip SSEA-1 one anti-diluent, 4 ℃ are spent the night, and Dilution ratio is respectively: 1. number pipe is 1: 25, and 2. number pipe is 1: 50, and 3. number pipe is 1: 100, and 4. number pipe is 1: 500, and 5. number pipe is 1: 1000.Next day, PBS solution cleans 3 times; Drip the fluorescence two anti-FITC working fluids of dilution in 1: 100, lucifuge, incubated at room 2h; PBS solution cleans 3 times; Cell behind the mark finally is resuspended in the 500 μ lPBS solution, does flow cytometry.
3. select suitable SSEA-1 activity according to the experimental result of previous step, mESC is done the SSEA-1 antibodies analysis of different action times, understand the action time of SSEA-1 the best: mESC discards nutrient solution, 0.01MPBS wash one time, 0.25% trysinization, centrifugal, 300g, 3min; 4% Paraformaldehyde 96 re-suspended cell is in 4 ℃ of 20min; 0.01M PBS solution is washed one time; Drip an anti-SSEA-1 working fluid of dilution in 1: 100, respectively at 4 ℃ be respectively 37 ℃ of action times: 1. number pipe 5min, 2. number pipe 10min, 3. number pipe 15min, 5. 4. number pipe 30min number manages 60min; The cell crossed of mark is resuspended in the 500 μ l PBS solution the most at last, does flow cytometry.
Two. the result
1. best SSEA-1 activity: different SSEA-1 activity ratios are as Fig. 1.The result shows that SSEA-1 presses 1: 100 dilution proportion, can reach ideal effect.
2. best SSEA-1 operative temperature and time: the result of different SSEA-1 action time is relatively as Fig. 2, and 3.The result shows that 37 ℃ of 5min or 4 ℃ of 10min can obtain ideal effect.
Embodiment 3. separates, the purifying noble cells
One. method
1. preparation single cell suspension: get the differentiation phase cell that embodiment 1 obtains, 0.25% trysinization 3min, increase serum stops digestion, and the pasteur pipe is blown and beaten about 20 times gently, and is centrifugal, 1000rpm, 3min abandons supernatant.With MACS damping fluid re-suspended cell, cross 40 μ m filter screens to obtain single cell suspension.Mirror is counting and adjustment cell density to 10 down
7/ ml.
2.MACS separation and purification noble cells: add mouse anti mouse SSEA-1IgM antibody by 1: 100 Dilution ratio, making cumulative volume is 500 μ l, hatches 10min for 4 ℃; With 2mlMACS damping fluid washed cell twice, 1000rpm, 3min is to remove unnecessary antibody; With 80 μ l MACS damping fluid re-suspended cells, add bag by the immunomagnetic beads 20 μ l of goat-anti mouse IgM antibody, place 15min for 4 ℃; Add the washing of MACS damping fluid, 1000rpm, 3min abandons supernatant, uses 500 μ l damping fluid re-suspended cells again; Behind the 2ml MACS damping fluid prewashing LD post (MACS accessory, MiltenyiBiotech company, Germany), 500 μ l cell suspensions are crossed post; The 1ml damping fluid is washed post twice, to remove the non-marked negative cells; Move post and go out magnetic field, the 3ml damping fluid is washed post, collects washing lotion and is positive cell.Respectively by 2 * 10
4/ cm
2Cell density is inoculated in the culture dish, collects all the other cells, subsequent experimental to be done.
3. cytoactive detects: after getting 10 μ l cell suspensions respectively and 10 μ l, 0.4% trypan blue solution mixes before and after the purifying, splash into cell counting count board and place microscopically to observe.Painted, not shinny is viable cell, and painted, the cell space person of expanding is a dead cell, counting cells sum and viable cell per-cent.
4. the flow cytometer behind the cell sub-sieve detects: the SSEA-1 negative cells and the positive cell of wash-out is centrifugal respectively, and 1000rpm, 3min; The Paraformaldehyde 96 of 4% precooling is fixed 20 minutes, and PBS washes twice; The anti-mouse IgM of the FITC bonded fluorescence two anti-incubated at room 2 hours that add dilution in 1: 100; PBS washes twice; 500 μ l PBS re-suspended cells, last machine testing.Adopt SPSS 10.0 softwares to carry out the t check of two sample means, analyze the purity of purifying front and back SSEA-1 negative cells and the difference of cell viability.
Two. the result
1. ratio average out to (11.61 ± 5.34) % of initiating cell in the noble cells before the purifying, and ratio average out to (0.325 ± 0.255) % (P＜0.01) of initiating cell in the SSEA-1 negative cells behind the purifying (Fig. 4-A to Fig. 4-C)
2. cell survival rate is (93.0 ± 2.5) % before the purifying, be (92.6 ± 0.7) % (P＞0.05) behind the purifying, the SSEA-1 positive cell that this explanation application Mini MACS screens out in the noble cells can obviously improve the purity of noble cells and not influence cell viability substantially.
Claims (3)
1. immunological magnetic bead sorting is removed the method for mouse embryonic stem cell in the differentiated system, it is characterized in that comprising the steps:
(1) preparation single cell suspension: get the mouse embryonic stem cell differentiation phase cell of cultivation, digest 2-4min with 0.25% trypsinase-EDTA, increase serum stops digestion, and is centrifugal after breaing up, and abandons supernatant; With MACS damping fluid re-suspended cell, cross 40 μ m filter screens and obtain single cell suspension, adjust cell density to 107/ml;
(2) MACS separation and purification noble cells: add mouse anti mouse SSEA-1 IgM antibody by 1: 100 Dilution ratio, making cumulative volume is 500 μ l, hatches 10min for 4 ℃; With 2ml MACS damping fluid washed cell two times centrifugal, remove unnecessary antibody; With 80 μ l MACS damping fluid re-suspended cells, add bag by the immunomagnetic beads 20 μ l of goat-anti mouse IgM antibody, place 15min for 4 ℃; It is centrifugal to add the washing of MACS damping fluid, abandons supernatant, uses 500 μ l MACS damping fluid re-suspended cells again; Behind the 2ml MACS damping fluid prewashing LD post, 500 μ l cell suspensions are crossed post; 1ml MACS damping fluid is washed post twice, removes the non-marked negative cells; Move post and go out magnetic field, wash post, collect washing lotion and be positive cell with damping fluid;
(3) screen out the antibodies positive cells, the negative cells behind the reservation purifying.
2. immunological magnetic bead sorting according to claim 1 is removed the method for mouse embryonic stem cell in the differentiated system, it is characterized in that: the time of described trypsinase-EDTA digestion is 3min.
3. immunological magnetic bead sorting according to claim 1 is removed the method for mouse embryonic stem cell in the differentiated system, and it is characterized in that: centrifugal speed and time in described step (1) and the step (2) are 1000rpm, 3min.
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