CN104357396A - Method for extracting early hematopoietic progenitor stem cells and application thereof - Google Patents

Method for extracting early hematopoietic progenitor stem cells and application thereof Download PDF

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Publication number
CN104357396A
CN104357396A CN201410610588.2A CN201410610588A CN104357396A CN 104357396 A CN104357396 A CN 104357396A CN 201410610588 A CN201410610588 A CN 201410610588A CN 104357396 A CN104357396 A CN 104357396A
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cell
placenta
stem cell
mononuclearcell
early stage
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周胜利
郑超
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Sail Parke (jiangsu) Stem Cell Biology Engineering Co Ltd
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Sail Parke (jiangsu) Stem Cell Biology Engineering Co Ltd
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Abstract

The invention discloses a method for extracting early hematopoietic progenitor stem cells and application thereof. The method for massively extracting the hematopoietic progenitor stem cells comprises the following steps: (1) mononuclear cells are separated from a placenta; (2) CD133<+> positive cell populations are separated from the mononuclear cells, then CD133<+>CD34<+> cell populations are separated from the CD133<+> positive cell populations, and cell populations flowing out are the CD133<+>CD34<-> hematopoietic progenitor stem cells. The method finds that the concentration of CD133<+>CD34<-> stem cell populations in the placenta is more than ten times higher than that of CD133<+>CD34<-> stem cell populations in umbilical cord blood, and the content of the CD133<+>CD34<-> stem cell populations in the placenta is more than thirty times higher than that of CD133<+>CD34<-> stem cell populations in umbilical cord blood; the CD133<+>CD34<-> stem cell populations are initiating cells of hematopoietic stem/progenitor cells, have the capacity of long-term repopulation hematopoietic cells, and are stem cells which can survive for a long term after being transplanted to a host; the CD133<+>CD34<-> stem cell populations have higher plasticity and have wide application prospects in regenerative medicine and curing diseases such as cellular damage.

Description

A kind of method and application thereof of extracting early stage hemopoietic precursors stem cell
Technical field
The invention belongs to field of biological pharmacy, relate to method and the application thereof of a kind of a large amount of extraction hemopoietic precursors stem cell, be specifically related to a kind of a large amount of method and application thereof of extracting early stage hemopoietic precursors stem cell from HUMAN HEALTH placenta.
Background technology
Along with the progress of science and technology, it is found that stem cell under given conditions can through being induced to differentiate into other cell types, people expect to carry out tissue repair in this way in the future, are used for the treatment of parkinsonism, diabetes, senile dementia, heart trouble, Spinal injury etc.Our patent " extracts hemopoietic stem cell for setting up the method for hemopoietic stem cell bank " from placenta tissue, the patent No.: ZL011311908, international monopoly Main classification number: point out in C12N5/08 in placenta containing abundant hemopoietic stem cell, therefore allosome marrow can be replaced to carry out the marrow of restoration and reconstruction patient, in other words, namely marrow is replaced with it.In our another section of patent, it is also proposed " one extracts original mesenchyma and hematopoietic stem/progenitor method from c-section placenta tissue " (patent No.: 200610098886) simultaneously.This method can be used for collecting the mononuclearcell coming from placenta tissue and go to set up hemopoietic precursors stem cell bank.Apply these methods can collect and the intraplacental cell of newborn infant discarded now of Long-term Cryopreservation, hemopoietic stem cell in placenta tissue, set up placenta hemopoietic precursors stem cell storage vault that is autologous or allosome, provide the whole nation and even whole world patient to select placenta hemopoietic precursors stem cell to be cell therapy application.
CD34 molecule is considered to the surface markers of HSC/ hemopoietic progenitor cell (HPC) always.Blood circle is up to the present all with CD34 +the content of HSC as transplanting the value mark of success.Scientist within 1997, is had to find to there is a group CD34 in mammalian body -cell also has the function of reconstitute hematopoiesis, shows CD34 +be comparatively ripe cell, and CD34-cell is more original HSC.Later someone proposes to express CD133 again, no matter be that the HSC/HPC of CD34 positive or negative is transplanted to immunodeficient mouse and can forms ectopic hemopoiesis tissue.Find CD133 simultaneously +hSC has the angiopoietic effect of stronger promotion.Bleeding of the umbilicus CD133 +(nobody recognized Cord blood only containing a small amount of CD34-cell in fact at that time, and major part is CD34 to be not only rich in HSC in cell mass +cell), and this group cell has stronger proliferation potential and long term hematopoietic Reconstruction of The Function, and these promptings CD133 is inmatureer stem-cell marker more original in CD34.Research confirms, is in G 0the CD133 of phase +cell is comparatively in G 1the CD133 of phase -cell more easily a large amount of enrichment and the participation hematopoiesis that is activated, its long term hematopoietic function is better than CD34 +cell mass.
Since discovery, purifying, mark AntiCD3 McAb 4 monoclonal antibody start, the CD34 Positive Hematopoietic Stem Cells of fluidic cell detection by quantitative in umbilical cord and placental blood or marrow is made to become possibility.CD34 antigen is present on various progenitor cell, comprising multipotential stem cell and mescenchymal stem cell.The molecular weight of CD34 antigen is 105-120KD, has the structure of the mucoid of a high glycosylation.In flow cytomery, haemolysis is carried out to whole blood and removes red corpuscle, directly add fluorescently-labeled CD34 antibody in sample and dye, then carry out analysis and calculation with streaming machine.Key cells when people CD34 positive cell is hematopoietic stem cell transplantation, decides the essential condition of transplanting success or not.At present, be all unique major criterion with CD34 positive cell quantity to whether transplanting clinically.People CD133 antigen is 5 transmembrane glycoproteins, and the homologous antigen found at present has CD133_1 and CD133_2.Its gene is positioned on No. 4 of the mankind and No. 2 karyomit(e)s respectively.CD133 is found as hematopoietic stem/progenitor surface marker, and is considered to the surface antigen more early stage than CD34.Utilize and take from the CD133+ of human marrow blood after hyperplasia, with the cell of this mark also referred to as blood vessel Pioneering cells.Cell directly injects the rat heart of myocardial infarction, has the function that myocardial cell recovers.From peripheral blood, also can isolate CD34+ cell, in vitro by its differentiation-inducing one-tenth endotheliocyte, implant in animal lower limb ischemia model and be partially formed new vessel.Also may treat ischemic disease as myocardial infarction, atherosclerotic occlusive disease, diabetes etc., these cause the broad interest of people.Recent discovery CD133 molecule is HSCs (hemopoietic stem cell) specificity marker.CD133 is a newfound hemopoietic stem cell surface mark, and in HSCs differentiation and maturation process, the content of CD133 reduces rapidly, and CD34 antigen starts to express, and when to blood cell differentiation phase, CD34 antigen presentation starts to disappear.
Above-mentioned a large amount of clinical practice confirms, comes from marrow, mobilizes containing a large amount of hematopoietic stem/progenitor in Peripheral blood and Cord blood, be i.e. CD34 +cell, the hemopoietic tissue that this cell can rebuild damaged recovers hemopoietic function.And this CD34 +cell comes from its more early stage cell, namely from CD133 positive cells differentiate, and this CD133 just +cD34 +or CD33 +cD34 -population transplant can plant work for a long time to after host.Only containing a small amount of CD133 in Cord blood +cD34 -cell, after this also causes Umbilical Cord Blood Transplantation, part hemopoietic stem cell not easily plants work, or needs one of major reason of planting time length alive.
Summary of the invention
First the present invention finds in Cord blood not containing CD33 +cD34 -cell, and placental blood and placenta tissue are rich in this kind of cell, only cannon born marrow also has this kind of cell, but marrow can not scale extract.The object of the invention is the above-mentioned deficiency for prior art, a kind of method extracting early stage hemopoietic precursors stem cell from placenta is in a large number provided.
Another object of the present invention is to provide the application of placenta hemopoietic precursors stem cell.
Object of the present invention realizes by following technical scheme:
The present invention finds a kind of novel population of stem cells from placental blood and placenta tissue, the early stage hemopoietic precursors stem cell of called after placenta:
(1) this cell mass come from remove remain in intraplacental blood after Cord blood or from placenta tissue through mechanical treatment (rinse, extrude, mill), or the mononuclearcell that enzymic digestion (pancreatin, collagenase, papain) etc. obtains.
(2) this cell mass is CD133 in the detection of flow cytometer (FACS) +cD34 -cD101 -cD29 -cD31 -, be present in hardly in normal people's Peripheral blood and Cord blood.
(3) hemopoietic precursors stem cell is a group CD133 +cD34 -and CD133 +cD34 +population of stem cells, and CD133 +cD34 -for early stage hemopoietic precursors stem cell, this stem cell is present in the placenta stem-cell of extraction, and it is preserved for a long time after can being concentrated by placenta separation in liquid nitrogen.
(4) this cell mass is in stem cell colonies culture experiment, and it forms granulocyte colony (namely each cell can increase into more than 50 cells) far beyond CD133-CD34 +cell is evening, and after incubation 24 hours, the latter can form the colony of cell mass in heaps in a large number, and early stage hemopoietic precursors stem cells needs more than 96 hours.CD133-CD34 +cell can generate early stage colony, but not easily long term growth, but CD33 +cell can steady in a long-term grow, and the standard of transplanting should with degree CD133 +content how much predict graft plant active fruit
(5) this cell mass can with the co-cultivation such as stem cell factor (G-CSF, GM-CSF, SCF etc.) or vascular endothelial growth factor (VEGF), this cell mass can adherent growth, there is obvious transport property, form monolayer cell, have the function to endothelial cell differentiation, namely this kind of cell can be divided into CD133 +blood vessel endothelium ancestral cells.
(6) this kind of cell is having stem cell stimulating factors (G-CSF, EPO) in nutrient solution, there is the function of a large amount of propagation, more than 1000 times can be increased, end non-cell after its amplification is mainly erythron, (under the stimulation of EPO), if but and leukocyte stimulation factor co-cultivation, also can increase as a large amount of white corpuscles.
(7) this kind of cell has larger plasticity-, in vitro in culture systems, after this kind of cell attachment growth, adds the corresponding stimulating factor of histocyte, has to liver cell, osteocyte, heart cell, the ability that neurocyte etc. are formed.
(8) in transplantation immunity immunodeficient disease mouse model, with Isodose, with the early stage hemopoietic precursors stem cell CD133 from placenta +cD34 -cell mass and CD133 +cD34 +cell mass is administered to stem cell transplantation mouse species, and early stage hemopoietic stem cell has plants active energy for a long time.
(9) early stage hemopoietic precursors stem cell be masked as CD133 +cD34 -, and CD133 molecular weight is 120KDa, has 5 cross-film districts, during it is cultivated in vitro, can form various red corpuscle, (CFU-E), white corpuscle (CFU-GM and CFU-GEMN) colony group.The method of MACS magnetic bead sorting can be adopted, this hemopoietic precursors stem cell of purification.
A method for the early stage hemopoietic precursors stem cell of a large amount of extraction, preferably comprises following steps:
(1) from placenta tissue or placental blood, mononuclearcell is separated;
(2) from the early stage hemopoietic precursors stem cell mononuclearcell purifying CD133 positive cell.
Wherein, the described mononuclearcell be separated from placenta preferably carry out concentrated after carrying out sorting.
Step (2) from the preferred following either method of early stage hemopoietic precursors stem cell mononuclearcell purifying CD133 positive cell, further preferably following paramagnetic particle method.
(1) paramagnetic particle method, (2) streaming method, (3) double antibody method, (4) live body culture method
Paramagnetic particle method: adopt the magnetic bead of the anti-CD133 antibody of band and mix from the mononuclearcell of placenta, hatching, then by the separator column that and magnetic bead combine, all cells with CD133 antigen are all combined on pillar, and other cell all rinses out.Then the wash buffer changing potential of hydrogen remains in the cell of the band CD133 antigen in pillar, and this cell mass is all CD133 +cell, this method rate of recovery is more than 80%, and purity can up to 90%.The CD133 positive cell after concentrated, then through the magnetic bead containing AntiCD3 McAb 4 antibody, use the same method separation, all CD133 +cD34 -cell elutes in advance, remain in pillar body for CD133 +cD34 +cell mass.
Selected by flow cytometry apoptosis method: adopt the various fluorescence labeling method of flow cytometer double antibody band, can from the cell required for individual cells sorting, what adopt at this is the double antibody of anti-CD133 antigen and AntiCD3 McAb 4 antigen, can from the CD133 required for the mononuclearcell sorting after placenta isolation and identification +cD34 -and CD133 +cD34 +cell mass.
Double antibody method: now adopt when antibody and there is this antigen cell to conjunction after, then add the second antibody of anti-first antibody, then this cell can be shunted out from solution.Also be adopt as mouse-anti people CD133 antibody here, mouse antihuman CD 34 antibody, and then the antibody adding sheep anti-mouse igg, then can successfully required cellular segregation out.
Live body culture method: adopt the mouse without immunologic function, being separated from placenta in a large number, mononuclearcell infusion after concentrated is to mouse, in the regular hour, as 2,4 weeks, stem cell is extracted from mouse bone marrow cells, plant hemopoietic stem cell alive for a long time containing a large amount of maintaining of people source in it, and then combine magnetic bead or airflow classification method, sub-elect and there is CD133 +cell.
The method being separated mononuclearcell from placental blood and placenta tissue described in step (1) is preferred:
(1) collect placenta, inject containing 25 to 50 milliliters of antithrombotics and antibiotic physiology isotonic solution or damping fluid in umbilical vein, adopt umbilical cord artery and vein perfusion to collect placental blood.
(2) adopt organic solvent and physiological buffer to remove the hyaluronic mucus of placenta tissue and umbilical cord appearance, the method adopting mechanical presses and enzyme and physiological buffer to rinse, rinses out any residual blood;
(3) collected by centrifugation placenta tissue stem cell: shred after placenta tissue, amnion and chorion tissue brine, adopt the pancreatin of 0.25%, collagenase II, 0.5%Dispense II enzyme of 0.1-1%, or add the EDTA of 0.05 to 0.2%, in 37 DEG C of incubators, hatch or stir 30 to 60min, the cell of collection, overanxious, centrifugal acquisition is placenta mononuclearcell.
Wherein, the filtration described in step (3), centrifugal kettle method are:
Filter: the placental blood after collection and tissue stem cell are all by each layer nylon or Steel net, and that minimum is 50um, and that maximum is 100mm, collect mononuclearcell;
Centrifugal: by above cell harvesting in the centrifuge tube of 50 milliliters, with 1,500g centrifugal 10 minutes in centrifuges, remove the liquid of 70%, then process according to following either method:
A cell that () collects and lymphocyte separation medium mix in the ratio of 1:4, with 2500g centrifugal 30 minutes in centrifuges, remove the granulocyte of remaining red corpuscle and maturation, and collect the cell between liquid level, cell RPMI-1640 washes secondary;
B the contain placenta tissue or placental blood stem cell solution and 6% polymer hydroxyethylamyle that extract mix in 1:3 to 1:6 ratio by (), 1500rpm, 4 DEG C centrifugal 5 to 20min, discard red corpuscle, retain supernatant liquor, then use low molecular dextran (saliferous) to be washed three times, collected cell is made mononuclearcell suspension.
The placenta CD133 extracted according to the method described in the present invention +cD34 -early stage hemopoietic precursors stem cell helps in preparation and promotes the application in the medicine of stem cell regenerating.Adopt the stem cell transplantation of placenta tissue hemopoietic precursors can help, accelerate the survival of stem cell transplantation.
The early stage CD133 of the placenta extracted according to the method described in the present invention +cD34 -the application of hemopoietic precursors stem cell in the medicine of preparation treatment hydrocephalus, cardiovascular and cerebrovascular diseases, hepatic necrosis, liver cirrhosis, diabetes, sacroiliitis, pulmonary fibrosis (pneumoconiosis) autoimmunity venereal disease, spinal cord injury disease, lupus erythematosus, scleroderma, ephritis, sexual cell functional disorder disease, congenital or acquired disease.
The preferred Intracerebral hemorrhage of described cardiovascular and cerebrovascular diseases, any one in cerebral thrombosis, myocardial infarction, coronary heart disease; The preferred Severe Diabetes Mellitus of described diabetes, or insulin-dependent diabetes; The osteoarthritis of the preferred degenerative joint of described sacroiliitis; Described sexual cell functional disorder disease preferable indifference or impotence; Any one in the preferred muscle hypertrophy disease of described congenital or acquired disease, encephalatrophy or senile dementia.
In hematopoietic stem cell transplantation, the precursor hemopoietic stem cell that tissue matching conforms to can infusion be to patient with Cord blood, placenta mesenchyma stem cell, marrow blood or the while of mobilizing Peripheral blood stem cell, and that accelerates hemopoietic stem cell plants work.
When being applied to clinical, fresh human placenta precursor hemopoietic precursors stem cell can being adopted, also can adopt freezen protective, then melt, then the precursor hemopoietic precursors stem cell cultivated.
When adopting placenta early stage hemopoietic precursors stem cell to be used for clinical, or only can need to do the low resolution distribution type of HLA, do not have rejection, can in patient body long-term surviving, have and plant effect alive for a long time.
The early stage hemopoietic precursors stem cell of placenta is applied to patient after can cultivating in vitro.Be applied as 1 × 10 at every turn 5/ KG to 5 × 10 7/ KG body weight.
The early stage hemopoietic precursors stem cell of the placenta adopted can be prepared into injection, packed venous transfusion bag, also can be prepared into bottled infusion bottle.When patients clinical is applied, be directly prepared into various pin, aqua by my company, directly for patient application, also can freezing bag under the condition of-80 DEG C, be transported to patient place hospital application.If directly apply for patient to hospital with the form of freezing bag or storage bag, according to the specification sheets of my company, need namely use the physiological saline of 4 DEG C, isotonic Glucose Liquid, DMEM nutrient solution or dilute containing the PBS (phosphate buffered saline buffer) of hypertonic glucose, could apply after centrifugal.
Detailed Description Of The Invention
Collect placenta: placenta is put in sterile cassette and is transported to aseptic, inject in umbilical vein containing 25 ~ 50 milliliters of antithrombotics and antibiotic solution, this antithrombotics can be heparin, or Sodium Citrate, microbiotic is the penicillin of 1%, Streptomycin sulphate, gentamicin, also nystatin can be added, solution can be the isotonic phosphoric acid buffer of physiological saline or the DMEM cell culture fluid of 0.9%, then clamp umbilical cord with clip, hold up umbilical cord one minute and (smooth out with the fingers downwards toward placenta place along umbilical cord with hand, allow solution enter placenta.Aseptic for whole placenta is put into sterile chamber and collector tank.
Collect placental blood: in an aseptic environment, inject containing microbiotic in umbilical vein, as the penicillin of 1%, Streptomycin sulphate, gentamicin, also can add nystatin, solution can be the isotonic phosphoric acid buffer of physiological saline or the DMEM cell culture fluid of 0.9%, right connection umbilical artery, middle connection bio-pump, forms circulation, time was at 1 to 24 hours, collection comes from intraplacental blood, called after placental blood, and the cell in placental blood is mononuclearcell.
Cleaning placenta tissue: after placenta enters gnotobasis, adopts organic solvent and physiological buffer to remove the hyaluronic mucus of placenta tissue and umbilical cord appearance and female blood.Adopt No. 1 sterilizing agent (alcohol, sodium-chlor, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC and iodine), in this sterilizing agent, ethanol concn is 50% ~ 75% and contains the iodine of 0.1 ~ 0.5% and the phosphoric acid buffer of 0.1 to 0.25M, pH is between 7.2 to 7.6, for cleaning placenta and umbilical cord surface, kill any microbial contamination that may exist on placental membrane and umbilical cord surface, remove the hyaluronic mucus of placenta tissue and umbilical cord appearance.Adopt No. 2 organic solvents (deduct iodine on the basis of No. 1 sterilizing agent, in this sterilizing agent, ethanol concn is 50% ~ 75%, and containing 0.1 ~ 0.25M phosphoric acid buffer, pH is between 7.2 to 7.6) for cleaning placenta and umbilical cord surface.No. 3 sterilizing agent cleaning liquors (alcohol and 0.9% sodium-chlor, wherein, ethanol concn is 50% ~ 75%, sodium chloride concentration is 0.9%, pH is between 7.2 to 7.6) for cleaning placenta and umbilical cord surface, phosphoric acid buffer (pH7.4) or low molecular dextran or cell culture fluid (DMEM) also can be used to clean.The method adopting mechanical presses and enzyme and physiological buffer to rinse, rinses out any residual blood.This damping fluid can contain antibiotic, and as penicillin (100U/ml), Streptomycin sulphate (100g/ml) also can contain the heparin of the preservative-free of 2500 to 10,000 unit; The composition of this physiological buffer can be physiological saline, low molecule dextrose is sweet, phosphoric acid buffer (phosphate-buffered saline) or cell culture fluid, picture 1640, DMEM (Dulbecco ' s modified Eagle ' s medium), contained by it, the cell of single core is called mononuclearcell, and cell total amount is 0.3 to 10.5X10 9between.
Collect placenta tissue mononuclearcell cell: adopt transfusion device (can with syringe or infusion pump) to inject 0.25% pancreatin (Trypsion) from umbilical vein or umbilical artery, or 0.1-1% collagenase II (Collegenase), or 0.5%Dispense II enzyme, also can add (Ethylene diamine tetra acetic acid) (EDTA) of 0.05 to 0.2%, the placenta 30 minutes to 90 minutes being separated enzyme containing histocyte is hatched in 37 DEG C of incubators, then in umbilical vein or umbilical artery, pour into physiological buffer rinse, the composition of this physiological buffer can be physiological saline, low molecule dextrose is sweet, phosphoric acid buffer (phosphate-buffered saline) or cell culture fluid, picture 1640, DMEM (Dulbecco ' s modifiedEagle ' s medium), often rinse once, then extrude once, collect the mixed solution contained based on the blood of placenta mononuclearcell and tissue juice, rinse, extrude 3 to 5 times, time is between 30 to 90 minutes, the mixture of the damping fluid collected like this is for containing placenta mononuclearcell, its volume is between 100 to 800 milliliters.This damping fluid can contain antibiotic, and as penicillin (100U/ml), Streptomycin sulphate (100g/ml) also can contain the heparin of the preservative-free of 2500 to 10,000 unit.This enzymic digestion, stirs, extruding, and the cell that the methods such as washing are collected is called placenta tissue mononuclearcell.
Collect placenta tissue (film) mononuclearcell: be separated amnion and chorion tissue from placenta tissue, shred with after brine, adopt the pancreatin (Trypsion) of 0.25% or the collagenase II (Collegenase) of 0.1-1%, or 0.5%Dispense II enzyme, or add (Ethylene diamine tetra acetic acid) (EDTA) of 0.05 to 0.2%, hatch in 37 degree of incubators or stir 30 to 60 minutes, the cell of collection, overanxious, centrifugal acquisition is placenta tissue mononuclearcell.(cell obtained together in film or placenta tissue is referred to as placenta tissue mononuclearcell) Fig. 1 can be clearly seen that dyeing mononuclearcell.
The placenta mononuclearcell collected is all by following treatment process:
1) placental blood collected or tissue stem cell are all by each layer nylon or Steel net, and that minimum is 50um, and that maximum is 100mm, collect mononuclearcell.
2) by the centrifuge tube of the cell harvesting to 50 milliliters that come from placenta, with 1,500g centrifugal 10 minutes in centrifuges, the liquid of 70% is removed.Then following two kinds of methods can be had.
2.1) cell collected and lymphocyte separation medium mix in the ratio of 1:4.With 2500g centrifugal 30 minutes in centrifuges, remove the granulocyte of remaining red corpuscle and maturation, collect the cell between liquid level, cell RPMI-1640 washes secondary.
2.2) another kind of method be by extract containing placenta tissue original mesenchyma and hematopoietic stem/progenitor solution and 6% polymer hydroxyethylamyle (Hespan, be greater than 60, 000 dalton) mix in 1:3 to 1:6 ratio, 1500rpm (500 to 1, 000g), 4 degree centrifugal 5 to 20 minutes, discard red corpuscle, retain supernatant liquor, then low molecular dextran (saliferous) is used to be washed three times, collected cell for concentrating mononuclearcell, be made into mononuclearcell suspension, and do mononuclearcell counting, measure cytoactive, calculate CD34, CD4, cd8 cell content and do the white corpuscle colony (CFU-GM) of vitro culture, red, white corpuscle colony (CFU-GEMM) and mesenchyme colony (CFU-F).Fig. 2 is the placenta cells after concentrating, and the cell of dyeing is mononuclearcell.
To finish various detection, and comprise, virus, the Cord blood of microbial culture and tissue matching (be adopt Cord blood to detect instead of for extracting), placental blood and placenta tissue stem cell make mononuclearcell, according to the method described above respectively with 1X10 7to 1X10 8the concentration of/ml and DMSO (methyl-sulphoxide) mixing, and with 10% final concentration DMSO and low molecular dextran cool to subzero 80 DEG C, then profound hypothermia preservation respectively in liquid nitrogen (-186 DEG C) liquid phase is transferred to, application during in order to transplanting.
Extract placenta hemopoietic precursors stem cell: technology has become cell magnetic can the cell gold standard of the positive and CD133 positive of sorting CD34, the advantage having it powerful and widely purposes: from being sorted into extensive sorting in a small amount, from high frequency cell to the cell sorting of rare subgroup, can efficiently, repeatably and high-quality cellular segregation result come from intraplacental population of stem cells, the full-automatic sorting of cell can be realized, and do not need special personnel or skill.Cell sorting reagent can obtain the cell of nearly all type from various species and sample, selects post for quick sorting by the cell of MACS marked by magnetic bead, and the magnetic field that cell can be sorted post amplification is caught.Gentle sorting environment; while the rate of recovery ensureing cell and purity, more protect cell-surface antigens epi-position, therefore; the cell epitope that sorting obtains is intact; cell function is unaffected, for carrying out the negative sorting of cell in the whole blood from anti-freezing process fast, and maximum primary treatment 30mL whole blood; without the need to steps such as density gradient centrifugations; erythrocytic sedimentation can be accelerated, to remove non-object cell, object cell can be obtained easily from supernatant liquor.First adopt the magnetic bead antibody concentrated and purified cell containing CD133 from placenta stem-cell containing anti-CD133, then isolate CD133 with AntiCD3 McAb 4 magnetic bead antibody from containing in the cell mass of CD133 +cD34 -population of stem cells, also referred to as early stage hemopoietic precursors stem cell, shown in Fig. 2, adopt the cell dyeing after magnetic beads for purifying, pigmented cells is large, and nucleus almost occupies whole cell, and tenuigenin loosens.
The cultivation of the early stage hemopoietic precursors stem cell of placenta: get plastics 100cm 2or 75cm 2culture dish or culturing bottle, the nutrient solution of 1-5ml is put in culture dish, nutrient solution can with DMEM or 2-Mebicm, or RPMI-1640, wherein contain the serum of 10%, this serum can be Trace element (FBS), also can be serum-free medium, containing cell stimulating factor, as fibroblast growth factor, white blood cell growth factors etc., incubation time is change liquid once in every 3 days, and now, cell is after with nutrient solution cleaning, again can cultivate and again preserve, placenta early stage hemopoietic precursors stem cell still keeps original characteristic.
Detection to the early stage hemopoietic precursors stem cell of placenta: placenta is after entering laboratory or company, first the supposition of various virus will be done, picture hepatitis B surface antibody, antibody of HCV, virus of AIDS antibody, cytomegalovirus antibody and syphilis antibody, only various be detected as feminine gender time, the placenta that just can carry out next step rinses and removes residual red blood cells program.After acquisition placenta hemopoietic precursors stem cell, first placenta mycotic culture will be carried out, only after various cultivation feminine gender, the placenta early stage hemopoietic precursors stem cell of extracting need carry out that the molecular biology of tissue matching (HLA) is low, high resolution detection, HLA mono-class antigen (A, B) and HLA bis-class antigen (DR), all data will by computer software and barcode control and management.Placenta hemopoietic precursors stem cell after all cultivations needs flow cytomery.Fig. 3, for adopting the placenta hemopoietic forebody cell flow cytometer detection after Beads enrichment.This figure is the cell after the anti-CD133 antibody purification of magnetic bead, has the two positive cell of CD133 and CD34 as seen, and Fig. 4 adopts AntiCD3 McAb 4 magnetic bead antibody purification to remove CD34 positive cell again, is the cell mass of the CD133 positive.Fig. 5 is that white corpuscle colony detects (CFU-GM) and Fig. 6 is that red-white corpuscle colony detects (CFU-GEMM), and every a pile cell is all come from an early stage hemopoietic precursors stem cell.
The clinical indication of placenta hemopoietic precursors stem cell: described in indulging above, the cure mechanism of placenta hemopoietic precursors stem cell, one is the multipotency differentiated utilizing this stem cell, he can be under particular circumstances, be divided into the various cells of tissue, picture is neural, skin, bone, cartilage, muscle, liver, heart, fat and islet cells, two is utilize this cell can break up various cell chemotactic factor and cell stimulating factor to help damaged cell core tissue repair and help damaged cell regeneration, and this stem cell can be used for the treatment of following disease clinically accordingly:
Cerebro-vascular diseases: hematencephalon, brain thromboembolism, senile dementia etc.
Cardiovascular diseases: myocardial infarction, stenocardia, myocardosis
Interior point voltinism disease: diabetes
Digestion disease: liver cirrhosis
Respiratory system disease: pneumosclerosis tuberculosis
Autoimmune disease: erythrocyte disorder pulmonary fibrosis, lupus erythematosus, scleroderma, glomerulonephritis
Immunological rejection disease: graft versus host disease (GVH disease)
Hemopoietic precursors stem cell method in clinical application: the placenta hemopoietic precursors stem cell after cultivating and collecting, before Clinical practice, needs again to carry out following detection:
1. flow cytometry analysis: more than 70% of whole cell colony need be accounted for for CD133+ and CD34-negative cells in its early stage hemopoietic precursors stem cell.
2. the supernatant liquor cultivating placenta hemopoietic precursors stem cell need be cooked microbial culture again, must confirm without any microorganism growth and foreign animal proteins.
3. check HLA distribution type, need the low resolution of its HLA of placenta hemopoietic precursors stem cell in I class more than 4 must be had to conform to the HLA distribution type of user arbitrarily with class Ⅱ antigens.
4. detect the activity of placenta hemopoietic precursors stem cell, require that, when using, its activity is greater than more than 90%.
5. detect the quantity of placenta hemopoietic precursors stem cell, require that the hemopoietic precursors stem cell number again giving patient is greater than 1.0 × 10 5/ kg body weight.
The infusion bag of infusion placenta hemopoietic precursors stem cell, requirement is aseptic, nontoxic, meet health ministry requirement, infusion bag will have obvious label, the content of XX placenta hemopoietic precursors stem cell, active and HLA distribution type and manufacturer and date manufactured, the Expiration Date etc.
The patent provides and a kind of adopt placenta hemopoietic precursors stem cell for immunoregulatory method, describe and how to produce and cultivate, screen activated placenta hemopoietic precursors stem cell.Come from the hemopoietic precursors stem cell of placenta, in vitro by cultivating and increasing, at least there is following cell surface marker: CD133+, and not there is CD34, CD45.Plastic infusion bag containing the early stage hemopoietic precursors stem cell of placenta, this infusion bag for sealing and aseptic, there is pipeline centre for intravenous infusion.This patent provides one and extracts from placenta, qualification, cultivates, cryopreservation and cultivation, amplification, the early stage hemopoietic precursors stem cell of placenta of preservation.
Placenta hemopoietic precursors stem cell has the function of various stimulating factor in strong cytodifferentiation and body secretion, can be used for treating various chronic inflammatory pain, cardiovascular disorder and liver cirrhosis, diabetes and autoimmune disease.
According to our method, the early stage hemopoietic precursors stem cell of placenta of extraction is all that activity is greater than more than 90%.Cytokines (TL-1, alpla, IL-1, beta, IFN-gamma, TFN, protein micromolecular hormone (Hormones, androgen, oestrogenic hormon, Regular Insulin, prolectin element, collagen protein etc. can be secreted.At least 1.7 × 10 5/ kg, preferably 2.5 × 10 6/ kg hemopoietic precursors stem cell can be used for the effect of anti-inflammatory (anti-inflammation), can be used for any inflammatory disease, and this inflammation can show any tissue, any organ, picture muscle, neural, brain, spinal cord, peripheral nerve, vascular tissue, heart, pancreas islet, intestines, digestive tube, liver, lung, kidney, reproductive system, endothelium and endocrine system, also can be used for treating autoimmune disease, especially time with inflammation, picture diabetes, amylotrophic lateral sclerosis, myasthenia fravis diabeticmeutropaathy, inpus, Alzheimer ' s, Pakinson ' s.sickle cell anemia, pompe ' s diseases, the early stage hemopoietic precursors stem cell of phenylketonuria (PKU) placenta is applicable to after wound very much, the recovery of biological cells and tissues, these wounds include but not limited to that central nervous system disease is damaged, picture hematencephalon, cerebral thrombosis, spinal cord or peripheral nerve injury, myocardial damage, picture myocardial infarction, myocardial ischemia, muscle or skeletal injury (as muscle or ligament injury), fracture, digestive system injury, picture liver cirrhosis, hepatitis, colitis, reproductive system damage, picture infertility, urinary injuries, picture glomerulonephritis, endocrine system is damaged, picture insulin-dependent diabetes mellitus, and all are due to physicogenic skin or mucosa injury, picture burn, core radiates, frostbite etc.This early stage hemopoietic precursors stem cell is also very suitable for senile cell regeneration and becomes and the disease of initiation, as senile arthritis, and the sick ACS of A Mo Er Shi, parkinsonism (Parkinson ' s).
The early stage hemopoietic precursors stem-cell therapy of placenta that this patent proposition HLA conforms to can strengthen the curative effect maintaining stem cell greatly, thus the early stage hemopoietic precursors stem cell of autologous placenta is adopted to preserve, not only can maintain the application of autologous depositary, and whole family can be benefited, for the application of the early stage hemopoietic precursors stem cell of allosome, also be to set up a huge early stage hemopoietic precursors stem cell bank of allosome, and the early stage hemopoietic precursors stem cell HLA distribution type to autologous each example of preservation, when Clinical practice, HLA has the low resolution of molecular biology more than 4 sites to conform to (HLA:A, B, DR) the early stage hemopoietic precursors stem cell of placenta storage temperature before use, if preserve when freezing, should transport with dry ice form, also can in small-sized liquid nitrogen container, if the cell of having survived in nutrient solution, then 4-10 DEG C should be kept at.According to cell state, the shelf time differs; Can preserve for a long time in liquid nitrogen pipe, can 18-21 days be preserved in dry ice, and in nutrient solution, be 24-48 hour.
The using dosage of the early stage hemopoietic precursors stem cell of placenta: application placenta hemopoietic precursors stem cell is as cell therapy, the cell competence exertion curative effect of patient's high dosage must be given, low dose of injection can only be harmful to patient, make patient that immune response occur, produce excessive stress situation, we adopt the dosage of the hematopoietic stem cell transplantation of more than 2 times, as applied 1 × 10 when hematopoietic stem cell transplantation 9mononuclearcell, if as come from marrow blood, be then equivalent to 0.1-0.6 × 10 7hemopoietic stem cell, and the content of the placenta hemopoietic precursors stem cell that we adopt is 1-10 × 10 5the usage quantity of/every body weight kilogram placenta hemopoietic precursors stem cell, these stem cells can disposablely be injected, and also can inject several times in space-number sky.The position of injection is many by PeV infusion, but according to the state of an illness, also can topical application, picture Peripheral Venous Injection, spinal cavity is injected, brain is injected, intraarticular injection or heart coronary artery injection, also any tissue or organ damage injection location can contain the infusion bag of placenta hemopoietic precursors stem cell in vivo.Cultivate, the early stage hemopoietic precursors stem cell of placenta after amplification is kept in aseptic transfusion bag, and the length of this sack is 18, and wide is 15, height is 10, be that the outside of this sack of infusion bag of a transparent nontoxic aseptic national health department approved applications and human vein's infusion indicates the unit that dispatches from the factory, the time, the quantity of placenta hemopoietic precursors stem cell in bag, active, title, storage temperature, shelf time and blood group and HLA distribution type.The infusion bag of placenta hemopoietic precursors stem cell need be contained in one and not be afraid of in the aluminium box of damage, to ensure that this infusion bag can not be damaged in transportation, this aluminium box, long 19, wide by 19, high by 12, infusion bag can be put into after opening aluminium box, then cover aluminium box, there is an opening top of box, can observe the situation in infusion bag.In this infusion bag except containing except placenta hemopoietic precursors stem cell, also cell stimulating factor can be contained, picture GM-CSF, red corpuscle stimulating factor (EPO), stem cell stimulating factors (FIT) etc., also various interleukin stimulating factor can be contained, picture IL-2, IL-4, IL-6, CD401, IFN-alpha, TNF-alpha, IFN-beta-3, also the stem cell stimulating factors of various cell tissue can be contained, picture hepatocyte-stimulating factor (Hepatocyte growth factor), myocardial cell's stimulating factor (cardiotropin-1), vasotonia stimulating factor I, II, III (angiotensin), IFN-famma, endotheliocyte stimulating factor, (Epidermalfrowth factor), fibrocyte stimulating factor (basic fibroblast growth factor), also antibiotic can be contained, the penicillin of picture 10-100 unit/ml and the Streptomycin sulphate of 10-100 microgram.
Freeze thawing and hemopoietic precursors cellular replacement therapy: when clinical confirmation need apply certain routine placenta stem-cell, in liquid nitrogen, stem cell is taken out before patient bed, melt in 37 DEG C of electric water bath cabinets, when hemocyte is almost complete by freeze thawing, add low molecular dextran balanced mix, give patient's infusion in order.
According to the state of an illness needs of patient, the stem cell in the multiple source of freeze thawing can be distinguished, also disposable freeze thawing various kinds of cell can combine and apply to patient.Patient body weight being less than to 10 kilograms can only with one bag of Cord blood, 10 kilograms of patients being less than 30 kilograms are greater than to body weight and can use Cord blood and placental blood stem cell, body weight is greater than to patient's Cord blood, placental blood hemopoietic stem cell and the placenta tissue hemopoietic precursors stem cell of more than 30 kilograms.
The early stage hemopoietic precursors stem cell of placenta tissue prepared by the present invention is generally input as master with vein, but also can directly enter in myeloid tissue local, also by other approach any, as method infusions such as artery injections to patient.The Pharmaceutical Compositions that the present invention mainly applies or containing physiological saline, low molecular dextran, umbilical cord blood plasma, DMEM (Dulbecco ' s Modified Essential Medium) nutrient solution, water for injection etc.Solution used is all aseptic, that prepared by pyrogen-free and useful clinical is transplanted patients above.
Beneficial effect:
Stem cell has self-replacation and the initiating cell to various cytodifferentiation potential, is the cells of origin forming each histoorgan of viable organism.The actual essence of growth course of human individual is exactly the self of stem cell and the process of proliferation and differentiation, and the cell of each organ is also the stem cell deriving from each organ respectively, as liver, neural, cerebral tissue, pancreas, skin etc.Have by the kind of stem cell:
1. embryonic stem cell
Embryonic stem cell is the most original, can the highest cell of differentiation degree, and be also the starting stage forming embryo after zygote combines, it can be divided into various cell, even can take the method for nuclear transplantation, utilizes autologous somatic differentiation to become various cell.Must embryo be destroyed during clinical application, allow it be divided into other cell, because the height of cell divides voltinism, easily form tumour cell, thus can not with any hemopoietic stem cell of this kind of cell extraction.
2 transgenosis multipotential stem cells
Transgenosis multipotential stem cell, the function that the autologous adult cell first found by Japanese Scientists (the acquisition Nobel prize) has embryonic stem cell after gene transfection also referred to as induced pluripotent stem cell (iPS), but maximum query transfectional cell also can have the possibility of tumour cell simultaneously, want overall application in clinical, also need a large amount of scientific researches and time, thus can not with any hemopoietic stem cell of this kind of cell extraction, its main application is still for tissue repair.
3 adult stem cells:
Adult stem cell is after people is born, and each organ also retains the cell that can regenerate voluntarily.This stem cell is younger body, and content is abundanter, can differentiation degree higher.1998, first find also to have in Adult Human Bone Marrow in Wisconsin university of the U.S. and can be divided into various histiocytic stem cell, people recognize that in human body, various histocyte all also exists this cell subsequently, be called adult stem (ASC cell), it has the function same with embryonic stem cell.Adult stem cell can be divided into:
3.1 Adult Human Bone Marrow stem cells: mainly containing the stem cell that can be divided into human blood cells, as white corpuscle, red corpuscle, thrombocyte etc.Someone adopts marrow to study, CD133 after MACS sorting +cell function Journal of Sex Research shows, CD133 +cell has long-term culture-initiating cell (Long-term culture-initiating cells, and long-term reconstitute hematopoiesis cell (Long term repopulation cells LTC-IC), LTRC) ability, for the most original hematopoietic cell, transplant and can plant work for a long time to after host.In addition, research recently finds that LTC-IC is mainly seen in AC133 +cD34 +subgroup, a small set of CD133 +/ CD34 -cell also has Long-term Proliferation potential.Obviously, due to the difficulty in source, adopt marrow to be used for scientific research, extensive extraction can not be used for.
3.2 Peripheral blood stem cells; In normal people's Peripheral blood not containing or have the stem cell of seldom amount, but apply a kind of be called the medicine of the leukocyte stimulation factor after, the stem cell in marrow blood can be discharged in Peripheral blood, so also referred to as mobilizing hemocytoblast around, this kind of cell is with CD34 +cell is main, only containing a small amount of CD133 +cell, the same with marrow, due to the difficulty in source, adopt Peripheral blood stem cell to be used for scientific research, extensive extraction can not be used for.
3.3 cord blood stem cell.Be after newborn infant is born, the stem cell in Cord blood, it contains abundant hemopoietic stem cell.Due to convenient sources, draw materials easily, so the most extensive to its research, the cord blood CD 34 done and CD133 sorting research find the CD34 with a sample +cell amplification potential is lower than CD133 +cell.Therefore AC133 sorting may be better than CD34 sorting.But as pointing out in our table 1, containing abundant CD133 in Cord blood +cD34 +cell, but hardly containing CD133 +cD34 -cell, and placental blood is just in time contrary, containing abundant CD133 in blood +cD34 -cell, but only containing the CD133 only accounting for about 10% +cD34 +cell, thus, the work of Cord blood is only hemopoietic precursors stem cell, instead of early stage hemopoietic precursors stem cell.So, why nobody studies placenta stem-cell; This is mainly:
1) science and technology of cord blood stem cell is taken to domesticly to be developed first at home in 1996 by the present inventor.
2) find that the domestic and international first section of paper containing abundant hemopoietic stem cell in placental blood is also that first this author delivered in 2004.
3) this author applied for placenta stem-cell patent in 2005.
4) the more important thing is it is how aseptic that this author grasps, ensure cytoactive, it is the key extracting placenta stem-cell, and this just this author succeed, on this basis, before this, nobody is expected and is found that Cord blood and placental blood have so huge difference in this respect, we have found in placenta containing the early stage hemopoietic precursors stem cell do not contained in Cord blood or seldom measure.
Table 1: the comparison of Cord blood and placental blood component
Wherein: UCB: Cord blood; UPB: placental blood; Mean: mean value; SEM: standard error; Min: minimum value; Max: maximum value;
The content of flow cytomery placental blood and Cord blood mid-early stage hemopoietic stem cell is adopted from Figure 10, then statistical analysis is carried out, and P " 0.001, there is significant difference, show placental blood than Cord blood early stage hemopoietic stem cell content clearly raise, corresponding CD133 +cD34 +cell indifference, P " 0.5.
Accompanying drawing explanation
Fig. 1, from placenta the placenta mononuclearcell of extraction and concentration.Adopt Gimsa dyeing, have nucleus painted for mononuclearcell, undyed is red corpuscle.
Fig. 2, come from concentrated after placenta mononuclearcell, adopt separafor purifying, then with Gimasa dyeing, have nucleus painted for placenta hemopoietic precursors stem cell.
Fig. 3, for flow cytometer two positive to placenta hemopoietic precursors stem cell, namely after CD133 magnetic bead sorting, the detection of CD133 and CD34: ordinate zou is anti-CD133 antibody, X-coordinate is AntiCD3 McAb 4 antibody cell to be detected, for CD133 is positive and CD34 is positive and the mixing group of feminine gender.
Fig. 4, flow cytometer are single positive to placenta hemopoietic precursors stem cell, and namely carry out cell detection with anti-CD133 antibody: ordinate zou is ssc side scattered light, X-coordinate is anti-CD133 antibody, irises out the cell mass of the CD133 positive.
Fig. 5, early stage hemopoietic precursors stem cell grow in CFU-GM substratum, by the white corpuscle colony group that individual cells propagation is more than more than 50 after 7 days.
Fig. 6, early stage hemopoietic precursors stem cell grow in CFU-GEMM substratum, by the red white corpuscle colony group that individual cells propagation is more than more than 50 after 7 days.
Early stage hemopoietic precursors stem cell after Fig. 7 is separated and non-early stage hemopoietic precursors stem cell grow in granulocyte colony substratum.
CFU-GM.CD133+CD34_ cell colony formation time is slow, form huge colony, and CD133-cell colony formation time is fast, but colony disappears, and shows that colony can not be lasting to the 25th talent also fast.Huge colony is formed to the 25th talent,
Early stage hemopoietic precursors stem cell after Fig. 8 is separated and non-early stage hemopoietic precursors stem cell grow in granulocyte colony substratum.
CFU-GEMM.CD133+CD34_ cell colony formation time is slow, form huge colony, and CD133-cell colony formation time is fast, but colony disappears, and shows that colony can not be lasting to the 25th talent also fast.Huge colony is formed to the 25th talent.
Hemopoietic stem cell in early stage after Fig. 9 is separated is rounded, is placed in and can cultivates by adherent environment, within every 3 days, change liquid, discard non-adherent cell, and visible a small amount of spindle cell occurs.Can microcolony be there is after 10 days, and form typical colony gradually.
Figure 10: be Cord blood and the comparison coming from placental blood or placenta tissue employing flow cytomery hemopoietic stem cell content in early stage.Statistical analysis: P " 0.001, there is significant difference; Wherein Cord blood represents Cord blood; Placental represents placental blood.
Embodiment
Embodiment 1 collects placenta immediate precursors hemopoietic stem cell
First, obtain after pregnant woman or its direct relatives agree to contribute placenta tissue, consult all survey reports of pregnant woman, confirm without the virus infection relevant with blood such as any virus, syphilis, then the medical history such as fertility, disease, heredity, infection of pregnant woman is inquired in detail, be normally at all, after pregnant woman childbirth, sterile collection placenta and Cord blood.Cord blood after collecting and placenta tissue, all with bar coded sticker, deposit in 4 DEG C of refrigerators.Shelf-time is generally no more than 48 hours.The Cord blood collected adopts the content of flow cytomery hemopoietic stem cell marker CD34 and the Colony cultivation of hemopoietic stem cell.
Collect the agreement that placenta must obtain pregnant woman, and sign letter of consent, pregnant woman and its relatives must not have various transmissible disease, picture hepatitis, acquired immune deficiency syndrome (AIDS) (AIDS) etc., after puerpera overflows fetus, mother and fetus treatment must not be disturbed, after fetus spilling, aseptic acquisition placenta, aseptic being saved in placenta box is transported to company with aseptic.Transport time temperature be 4-20 DEG C, the time within 36 hours, after placenta to company, placenta is opened in sterile environment, adopt stroke-physiological saline solution, and put the flushing placentas such as physiological saline, phosphoric acid buffer, DMEM or RPMI-1640, until extraplacental residue blood all rinses complete.At umbilical vein in placenta tissue, temperature is with 20-40 DEG C of requirement, can know and see that conduit enters into placenta Ink vessel transfusing, then fix at umbilical vein and conduit place with strile gauze dressing, adopt and use the same method, insert respectively in Umbilical artery with two conduits, and with fixing, then the external infusion bag of these conduits, clean placenta 24-36 hour, in the process, constantly can extrude placenta, intraplacental liquid is constantly flow in receiving flask, and the liquid volume in receiving flask is generally between 300-2000 milliliter.Collect the heparin that can contain the penicillin of 50-100/ml unit or the Streptomycin sulphate of 50-100 microgram/ml and 1-10 unit/ml in liquid.Scavenging solution is physiological saline, phosphate buffered saline buffer or DMEM-1640 nutrient solution, and deriving flow velocity is per minute 10-50 milliliter, and extruding placenta speed can be accelerated after beginning is comparatively slow.When effluent liquid is no longer rubescent, or cell count lower than 100/ milliliter time, the liquid now collected is placental blood, and the cell of the inside is placental blood cell.And then to use temperature instead be 37 DEG C, 0.05% pancreatin, 0.29%EDTA, also collagenase can be added, its concentration is 5-50 units per ml, be injected into continuously in placenta, holding temperature, between 35-38 DEG C, is poured into latter 30 minutes at enzyme, start to adopt and again do not pour into until 24 hours containing the solution of enzyme, can stir when collecting, extruding placenta tissue, receiving flask does not wash, maintain the temperature at 4 DEG C, and be dispensed in 50 milliliters of centrifuge tubes, 3000-5000 turn 4 DEG C centrifugal 15 minutes, the mixture after centrifugal is for containing placenta tissue stem cell.
The separation of placenta tissue hemopoietic precursors stem cell, purifying also can be alternatively as follows: come from the placenta of hospital in sterile chamber, need to adopt stroke-physiological saline solution after company, PBS, or cell culture fluid (DMEM, 1640) thoroughly cleaning is comprehensively carried out, placenta after cleaning is put in generatrix upward, the ventricumbent position of son, by sterile scissors, or sterile razor blade, cut off the generatrix of about 1-2 cm thick, then placenta (amnion employing same method) is cut into 1-3 cubic centimetre size, and then cut 1 cubic centimetre of size, the placenta tissue shredded is put into a container, as centrifuge tube, in beaker or flask, add collagenase (collagenase) I or II in a reservoir, concentration is 0.5-5 milligram/ml, also can with the pancreatin %-EDTA of 0.25, hatch 3 minutes for 37 DEG C, also can collagenase digesting complete after, use trysinization again 10 minutes, after this cell of digestion is collected, 400g in whizzer, 10 minutes, the cell that room temperature is collected uses PBS again, physiological saline or cell culture fluid washing, the cell counting of collecting, survey activity, activity need be greater than 90%, gained cell is just mononuclearcell.
Come from the mononuclearcell of placental blood and placenta tissue, then adopt the sorting of MACS cell magnetic bead separating method to come from the hemopoietic precursors stem cell of placenta.Its principle be adopt and the AntiCD3 McAb 4 that combines of magnetic bead or anti-CD133 monoclonal antibody to carry out CD133 +cD34 -the sorting of hemopoietic precursors stem cell.Because MACS cell magnetic bead separating method has the magnetic capture of amplification, gentle sorting environment, the activity of cell and the rate of recovery and purity can be ensured, thus the present invention with it for representative carrys out sorting, purifying comes from the hemopoietic precursors stem cell of placenta, its method is divided into two parts: first adopt magnetic bead and anti-CD133 antibody (manufacturer: Miltenyi Biotec, Germany, buys producer: Qiang Zhi bio tech ltd, Shanghai 021-50937010) from the placental stem cell populations after concentrated, isolate the cell of all CD133 positives.Total cell count of placental cell populations is 0.5 to 12.5 × 10 9, therefrom separable go out CD133 positive cell, comprise CD133 +cD34 +and CD133 +cD34 -, its minimum value 0.01X10 8be 9.0 × 10 to maximum value 8.And then with magnetic bead and AntiCD3 McAb 4 antibody (manufacturer: Miltenyi Biotec Germany, buy producer: Qiang Zhi bio tech ltd, Shanghai 021-50937010) and the reaction of isolated CD133 positive cell, isolate the cell of wherein CD34 feminine gender, the CD133 of acquisition +cD34 -its minimum value 0.05X10 7be 1.0 × 10 to maximum value 7.Below for duckpin: be separated mononuclearcell in placental blood or placenta tissue in 1: 2 ratio be added to lymphocytes separating solution surface, the centrifugal 30min of 400g normal temperature, in the middle of careful absorption, the mononuclearcell of leukocytic cream is in 50ml centrifuge tube, adds phosphoric acid buffer and washes 3 times.
Adopt purchased from the magnetic bead antibody labeling method sorting CD133 of MACS company +cD34 +precursor hemopoietic stem cell.Come from add phosphoric acid buffer wash after cell, use the buffering liquid containing bovine serum albumin instead, mononuclearcell adjusts to 1X10 8to 10X10 9between, add FcR blocker fully to mix latter 4 DEG C and hatch 30min, and then add the reagent (manufacturer: Miltenyi Biotec combined with mouse-anti people CDl33 monoclonal antibody and magnetic bead, Germany buys producer: Qiang Zhi bio tech ltd, Shanghai 021-50937010), 60min is hatched in latter 4 DEG C of abundant mixing, period constantly mixes repeatedly, is put into one and has the CDl33 of stabilizing magnetic field background by force after taking-up +sorting post, all liquid by pillar, and the anti-CDl33 that magnetic combines +cell antibody combines with the cell with CD133 antigen and is trapped in post, and the cell without CD133 antigen can not combine with magnetic bead and be washed out, here containing the CD133 that people often mention -cD34 +cell.Sorting post is shifted out magnetic field, because without magnetic field, can with damping fluid the CDl33 be trapped in post +cell elutes, and comprises CD133 here +cD34 +and CD133 +cD34 -cell.Apply above same principle, and then what add is the reagent (manufacturer: Miltenyi Biotec combined with mouse antihuman CD 34 monoclonal antibody and magnetic bead, Germany buys producer: Qiang Zhi bio tech ltd, Shanghai 021-50937010), and the cell starting to hatch is CD133 +cD34 +and CD133 +cD34 -cell.The cell of wash-out is CD133 +cD34 -, and be detained in pillar, then that wash-out is CD133 +cD34 +cell, the rate of recovery is to CDl33 +cell, greatly about 1/50 to 1000th, reclaims cell greatly about 1/10 to 50th to mouse antihuman CD 34 monoclonal antibody and magnetic bead pillar.
The feature that embodiment 2 placenta early stage hemopoietic precursors stem cell streaming and stem cell detect
Placenta hemopoietic precursors stem cell all needs the feature of observation of cell in surface markers after concentrating and separating, conventional technology has flow cytometer (Flowcytometry) and immunocytochemistry (immunocytochemistry) and Protocols in Molecular Biology to check cell DNA or the isogenic change of RNA, the methods such as normal employing PCR or RT-PCR, the normal surface markers finding this kind of hemopoietic precursors stem cell is CD133 +cD34 +, and the surface markers of early stage hemopoietic precursors stem cell is CD133 +cD34 -, comparatively generally placenta hemopoietic precursors stem cell is CD34 being about to surface marker when being divided into unidirectional stem cell -, CD38 -, and CD133 +if during to blood system cytodifferentiation, its surface marker is characterized as CD34 further +, CD38 +, and CD133 -.Fig. 5 and Fig. 6 is respectively early stage hemopoietic precursors stem cell at CFU-GM and CFU---grow in GEMM substratum, breed as more than the white corpuscle of more than 50 or red white corpuscle colony group by individual cells after 7 days.Show that placenta hemopoietic precursors stem cell has stem cells hyperplasia and differential growth ability.Fig. 7 and Fig. 8 is respectively early stage hemopoietic precursors stem cell at CFU-GM and CFU---grow GEMM substratum in, being bred by individual cells after 10 days is white corpuscle more than more than 50 or red white corpuscle colony group.Non-early stage hemopoietic precursors stem cell is to impinging upon CFU-GM and CFU---and in GEMM substratum, growth is fast, just Clone formation is had at second day, but clone progressively desolate moving back, and placenta early stage hemopoietic precursors stem cell clone forms late, clone has very strong propagation and differential growth ability after 10 days.
The freezen protective of the early stage hemopoietic precursors stem cell of embodiment 3 placenta
The early stage hemopoietic precursors stem cell of placenta is kept at-196 DEG C, namely in liquid nitrogen, can avoid necrocytosis, downright bad (necrosis) or apoptosis (apoptosis).Conventional method is adjustment cell concn 1-10 × 10 6/ ml, the apparatus stored mostly is the freezing pipe of 2-100 milliliter or 50 milliliters of freezing bags, the preservatives of stored frozen has PBS (phosphate buffered saline buffer), cell culture fluid (1640, DMEM or α-nutrient solution), foetal calf serum, human serum, physiological saline, wherein important cell-preservation liquid is dimethyl second sulfone (dimethyl sulfoxide, DMSO), concentration is 5-10%, after adding DMSO and the mixing of placenta hemopoietic precursors stem cell, put in Programmed freezing instrument, with per minute 1 DEG C cooling, drop to-90 DEG C always, then between liquid nitrogen gas phase-120 DEG C-140 DEG C, also can directly be put in liquid nitrogen liquid phase, temperature is at-196 DEG C.The early stage hemopoietic precursors stem cell of all placentas divides the time, source, the various information gone down to posterity all are stored in computer software computer, when needed, patient can be taken out easily and make the placenta hemopoietic precursors stem cell sample needed, within 37 DEG C-40 DEG C, generally with 37 DEG C of these stem cells of fast freeze-thaw, after aseptic washing, be placed in culture dish (bottle) and cultivate and amplification placenta hemopoietic precursors stem cell.Each early stage hemopoietic precursors stem cell of placenta of collecting needs to do hepatitis B (hepatitis B), third liver (hepatitis C) virus of AIDS antibody (human immunodeficiency virus) types I and II), cytomegalovirus (cytomegalovirus) etc.Before use, bacterium and mycotic culture, only have feminine gender just can apply.Cultivate, increase and the placenta hemopoietic precursors stem cell of collecting with 1-10 × 10 6the cell concn of/ml and the dimethyl second sulfone (DMSO) of 10% volume, mix mutually, and the freezing pipe that mixed cell suspension is placed in 2-10 milliliter drops to subzero 80 DEG C, then, transfers in the gas phase of liquid nitrogen (subzero 186 DEG C) and preserve.
The recovery of the early stage hemopoietic precursors stem cell of placenta and cultivation, thaw rapidly in 37 DEG C of water-baths the placenta hemopoietic precursors stem cell of stored frozen, aseptically operate, extract the cell suspension in freezing pipe, with sterile phosphate buffer (PBS), DMEM or RPMI-1640 washed cell suspension, then with 1-5 × 10 6the concentration of/ml cultivates placenta hemopoietic precursors stem cell again.
The early stage hemopoietic precursors stem cell of embodiment 4 placenta is to the differentiation of other clone
Placenta early stage hemopoietic precursors stem cell is under certain internal milieu, or under certain stimulation or somatomedin condition, cell specially in adult can be broken up, the factor for breaking up includes but not limited to the factor, picture GM-CSF, IL-2, IL-3, IL-4, Fit32, CD40L, IFN-alpha, TNF-alpha, IFN-gamma, IL-6, folic acid, scleroproein somatomedin (hasicfibroblst growth factor), TGF-beta-1, TGF-beta-3, liver, cell growth factor (epodermal growth factor), cardiotropin-1, angiotensinogen cngiotensin I (AI), angiotensin II, Deng.
In differentiation-inducing, first cultivate the early stage hemopoietic precursors stem cell of placenta, make it become attached cell, as shown in Figure 9, the CDl33 be just separated +mononuclearcell, rounded, suspend distribution equably.Discard non-adherent cell after 3d, visible a small amount of spindle cell occurs.Within about 10 days, there is microcolony.Within about 14 days, microcolony forms typical colony gradually, and then adopts following method:
1. placenta hemopoietic precursors stem cell is at DMEM, and 1640,2-Medium contains 100 units of Penicillin, and the Streptomycin sulphate of 100 micrograms and 20% foetal calf serum are cultivated.
2., when Growth of Cells is to the culture dish of 60-80%, collect nutrient solution, wash three times with aseptic PBS.
3. time for neural cellular differentiation, DMEM nutrient solution can be adopted to add 1-10ml beta-mercaptoectanol simultaneously, 2%DMSO also can be used to add butylatedhydroxyanisole
4.1. the early stage hemopoietic precursors stem cell of placenta is to the differentiation of adipocyte, first, the early stage hemopoietic precursors stem cell of placenta is at DMEM, 1640 or 2-Medium contain 100 units of Penicillin, 100 microgram Streptomycin sulphates and 20% foetal calf serum cultivate, after cell is covered with culture dish (bottle) 3 days, use nutrient solution instead, this nutrient solution adds the Dexamethasone (dexamethasone) of 1mm, the Indomethacin of 0.2mm, 0.01mg/ml Regular Insulin (Insulin), 0.5mm IBMX, DMEM nutrient solution, within 3 days, change ordinary nutritional liquid into later, then liquid was changed every 3 days, after the 7th day, can be easy to observe the early stage hemopoietic precursors differentiation of stem cells of placenta and become embryonic cell, and available Redo dyestuff, fat particle in transfect cell is red, 107/PCR also reflects the expression of tire enzyme and lipase binding-protein gene.
4.2. the early stage hemopoietic precursors stem cell of placenta is to Chondrocyte Differentiation, placenta early stage hemopoietic precursors is cultivated to above-mentioned cell mass by stem cell, then the early stage hemopoietic precursors stem cell of placenta is collected, wash three times with PBS, again get in culture dish (bottle) and cultivate, nutrient solution used contains 0.01 milligram/ml TGF-beta-3, within every 3 days, change liquid once, after 2-3 week cultivates, observation of cell morphology, as cartilage group cell, adopts RT/PCR method can measure cell expressing collagen protein 2 and 9.
4.3. the early stage hemopoietic precursors stem cell to osteoblast differentiation of placenta
First, the early stage hemopoietic precursors stem cell of placenta is cultivated as above-mentioned, then use instead in nutrient solution containing 0.1mM Decalix, 0.05mM ascorbic acid-2-phosphate, 10Mm beta-glycerophospate, cell changed liquid once every 3 days, after 1-2 week, can see that cell is to osteoblast differentiation from morphology, also can adopt the expression of (corlcium-specific stain) or employing RT/PCR detection of alkaline phosphatase gene.
4.4. placenta hemopoietic precursors stem cell is to the differentiation of liver cell, first, as above-mentioned cell cultures, then liver cell stimulating factor 20 pico-gram (ug/ml) and epithelial cell stimulating factor 100 pico-gram (ug/ul) is contained in nutrient solution, or add IL-6 50 pico-grams/ul again, liquid is changed once, until morphologic differentiation of stem cells becomes liver cell every 3triangle.
4.5. the differentiation of placenta early stage hemopoietic precursors stem cell to pancreatic islet cell.Basic fibroblst growth factor 10 pico-gram (ug/ml) is added in nutrient solution, transformiy growth factor beta-1, I pico-gram/ml, liquid is changed once every 3 days, cultivate 14-28 days, the pancreatic stem cell after differentiation or can adopt RT-PCR to detect the expression of pancreas islet gene by (inswlinprolein) of islet proteins.
4.6. the differentiation of placenta early stage hemopoietic precursors stem cell cells into cardiomyocytes.Adopt above-mentioned cell cultures, and adopting human heart tissue extract simultaneously: this extracting solution adopts heart tissue, grinding pulping, then centrifugal with 8000g, filter, Aseptic sterilisation, be prepared into heart extracting solution, concentration with 10% and the mixing of above-mentioned nutrient solution, changed liquid every 3 days 1 time, the cell of differentiation can adopt the genetic expression of cardiac proteins (cardice actin) or RT-PCR to confirm.
The summary of this patent
Adopt nontoxicity, the early stage hemopoietic precursors stem cell of the placenta that has no side effect is for clinical treatment, break the concept of current a lot of incurable disease, for human health provides guarantee, the minimus stem cell of life is isolated from placenta, a series of inspection is adopted to confirm to there are not virus and bacterium etc., the cell be separated can be cultivated again, stored frozen again, almost can infinitely apply, and its range of application recovers or alleviate existing a variety of causes (pathology in human body, decay, burn etc.), the cell or tissue damaged, so its therapeutic domain almost can contain human body all chronic diseases existing at present.This patent also provides one and how to be separated and to cultivate, until the method for clinical application patient, adopt our method, can be separated from healthy placenta, purification of hematopoietic precursor stem cell, and this stem cell can be kept at the gas phase of liquid nitrogen container, can get again at any time, then preserve, then cultivate, circulate it again, almost limitless application.According to the proposition of this patent, when the stem cell clinical application of placenta hemopoietic precursors, its therapeutic dose must be abundant, and at least often secondary 1 × 10 7be the adult of 90 kilograms to an individual weight, treatment number of times, as many as 3 times, adopts hemopoietic precursors stem cell must the HLA early stage hemopoietic precursors stem cell of placenta that has at least more than 4 low recognition site to conform to.

Claims (10)

1. a large amount of method extracting early stage hemopoietic precursors stem cell from HUMAN HEALTH placenta, is characterized by: described early stage hemopoietic precursors stem cell is a group CD133 +cD34 -cell mass, derive from the mononuclearcell removed and remain in intraplacental blood or obtain through mechanical treatment or enzymic digestion from placenta tissue after Cord blood, be CD133 in the detection of flow cytometer +cD34 -cD105 -cD29 -cD31 -, be present in hardly in normal people's Peripheral blood and Cord blood.
2. method according to claim 1, is characterized in that comprising following steps:
(1) from placental blood or placenta tissue, mononuclearcell is separated;
(2) from mononuclearcell, first CD133 is sub-elected +positive cell group, and then from CD133 +cD133 is wherein sub-elected in positive cell group +cD34 +cell mass, and the cell mass flowed out is CD133 +cD34 -hemopoietic precursors stem cell.
3. method according to claim 2, it is characterized in that the described mononuclearcell be separated from placenta carry out concentrated after carry out sorting again.
4. method according to claim 3, is characterized in that step (2) by adopting MACS magnetic bead sorting to go out CD133 from the placenta mononuclearcell after concentrated +cD34 -early stage hemopoietic precursors stem cell, or adopt the antibody of any AntiCD3 McAb 4 or anti-CD133 and biological respinse element to combine from the placenta mononuclearcell after concentrated, or and second antibody combine and remove sorting CD133 +cD34 -early stage hemopoietic precursors stem cell.
5. method according to claim 4, is characterized in that the method adopting MACS magnetic bead sorting, first sub-elects CD133 by anti-CD133 magnetic bead monoclonal antibody from mononuclearcell +positive cell group, and then from CD133 +apply AntiCD3 McAb 4 magnetic bead monoclonal antibody in positive cell group and sub-elect CD133 wherein +cD34 +cell mass, and the cell mass flowed out is CD133 +cD34 -early stage hemopoietic precursors stem cell.
6. method according to claim 2, is characterized in that the described method being separated mononuclearcell from placental blood or placenta tissue is:
(1) collect placenta, inject containing 25 ~ 50 milliliters of antithrombotics and antibiotic physiology isotonic solution or damping fluid in umbilical vein; Umbilical cord artery and vein lavage is adopted to collect placental blood; Wherein, described microbiotic is selected from penicillin, Streptomycin sulphate or nystatin;
(2) adopt organic solvent and physiological buffer to remove the hyaluronic mucus of placenta tissue and umbilical cord appearance, the method adopting mechanical presses and enzyme and physiological buffer to rinse, rinses out any residual blood;
(3) collected by centrifugation placenta tissue stem cell: shred after placenta tissue, amnion and chorion tissue brine, adopt the pancreatin of 0.25%, collagenase II, 0.5%Dispense II enzyme of 0.1-1%, or add the EDTA of 0.05 to 0.2%, in 37 DEG C of incubators, hatch or stir 30 to 90min, the cell of collection, filtration, centrifugal acquisition is placenta tissue stem cell.
7. method according to claim 6, is characterized in that the filtration described in step (3), centrifugal concrete grammar is:
The placental blood stem cell of collecting and placenta tissue stem cell are all by each layer nylon or Steel net, and that minimum is 50um, and that maximum is 100mm, collect mononuclearcell;
The mononuclearcell coming from placenta or placental blood is collected in the centrifuge tube of 50 milliliters, with 1,500g centrifugal 10 minutes in centrifuges, removes the liquid of 70%, then process according to one of following two kinds of methods:
A cell that () collects and lymphocyte separation medium mix in the ratio of 1:4, with 2500g centrifugal 30 minutes in centrifuges, remove the granulocyte of remaining red corpuscle and maturation, collect the cell between liquid level, cell RPMI-1640 washes secondary, obtains the placenta mononuclearcell after concentrating;
(b) by extract concentrated after placenta mononuclearcell and 6% polymer hydroxyethylamyle mix in 1:3 to 1:6 ratio, 1500rpm, 4 DEG C centrifugal 5 to 20min, discard red corpuscle, retain supernatant liquor, then use low molecular dextran (saliferous) to be washed three times, collected cell is made mononuclearcell suspension.
8. the early stage hemopoietic precursors stem cell of extracting according to the method according to any one of claim 1 ~ 7 is preparing the application helping and promote in the medicine of stem cell regenerating.
9. according to the application of phase hemopoietic precursors stem cell in the medicine of preparation treatment hydrocephalus, cardiovascular and cerebrovascular diseases, hepatic necrosis, liver cirrhosis, diabetes, sacroiliitis, pulmonary fibrosis (pneumoconiosis) autoimmunity venereal disease, spinal cord injury disease, lupus erythematosus, scleroderma, ephritis, sexual cell functional disorder disease, congenital or acquired disease that the method according to any one of claim 1 ~ 8 is extracted.
10. application according to claim 9, is characterized in that described cardiovascular and cerebrovascular diseases is Intracerebral hemorrhage, any one in cerebral thrombosis, myocardial infarction, coronary heart disease; Described diabetes are Severe Diabetes Mellitus, or insulin-dependent diabetes; Described sacroiliitis is the osteoarthritis of degenerative joint; Described sexual cell functional disorder disease is hyposexuality or impotence; Described congenital or acquired disease be selected from muscle hypertrophy disease, encephalatrophy or senile dementia any one.
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CN104877965A (en) * 2015-05-27 2015-09-02 赛欧帕克（江苏）干细胞生物工程有限公司 Method for preparing mature erythrocyte
CN109475581A (en) * 2016-05-22 2019-03-15 耶达研究及发展有限公司 In order to transplant and inducing tolerance and use pneumonocyte method
CN110996978A (en) * 2017-05-16 2020-04-10 盖米达细胞有限公司 Selection method and use of cord blood cell fraction suitable for transplantation
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CN109735492A (en) * 2019-03-06 2019-05-10 福建省海西细胞生物工程有限公司 A kind of candidate stem cell extracting method
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CN113966466A (en) * 2019-04-15 2022-01-21 奥瑟姆健康公司 System and method for bone marrow extraction and cryopreservation
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CN115011556B (en) * 2022-08-10 2022-10-25 山东省齐鲁干细胞工程有限公司 Separation method of umbilical cord blood hematopoietic stem cells

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