CN104894062A - Stem cell exosome patch and preparation method and application thereof - Google Patents
Stem cell exosome patch and preparation method and application thereof Download PDFInfo
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- CN104894062A CN104894062A CN201510260758.3A CN201510260758A CN104894062A CN 104894062 A CN104894062 A CN 104894062A CN 201510260758 A CN201510260758 A CN 201510260758A CN 104894062 A CN104894062 A CN 104894062A
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Abstract
The invention discloses a stem cell exosome patch and its preparation method and application and belongs to the field of tissue engineering. The stem cell exosome patch comprises the following steps: stem cell is cultured and stem cell exosome is collected; a patch scaffold is prepared; and the exosome obtained is attached to the patch scaffold to prepare the stem cell exosome patch. the stem cell exosome patch obtained has natural and easily available drug sources; action site accuracy of the exosome patch can be guaranteed; effective acting time of the patch after implantation can be prolonged; and potential safety hazard can be avoided to a great extent. Through the method, the stem cell exosome patch with good biological characteristics can be obtained. The invention is a new breakthrough of establishing a biological patch in the field of tissue engineering. Meanwhile, the product also provides a feasible scheme for clinical treatment of diseases. The principle of the invention is scientific and reliable, and the technology is simple and flexible.
Description
Technical field
The invention belongs to field of tissue engineering technology, particularly outside a kind of stem cell, secrete body (stem cell-derived exosome) sticking patch and preparation method thereof and application.
Background technology
Stem cell, as having propagation, differentiation potential in organism, possesses self replication, can well differentiated functioning cell, is research material popular in regenerative medicine cell therapy field always.Along with going deep into of cell therapy research, the stem cell sticking patch prepared by various method shows therapeutic action to a certain extent to the tissues such as treatment myocardial infarction, skin wound, necrosis of femoral head, diabetes or the disease such as organ damage, dysfunction, and many animals or clinical and experimental study also demonstrate stem cell and can play promotion necrotic tissue Regeneration and Repair, improve the effects such as related tissue's organ dysfunction.
Conventional cell treatment normal in the mode of direct injection stem cell transplantation to site of tissue damage, afterwards, many Tissue Engineering Study persons utilized different construction processs to become cell sheet to carry out transplanting in body stem cell constructing again.Compared to the stem cell therapy of traditional direct injection cell suspension, the advantage that stem cell sheet is transplanted is: 1. stem cell forms the weave construction of two dimension or three-dimensional through external structure, still can keep cell and iuntercellular and the connection between cell and kytoplasm after in cell sheet transplant, be conducive to the maintenance of stem cell biology activity; 2. can make stem cell directional implant object region, not easily run off, and in building process manual control cell derived, planting density, construction process, greatly promote the process of cell therapy; 3. cell sheet transplantation treatment can obviously promote surrounding tissue, cell neogenesis, more obvious to the injury repairing effect of histoorgan.
Although the method for being transplanted by stem cell sticking patch carries out treating the repair that to a certain degree can improve damaged tissue organ, but still there is certain defect in the methods for the treatment of of carrying out stem cell transplantation in vivo: 1. quantity source is in short supply: from the angle of clinical application, except hemopoietic stem cell and mescenchymal stem cell, the high stem cell of many types clinical value is owing to extracting difficulty or ethics problem limits its clinical application, and after the stem cell transplantation of xenogenic origin, easily produce immunological rejection, as skin progenitor cell, limbal stem cell, cardiac stem cells, embryonic stem cell etc., 2. the biologic activity of stem cell in vitro is difficult to maintain: in current various cell sheet constructing technology, its research emphasis is how to build more complete cell sheet, do not relate to the Concept of Maintenance of Stem Cell Activity, comprise use at present the most maturation widely cell sheet build temperature sensitive culture method.Therefore, carry out stem cell sticking patch structure in these methods of use and easily cause stem cell to occur differentiating phenomenon in building process, reduce the biologic activity such as its propagation, paracrine, be unfavorable for that stem cell plays the effect of tissue repair in vivo; 3. stem cell sticking patch construction schedule is longer: at present stem cell sticking patch construction strategy is cultivated mainly through stem cell high-density planting, treats to set up between cell cell and is linked to be sheet and carries out gathering in the crops and subsequent machining operations again.This type of cell sticking patch construction schedule is longer, general needs 7 to 14 days, and this process easily reduces stem cell biology activity, does not also utilize the industrialization of stem cell sticking patch to produce and preserves; 4. transplant in stem cell body and there is risk and X factor: stem cell is the cell that a class has strong self-renewal capacity, and being migrated to knurl risk in its body is one of focal point of current stem-cell therapy.In addition, because stem-cell therapy mechanism still imperfectly understands, hyper-proliferative may be there is, not as uncontrollable situations such as anticipated orientation differentiation, migration, apoptosis, immunological rejections, affect result for the treatment of to a great extent because of surrounding environment effect after transplanting in stem cell body.
Along with going deep into of stem cell study on mechanism, multinomial research show stem cell in vivo majority be play therapeutic action by the material such as paracrine somatomedin and cytokine, and secrete the important transport vehicle of body this kind of material just outward.In recent years, research finds to be played an important role in information transmission, regulation and control, transhipment, immunity by the outer body of secreting of stem cell natural secretion, can be widely used in the aspect such as clinical treatment, beauty treatment.Secrete the film vesicles that body is stem cell secretion outward, obtain its membranin composition outward when derived cell is arranged, optionally protein, nucleic acid etc. that derived cell inside is contained are transported to recipient cell, regulate intercellular signal conduction.
Compared to the tissue engineering product building stem cells sticking patch, the acellular product secreting body sticking patch outside stem cell has clear superiority, specifically be: 1. wide material sources, be easy to collect and store: outside most stem cell can both constantly secrete under certain environment condition, secrete body, and outer body difference of secreting is small between allogeneic, thus body is secreted outside constantly can extracting in cell cultivation process, and by centrifugal, filter, chromatogram, chromatography, body is secreted outside needed for the common methods such as physical adsorption obtain, collect the outer body of secreting obtained to preserve for a long time in 4 DEG C of short-term preservations or-80 DEG C, 2. avoid stem cell directly transplanting: secrete body patch structure outside stem cell and stablize, the possibility occurring to transplant rear cell migration, inefficacy, non-directional differentiation in similar stem cell sticking patch body can be avoided largely, improve functioning efficiency, 3. content enriches, result for the treatment of is similar to stem cell directly transplanting: secreting body is outward to external cystic structures by emiocytosis, comprise multiple from intracellular protein as somatomedin, angiogenic factors etc., and nucleic acid, lipid material, be composition important in known at present stem cell paracrine action, 4. implant outside raising with sticking patch form and secrete body effect targeting: in current research, secrete body suspension outside the many employings of investigator and be injected directly into method in tissue or organ, although also there is certain result for the treatment of, but easily cause outer loss of secreting body, and outer secrete body sticking patch implant after directive action in object region, the outer material secreted in body can be discharged in this region for a long time, extend the outer body action time of secreting, 5. be easy to standardization produce: secrete body sticking patch outside stem cell and be more easy to stdn, can the relevant metering of Accurate Determining and biologic activity, its charge capacity can manual control, and sticking patch can be produced in a large number, and cost is lower, technique is simple.
Because stem cell sticking patch utilizes PD stem cell as seed cell more, it requires high for living environment, is difficult to the differentiated result reaching expection, affects result for the treatment of after usually implanting.Therefore source of human stem cell outer can be secreted body exploitation and become a kind of action character both with stem cell, the novel therapeutic means that its bad differentiation and tumour form defect can be evaded again.Thus obtain in vitro to secrete body and make sticking patch with this outside stem cell and implant, will the revolutionary approach of cell therapy be become.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is to provide the preparation method secreting body (stem cell-derived exosome) sticking patch outside a kind of stem cell.The method can provide the effect being better than cell sheet treatment on the basis avoiding cell sheet Treatment defect.
Another object of the present invention is to provide outside the stem cell that prepared by above-mentioned preparation method and secrete body sticking patch.
Another object of the present invention is the application of secreting body sticking patch outside above-mentioned stem cell.
Object of the present invention is achieved through the following technical solutions:
Secrete a preparation method for body sticking patch outside stem cell, comprise the following steps:
(1) culturing stem cells collect the outer of stem cell and secrete body;
(2) sticking patch support is prepared;
(3) the outer body of secreting step (1) obtained is attached on sticking patch support, secretes body sticking patch outside obtained stem cell.
Described stem cell be preferably in myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell one or more;
Described stem cell is preferably embryonic stem cell, inducibility pluripotent differentiation cell, mescenchymal stem cell, fat stem cell, amnion stem cell, limbal stem cell, cardiac stem cells, skin progenitor cell, endothelial progenitor cell, neural stem cell, hemopoietic stem cell, adult stem cell and one or more in hemocytoblast;
Described stem cell outer secretes the ubcellular bilayer vesicles film bubble that molecular diameter that body refers to stem cell secretion is 30 ~ 100nm;
The method of described collection refers to by collecting culture supernatant, and one or more methods in centrifuging, chromatography, filtration method, immunomagnetic beads method and physisorphtion carry out outer collection of secreting body;
Described centrifuging is preferably that ultracentrifugation, differential centrifugation, ultrafiltration are centrifugal, one or more method in density gradient centrifugation and rotation ultrafiltration carries out outer collection of secreting body;
Described chromatography refers to utilize one or more the method in gel chromatography, adsorption chromatography and ion exchange method to isolate complete outer collection method of secreting body;
Described immunomagnetic beads method refer to by containing to secrete outward body related antigen antibody immunomagnetic beads with secrete body outward and jointly hatch, after distilled water flushing, put into the collection method of PBS damping fluid;
Described antibody be preferably in CD63, CD81, CD90, CD73, CD105, CD29, CD166 and CD9 one or more;
Described physisorphtion is preferably one or more the collection method in electrostatic adhesion and magnetic-adsorption;
Described filtration method refers to utilize one or more the film in nanofiltration membrane and ultra-filtration membrane that the molecular separation of different molecular weight or particle diameter is obtained outer method of secreting body;
Described stem cell outer is secreted body and is referred to be formed and the molecule that can secrete through a series of regulation processes such as stem cells " endocytosis-fusion-arrange outward ", in vivo or external performance information is transmitted, regulation and control, transhipment, any one of immunity or more than one function;
The outer body of secreting of described stem cell includes for one or more in body or in the transmission of external performance information, regulation and control, transhipment, immune albumen, nucleic acid, enzyme and cytokine;
The carrier of described sticking patch is natural carrier or synthetic vectors;
Described natural carrier is preferably prepared by matrigel, collagen gel, chitosan, gelatin, acellular matrix, animal peritoneal, glycosaminoglycan any one or more than one natural materials;
Described synthetic vectors is preferably by ethylene-vinyl acetate copolymer (Ethylene Vinyl Acetate, EVA), Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid), PLGA) one or more the synthetic materials, in poly(lactic acid) (polylacticacid, PLA), alginate and polycaprolactone (polycaprolactone average) prepares;
Described acellular matrix refers to the natural carrier material utilizing natural tissues to be formed, and is preferably cell-eliminating coanea matrix, de-cell dermal matrix or the substrate formed natural carrier material of de-cell heart;
Described outer body of secreting is attached on sticking patch support and refers to utilize one or more the method in gel suspendible method, Crosslink bond type, charged absorption method and pore injection method to prepare;
Described gel refer in nanofiber gel, nanometer polypeptide gel, fibrin gel, sephadex, collagen gel, gelatin, chitosan gel rubber and sepharose one or more;
Described linking agent refers to 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, EDAC), N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS) one or more, in glycosaminoglycan (glycosaminoglycan), genipin (Genipin) and glutaraldehyde (glutaraldehyde, GA);
Described charged absorption method refers to and utilizes outer characteristic of secreting physique film surface charging to be adsorbed onto method on the contrary charged sticking patch carrier of charged situation;
Described pore injection method refers to utilize pore-creating agent to make sticking patch carrier inside pore-forming, remove pore-creating agent by one or more the method in over-saturation gas method, supercutical fluid pore method, thermally induced phase separation and microemulsion polymerization method again, and make outer method of secreting in the hole that body stays in sticking patch carrier;
Described charged sticking patch carrier refers to the sticking patch carrier natural charged chitosan, heparin etc. being attached to the formation of sticking patch carrier;
Described pore-creating agent is preferably nitrogen, carbonic acid gas, water, polyvinyl alcohol (polyvinyl alcohol, PVA), polyvinylpyrrolidone (polyvinyl pyrrolidone, PVP) one or more and in polyoxyethylene glycol (polyethylene glycol, PEG) etc.
Secrete body sticking patch outside a kind of stem cell, prepared by above-mentioned preparation method.
The application of body sticking patch in field of tissue engineering technology is secreted outside described stem cell.
The present invention selects and to secrete body outside stem cell and build outside stem cell and secrete body sticking patch, according to different therapeutic purpose, the stem cell of different sources can be selected, by directly collecting or inducing or in-vitro simulated microenvironment is impelled it to produce outer to secrete body, and multiple method can be selected to prepare required sticking patch flexibly.
The present invention has following advantage and effect relative to prior art:
(1) medicament sources is natural: secrete the natural microballoon of one that body is emiocytosis outward, the many kinds of substances such as inner protein, lipid, nucleic acid, and have good targeting, and after implanting, rapid aggregation is in object region, sustained release high concentration nutritional or all kinds of factor.Compared to cell sheet, this sticking patch cost is low, and efficiency is high, long action time, better effects if, and immunological rejection possibility is extremely low.Can for different tissues or impaired organ's situation control dose, the stem cell avoiding dryness high becomes the possibility of knurl in vivo, and the ethics morals problem avoiding embryonic stem cell to apply.
(2) easily obtain: current existing outer to secrete body acquisition methods many, and process is simple and easy, convenient, secrete body amount to obtain outward greatly, and preliminary preparation is light.In addition, a large amount of production application of mode by induction or in-vitro simulated microenvironment secretes body in the outer of different situations, is convenient to later stage preservation, configuration.Known secrete body wide material sources outward, be not limited to human stem cell, and different biogenetic derivation outer to secrete difference between body small, not easily there is allosome rejection.
(3) can ensure and secrete body sticking patch site of action accuracy outward: compared to the direct injection secreting body suspension outward, sticking patch is fixed on certain position as good carrier by secreting body outward, secrete the fixed point directive action after body implantation outside ensureing, greatly reduce the outer possibility of secreting body and running off.In addition, sticking patch support, as the storage vessel secreting body outward, can protect outer body of secreting be not subject to or reduce extraneous detrimentally affect to a certain extent.
(4) can extend sticking patch implant after effective acting time: natural outer to secrete body be ubcellular bilayer vesicles film bubble, and its membrane structure, from cytoplasmic membrane, can protect inner material, by fast degradation after avoiding implanting.And not there is bio-toxicity or bio-toxicity is less, not easily affect by internal milieu and unpredictable large change in form, character etc. occurs.The natural outer double membrane structure secreting body makes the release of its inner material to a certain extent by effect microenvironment, and can make the sticking patch of slow releasing function, is natural vehicle, nutrition slow-release system, regulation system.
(5) can avoid potential safety hazard to a great extent: natural outer collection of secreting body is batch capture, source, maturity are similar or identical, ensure that its action effect is same or similar, avoid the generation as bad Influence on test result such as non-directional differentiation occurs.And it is simple and easy compared to stem cells sticking patch to secrete body sticking patch outside structure stem cell, reduces the demand of foreign matter, more close to natural component.By method of the present invention, can obtain there is good biological characteristic stem cell outside secrete body sticking patch, be that field of tissue engineering technology builds the new breakthrough of biological sticking patch, this product also provides feasible scheme for solving clinical disease treatment simultaneously.Principle of the invention science is reliable, technique simple and flexible.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 is to secrete body sticking patch for myocardial infarction treatment outside the de-cell pericardium cardiac stem cells that is carrier
(1) experimental principle
With natural de-cell pericardium for sticking patch carrier, utilize filter centrifugation method extract vitro culture to the 3rd generation cardiac stem cells secreted by cardiac stem cells outside secrete body, being attached to sticking patch support on by fibrin gel suspendible method by secreting body outward, replacing cardiac stem cells sticking patch but can reach the effect that cell sticking patch improves myocardial infarction situation largely to being prepared as.
(2) experiment appliance
Instrument and reagent: liquid-transfering gun, centrifuge tube, 100-kDa MWCO hollow-fibre membrane (Millipore), PBS phosphate buffer solution (Sigma), 30% sucrose/D
2o damping fluid (density 1.210g/cm
3), low temperature ultracentrifuge, 100-kDa MWCO Ultrafree-15 capsule (Millipore), containing microbiotic (100U/mL penicillin G, 100 μ g/mL Vetstreps, Sigma) carbonate buffer solution (0.05mol/L, pH=9.0 ~ 9.6), sterile pure water, water bath, containing the aseptic PBS salts solution (0.01mol/L of 0.3% (w/v) sodium lauryl sulphate, pH=6 ~ 8), magnetic stirring apparatus, the trepan of diameter 12mm, containing the PBS salt buffer (0.01mol/L of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) 24mg/mL and N-hydroxysuccinimide (NHS) 60mg/mL, pH=7 ~ 8), fibrin gel, incubator.
Experiment material: (7 ~ 8) × 10 of purifying
6individual/cm
2the cardiac stem cells in the 3rd generation, fresh pig pericardium (25cm
2).
(3) experimental procedure
A. culturing stem cells extract the outer of described stem cell and secrete body:
(7 ~ 8) × 10 of purifying are collected under aseptic condition
6individual/cm
2reach the culture supernatant 10mL of the cardiac stem cells in the 3rd generation through original cuiture, remove cell debris with the centrifugal 20min of 2000g.Supernatant liquor after concentrated uses 100-kDa MWCO hollow-fibre membrane (Millipore) in the centrifugal 30min of 1000g further, 2 times, is diluted in by the supernatant liquor of acquisition in PBS phosphate buffer solution (Sigma).Then solution is moved in centrifuge tube, with 30% sucrose/D
2o damping fluid (density 1.210g/cm
3) together at 4 DEG C, centrifugal 1h under 100000g.Finally, with PBS solution in 100-kDa MWCO Ultrafree-15 capsule (Millipore) with the centrifugal 30min of 1000g, 3 times.(method reference: Lai RC, Arslan F, Lee MM, et al.Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.Stem Cell Res, 2010,4:214-222)
B. sticking patch support is prepared:
Under room temperature, normal sterile operation, by fresh pig pericardium (25cm
2) repeatedly clean by stroke-physiological saline solution, removing clot and slurries are to clean, be placed in aseptic square plate, use containing microbiotic (100U/mL penicillin G, 100 μ g/mL Vetstreps, Sigma) carbonate buffer solution (0.05mol/L, pH=9.0 ~ 9.6) soaks 2 times, each 5 minutes.Pericardium is inserted in sterile pure water, soak 30 minutes in 37 DEG C of water-baths.10mL is inserted containing in the aseptic PBS salts solution (0.01mol/L, pH=6 ~ 8) of 0.3% (w/v) sodium lauryl sulphate, concussion process (rotating speed=185rpm) 10 ~ 12 hours in 37 DEG C of water-baths after immersion.After insert 50ml containing microbiotic (100U/mL penicillin G, 100 μ g/mL Vetstreps) carbonate buffer solution (0.05mol/L, pH=9.0 ~ 9.6) in, in 25 DEG C of water-baths, shake (rotating speed=185rpm) rinse 6 times, each 60 minutes, obtain the de-cell Pigs Hearts coating after cleaning.Drill through 12mm diameter with the trepan of diameter 12mm and take off cell Pigs Hearts coated fertilizer, be immersed in PBS salt buffer (0.01mol/L, pH=7 ~ 8 containing 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) 24mg/mL and N-hydroxysuccinimide (NHS) 60mg/mL, Sigma) reaction 20 minutes in, 6 times are softly cleaned, each 30 minutes with distilled water.De-cell pericardium after cleaning is sealed in aseptic plastic bag, after Co 60 (15kGy) radiation sterilization, saves backup at 4 DEG C.
C. outer secrete body by described and be attached on sticking patch support, outside obtained stem cell, secrete body sticking patch:
The outer body of secreting obtained is mixed with 20 μ L fibrin gels, and drips the de-cell pericardium surface in preparation, put into 37 DEG C of incubators and leave standstill 5min, make it solidify.
(4) experimental result
Immunofluorescence dyeing shows that secreting body sticking patch outside cardiac stem cells promotes neovascularization, and vessel density reaches about 150 ~ 200/ infarct size (mm
2), be conducive to the regeneration of infarcted region myocardial cell.After transplanting 1 week, ultrasonic cardiogram shows that left ventricular pressure declines about 8% ~ 10%.Left ventricular ejection fraction (LVEF) improves degree and improves 5 ~ 8% compared to cardiac stem cells patch treatment.Sticking patch is taken out after transplanting one week, sticking patch bFGF (b fibroblast growth factor is shown by elisa assay, bFGF), heparin somatomedin (hyparin growth factor, HGF), type-1 insulin like growth factor (insulin-like growth factor-1, IGF-1) content declines 20% before comparatively transplanting, illustrate to secrete outside cardiac stem cells the body sticking patch time length can reach one week and more than.It is thinning that pathological section shows effectively to suppress left ventricular wall.
Embodiment 2 is to secrete body sticking patch for femoral head necrosis therapeutic outside the mesenchymal stem cells MSCs of poly(lactic acid) (polylacticacid, PLA) for sticking patch carrier
(1) experimental principle
With the poly(lactic acid) (polylacticacid of synthesis, PLA) be sticking patch carrier, utilize immunomagnetic beads method extract vitro culture to the 3rd generation mesenchymal stem cells MSCs secreted by mesenchymal stem cells MSCs outside secrete body, being attached to sticking patch support on by nanometer polypeptide gel suspendible method by secreting body outward, replacing mesenchymal stem cells MSCs sticking patch but can reach the effect that cell sticking patch improves necrosis of femoral head situation largely to being prepared as.
(2) experiment appliance
Instrument and reagent: PBS phosphoric acid buffer (Sigma), serum-free and remove the outer BMSC nutrient solution secreting body, incubator, ultracentrifuge, SW28 rotor (Beckman Coulter Instruments, Fullerton, CA), 0.32M sucrose, SW60rotor (Beckman Coulter Instruments), immunomagnetic beads (Miltenyi), goat anti-mouse immunoglobulin G (Invitrogen Dynal AS, Oslo, Nroway), anti-CD9 monoclonal antibody (clone MM2/57, Chemicon International, London, UK), the trepan of 12nm, nanometer polypeptide gel (acetyl-(Arg-Ala-Asp-Ala) 4-amide4HCL, BD BiocaotPuraMatrix).
Experiment material: (7 ~ 8) × 10 of purifying
6individual/cm
23rd generation mesenchymal stem cells MSCs, poly(lactic acid) (polylacticacid, PLA).
(3) experimental procedure
A. culturing stem cells extract the outer of described stem cell and secrete body:
(7 ~ 8) × 10 of purifying are collected under aseptic condition
6individual/cm
2reached for the 3rd generation mesenchymal stem cells MSCs (BMSC) through original cuiture and clean culture dish 2 times with PBS phosphoric acid buffer (Sigma), remove outer to secrete in the BMSC nutrient solution of body after 37 DEG C of incubators hatch 48h at serum-free, supernatant liquor is through 400 × g (10min), 3000 × g (20min), 10000 × g (30min) continuously centrifuged.Secrete body outward to concentrate in 64000 × g (110min) SW28 rotor (Beckman Coulter Instruments, Fullerton, CA).Secreting body is outward deposited in 0.32M sucrose resuspended, then the centrifugal 1h of 100000 × g (SW60 rotor, Beckman Coulter Instruments).Secrete body to be outward further purified by immunomagnetic beads (Miltenyi).25 μ L scribble goat anti-mouse immunoglobulin G (Invitrogen Dynal AS in advance, Oslo, Nroway) immunomagnetic beads and the anti-CD9 monoclonal antibody of 30 μ L (clone MM2/57, Chemicon International, London, UK) hatch 1h.Thereafter, body is secreted at 4 DEG C of suspendible 1h outside magnetic bead and 20 μ g.After cleaning 4 times, secrete body outward and magnetic bead is again resuspended in PBS.(method reference: Jansen FH, Krijgsveld J, van Rijswijk A, et al.Exosomal Secretion of Cytoplasmic Prostate Cancer Xenograft-derived Proteins.Mol Cell Proteomics, 2009,8:1192-1205)
B. sticking patch support is prepared:
Be that on ready-made PLA sticking patch, drill through diameter be that the PLA sticking patch carrier of 12nm is for subsequent use for the trepan of 12nm with diameter.
C. outer secrete body by described and be attached on sticking patch support, outside obtained stem cell, secrete body sticking patch:
Outer nanometer polypeptide gelating soln (acetyl-(Arg-Ala-Asp-Ala) 4-amide4HCL secreting body suspension and 20 μ L and prepared obtained there being above-mentioned steps, BD BiocaotPuraMatrix) mix, and the PLA sticking patch carrier surface dripped in preparation, put into 37 DEG C of incubators and leave standstill 5min, at mesenchymal stem cells MSCs nutrient solution (DMEM basic culture solution (Corning), 10% (v/v) has secreted foetal calf serum (Corning Cellgro)+1% (w/v) glutamine (Corning Cellgro)+100U/mL penicillin G (Sigma)+100 μ g/mL Vetstrep (Sigma) of body outside filtering) in leave standstill 5min, make it solidify.
(4) experimental result
Implanted unit head necrosis animal model is after 1 week, and bone density improves 12% ~ 18%, and function of joint improves 40%, and histopathology shows as sky bone lacuna and reduces, and scleroblast increases, and in addition, vessel density improves 15% ~ 20%.In addition, the outer of mesenchymal stem cells MSCs source secretes the excellence effect that body shows the treatment to cancer, lethality heart disease, Immunological diseases.
Embodiment 3 is secrete body sticking patch for skin wound treatment outside the skin progenitor cell of carrier with alginate (Alginates) sticking patch
(1) experimental principle
With natural alginate (Alginates) for sticking patch carrier, utilize rotate ultrafiltration process extract vitro culture to the 3rd generation skin progenitor cell secreted by skin progenitor cell outside secrete body, being attached to sticking patch support on by nanometer polypeptide gel suspendible method by secreting body outward, replacing skin progenitor cell sticking patch but can reach the effect that cell sticking patch improves skin wound situation largely to being prepared as.
(2) laboratory apparatus
Instrument and reagent: liquid-transfering gun, refrigerated centrifuge, centrifuge tube, 0.22 μm of filter membrane (Millex-GP, Millipore), aseptic PBS phosphoric acid buffer (Sigma), 100nm ultra-filtration centrifuge tube (Amicon Ultra-4, Millipore), 0.22 μm of ultra-filtration centrifuge tube (Millipore), maltonic acid half calcium salt (1.08% (w/v), Sigma), bi-distilled water, 96-orifice plate (100 μ L/ hole), 1.2mg mL
-1dMEM nutrient solution (Corning), NaOH solution.
Experiment material: (7 ~ 8) × 10 of purifying
6individual/cm
23rd generation skin progenitor cell, alginate.
(3) experimental procedure
A. culturing stem cells extract the outer of described stem cell and secrete body:
(7-8) × 10 of purifying are collected under aseptic condition
6individual/cm
2through original cuiture reach the 3rd generation skin progenitor cell culture supernatant 10mL, with 300r/min after centrifugal 10 minutes at 4 DEG C, Aspirate supernatant is in another totally aseptic 15mL centrifuge tube.At 4 DEG C with 2000r/min centrifugal 20 minutes, Aspirate supernatant was in another totally aseptic 15mL centrifuge tube.In Bechtop, filter collecting the liquid obtained with 0.22 μm of filter membrane (Millex-GP, Millipore).Collect liquid after filter, at 4 DEG C with 100000r/min centrifugal 70 minutes, obtain white precipitate and be and secrete body outward.Suck supernatant liquor, add the washing of aseptic PBS phosphoric acid buffer (Sigma), at 4 DEG C with 100000r/min centrifugal 70 minutes, secrete body outward with the aseptic PBS of 1mL is resuspended.Use 100nm ultra-filtration centrifuge tube (Amicon Ultra-4 again, Millipore) within centrifugal 30 minutes, filter with 300r/min at 4 DEG C, use 0.22 μm of ultra-filtration centrifuge tube (Millipore) for subsequent use to secrete body outside 300r/min filtration in centrifugal 30 minutes at 4 DEG C afterwards.
B. sticking patch support is prepared:
Alginate are dissolved in bi-distilled water and obtain 1.2% solution (w/v), when exciting the alginate forming uniqueness to be cross-linked, then adding maltonic acid half calcium salt (1.08% (w/v), Sigma) and being cross-linked.Crosslinking alginate solution pours 96-orifice plate (100 μ L/ hole) into, and frozen overnight at 2 ~ 8 DEG C is freezing 24 hours at-20 DEG C, and freeze-drying.Support is exposed to sterilization 20min (method reference: Y.Sapir et al.The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds.Biomaterials 33 (2012) 4100e4109) under UV.
C. outer secrete body by described and be attached on sticking patch support, outside obtained stem cell, secrete body sticking patch:
The preparation of collagen matrices: collagen is distributed to 1.2mg mL
-1in DMEM nutrient solution (Corning) and with NaOH solution adjust pH to 7.4.
The outer body of secreting obtained is mixed with 20 μ L collagen matrices, and drips the alginate sticking patch carrier surface in preparation, cultivate 45min at 37 DEG C, make it solidify.
(4) experimental result
After implanting wound, damaged part, wound recovery rate improves 10% ~ 18.4%, does not occur inflammatory phenomena.After 1 week, wound shrinking percentage reaches 17.5% ~ 25%, and wound site neonatal cell is active good.ELISA detects obvious 15% ~ 20%, the SP immunohistochemical method that rises of Urogastron (EGF) expression can detect wound area β 1 integrin, Keratin 19 (K19) increasing expression.HE stained finds that newborn epidermis is thicker, and the cell number of plies is more and layering is comparatively obvious, and wound site healing better.
Embodiment 4 is secrete body sticking patch outside the embryonic stem cell of carrier to treat for chronic liver failure with ethylene-vinyl acetate copolymer (Ethylene Vinyl Acetate, EVA) sticking patch
(1) experimental principle
With ethylene-vinyl acetate copolymer (the Ethylene Vinyl Acetate of synthesis, EVA) be sticking patch carrier, utilize filter centrifugation method extract vitro culture to the 3rd generation embryonic stem cell secreted by embryonic stem cell outside secrete body, being attached to sticking patch support on by EVA by secreting body outward, replacing embryonic stem cell sticking patch but can reach the effect that cell sticking patch improves chronic liver failure situation largely to being prepared as.
(2) experiment appliance
Instrument and reagent: 0.22 μm of filter membrane (Millex-GP, Millipore), the Model 8050 that 100 000 NWCO ultra-filtration membranes are housed rotate ultrafiltration instrument (Millipore), nitrogen, magnetic stirring apparatus, PBS phosphoric acid buffer (Sigma), the trepan of 12nm, cyclohexane solution (Sigma).
Experiment material: (7 ~ 8) × 10
6individual/cm
2the embryonic stem cell in the 3rd generation, ethylene-vinyl acetate copolymer (Ethylene Vinyl Acetate, EVA).
(3) experimental procedure
A. culturing stem cells extract the outer of described stem cell and secrete body:
By (7 ~ 8) × 10 of purifying
6individual/cm
2after the embryo-stem cell into hepatocyte direction induction success that original cuiture reached for the 3rd generation, under aseptic condition, collect its culture supernatant 50mL, filter with 0.22 μm of filter membrane (Millex-GP, Millipore) in Bechtop.The supernatant liquor collected being added the Model 8050 that 100000NWCO ultra-filtration membrane is housed rotates in ultrafiltration instrument (Millipore), connect nitrogen by full admission pressure-controlling at below 517.125kPa, open magnetic stirring apparatus (IKA), make vortex height be 1/3 of liquid height.After treating supernatant liquor ultrafiltration, add 50mL PBS phosphoric acid buffer (Sigma) and ultrafiltration again, repeat 3 times.After ultrafiltration, secrete body with outer on 0.5mL PBS phosphoric acid buffer suspension ultra-filtration membrane stand-by.(method reference: Hu Guowen etc. rotates ultrafiltration: a kind ofly extract the novel method that body is secreted in extracellular, Acad J Sec Mil Med Univ, 2014,35 (6): 59860)
B. sticking patch support is prepared:
Be that on ready-made EVA sticking patch, drill through diameter be that the EVA sticking patch carrier of 12nm is for subsequent use for the trepan of 12nm with diameter.
C. outer secrete body by described and be attached on sticking patch support, outside obtained stem cell, secrete body sticking patch:
Smear in the EVA hexanaphthene molten (Sigma) of 0.3% at EVA patch faces, be placed in room temperature volatilization, before complete drying, make outer body suspension of secreting slowly drop in EVA sticking patch carrier surface and make it be attached to EVA sticking patch carrier surface, the cyclohexane solution of EVA is put into tetrafluoroethylene (Sigma) groove at room temperature to dry, the eva film of preparation floats on the surface again.By sticking patch natural air drying, obtain the EVA sticking patch with secreting body outside embryonic stem cell.
(4) experimental result
After inducing embryo stem cell changes to liver cell direction, detect that the expression amount of stem cell markers alpha-fetoglobulin (AFP), albumin (ALB) obviously increases along with the prolongation of time by RT-PCR and Western blot method.Sticking patch is transplanted to chronic liver failure animal model after 7 days, liver cell double-core increases, and occurs inflammatory cell infiltration, and necrosis region obviously reduces, and congested, bleeding alleviates.The expression of model group liver function index pyruvic transaminase (ALT), glutamic-oxal(o)acetic transaminase (AST), alkaline phosphatase (ALP) all declines 8.7% ~ 15%, and albumin (ALB) is expressed without considerable change.
The body sticking patch of secreting outside hemocytoblast that embodiment 5 is carrier with de-cell amnion is treated for type 1 diabetes
(1) experimental principle
With natural de-cell amnion for sticking patch carrier, utilize rotate ultrafiltration process extract vitro culture to the 3rd generation through secreting body secreted by hemocytoblast outside hemocytoblast, being attached to sticking patch support on by crosslinking by secreting body outward, replacing but to reach the effect that cell sticking patch improves type 1 diabetes situation largely through hemocytoblast sticking patch to being prepared as.
(2) experiment appliance
Instrument and reagent: refrigerated centrifuge, centrifuge tube, 0.22 μm of filter membrane (Millex-GP, Millipore), aseptic PBS phosphoric acid buffer (Sigma), 100nm ultra-filtration centrifuge tube (Amicon Ultra-4, Millipore), 0.22 μm of ultra-filtration centrifuge tube (Millipore), containing microbiotic (100U/mL penicillin G and 100 μ g/mL Vetstreps, Sigma) carbonate buffer solution (0.05mol/L, pH=9.0 ~ 9.6, Sigma), sterile pure water, water bath, (use the preparation of 0.05mol/L carbonate buffer solution, pH=8 ~ 9, phospholipase A1=200U/mL, Phospholipase A2=250U/mL, Sigma), trepan, 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC of 12mm, Sigma), N-hydroxysuccinimide (NHS, Sigma) a water morpholino b acid (MES, Sigma) damping fluid (0.1mol/L, pH=6.0), shaking table, disodium phosphate soln (0.1mol/L, Sigma), sodium chloride solution (4mol/L, Sigma).
Experiment material: (7 ~ 8) × 10 of purifying
6individual/cm
2in 3rd generation, is through hemocytoblast, de-cell amnion.
(3) experimental procedure
A. culturing stem cells extract outside described stem cell and secrete body:
(7 ~ 8) × 10 of purifying are collected under aseptic condition
6individual/cm
2reach the culture supernatant 10mL of the 3rd generation through hemocytoblast through original cuiture, with 300r/min after centrifugal 10 minutes at 4 DEG C, Aspirate supernatant is in another totally aseptic 15mL centrifuge tube.At 4 DEG C with 2000r/min centrifugal 20 minutes, Aspirate supernatant was in another totally aseptic 15mL centrifuge tube.In Bechtop, filter collecting the liquid obtained with 0.22 μm of filter membrane (Millex-GP, Millipore).Collect liquid after filter, at 4 DEG C with 100000r/min centrifugal 70 minutes, obtain white precipitate and be and secrete body outward.Suck supernatant liquor, add the washing of aseptic PBS phosphoric acid buffer (Sigma), at 4 DEG C with 100000r/min centrifugal 70 minutes, secrete body outward with the aseptic PBS of 1mL is resuspended.100nm ultra-filtration centrifuge tube (Amicon Ultra-4, Millipore) is used to secrete body so that 300r/min filtration in centrifugal 30 minutes is outer again at 4 DEG C.0.22 μm of ultra-filtration centrifuge tube (Millipore) is used to secrete body so that 300r/min filtration in centrifugal 30 minutes is outer again at 4 DEG C.
B. sticking patch support is prepared:
In room temperature, under normal sterile operation, by fresh amnion (25cm
2) repeatedly clean by stroke-physiological saline solution, removing clot and slurries are to clean, be placed in aseptic square plate, use containing microbiotic (100U/mL penicillin G and 100 μ g/mL Vetstreps, Sigma) carbonate buffer solution (0.05mol/L, pH=9.0 ~ 9.6) soaks 5 times, each 10 minutes.Amnion is inserted in sterile pure water, soak 60 minutes in 37 DEG C of water-baths.Inserted the aseptic Phospholipid hydrolase solution of 10mL after immersion and (used the preparation of 0.05mol/L carbonate buffer solution, pH=8 ~ 9; Phospholipase A1=200U/mL, Phospholipase A2=250U/mL, Sigma) in, concussion process (rotating speed=185rpm) 6 hours in 37 DEG C of water-baths.After insert 50mL containing microbiotic (100U/mL penicillin G and 100 μ g/mL Vetstreps, Sigma) carbonate buffer solution (0.05mol/L, pH=9.0 ~ 9.6, Sigma) in, in 25 DEG C of water-baths, shake (rotating speed=185rpm) rinse 6 times, each 60 minutes, obtain de-cell amnion.
C. outer secrete body by described and be attached on sticking patch support, outside obtained stem cell, secrete body sticking patch:
At room temperature, under normal sterile operation, drill through 12mm diameter with the trepan of diameter 12mm and take off cell amniotic material, be immersed in outside 2mL and secreted body suspension, 3mmol/L 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC, and 5mmol/L N-hydroxysuccinimide (NHS Sigma), Sigma) a water morpholino b acid (MES, Sigma) damping fluid (0.1mol/L, pH=6.0) in, after on shaking table, (rotating speed=40rpm) reacts 24 ~ 72 hours, use disodium phosphate soln (0.1mol/L successively, Sigma) clean 6 times, each 30 minutes, sodium chloride solution (4mol/L, Sigma) cleans 6 times, each 30 minutes, distilled water softly cleans 6 times, each 30 minutes, and what to obtain with de-cell amnion be carrier secretes body sticking patch outside hemocytoblast.
(4) experimental result
Sticking patch is implanted type 1 diabetes mouse model after 1 week, Immunofluorescence test finds that sticking patch promotes the expression of beta Cell of islet precursor markers thing Ngn3, and finds that Ngn3 positive cell is organized all can detect at ductal epithelial cell, pancreas islet, external secretion.In addition, detecting β development and cell differentiation gene by real-time quantitative PCR and obviously raise 13%, illustrating through hemocytoblast (mentrual blood progenitor cells, MBPCs) by promoting that the differentiation of pancreas endogenous stem cells improves type 1 diabetes.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. secrete a preparation method for body sticking patch outside stem cell, it is characterized in that comprising the following steps:
(1) culturing stem cells collect the outer of stem cell and secrete body;
(2) sticking patch support is prepared;
(3) the outer body of secreting step (1) obtained is attached on sticking patch support, secretes body sticking patch outside obtained stem cell.
2. preparation method according to claim 1, is characterized in that:
Described stem cell is one or more in myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell;
Described stem cell outer secretes the ubcellular bilayer vesicles film bubble that molecular diameter that body refers to stem cell secretion is 30 ~ 100nm.
3. preparation method according to claim 1 and 2, is characterized in that:
Described stem cell is embryonic stem cell, inducibility pluripotent differentiation cell, mescenchymal stem cell, fat stem cell, amnion stem cell, limbal stem cell, cardiac stem cells, skin progenitor cell, endothelial progenitor cell, neural stem cell, hemopoietic stem cell, adult stem cell and one or more in hemocytoblast;
The outer body of secreting of described stem cell includes for one or more in body or in the transmission of external performance information, regulation and control, transhipment, immune albumen, nucleic acid, enzyme and cytokine.
4. preparation method according to claim 1, is characterized in that:
The method of described collection refers to by collecting culture supernatant, and one or more methods in centrifuging, chromatography, filtration method, immunomagnetic beads method and physisorphtion carry out outer collection of secreting body;
The carrier of described sticking patch is natural carrier or synthetic vectors;
Described outer body of secreting is attached on sticking patch support and refers to utilize one or more the method in gel suspendible method, Crosslink bond type, charged absorption method and pore injection method to prepare.
5. preparation method according to claim 4, is characterized in that:
Described centrifuging is ultracentrifugation, differential centrifugation, ultrafiltration are centrifugal, one or more method in density gradient centrifugation and rotation ultrafiltration carries out outer collection of secreting body;
Described chromatography refers to utilize one or more the method in gel chromatography, adsorption chromatography and ion exchange method to isolate complete outer collection method of secreting body;
Described immunomagnetic beads method refer to by containing to secrete outward body related antigen antibody immunomagnetic beads with secrete body outward and jointly hatch, after distilled water flushing, put into the collection method of PBS damping fluid;
Described antibody is one or more in CD63, CD81, CD90, CD73, CD105, CD29, CD166 and CD9;
Described physisorphtion is one or more the collection method in electrostatic adhesion and magnetic-adsorption;
Described filtration method refers to utilize one or more the film in nanofiltration membrane and ultra-filtration membrane that the molecular separation of different molecular weight or particle diameter is obtained outer method of secreting body.
6. preparation method according to claim 4, is characterized in that:
Described natural carrier is prepared by matrigel, collagen gel, chitosan, gelatin, acellular matrix, animal peritoneal, glycosaminoglycan any one or more than one natural materials;
Described acellular matrix is cell-eliminating coanea matrix, de-cell dermal matrix or the substrate formed natural carrier material of de-cell heart;
Described synthetic vectors is prepared by the synthetic materials of one or more in ethylene-vinyl acetate copolymer, Poly(D,L-lactide-co-glycolide, poly(lactic acid), alginate and polycaprolactone.
7. preparation method according to claim 4, is characterized in that:
Described gel refer in nanofiber gel, nanometer polypeptide gel, fibrin gel, sephadex, collagen gel, gelatin, chitosan gel rubber and sepharose one or more;
Described linking agent refer in 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine, N-hydroxy-succinamide, glycosaminoglycan, genipin and glutaraldehyde one or more;
Described charged absorption method refers to and utilizes outer characteristic of secreting physique film surface charging to be adsorbed onto method on the contrary charged sticking patch carrier of charged situation;
Described pore injection method refers to utilize pore-creating agent to make sticking patch carrier inside pore-forming, remove pore-creating agent by one or more the method in over-saturation gas method, supercutical fluid pore method, thermally induced phase separation and microemulsion polymerization method again, and make outer method of secreting in the hole that body stays in sticking patch carrier.
8. preparation method according to claim 7, is characterized in that:
Described charged sticking patch carrier refers to and natural charged chitosan, heparin is attached to the sticking patch carrier that sticking patch carrier is formed;
Described pore-creating agent is one or more in nitrogen, carbonic acid gas, water, polyvinyl alcohol, polyvinylpyrrolidone and polyoxyethylene glycol.
9. secrete a body sticking patch outside stem cell, prepared by the preparation method described in any one of claim 1 ~ 8.
10. secrete the application of body sticking patch in field of tissue engineering technology outside stem cell according to claim 9.
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