CN107988153A

CN107988153A - The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used

Info

Publication number: CN107988153A
Application number: CN201711352019.2A
Authority: CN
Inventors: 冯迹
Original assignee: British Liberal Life Science & Technology Co Ltd
Current assignee: British Liberal Life Science & Technology Co Ltd
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2018-05-04
Anticipated expiration: 2037-12-15
Also published as: CN107988153B

Abstract

The present invention relates to the reagent that mesenchymal stem cells derived from human umbilical blood source separates the method for excretion body and uses.Specifically, the method for the present invention includes：(1), the culture of mesenchymal stem cells in umbilical cord blood, and (2), the extraction of Cord blood MSC excretion bodies；Wherein, the culture of the mesenchymal stem cells in umbilical cord blood is to make mesenchymal stem cells in umbilical cord blood culture to P3 generations；The extraction of the Cord blood MSC excretion bodies includes the following steps：Excretion body suspension pre-processes, and the extraction of excretion body.The reagent that the present invention uses is related to the solution as cushion pad with special formulation.The method of the present invention has excellent technique effect as used in the description.

Description

The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used

Technical field

The invention belongs to biological technical field, is related to a kind of method for the film bubble for obtaining and participating in cell biological activity, especially It is related to one kind to be separated by mesenchymal stem cells derived from human umbilical blood (MSC) and obtain excretion body (Exosome, or be denoted as Exosomes) Method, and its application in cell repair.Especially, the present invention relates to mesenchymal stem cells derived from human umbilical blood source to separate excretion The method of body, moreover, it relates to the reagent used.

Background technology

Mescenchymal stem cell (mesenchymal stem cells, MSCs) is derived from the non-hematopoiesis system of mesoblastic one kind System multipotential stem cell, has confirmed MSCs in addition to self-renewing and multi-lineage potential, has also had very strong anti-inflammatory and suppression The ability of panimmunity cell processed, and being capable of inducing peripheral immune tolerance.Research shows that MSCs is adjusted by secreting panimmunity The factor, such as IFN-γ, PGE2 (reference can be made to：Polchert D,Sobinsky J,Douglas G,Kidd M,Moadsiri A,Reina E,et al.IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease.European journal of immunology.2008；38(6):1745-55. and Spaggiari GM, Abdelrazik H, Becchetti F, Moretta L.MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs:central role of MSC-derived prostaglandin E2.Blood.2009；113(26):6576-83.), so as to suppress immunocyte (such as T cell, B cell, NK cells, antigen presenting cell etc.) function (reference can be made to：Corcione A,Benvenuto F, Ferretti E,Giunti D,Cappiello V,Cazzanti F,et al.Human mesenchymal stem cells modulate B-cell functions.Blood.2006；107(1):367-72. and Di Nicola M, CarloStella C,Magni M,Milanesi M,Longoni PD,Matteucci P,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood.2002；99(10):3838-43.).

Excretion body (exosome) is a kind of diameter about 30~100nm secreted by cell, density range 1.13~ Film microcapsule bubble between 1.19g/ml.Excretion body can carry a variety of protein similar to source cell, mRNA, miRNA, participate in The processes such as immunological regulation, cell communication, cell migration, angiogenesis (reference can be made to：Yanez-Mo M,Siljander PR, Andreu Z,Zavec AB,Borras FE,Buzas EI,et al.Biological properties of extracellular vesicles and their physiological functions.Journal of extracellular vesicles.2015；4:27066.).Lai etc. has found that the excretion body in MSCs sources can reduce myocardial ischemia Reperfusion injury, and confirm that the miRNA of excretion body can promote angiogenesis, therefore excretion body is likely to become treatment angiocarpy Disease a new direction (reference can be made to：Lai RC,Arslan F,Lee MM,Sze NS,Choo A,Chen TS,et al.Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.Stem cell research.2010；4(3):214-22.).Xin etc. has found the excretion body in MSCs sources by turning MiR-133b is moved to nerve cell, can promote neural axon growth (reference can be made to：Xin H,Li Y,Buller B, Katakowski M,Zhang Y,Wang X,et al.Exosome mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.Stem cells.2012；30(7):1556-64.).Filipazzi etc. has found the excretion body in tumour cell source T cell and NK cells can be suppressed by NK cell-stimulating receptors NKG2D (naturalkiller group 2, member D) Cytotoxicity so that influence host immune system (reference can be made to：Filipazzi P,Burdek M,Villa A, Rivoltini L,Huber V.Recent advances on the role of tumor exosomes in immunosuppression and disease progression.Seminars in cancer biology.2012；22 (4):342-9.)。

This film bubble secreted by living cells of excretion body (exosome), is most found earlier than nineteen eighty-three.As research is deep Enter, it is found that it has the transport for performing albumen, nucleic acid, specifically target recipient cell, exchanger and lipid or trigger downstream letter Number event, participates in the functions such as intercellular communication, and is constantly taken seriously.It is non-specific including source cell that excretion body carries protein Property and specific two proteinaceous molecules of source cell.The former may occur with the biology of excretion body and biological action is related, mainly Including：Cytosol albumen, albumen, various metabolic enzymes, heat shock protein and the four transmembrane proteins for participating in intracellular signal transduction； Another kind of is specific proteins, this proteinoid exists only in the excretion body of certain special cell secretion, and these certain details The excretion body in born of the same parents source has close ties with its biological function, such as：Contain mhc class ii point on the excretion body of molecular origin Son.Therefore, the signaling molecule entrained by the excretion body of different cell sources is different, and the function of performance is also not quite similar.Such as：It is swollen The excretion body of oncocyte secretion can mediate vascular recurrent tumors cell Proliferation and immunologic escape, and Dendritic Cells source property excretion Body can then cause body effective antitumour immune response.It has now been found that containing related to cell derived in excretion body Protein rRNA and microRNA, and excretion body can by biological barrier, in intercellular trafficking functional nucleic acid, So as to play various biological functions, therefore excretion body is expected to become a kind of novel medicine feeding approach and gene therapy vector.

It has now been found that the excretion body in mesenchymal stem cells in umbilical cord blood source carry a variety of effective cell factors, Albumen and small molecular core acid substance, can effectively mediated cell propagation, apoptosis and the Function Regulation.Research is found, in excretion body Contain vascular endothelial growth factor, fibroblast growth factor, the platelet proliferation factor, tumor necrosis factor and tumour Growth factor etc., has and suppresses Apoptosis, the fibrosis of cell, promotes angiogenesis mitosis and mediated immunity anti- Function should be waited.It is demonstrated experimentally that in the insulin-like growth factor and blood that are carried using the excretion body secreted by mescenchymal stem cell Endothelial cell growth factor is to treat the crucial dominant factor of acute injury of kidney.In immunology aspect, mescenchymal stem cell point The excretion body fat Membrane surface expression secreted has a variety of memebrane proteins, such as：Clotting factor, tumor necrosis factor, MHC I/II molecules and CCR5 chemokine receptors etc., these adipose membrane surface proteins have the function that resistance inflammation important.

It is also varied that mode and the approach of excretion body extraction are carried out at present, such as：Extracted in marrow in extraction, peripheral blood The approach obtained Deng, these excretion bodies has been required to traumatic cell or tissue source.Excretion body extracting method mainly uses The kit of supercentrifugation or costliness crosses pillar method, however excretion weight heterogeneity acquired in supercentrifugation, cannot Ensure the acquired amount of excretion body, centrifugation time is tediously long step by step, even centrifugation time or centrifugal speed reason and final nothing sometimes Method is isolated, loses time and cost.And kit crosses pillar method and is typically only capable to be suitable for the acquisition of small amount of excretion body, and It is and costly.

The prior art has some reports on the separating and extracting process of excretion body.For example, CN106282107A (China Number of patent application 201610779165.2) method that discloses a kind of Human plactnta mescenchymal stem cell source separation excretion body, P2~ When P3 placenta mesenchyma stem cells converge rate up to 85%~90%, culture medium is collected, obtains cell culture supernatant, and carry out film After filtering, filtrate is collected；Filtrate is centrifuged again, collects centrifuged supernatant, 10w/ is added in 1: 1 ratio of centrifuged supernatant volume The medium supernatant solution of v%~12w/v% polyethylene glycol, and centrifuged for the second time after fully mixing, last gained precipitation is For excretion body.It is believed that the method for the invention, convenient material drawing, are easy to Enrichment Amplification culture, P2~P3 is done carefully for placenta mesenchyma Born of the same parents' culture medium supernatant, which recycles, obtains excretion body, is not related to medical ethics problems.In excretion body separation process using membrane filtration, from The means such as the heart and addition polyglycol solution are effectively increased the enrichment of excretion body, and time-consuming short, cost is low.

CN105708861A (Chinese Patent Application No. 201610149852.6) discloses the outer of source for mesenchymal stem cells Application of the body in the medicine for preparing treatment ankylosing spondylitis is secreted, the extracting method of wherein excretion body is：Cultivate enough bones Bone marrow-drived mesenchymal stem, the 48h before stem cell excretion body is extracted, with PBS cleaning cell, replaces serum free medium, continues to train Support 48h；Serum free medium is collected, 300 × g centrifugation 10min, take supernatant；2000 × g centrifuges 10min, takes supernatant；10000× G centrifuges 30min, takes supernatant；100000 × g centrifuges 70min, takes precipitation；Precipitation is resuspended in PBS, and 100000 × g centrifugation 70min, are received Collect and precipitate up to the excretion body.

CN105267240A (Chinese Patent Application No. 201410781765.3) discloses the outer of source for mesenchymal stem cells The purposes of body is secreted, wherein being prepared by the method included the following steps：(1) mescenchymal stem cell is separately cultured：According to Mesenchymal stem cells source is different, specific as follows using the different modes that is separately cultured：1. the separation of human umbilical cord mesenchymal stem cells Culture：The fresh umbilical cord of sterile neonate is taken, after phosphate buffer (PBS) rinses repeatedly, is cut into the tissue of diameter about 1-2mm Block；After 2 Collagenase Types and pancreatin digestion successively, supernatant is centrifuged, takes cell precipitation to be put into blake bottle, using containing 10% tire Cow's serum DMEM/F12 culture mediums, 5%CO2,37 DEG C of saturated humidity cultures；Non-adherent cell is removed, is melted in attached cell 80% 0.25% Trypsin Induced carries out secondary culture after conjunction；2. Human plactnta mescenchymal stem cell is separately cultured：Clip placenta suede The tissue of trichilemma face 1-2cm thickness, is cut into the fragment of 1-2mm, is rinsed with phosphate buffer to colourless, with DispaseII type enzymes With 4 Collagenase Type, 37 DEG C of water-baths digestion 1 it is small when, stood after vibration, liquid is divided into three layers naturally, draws intermediate layer suspension in centrifugation Guan Zhong, adds after phosphate buffer mixes and centrifuges, abandon supernatant, take cell precipitation to be put into blake bottle, using containing 10% tire ox blood Clear DMEM culture mediums, 5%CO2,37 DEG C of saturated humidity cultures；Non-adherent cell is removed, after attached cell 80% is merged 0.25% Trypsin Induced carries out secondary culture；3. human adipose mesenchymal stem cells are separately cultured：Sterile aspirating adipose is taken out Extract, is rinsed for several times with phosphate buffer.Medicine and haemocyte used in liposuction procedures are removed, is disappeared for 37 DEG C with 1 Collagenase Type Change 1 it is small when, or stirring；Upper-layer fat is discarded after centrifugation, is mixed, with 200 mesh sieve net filtrations, by the cell suspension obtained from The heart, cell precipitation are washed with phosphate buffer, are suspended, are transferred in blake bottle with the α-MEM nutrient solutions of 10% hyclone, 5%CO2,37 DEG C of saturated humidity cultures；0.25% Trypsin Induced carries out secondary culture after attached cell 80% is merged. (2) collection of mescenchymal stem cell conditioned medium：Take the mesenchymal stem cell serum-free culture 48h in 3-5 generations；Collect in culture Clear liquid；0.22 μm of non-velum filteration obtains mescenchymal stem cell conditioned medium.(3) excretion body isolate and purify including：Collected 4 DEG C of the mescenchymal stem cell conditioned medium of filter, 1000g centrifugation 10min, collects supernatant；4 DEG C of the supernatant of collection, 2000g from Heart 20min, collects supernatant；4 DEG C of the supernatant of collection, 10000g centrifugation 30min, collects supernatant；The supernatant of collection, 110000g centrifuges 70min, abandons supernatant, is resuspended and precipitated using phosphate buffer；110000g centrifuges 70min again, abandons supernatant, Precipitation is resuspended in a small amount of phosphate buffer, and 0.22 μm of membrane filtration is degerming, obtains source for mesenchymal stem cells excretion body.

CN104382827A (Chinese Patent Application No. 201410705462.3) is disclosed outside human amnion mesenchymal stem cell The purposes of body is secreted, excretion preparation therein is as follows：1st, the culture of human amnion mesenchymal stem cell：Take well-grown people Amnion mesenchymal stem cell, with serum free medium culture, 0.25% pancreatin had digestive transfer culture, reaches P2 generations, treats its growth to melting Right 80%, culture supernatant is abandoned, is washed three times with PBS, 1640 basal mediums is added, collects culture supernatant daily, and more renew 1640 fresh culture mediums, continuous collection culture collect the extraction that culture supernatant is used for excretion body after 3 to 5 days.2nd, excretion body carries Take (extraction overall process unless otherwise indicated, carries out at 4 DEG C)：All culture supernatant 300g of collection are centrifuged into 10min, are protected Supernatant is stayed, abandons precipitation, removes the cell in nutrient solution；Supernatant 2000g~3000g centrifuges 20min~30min, in reservation Clear liquid, abandons precipitation, removes cell fragment；Supernatant 10000g centrifuges 60min~100min, retains supernatant, abandons precipitation, again Remove cell fragment；Supernatant 100000g centrifuges 60min~120min, abandons supernatant, retains precipitation；Precipitation adds PBS and is resuspended Afterwards, 100000g centrifuges 60min~120min and abandons supernatant, retains precipitation, removes protein in culture medium, obtains as after purification Human amnion mesenchymal stem cell excretion body, be resuspended with PBS buffer, with BCA kits survey protein concentration.

CN103767985A (Chinese Patent Application No. 201210402915.6) discloses people source blood or mesenchyma is done carefully The preparation and application of intracrine excretion body, wherein excretion body are made by the following method：1) blood → differential centrifugation separation is gathered The excretion body that → low temperature hypervelocity purifying → acquisition high-purity serum contains；2) mescenchymal stem cell → in vitro culture, amplification are extracted → collect cell culture supernatant → thawing culture supernatant and mix → differential centrifugation separation → low temperature hypervelocity purifying → acquisition The excretion body of high-purity stem cell secretion；3) mescenchymal stem cell → in vitro culture, amplification → collection cells and supernatant are extracted Liquid → thawing culture supernatant simultaneously mixes → low-temperature centrifugation, immunoabsorption purify → and obtains the secretion of high-purity mescenchymal stem cell Excretion body.

Excretion body disclosed in CN105861430A (Chinese Patent Application No. 201610279473.9) is prepared by following methods： (1) human umbilical cord mesenchymal stem cells are separately cultured；(2) IL-1 β optimization processing human umbilical cord mesenchymal stem cells are used；(3) step is collected Suddenly the medium supernatant in (2), it is to obtain the human umbilical cord mesenchymal of IL-1 β optimizations to collect excretion body with the method for differential centrifugation The excretion body of source of human stem cell.

However, when cultivating mescenchymal stem cell from placenta, Cord blood, marrow, fat in the prior art, using these The culture medium supernatant of MSC extracts excretion body, these deficiencies that aspect is relatively low there are yield and gained excretion body purity is relatively low. Therefore, this area still expects have new method to carry out separation and Extraction excretion body, particularly with high yield and high-purity from people's umbilical cord Blood mescenchymal stem cell secretion extraction excretion body.

The content of the invention

It is an object of the invention to provide a kind of new method to carry out separation and Extraction excretion body, and particularly this method can be with High yield and high-purity secrete extraction excretion body from mesenchymal stem cells derived from human umbilical blood.Have now surprisingly been found that by this hair Bright method can be beneficial realize above-mentioned purpose.The present invention is accomplished based on this discovery.

For this reason, first aspect present invention provides a kind of separation and Extraction excretion body from mesenchymal stem cells derived from human umbilical blood Method, this method include：

1st, the culture of mesenchymal stem cells in umbilical cord blood, and

2nd, the extraction of Cord blood MSC excretions body；Wherein

The culture of the mesenchymal stem cells in umbilical cord blood is to make mesenchymal stem cells in umbilical cord blood culture to P3 generations；

The extraction of the Cord blood MSC excretion bodies includes the following steps：

2.1st, excretion body suspension pre-processes

2.1.1, excretion body source one：When P3 converges rate for Cord blood MSC and reaches 75%~85%, culture is collected as far as possible Culture medium in bottle, it is stand-by to be put into 4 DEG C of refrigerators；

2.1.2, excretion body source two：The blake bottle for having removed culture medium is paved with PBS, by the Cord blood MSC equipped with PBS P3 be put into volume fraction for Tissue Culture Flask to be cultivated in 5%CO2 saturated humidity incubators, hungry Cord blood MSC is thin Born of the same parents 24h, collects the PBS in blake bottle afterwards；

2.1.3, excretion body source three：

2.1.3.1, the P3 after PBS starvation is disappeared for Cord blood MSC by adding 0.25% trypsase -0.01%EDTA Change liquid, the cell dissociation in blake bottle is got off and is collected into new centrifuge tube, dilute constant volume with PBS, take 500 μ l cell suspensions Hematometer counts；

2.1.3.2 the P3 cells of Cord blood MSC, are taken, are transferred in new centrifuge tube, carry out centrifugation 300*g, 10min, are risen Speed 9, reduction of speed degree 7；

2.1.3.3 supernatant, is removed, cell is resuspended with PBS, centrifuges and repeats to add PBS washings；

2.1.3.4 PBS is removed after, washing, the cell pyrolysis liquid with cushioning effect is added to cell precipitation, and low 30min is incubated on warm ice bag, during which every 5min, 1min is mixed on rotation nest oscillator, resonance is swung 4 times；

2.1.3.5, it is incubated and terminates, centrifuges 20000*g again, 30min, lifting speed 9, reduction of speed degree 7, extracts whole supernatants It is transferred in new centrifuge tube, is diluted with PBS, is put into -80 DEG C of refrigerator Cord blood at least 24h；

2.1.4, by the suspension recovery in 2.1.3.5 and with the culture medium in above 2.1.1 steps, above 2.1.2 is from training Support the PBS collected in bottle to be mixed, as excretion body source mixed liquor, for extracting excretion body jointly；

2.2nd, excretion body extracts

2.2.1 volume ratio 1, is carried out to excretion body source mixed liquor with PBS:1 dilution, trim, is centrifuged, rotating speed 350* G, 15min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unnecessary MSC cells；

2.2.2, carefully the supernatant after centrifugation is collected into new centrifuge tube, removes precipitation, is centrifuged, rotating speed 2500*g, 20min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unnecessary dead cell；

2.2.3, supernatant is collected into new centrifuge tube again, abandons precipitation, and centrifuges 13000*g, 30min again, is risen Speed 9,7,4 DEG C of reduction of speed degree, to remove unwanted cell fragment；

2.2.4, gently the supernatant after centrifugation is collected into new centrifuge tube, and carries out ultracentrifugation, rotating speed 110000*g, 2h, lifting speed 9,7,4 DEG C of reduction of speed degree, gained sediment are excretion body intermediate product；

2.2.5 supernatant, is abandoned, dilutes sediment with PBS, as excretion body intermediate product, is filtered by 0.22 μm of filter Suspension；

2.2.6 the TRIS/ sucrose/D2O solution configured, is added in a new 50ml centrifugation bottom of the tube, is formed slow Punching pad；Suspension is gently added in above cushion pad with 5~6 times of cushion pad volumes, avoids breaking through liquid level；Again carry out ultrahigh speed from The heart, rotating speed 100000*g, 80min, lifting speed 9,9,4 DEG C of reduction of speed degree；

2.2.7 supernatant, is abandoned, contains the cushion pad of excretion body with the 5ml syringe collectings of 18G syringe needles, by cushion pad PBS 10 times of dilution, carries out high speed centrifugation 100000*g, 60min, lifting speed 9, by 7,4 DEG C of speed；Supernatant is removed after centrifugation as far as possible, Precipitation is excretion body, and excretion body, which is resuspended, with PBS is cleaned, and carries out high speed centrifugation 100000*g, 20min, removes supernatant Liquid, then with PBS repeated washing excretions body, as above high speed centrifugation 2-3 times；Finally diluted and mixed with PBS, obtain final excretion Body, optional puts it into -80 DEG C of refrigerator Cord bloods.

Method according to a first aspect of the present invention, wherein the incubation of the mesenchymal stem cells in umbilical cord blood uses ability The general method in domain carries out.Such as these methods be well known to a person skilled in the art.

Method according to a first aspect of the present invention, wherein the culture of the mesenchymal stem cells in umbilical cord blood includes following step Suddenly：

1.1st, the collection of Cord blood：

Choose the umbilical cord of term neonatal, by umbilical cord blood collection therein into disposable sterilized plastic blood sampling bags, low temperature Fresh-keeping storing；

1.2nd, Cord blood pre-processes：The Cord blood that will be collected, new centrifugation is transferred to by the filter in 100 μm of apertures Guan Zhong, to remove unnecessary clot；

1.3rd, human umbilical cord blood mononuclear cell extracts：

1.3.1 the Cord blood after filtering and PBS, are pressed 1:1 ratio is uniformly mixed so as to obtain cell suspension, then takes cell suspension to add In centrifuge tube equipped with Ficoll separating liquids, cell suspension is 5 with Ficoll separating liquids volume ratio：3, carry out centrifugation 2000rpm/ Min, 30min, 4 DEG C, lifting speed 1, reduction of speed degree 0；

After the centrifugation of Ficoll separating liquids, liquid level is divided into four layers, is successively：Red blood cell and granulocyte layer, separation of lymphocytes Liquid layer, human umbilical cord blood mononuclear cell layer, plasma layer；

1.3.2 human umbilical cord blood mononuclear cell layer, is exhausted as far as possible with pipette, is transferred to equipped with Cord blood MSC culture mediums 1 In centrifuge tube and mix, obtain suspension；In addition plasma layer is extracted it is spare in another centrifuge tube, for MSC culture mediums configure；

1.3.3, suspension is filtered with the filter in 0.22 μm of aperture, to remove unnecessary lymphocyte and impurity, is obtained To cell suspension；

1.4th, the P0 of Cord blood MSC is commissioned to train foster

1.4.1, cell suspension inoculation is trained T25 in T25 blake bottles, adding 10ml Cord blood MSC culture mediums 1 Foster bottle is placed in the CO that volume fraction is 5%₂Cultivated in saturated humidity incubator；

1.4.2, treat primary cell inoculation 48h or so, 3ml Cord blood MSC culture mediums 1 are added into T25 blake bottles, afterwards Every 2-3 days, add a not good liquor, add 3-4 subcultures altogether；Afterwards every 3 days, original culture medium is removed, adds 15ml navels Band blood MSC culture mediums 1, carry out cell to change liquid processing, change liquid altogether 2-3 times；

1.5th, the P1-P3 of Cord blood MSC is commissioned to train foster

1.5.1, when Cord blood MSC primary cells converge rate and reach 40%-50%, P0 generation biography P1 generation processing is done, suction is abandoned Original culture medium in T25 bottles, the PBS buffer for drawing 10ml gently add washing in blake bottle, and washing finishes removal PBS and delays Fliud flushing；Add 0.25% trypsase -0.01%EDTA digestive juice 1ml, soak unrestrained covering T25 blake bottles bottom, then by blake bottle It is positioned over CO₂30-60s is incubated in incubator, blake bottle side is gently patted, is observed under inverted microscope, treat MSC cell shrinkages Into circle, when being floated into quicksand like, add 5ml Cord blood MSC culture mediums 1 and carry out termination digestion, fully blown and beaten with 5ml pipettes Cell simultaneously is collected in 15ml centrifuge tubes in blake bottle bottom；Carry out centrifugation 1400rpm/min, 5min, 4 DEG C, lifting speed 9, reduction of speed Degree 7；

1.5.2 after, centrifuging, supernatant discarding, is resuspended cell with the IMDM basal mediums of 5ml, draws 200 μ l cell suspensions And counted with ADAM calculating instruments, according to 3500-5000 cells/cm²Density is inoculated in T75 blake bottles, and adds new umbilical cord Blood MSC culture mediums 2, it is 5%CO to be placed in volume fraction₂Cultivated in saturated humidity incubator, be denoted as the P1 generations of Cord blood MSC Cell；

1.5.3, when cell confluency rate reaches 80-90%, aforesaid operations are repeated, it is thin that Cord blood MSC is carried out P2, P3 generation Born of the same parents expand.

Method according to a first aspect of the present invention, it includes the following steps：

1st, the culture of mesenchymal stem cells in umbilical cord blood, it includes the following steps：

1.1st, the collection of Cord blood：

1.3rd, human umbilical cord blood mononuclear cell extracts：

1.4th, the P0 of Cord blood MSC is commissioned to train foster

1.5, the P1-P3 of Cord blood MSC be commissioned to train it is foster

1.5.3, when cell confluency rate reaches 80-90%, aforesaid operations are repeated, it is thin that Cord blood MSC is carried out P2, P3 generation Born of the same parents expand；

2nd, the extraction of Cord blood MSC excretions body, it includes the following steps：

2.1st, excretion body suspension pre-processes

2.1.3, excretion body source three：

2.2nd, excretion body extracts

2.2.7 supernatant, is abandoned, contains the cushion pad of excretion body with the 5ml syringe collectings of 18G syringe needles, by cushion pad PBS 10 times of dilution, carries out high speed centrifugation 100000*g, 60min, lifting speed 9,7,4 DEG C of reduction of speed degree；Supernatant is removed after centrifugation as far as possible, Precipitation is excretion body, and excretion body, which is resuspended, with PBS is cleaned, and carries out high speed centrifugation 100000*g, 20min, removes supernatant Liquid, then with PBS repeated washing excretions body, as above high speed centrifugation 2-3 times；Finally diluted and mixed with PBS, obtain final excretion Body, optional puts it into -80 DEG C of refrigerator Cord bloods.

1.1st, the collection of Cord blood：

The umbilical cord of term neonatal is chosen, by umbilical cord blood collection therein into disposable sterilized plastic blood sampling bags, transport Whole preservation by low temperature；

1.2nd, Cord blood pre-processes：

The Cord blood that will be collected, is transferred to by the filter in 100 μm of apertures in new centrifuge tube, to remove unnecessary blood Block；

1.3rd, human umbilical cord blood mononuclear cell extracts：

1.3.1, by the Cord blood (such as taking 50ml) after filtering and PBS by 1:1 ratio mixes (cumulative volume 100ml) and obtains To cell suspension, then take cell suspension 15ml add equipped with Ficoll separating liquids 50ml centrifuge tubes in, cell suspension with Ficoll separating liquids volume ratio is 5：3, carry out centrifugation 2000rpm/min, 30min, 4 DEG C, lifting speed 1, reduction of speed degree 0；

1.3.2 human umbilical cord blood mononuclear cell layer, is exhausted as far as possible with pipette, is transferred to equipped with 5ml Cord blood MSC culture mediums In 1 50ml centrifuge tubes and mix, obtain suspension；In addition plasma layer is extracted it is spare in another 50ml centrifuge tube, for MSC Culture medium configures；

1.4th, the P0 of Cord blood MSC is commissioned to train foster

1.5th, the P1-P3 of Cord blood MSC is commissioned to train foster

2.1st, excretion body suspension pre-processes

2.1.2, excretion body source two：The blake bottle for having removed culture medium is paved with the PBS of 50ml, by the navel equipped with PBS It is 5%CO that P3 with blood MSC is put into volume fraction for Tissue Culture Flask₂Cultivated in saturated humidity incubator, hungry umbilical cord Blood MSC cell 24h, collect the PBS in blake bottle afterwards；

2.1.3, excretion body source three

2.1.3.1, the P3 after PBS starvation is disappeared for Cord blood MSC by adding 0.25% trypsase -0.01%EDTA Change liquid, the cell dissociation in blake bottle is got off and is collected into new 50ml centrifuge tubes, with PBS constant volumes to 20ml, takes 500 μ l Cell suspension Hematometer counts；

2.1.3.2 Cord blood MSC cell numbers, are calculated, take 6*10⁷The P3 cells of Cord blood MSC, are transferred to new In 15ml centrifuge tubes, centrifugation 300*g, 10min, lifting speed 9, reduction of speed degree 7 are carried out；

2.1.3.3 supernatant, is removed, cell is resuspended with the PBS of 3ml, centrifuges and repeats to add PBS washings；

2.1.3.4 PBS is removed after, washing, adding 1.5ml to cell precipitation has the cell pyrolysis liquid of cushioning effect, and And 30min is incubated on low temperature ice bag, during which every 5min, 1min is mixed on rotation nest oscillator, resonance is swung 4 times；

2.1.3.5, it is incubated and terminates, centrifuges 20000*g again, 30min, lifting speed 9, reduction of speed degree 7, extracts whole supernatants It is transferred in new 15ml centrifuge tubes, 10ml is diluted to PBS, is put into -80 DEG C of refrigerator Cord blood at least 24h；

2.2nd, excretion body extracts

2.2.1 (1, is diluted to excretion body source mixed liquor with PBS:1), trim, is centrifuged, rotating speed 350*g, 15min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unnecessary MSC cells；

2.2.5 supernatant, is abandoned, dilutes sediment with PBS, as excretion body intermediate product, is filtered by 0.22 μm of filter Suspension, by suspension constant volume to a 50ml centrifuge tube, fixing fabric structure is in 25ml；

2.2.6 TRIS/ sucrose/D of the 4.5ml configured, is added in a new 50ml centrifugation bottom of the tube₂O solution, shape Into cushion pad；25ml suspensions are gently added in above cushion pad, avoid breaking through liquid level；Ultracentrifugation, rotating speed are carried out again 100000*g, 80min, lifting speed 9,9,4 DEG C of reduction of speed degree；

2.2.7 supernatant, is abandoned, contains the cushion pad of excretion body with the 5ml syringe collectings of 18G syringe needles, by cushion pad PBS It is diluted to 50ml, carries out high speed centrifugation 100000*g, 60min, lifting speed 9, by 7,4 DEG C of speed；Supernatant is removed after centrifugation as far as possible Liquid, precipitation are excretion body, and excretion body, which is resuspended, with the PBS of 100ml is cleaned, and carries out high speed centrifugation 100000*g, 20min, removes supernatant, then PBS repeated washing excretions body with 100ml, as above high speed centrifugation 2-3 times；It is final to use 5ml's PBS is diluted and mixed, and obtains final excretion body, and optional puts it into -80 DEG C of refrigerator Cord bloods.

Method according to a first aspect of the present invention, wherein the composition of the Cord blood MSC culture mediums 1 includes：Volumetric concentration The GM-CSF of umbilical cord blood, 15ng/ml for 10%, volumetric concentration 2%HEPES, 1% green grass or young crops of volumetric concentration-streptomysin, volume Concentration 1%L- glutamine, surplus IMDM basal mediums.

Method according to a first aspect of the present invention, wherein the composition of the Cord blood MSC culture mediums 2 includes：Volumetric concentration IL-1 β of umbilical cord blood, 10ng/ml for 10%, volumetric concentration 2%HEPES, 1% mycillin of volumetric concentration, volume are dense Spend 1%L- glutamine, surplus IMDM basal mediums.

Method according to a first aspect of the present invention, wherein the formula composition of the cell pyrolysis liquid includes：300mmol/L's The Tris (pH7.4) of NaCl, 50mmol/L, 3mmol/L sodium pyrophosphates, 1% Triton X-100,0.1% dodecane Base sodium sulphate (SDS), the sodium β-glycerophosphate of the EDTA of 1.5mmol/L, 30mmol/L, are prepared with water.In an embodiment In, the cell pyrolysis liquid each component is diluted and dissolved with the purified water Jing Guo high temperature and pressure in proportion to be prepared.

Method according to a first aspect of the present invention, wherein the preparation method of the cushion pad is：30g is taken without protease sugarcane Sugar, the Tris alkali of 2.4g, the D of 50ml₂O, it is fully mixed, then adjusts PH to 7.4 with the HCL titration of 10mol/L, uses D₂O will Volume is modulated into 100ml, spare in 4 DEG C of refrigerator storages by 0.22 μm of filter filtering and autoclaving.

Method according to a first aspect of the present invention, wherein further including volumetric concentration in the Cord blood MSC culture mediums 2 0.2% glycine.Method according to a first aspect of the present invention, wherein further including 0.05w/v% calcium chloride in the cushion pad.Root According to the method for first aspect present invention, wherein further including 0.2% glycine of volumetric concentration, institute in the Cord blood MSC culture mediums 2 State and 0.05w/v% calcium chloride is further included in cushion pad.It has been had now surprisingly been found that, in Cord blood MSC culture mediums 2 and cushion pad (the two can be referred to as improvement Cord blood MSC culture mediums 2 and improvement cushion pad), Neng Gouxian after increase above two material respectively Write the yield for improving excretion body.Tested for example, referring to embodiment 1 and embodiment 2, but at the trial respectively using improvement Cord blood MSC culture mediums 2 and improvement cushion pad, products therefrom is when correspondingly carrying out the test of embodiment 3~5, except Fig. 2 results Variant outer, remaining result is consistent with the measurement result of 1~2 gained sample of embodiment；Such as a diameter of the 50 of excretion body to 120nm, separating obtained cell express mescenchymal stem cell surface antigen CD73, CD90, CD105 more than 99%, without Expression of HLA-DR, CD-34, CD-45, CD19, CD11b (are below 0.5%)；And using 2 He of improvement Cord blood MSC culture mediums In the experiment for improveing cushion pad, the excretion body yield (ug/10 of tetra- groups of experiments of A, B, C, D⁶Cell) respectively reach 3.13,3.28, 2.93rd, 2.27, it is significantly higher than 0.66~0.82ug/10 when not adding this two kinds of reagents in two kinds of solution⁶The excretion body of cell Yield.Regrettably, it has been found that, if using only one of above-mentioned improvement Cord blood MSC culture mediums 2 and improvement cushion pad Experiment (improvement Cord blood MSC culture mediums 2 being used only, or using only improvement cushion pad) in, although 3~5 side of embodiment Remaining result obtained by method is substantially essentially identical with 1~2 sample tests of embodiment, but four groups of experiments is outer in the case of two kinds Secrete body yield (ug/10⁶Cell) respectively in the range of 0.74~0.91 and in the range of 0.64~0.86, can not effectively it improve Excretion body yield.

Further, second aspect of the present invention provides a kind of Cord blood MSC culture mediums, its composition includes：Volumetric concentration The GM-CSF of umbilical cord blood, 15ng/ml for 10%, volumetric concentration 2%HEPES, 1% green grass or young crops of volumetric concentration-streptomysin, volume Concentration 1%L- glutamine, surplus IMDM basal mediums.

Further, second aspect of the present invention provides a kind of Cord blood MSC culture mediums, its composition includes：Volumetric concentration IL-1 β of umbilical cord blood, 10ng/ml for 10%, volumetric concentration 2%HEPES, 1% mycillin of volumetric concentration, volume are dense Spend 1%L- glutamine, surplus IMDM basal mediums.For example, wherein further include 0.2% glycine of volumetric concentration

Further, second aspect of the present invention provides a kind of cell pyrolysis liquid, its composition includes：300mmol/L's The Tris (pH7.4) of NaCl, 50mmol/L, 3mmol/L sodium pyrophosphates, 1% Triton X-100,0.1% dodecane Base sodium sulphate (SDS), the sodium β-glycerophosphate of the EDTA of 1.5mmol/L, 30mmol/L, are prepared with water.In an embodiment In, the cell pyrolysis liquid each component is diluted and dissolved with the purified water Jing Guo high temperature and pressure in proportion to be prepared.

Further, second aspect of the present invention provides a kind of cushion pad, its preparation method is：30g is taken without protease sugarcane Sugar, the Tris alkali of 2.4g, the D of 50ml₂O, it is fully mixed, then adjusts PH to 7.4 with the HCL titration of 10mol/L, uses D₂O will Volume is modulated into 100ml, spare in 4 DEG C of refrigerator storages by 0.22 μm of filter filtering and autoclaving.For example, wherein also Including 0.05w/v% calcium chloride.

The present invention is further illustrated below.Document cited in the present invention, and text cited in the document Offer, their full content is incorporated herein by reference.

In the present invention, in any technical solution of either side of the present invention, its any technical characteristic is equally applicable to this Any embodiment of the either side of invention, as long as they will not cause contradiction, and this be mutually applicable in when necessary may be used To be suitably modified.

In the present invention, term " mesenchymal stem cells in umbilical cord blood " refers to the mescenchymal stem cell from Cord blood.Cause This in the present invention, more particularly to the present invention linguistic context in, term " mesenchymal stem cells in umbilical cord blood " can with " Cord blood is done Cell ", " stem cell ", " mescenchymal stem cell " are used interchangeably, unless otherwise clearly indicating.

Brief description of the drawings

Fig. 1, mesenchymal stem cells derived from human umbilical blood metamorphosis, in figure：A is mesenchymal stem cells derived from human umbilical blood culture 7d, In fusiformis (inverted microscope, × 100)；B is mesenchymal stem cells derived from human umbilical blood culture 14d, and arrangement (is fallen in netted, radial Microscope is put, × 200)；C is the 3rd generation mesenchymal stem cells derived from human umbilical blood, and cell distribution is uniform, in fibroblast-like cells form (inverted microscope, × 200).

Fig. 2, general aqueous solution and D₂O excretion body yield comparison diagrams, in figure：4 groups of Cord blood MSC are chosen respectively to be extracted Excretion body, buffer solution prepares cushion pad based on general aqueous solution and heavy water is respectively adopted.It is it can be seen from the figure that identical thin Born of the same parents' number directly influences excretion body yield, D using different basis buffers₂O basis buffers are apparently higher than general aqueous solution. In figure, for tetra- groups of A, B, C, D, each left side compared with short column is the buffering that prepared by buffer solution based on general aqueous solution for it Pad, right side is compared with the cushion pad that long column is buffer solution preparation based on heavy water.

The flow cytometry figure of Fig. 3, mesenchymal stem cells derived from human umbilical blood surface antigen molecules, in figure：Express surface Antigens c D73, CD90, CD105 >=99%, does not express surface antigen HLA-DR, CD-34, CD-45, CD19, CD11b≤0.5%.

Excretion body is observed under Fig. 4, scanning electron microscope, it is seen that excretion body diameter is about 50~120nm, there is complete lipid film, Corpusculum center is in low electron density component, circular or ellipse film imitated vesicle structure.

Embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method that are arrived used in experiment general And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that But the present invention still makees description as detailed as possible herein.

Embodiment 1, Cord blood MSC cultures

1.1st, the collection of Cord blood：

The umbilical cord of term neonatal is chosen (it is required that the passing no major disease history of lineal relative, gatherer process need rigorous aseptic Collection, blood volume at least gather 50ml), by umbilical cord blood collection therein into disposable sterilized plastic blood sampling bags, transport is whole low Temperature is fresh-keeping.

1.2nd, Cord blood pre-processes：

The Cord blood that will be collected, is transferred to by the filter in 100 μm of apertures in new centrifuge tube, to remove unnecessary blood Block.

1.3rd, human umbilical cord blood mononuclear cell extracts：

[PBS is well known to those skilled in the art, and in the present invention, if not otherwise indicated, the PBS used is phosphate Buffer solution (PH6.8), it is prepared by the following method：0.2mol/L potassium dihydrogen phosphate 250ml are taken, add 0.2mol/L hydroxides Sodium solution 118ml, is diluted with water to 1000ml, shakes up, to obtain the final product.]

After the centrifugation of Ficoll separating liquids, liquid level is divided into four layers, is successively：Red blood cell and granulocyte layer, separation of lymphocytes Liquid layer, human umbilical cord blood mononuclear cell layer, plasma layer.

1.3.2 human umbilical cord blood mononuclear cell layer, is exhausted as far as possible with pipette, is transferred to equipped with 5ml Cord blood MSC culture mediums In 1 50ml centrifuge tubes and mix, obtain suspension；In addition plasma layer is extracted it is spare in another 50ml centrifuge tube, for MSC Culture medium configures.

1.3.3, suspension is filtered with the filter in 0.22 μm of aperture, to remove unnecessary lymphocyte and impurity, is obtained To cell suspension.

1.4th, the P0 of Cord blood MSC is commissioned to train foster

1.4.1, cell suspension inoculation is trained T25 in T25 blake bottles, adding 10ml Cord blood MSC culture mediums 1 Foster bottle is placed in the CO that volume fraction is 5%₂Cultivated in saturated humidity incubator.

1.4.2, treat primary cell inoculation 48h or so, 3ml Cord blood MSC culture mediums 1 are added into T25 blake bottles, afterwards Every 2-3 days, add a not good liquor, add 3-4 subcultures altogether；Afterwards every 3 days, original culture medium is removed, adds 15ml navels Band blood MSC culture mediums 1, carry out cell to change liquid processing, change liquid altogether 2-3 times.

[1 component of Cord blood MSC culture mediums that the present invention uses：IMDM basal mediums (GIBCO), volumetric concentration are 10% umbilical cord blood, the GM-CSF of 15ng/ml, volumetric concentration 2%HEPES, 1% green grass or young crops of volumetric concentration-streptomysin, volume are dense Spend 1%L- glutamine.1 advantage of Cord blood MSC culture mediums is that it is possible to fast and effectively promote Cord blood MSC primary cells Fast-growth, cell yield are higher.]

1.5th, the P1-P3 of Cord blood MSC is commissioned to train foster

1.5.1, when Cord blood MSC primary cells converge rate and reach 40%-50%, P0 generation biography P1 generation processing is done, suction is abandoned Original culture medium in T25 bottles, the PBS buffer for drawing 10ml gently add washing in blake bottle, and washing finishes removal PBS and delays Fliud flushing；Add 0.25% trypsase -0.01%EDTA digestive juice 1ml, soak unrestrained covering T25 blake bottles bottom, then by blake bottle It is positioned over CO₂30-60s is incubated in incubator, blake bottle side is gently patted, is observed under inverted microscope, treat MSC cell shrinkages Into circle, when being floated into quicksand like, add 5ml Cord blood MSC culture mediums 1 and carry out termination digestion, fully blown and beaten with 5ml pipettes Cell simultaneously is collected in 15ml centrifuge tubes in blake bottle bottom.Carry out centrifugation 1400rpm/min, 5min, 4 DEG C, lifting speed 9, reduction of speed Degree 7.

1.5.2 after, centrifuging, supernatant discarding, is resuspended cell with the IMDM basal mediums of 5ml, draws 200 μ l cell suspensions And counted with ADAM calculating instruments, according to 3500-5000 cells/cm²Density is inoculated in T75 blake bottles, and adds new umbilical cord Blood MSC culture mediums 2, it is 5%CO to be placed in volume fraction₂Cultivated in saturated humidity incubator, be denoted as the P1 generations of Cord blood MSC Cell.

[visible Fig. 1 of cellular morphology.

2 component of Cord blood MSC culture mediums that the present invention uses：IMDM basal mediums (GIBCO), volumetric concentration 10% Umbilical cord blood, IL-1 β of 10ng/ml, volumetric concentration 2%HEPES, 1% mycillin of volumetric concentration, volumetric concentration 1% L-Glutamine；The present invention removes the new culture medium added herein, it is therefore intended that, new culture medium can stimulate MSC cells Secrete more excretion bodies].

The extraction of embodiment 2, Cord blood MSC excretion bodies

The embodiment that continues 1 carries out the present embodiment 2.

2.1st, excretion body suspension pre-processes

2.1.1, excretion body source one

When P3 converges rate for Cord blood MSC and reaches 75%~85%, the culture medium in blake bottle is collected as far as possible, is put into 4 DEG C refrigerator is stand-by.

2.1.2, excretion body source two

The blake bottle for having removed culture medium is paved with the PBS of 50ml, the P3 of the Cord blood MSC equipped with PBS is trained for cell It is 5%CO that foster bottle, which is put into volume fraction,₂Cultivated in saturated humidity incubator, hungry Cord blood MSC cells 24h, Zhi Houshou Collect the PBS in blake bottle.

2.1.3, excretion body source three

2.1.3.1, the P3 after PBS starvation is disappeared for Cord blood MSC by adding 0.25% trypsase -0.01%EDTA Change liquid, the cell dissociation in blake bottle is got off and is collected into new 50ml centrifuge tubes, with PBS constant volumes to 20ml, takes 500 μ l Cell suspension Hematometer counts.

2.1.3.2 Cord blood MSC cell numbers, are calculated, take 6*10⁷The P3 cells of Cord blood MSC, are transferred to new In 15ml centrifuge tubes, centrifugation 300*g, 10min, lifting speed 9, reduction of speed degree 7 are carried out.

2.1.3.3 supernatant, is removed, cell is resuspended with the PBS of 3ml, centrifuges and repeats to add PBS washings.

2.1.3.4 PBS is removed after, washing, adding 1.5ml to cell precipitation has the cell pyrolysis liquid of cushioning effect, and And 30min is incubated on low temperature ice bag, during which every 5min, 1min is mixed on rotation nest oscillator, resonance is swung 4 times.

[this patent provides cell pyrolysis liquid recipe ingredient：The Tris (pH7.4) of the NaCl of 300mmol/L, 50mmol/L, 3mmol/L sodium pyrophosphates, 1% Triton X-100,0.1% lauryl sodium sulfate (SDS), 1.5mmol/L's The sodium β-glycerophosphate of EDTA, 30mmol/L.Diluted and dissolved in proportion with the purified water Jing Guo high temperature and pressure, room temperature storage 3 Used again within a month.]

2.1.3.5, it is incubated and terminates, centrifuges 20000*g again, 30min, lifting speed 9, reduction of speed degree 7, extracts whole supernatants It is transferred in new 15ml centrifuge tubes, 10ml is diluted to PBS, is put into -80 DEG C of refrigerator Cord blood at least 24h.

2.1.4, by the suspension recovery in 2.1.3.5 and with the culture medium in above 2.1.1 steps, above 2.1.2 is from training Support the PBS collected in bottle to be mixed, as excretion body source mixed liquor, for extracting excretion body jointly.

2.2nd, excretion body extracts

2.2.1 (1, is diluted to excretion body source mixed liquor with PBS:1), trim, is centrifuged, rotating speed 350*g, 15min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unnecessary MSC cells.

2.2.2, carefully the supernatant after centrifugation is collected into new centrifuge tube, removes precipitation, is centrifuged, rotating speed 2500*g, 20min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unnecessary dead cell.

2.2.3, supernatant is collected into new centrifuge tube again, abandons precipitation, and centrifuges 13000*g, 30min again, is risen Speed 9,7,4 DEG C of reduction of speed degree, to remove unwanted cell fragment.

2.2.4, gently the supernatant after centrifugation is collected into new centrifuge tube, and carries out ultracentrifugation, rotating speed 110000*g, 2h, lifting speed 9,7,4 DEG C of reduction of speed degree, gained sediment are excretion body intermediate product.

2.2.5 supernatant, is abandoned, it is outstanding by 0.22 μm of filter filtering with PBS dilution sediments (excretion body intermediate product) Liquid, by suspension constant volume to a 50ml centrifuge tube, fixing fabric structure is in 25ml.

2.2.6 TRIS/ sucrose/D of the 4.5ml configured, is added in a new 50ml centrifugation bottom of the tube₂O solution, shape Into cushion pad.25ml suspensions are gently added in above cushion pad, avoid breaking through liquid level.Ultracentrifugation, rotating speed are carried out again 100000*g, 80min, lifting speed 9,9,4 DEG C of reduction of speed degree；

2.2.7 supernatant, is abandoned, contains the cushion pad of excretion body with the 5ml syringe collectings of 18G syringe needles, by cushion pad PBS It is diluted to 50ml, carries out high speed centrifugation 100000*g, 60min, lifting speed 9, by 7,4 DEG C of speed.Supernatant is removed after centrifugation as far as possible Liquid, precipitation are excretion body, and excretion body, which is resuspended, with the PBS of 100ml is cleaned, and carries out high speed centrifugation 100000*g, 20min, removes supernatant, then PBS repeated washing excretions body with 100ml, as above high speed centrifugation 2-3 times.It is final to use 5ml's PBS is diluted and mixed, and obtains final excretion body, and optional puts it into -80 DEG C of refrigerator Cord bloods.

[present invention provides cushion pad collocation method, and collocation method is as follows：Prepare 30g in advance without protease sucrose, 2.4g The D of Tris alkali, 50ml₂O, it is fully mixed, then adjusts PH to 7.4 with the HCL titration of 10mol/L, uses D₂O modulates volume Into 100ml, by 0.22 μm of filter filtering and autoclaving, in 4 DEG C of refrigerator storages, used in 2 months.

Purpose with cushion pad extraction excretion body is the density contrast formed using excretion body suspension with cushion pad, then auxiliary With ultracentrifugation, the purity of excretion body is largely improved.This patent uses D₂O is that basic buffer solution extraction effect is bright It is aobvious to be better than general aqueous solution, choose 4 groups of Cord blood MSC respectively herein and carry out extraction excretion body, general aqueous solution is respectively adopted (H₂) and D O₂Buffer solution prepares cushion pad, correction data such as Fig. 2 based on O.It can be seen that the present invention uses D₂Buffered based on O Liquid, excretion body yield are significantly higher.

It is repeated several times with PBS and cleans final excretion body precipitation, D can be removed completely₂O is remained and other impurity.]

The experiment of example 3 below~5 is carried out to above-described embodiment 1 and 2 gained Cord blood MSC of embodiment and excretion body, And its result is recorded in embodiment 3~5.

Embodiment 3, Cord blood MSC flow cytometer detections

3.1st, when Cord blood mesenchyma P3 converges for cell growth to 75%~85%, original training in blake bottle is removed Support base, every bottle of PBS cleaning one time with 50ml, with 0.25% trypsase conventional digestion cell and is centrifuging, centrifuging 1400rpm/min, lifting speed 9, reduction of speed degree 9,10min.

3.2nd, P3 is resuspended to 1*10 for cell with PBS⁷Cell/ml, is separately added into mouse anti-human monoclonal's labelled antibody

CD90-PC5, CD105-PC7, CD73-FITC, HLA-DR/CD-34/CD-45/CD19/CD11b-PE, are resisted with mouse The igG1-FITC of people, igG1-PE, igG1-PC5, igG1-PC7 is as Isotype control.

3.3rd, room temperature lucifuge is incubated 20min, utilizes flow cytomery.Between the separating obtained cell expression of the results show Surface of mesenchymal stem cells antigens c D73, CD90, CD105 are more than 99%, without expression of HLA-DR, CD-34, CD-45, CD19, CD11b are below 0.5%, and the result is shown in Fig. 3.As seen from Figure 3, the Cord blood MSC purity of preparation is very high.

Embodiment 4, the identification of excretion volume morphing

4.1st, the PBS suspensions for taking 50 μ l to contain excretion body, are resuspended in 2% paraformaldehyde of 100 μ l.

4.2nd, from above-mentioned suspension, 20 μ l excretion body suspensions are taken to be added on carbon film load sample copper mesh.Close the lid, in dry ring Carbon film is allowed to absorb 20min in border.

4.3rd, the PBS of 300 μ l is instilled on a piece of parafilm.Copper mesh is transferred to (under carbon film is lateral) with clean tweezers To be washed under PBS drops.

4.4th, after PBS is washed, carbon film load sample copper mesh is transferred in 1% glutaraldehyde of 50 μ l and is incubated 5min.

4.5th, it is then transferred to after being incubated in 300 μ l distilled water, allows copper mesh to place 2 minutes.Repeat seven times, totally eight washings.

4.6th, copper mesh is transferred in the two uranic acid salting liquid of oxalic acid of the pH7 of 100 μ l and is incubated 5min.

4.7th, copper mesh is transferred to 150 μ l methylcellulose-UA (uranyl acetate) 20min again, is operated on ice. (methylcellulose-UA：2% methylcellulose and the mixing of 4% uranyl acetate)

4.8th, copper mesh is removed with stainless steel ring, and by the way that loop lightly is moved laterally to Whatman NO.1 filter paper, Allow filter paper gentle aspiration surplus liquid, leave a thin layer Methyl Cellulose Hydrogel Films.(stainless steel ring, slightly larger than copper mesh)

4.9th, observe under an electron microscope.

The excretion of negative staining is known from experience is shown as diameter 50 to the cup-shaped membrane vesicle of 120nm in electron microscope, has complete Lipid film, corpusculum center is in low electron density component, and circular or ellipse film imitated vesicle structure (Fig. 4), meets excretion body Morphological feature.

Embodiment 5, flow cytometer detection excretion body surface marker

5.1st, 500 μ l excretion body suspensions are taken, is resuspended to 2ml, is sub-packed in two streaming pipes (often pipe 1ml) with PBS dilutions.

5.2nd, mouse anti-human monoclonal's labelled antibody CD63-PE is separately added into, CD9-PE, room temperature lucifuge incubation 20min, is used PBS cleaning one time.

5.3rd, it is resuspended again with 1% paraformaldehyde, using flow cytomery, the results show Cord blood MSC comes Source excretion body surface reaches CD63 and CD9.

Claims

1. the method for separation and Extraction excretion body, this method include from mesenchymal stem cells derived from human umbilical blood：

(1), the culture of mesenchymal stem cells in umbilical cord blood, and

(2), the extraction of Cord blood MSC excretions body；Wherein

2.1st, excretion body suspension pre-processes

2.1.1, excretion body source one：When P3 converges rate for Cord blood MSC and reaches 75%~85%, collect as far as possible in blake bottle Culture medium, it is stand-by to be put into 4 DEG C of refrigerators；

2.1.2, excretion body source two：The blake bottle for having removed culture medium is paved with PBS, by the P3 of the Cord blood MSC equipped with PBS Volume fraction is put into be cultivated in 5%CO2 saturated humidity incubators for Tissue Culture Flask, hungry Cord blood MSC cells 24h, collects the PBS in blake bottle afterwards；

2.1.3, excretion body source three：

2.1.3.1, the P3 after PBS starvation is for Cord blood MSC, by adding 0.25% trypsase -0.01%EDTA digestive juices, Cell dissociation in blake bottle is got off and is collected into new centrifuge tube, constant volume is diluted with PBS, takes 500 μ l cell suspension blood cells Instrument meter number；

2.1.3.2 the P3 cells of Cord blood MSC, are taken, are transferred in new centrifuge tube, carry out centrifugation 300*g, 10min, lifting speed 9, reduction of speed degree 7；

2.1.3.4 PBS is removed after, washing, the cell pyrolysis liquid with cushioning effect is added to cell precipitation, and in Low-temperature Ice 30min is incubated on bag, during which every 5min, 1min is mixed on rotation nest oscillator, resonance is swung 4 times；

2.1.3.5, it is incubated and terminates, centrifuges 20000*g again, 30min, lifting speed 9, reduction of speed degree 7, extracts whole supernatant transfers To new centrifuge tube, diluted with PBS, be put into -80 DEG C of refrigerator Cord blood at least 24h；

2.1.4, by the suspension recovery in 2.1.3.5 and with the culture medium in above 2.1.1 steps, above 2.1.2 is from blake bottle The PBS of middle collection is mixed, as excretion body source mixed liquor, for extracting excretion body jointly；

2.2nd, excretion body extracts

2.2.1 volume ratio 1, is carried out to excretion body source mixed liquor with PBS:1 dilution, trim, is centrifuged, rotating speed 350*g, 15min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unnecessary MSC cells；

2.2.3, supernatant is collected into new centrifuge tube again, abandons precipitation, and centrifuges 13000*g again, 30min, lifting speed 9,7,4 DEG C of reduction of speed degree, to remove unwanted cell fragment；

2.2.5 supernatant, is abandoned, dilutes sediment with PBS, as excretion body intermediate product, suspension is filtered by 0.22 μm of filter；

2.2.6 the TRIS/ sucrose/D2O solution configured, is added in a new 50ml centrifugation bottom of the tube, forms cushion pad； Suspension is gently added in above cushion pad with 5~6 times of cushion pad volumes, avoids breaking through liquid level；Ultracentrifugation is carried out again, is turned Fast 100000*g, 80min, lifting speed 9,9,4 DEG C of reduction of speed degree；

2.2.7 supernatant, is abandoned, contains the cushion pad of excretion body with the 5ml syringe collectings of 18G syringe needles, cushion pad is diluted with PBS 10 times, carry out high speed centrifugation 100000*g, 60min, lifting speed 9, by 7,4 DEG C of speed；Supernatant, precipitation are removed after centrifugation as far as possible As excretion body, is resuspended excretion body with PBS and is cleaned, and carries out high speed centrifugation 100000*g, 20min, removes supernatant, then With PBS repeated washing excretions body, as above high speed centrifugation 2-3 times；Finally diluted and mixed with PBS, obtain final excretion body, optionally Put it into -80 DEG C of refrigerator Cord bloods.

2. the method according to claim 1, wherein the culture of the mesenchymal stem cells in umbilical cord blood includes the following steps：

1.1st, the collection of Cord blood：

Choose the umbilical cord of term neonatal, by umbilical cord blood collection therein into disposable sterilized plastic blood sampling bags, preservation by low temperature Storing；

1.2nd, Cord blood pre-processes：The Cord blood that will be collected, new centrifuge tube is transferred to by the filter in 100 μm of apertures In, to remove unnecessary clot；

1.3rd, human umbilical cord blood mononuclear cell extracts：

1.3.1 the Cord blood after filtering and PBS, are pressed 1:1 ratio is uniformly mixed so as to obtain cell suspension, then takes cell suspension to add and be equipped with In the centrifuge tube of Ficoll separating liquids, cell suspension is 5 with Ficoll separating liquids volume ratio：3, centrifugation 2000rpm/min is carried out, 30min, 4 DEG C, lifting speed 1, reduction of speed degree 0；

After the centrifugation of Ficoll separating liquids, liquid level is divided into four layers, is successively：Red blood cell and granulocyte layer, lymphocyte separation medium layer, Human umbilical cord blood mononuclear cell layer, plasma layer；

1.3.2 human umbilical cord blood mononuclear cell layer, is exhausted as far as possible with pipette, is transferred to the centrifugation equipped with Cord blood MSC culture mediums 1 In pipe and mix, obtain suspension；In addition plasma layer is extracted it is spare in another centrifuge tube, for MSC culture mediums configure；

1.3.3, suspension is filtered with the filter in 0.22 μm of aperture, to remove unnecessary lymphocyte and impurity, is obtained thin Born of the same parents' suspension；

1.4, the P0 of Cord blood MSC be commissioned to train it is foster

1.4.1, by cell suspension inoculation in T25 blake bottles, adding 10ml Cord blood MSC culture mediums 1, by T25 blake bottles It is placed in the CO that volume fraction is 5%₂Cultivated in saturated humidity incubator；

1.4.2, treat primary cell inoculation 48h or so, 3ml Cord blood MSC culture mediums 1 added into T25 blake bottles, afterwards every 2-3 days, add a not good liquor, add 3-4 subcultures altogether；Afterwards every 3 days, original culture medium is removed, adds 15ml Cord bloods MSC culture mediums 1, carry out cell to change liquid processing, change liquid altogether 2-3 times；

1.5th, the P1-P3 of Cord blood MSC is commissioned to train foster

1.5.1, when Cord blood MSC primary cells converge rate and reach 40%-50%, P0 generation biography P1 generation processing is done, suction abandons T25 bottles In original culture medium, the PBS buffer for drawing 10ml gently adds washing in blake bottle, and washing finishes removal PBS buffer； 0.25% trypsase -0.01%EDTA digestive juice 1ml are added, soak unrestrained covering T25 blake bottles bottom, then blake bottle is positioned over CO₂30-60s is incubated in incubator, blake bottle side is gently patted, is observed under inverted microscope, treat MSC cell shrinkages Cheng Yuan Shape, when being floated into quicksand like, adds 5ml Cord blood MSC culture mediums 1 and carries out termination digestion, culture is fully blown and beaten with 5ml pipettes Cell simultaneously is collected in 15ml centrifuge tubes in bottom of bottle portion；Carry out centrifugation 1400rpm/min, 5min, 4 DEG C, lifting speed 9, reduction of speed degree 7；

1.5.2 after, centrifuging, supernatant discarding, is resuspended cell with the IMDM basal mediums of 5ml, draws 200 μ l cell suspensions and be used in combination ADAM calculating instruments count, according to 3500-5000 cells/cm²Density is inoculated in T75 blake bottles, and adds new Cord blood MSC Culture medium 2, it is 5%CO to be placed in volume fraction₂Cultivated in saturated humidity incubator, be denoted as the P1 of Cord blood MSC for cell；

1.5.3, when cell confluency rate reaches 80-90%, aforesaid operations are repeated, Cord blood MSC is carried out P2, P3 expands for cell Increase.

3. the method according to claim 1, it includes the following steps：

(1), the culture of mesenchymal stem cells in umbilical cord blood, it includes the following steps：

1.1st, the collection of Cord blood：

1.3rd, human umbilical cord blood mononuclear cell extracts：

1.4th, the P0 of Cord blood MSC is commissioned to train foster

1.5th, the P1-P3 of Cord blood MSC is commissioned to train foster

1.5.3, when cell confluency rate reaches 80-90%, aforesaid operations are repeated, Cord blood MSC is carried out P2, P3 expands for cell Increase；

(2) extraction of Cord blood MSC excretions body, it includes the following steps：

2.1st, excretion body suspension pre-processes

2.1.3, excretion body source three：

2.2nd, excretion body extracts

4. the method according to claim 1, it includes the following steps：

1.1st, the collection of Cord blood：

The umbilical cord of term neonatal is chosen, by umbilical cord blood collection therein into disposable sterilized plastic blood sampling bags, transport is whole Preservation by low temperature；

1.2nd, Cord blood pre-processes：

The Cord blood that will be collected, is transferred to by the filter in 100 μm of apertures in new centrifuge tube, to remove unnecessary clot；

1.3rd, human umbilical cord blood mononuclear cell extracts：

1.3.1, by the Cord blood (such as taking 50ml) after filtering and PBS by 1:1 ratio mixes (cumulative volume 100ml) and obtains carefully Born of the same parents' suspension, then take cell suspension 15ml to add in the 50ml centrifuge tubes equipped with Ficoll separating liquids, cell suspension divides with Ficoll Chaotropic volume ratio is 5：3, carry out centrifugation 2000rpm/min, 30min, 4 DEG C, lifting speed 1, reduction of speed degree 0；

1.3.2 human umbilical cord blood mononuclear cell layer, is exhausted as far as possible with pipette, is transferred to equipped with 5ml Cord blood MSC culture mediums 1 In 50ml centrifuge tubes and mix, obtain suspension；In addition plasma layer is extracted it is spare in another 50ml centrifuge tube, for MSC train Foster basigamy is put；

1.4th, the P0 of Cord blood MSC is commissioned to train foster

1.5th, the P1-P3 of Cord blood MSC is commissioned to train foster

(2), the extraction of Cord blood MSC excretions body, it includes the following steps：

2.1st, excretion body suspension pre-processes

2.1.2, excretion body source two：The blake bottle for having removed culture medium is paved with the PBS of 50ml, by the Cord blood equipped with PBS It is 5%CO that the P3 of MSC is put into volume fraction for Tissue Culture Flask₂Cultivated in saturated humidity incubator, hungry Cord blood MSC Cell 24h, collects the PBS in blake bottle afterwards；

2.1.3, excretion body source three

2.1.3.1, the P3 after PBS starvation is for Cord blood MSC, by adding 0.25% trypsase -0.01%EDTA digestive juices, Cell dissociation in blake bottle is got off and is collected into new 50ml centrifuge tubes, with PBS constant volumes to 20ml, takes 500 μ l cells Suspension Hematometer counts；

2.1.3.2 Cord blood MSC cell numbers, are calculated, take 6*10⁷The P3 cells of Cord blood MSC, be transferred to new 15ml from In heart pipe, centrifugation 300*g, 10min, lifting speed 9, reduction of speed degree 7 are carried out；

2.1.3.4 PBS is removed after, washing, the cell pyrolysis liquid to cell precipitation addition 1.5ml with cushioning effect, and 30min is incubated on low temperature ice bag, during which every 5min, 1min is mixed on rotation nest oscillator, resonance is swung 4 times；

2.1.3.5, it is incubated and terminates, centrifuges 20000*g again, 30min, lifting speed 9, reduction of speed degree 7, extracts whole supernatant transfers To new 15ml centrifuge tubes, 10ml is diluted to PBS, is put into -80 DEG C of refrigerator Cord blood at least 24h；

2.2nd, excretion body extracts

2.2.5 supernatant, is abandoned, dilutes sediment with PBS, as excretion body intermediate product, suspension is filtered by 0.22 μm of filter, By in suspension constant volume to a 50ml centrifuge tube, fixing fabric structure is in 25ml；

2.2.6 TRIS/ sucrose/D of the 4.5ml configured, is added in a new 50ml centrifugation bottom of the tube₂O solution, forms slow Punching pad；25ml suspensions are gently added in above cushion pad, avoid breaking through liquid level；Ultracentrifugation, rotating speed 100000* are carried out again G, 80min, lifting speed 9,9,4 DEG C of reduction of speed degree；

2.2.7 supernatant, is abandoned, contains the cushion pad of excretion body with the 5ml syringe collectings of 18G syringe needles, cushion pad is diluted with PBS To 50ml, high speed centrifugation 100000*g is carried out, 60min, lifting speed 9, by 7,4 DEG C of speed；Supernatant is removed after centrifugation as far as possible, is sunk It is excretion body to form sediment, and excretion body, which is resuspended, with the PBS of 100ml is cleaned, and carries out high speed centrifugation 100000*g, 20min, is removed Supernatant, then PBS repeated washing excretions body with 100ml, as above high speed centrifugation 2-3 times；Finally diluted and mixed with the PBS of 5ml It is even, final excretion body is obtained, optional puts it into -80 DEG C of refrigerator Cord bloods.

5. the method according to claim 1, wherein the composition of the Cord blood MSC culture mediums 1 includes：Volumetric concentration is 10% Umbilical cord blood, the GM-CSF of 15ng/ml, volumetric concentration 2%HEPES, 1% green grass or young crops of volumetric concentration-streptomysin, volumetric concentration 1% L-Glutamine, surplus IMDM basal mediums.

6. the method according to claim 1, wherein the composition of the Cord blood MSC culture mediums 2 includes：Volumetric concentration is 10% Umbilical cord blood, IL-1 β of 10ng/ml, volumetric concentration 2%HEPES, 1% mycillin of volumetric concentration, volumetric concentration 1%L- Glutamine, surplus IMDM basal mediums.

7. the method according to claim 1, wherein the formula composition of the cell pyrolysis liquid includes：The NaCl of 300mmol/L, The Tris (pH7.4) of 50mmol/L, 3mmol/L sodium pyrophosphates, 1% Triton X-100,0.1% dodecyl sulphate Sodium (SDS), the sodium β-glycerophosphate of the EDTA of 1.5mmol/L, 30mmol/L, are prepared with water.

8. the method according to claim 1, the cell pyrolysis liquid each component is dilute in proportion with the purified water Jing Guo high temperature and pressure Release and dissolve and be prepared.

9. the method according to claim 1, wherein the preparation method of the cushion pad is：30g is taken without protease sucrose, 2.4g The D of Tris alkali, 50ml₂O, it is fully mixed, then adjusts PH to 7.4 with the HCL titration of 10mol/L, uses D₂O modulates volume It is spare in 4 DEG C of refrigerator storages by 0.22 μm of filter filtering and autoclaving into 100ml.

10. a kind of solution as cushion pad, its preparation method are：Take Tris alkali, 50ml of the 30g without protease sucrose, 2.4g D₂O, it is fully mixed, then adjusts PH to 7.4 with the HCL titration of 10mol/L, uses D₂Volume is modulated into 100ml by O, is passed through 0.22 μm of filter filtering and autoclaving, it is spare in 4 DEG C of refrigerator storages.