CN107858324A - A kind of method for the extracellular vesica including excretion body secreted based on anion exchange resin adsorbing separation cell to culture medium - Google Patents
A kind of method for the extracellular vesica including excretion body secreted based on anion exchange resin adsorbing separation cell to culture medium Download PDFInfo
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Abstract
The invention discloses a kind of method for including based on Anion exchange resin separation cell culture fluid the extracellular vesica including excretion body, by extracellular vesica caused by methods described including excretion body and application thereof.The method of the present invention is simple to operate, convenient, it is only necessary to uses chromatography pipe/post and liquid-transfering gun and other common laboratory consumptive materials, it is not necessary to using ultracentrifuge, saves expense and time；The extracellular vesica purity of acquisition is high, concentration is big and can be mass-produced, and is easy to clinically further apply.
Description
Technical field
It is thin including excretion body the present invention relates to being secreted by anion exchange resin adherent cell to culture medium
The method of extracellular vesica, application and treatment by excretion body caused by methods described and the extracellular vesica comprising excretion body
Product.
Background technology
Excretion body is secreted outward by cell, belongs to a kind of extracellular vesica, and vesicle diameter is about 30-150nm, previously finds
Important physiopathology function is played in a variety of biological aspects and disease.Excretion body structure is similar to membrane structure,
The phospholipid bilayer for being about 5nm by thickness is formed, and its composition mainly comprising ceramide, cholesterol, sphingolipid and contains length
The glycerophosphatide of saturated aliphatic chain.Excretion body is rich in various protein in vesicle surface and inside, and contains multiple nucleic acid etc.
Important large biological molecule material, these large biological molecule materials can be used to the detection reflection disease such as including tumour
State.The understanding of the biological function of excretion body at present is not also very abundant, but numerous experimental datas shows, excretion body exists
Played an important role in vital movement, such as cell-cell communication, antigen presentation and protein core acid substance exchange.Especially
Excretion body participate in cell communication in terms of it is particularly important, excretion body by participate in iuntercellular immune signal, angiogenesis, propagation and
The functions such as differentiation, directly affects the generation of cancer, nerve degenerative diseases etc..More and more evidence shows, excretion
Body directly take part in the generation of tumour, including angiogenesis, immunosupress, transfer etc..Therefore can be by holding in excretion body
The detection of thing, realize early diagnosis and prognosis evaluation of minimally invasive or noninvasive disease organism target signal etc..Wherein, stem cell
The excretion body of secretion finds there is therapeutic effect in multiple disease areas, or even has researcher to think stem cell mainly by outer
Secrete body and play its therapeutic action for the medicative cell factor of carrier secreting outside tool and non-coding nucleic acid etc..To sum up institute
State, how isolated enough and high purity excretion bodies turn into is asked primary face of its studies and clinical application at present
Topic.
Mescenchymal stem cell is a kind of multiple-effect stem cell, can directed differentiation be fat under certain cytokine environment
The various kinds of cell such as cell, osteocyte, cartilage cell, myocyte, nerve cell, cardiac muscle cell, beta Cell of islet, and can lead to
Cross secretory protein or cell factor and immunosupress plays a role indirectly, therefore turn into the focus in regenerative medicine always.Between
Mesenchymal stem cells can be divided into Gegenbaur's cell, and the marrow of allogeneic carries out the clinical examination of the infull disease of transplantation treatment Osteogenic developmental
Test and carried out.And the immunoregulation capability of mescenchymal stem cell is attributed to the fact that, in preclinical test and treatment cardiovascular disease, degenerate
Nerve disease, Crohn disease, graft versus host disease and autoimmune disease clinical test on widely apply.Mesh
Preceding discovery will not form tumour after transplantation treatment, it was demonstrated that the cell therapy of mescenchymal stem cell is effective and safe.In addition
In myocardial infarction, diabetes, spinal injury, osteoarthritis, multiple sclerosis, alpastic anemia, parkinson's syndrome, brain
Damage and other disease areas have also carried out relevant clinical experiment, prospect Worth Expecting.
The most frequently used separation method of extracellular vesica and excretion body of the prior art is ultracentrifugation either density
Gradient centrifugation, but these methods need to be equipped with the high scheming of various ultrahigh speeds, and this equipment and supporting hypervelocity from
The price of heart pipe is very expensive, and the time of centrifugation is also very long, it is necessary to Loss Research person's substantial amounts of time；Ultracentrifuge
Using needing individually to train user, and there is very strict operation sequence, improper use can cause sample even instrument
The damage of device；In addition, the excretion bulk concentration that simple hyperfiltration process is collected tends not to reach very high, for needing higher concentration
The detection of excretion body is also a kind of limitation with applications such as treatments.Therefore, it is also desirable to seek the separation of new low-cost high-efficiency
The method of the outer vesica of purifying cells and excretion body, to overcome defect present in prior art.
The general principle of ion exchange chromatography is as follows：The stationary phase of ion-exchange chromatography is ion-exchanger, it
It is that certain electric charge base in certain chemical reaction covalent bond is passed through by a kind of inert polymer polymer substrate not soluble in water
What group was formed.Ion-exchanger can be divided into three parts：High molecular polymer matrix, charge groups and ion balance.Electric charge base
Group and high molecular polymer covalent bond, form a powered ion-exchangeable group.Ion balance is to be incorporated into
Counter ion on charge groups, reversible exchange reaction can occurs with other ionic groups in solution in it.Ion balance band
With the ionic group of positively charged exchange interaction, referred to as cation-exchanger can occur for the ion-exchanger of positive electricity；Ion balance band
With negatively charged ionic exchange interaction, referred to as anionite occur for the ion-exchanger of negative electricity.Under certain condition,
Certain ionic group in solution can cement out ion balance, and be attached to by charge groups in stationary phase, and put down
Weighing apparatus ion then enters mobile phase, and here it is the basic displacement of ion-exchange chromatography reaction.Pass through repeatedly putting at different conditions
Change reaction, it is possible to which ionic group different in solution is separated.Simply introduced by taking anionite as an example below from
Sub- displacement chromatography is basically separated process.
The charge groups positively charged of anionite, fill column equilibration after, with the electronegative balance in cushioning liquid from
Son combines.May there are positive charged group, negative electricity group and neutral group in solution to be separated.After sample-adding, negative electricity group can be with putting down
The ion that weighs carries out reversible displacement reaction, and is attached on ion-exchanger.And positive charged group and neutral group then can not with from
Sub- exchanger combines, and flows out and is removed with mobile phase.By selecting suitable type of elution and eluent, it is strong such as to increase ion
The gradient elution of degree.With the increase of eluent ionic strength, the ion in eluent can be progressively with being incorporated in ion exchange
Various negative electricity groups in agent are swapped, and various negative electricity groups are cemented out, and are flowed out with eluent.With ion-exchanger
The small negative electricity group of adhesion first is replaced out, and the ionic strength ability that the needs strong with ion-exchanger adhesion are higher
It is replaced out, so various negative electricity groups will be progressively eluted by its order with ion-exchanger adhesion from small to large
Get off, so as to reach separation purpose.
The present invention, which wishes to be used as new low cost by anion chromatography method, easily isolates and purifies extracellular vesica
And the method for excretion body, the separation concentration of excretion body and further large-scale production excretion body are improved, thinks the excretion of next step
Body function is tested and possible clinical practice provides help.
The content of the invention
On the one hand, provide between separation includes and fill it is an object of the invention to overcome the shortcomings of the prior art part
The method for the extracellular vesica including excretion body that cell including matter stem cell is secreted to culture medium, method of the invention
The extracellular vesica that more easily can be secreted the cell of separate sources in serum free medium extracts separation.Wherein, no
With the cell in source, to include but is not limited to all kinds of mescenchymal stem cells, embryonic stem cell, inductive pluripotent stem cells and adult thin
At least one of born of the same parents.
The technical solution adopted by the present invention is：One kind is secreted based on anion exchange resin adsorbing separation cell to culture medium
The extracellular vesica including excretion body method, comprise the following steps：
1) 50-90% is reached using the cell of the outer vesica of cell culture complete medium culture secretory cell, cell density
When, culture medium is changed to serum free medium, collects the serum free medium rich in extracellular vesica；
2) using equilibrium liquid cleaning chromatography pipe, anion exchange resin is added into chromatography pipe, is formed in bottom and carries positive electricity
The beds of precipitation of lotus；Resin is balanced using equilibrium liquid；Resin is cleaned using cleaning fluid；The serum free medium of collection is added into chromatography
Post；Eluted using eluent, and collect eluent；
3) eluent of collection is dialysed, the extracellular vesica purified.
Preferably, cell culture complete medium is the serum added with 0.1-20% in the step (1), 0.1-20mmol/L
Glu and the α-MEM culture mediums of 50-1000000 units/L penicillin and 50-1000000 units/L streptomysins；Institute
It is fetal bovine serum to state serum.
Preferably, the cell of the outer vesica of secretory cell includes filling between multipotency induction source of human stem cell in the step (1)
Matter stem cell, the mescenchymal stem cell in human embryo stem cell source, human marrow mesenchymal stem cell and human umbilical cord mesenchymal are dry thin
At least one of born of the same parents, human adipose-derived stem cell, embryonic stem cell, inductive pluripotent stem cells and adult cell.
Preferably, the planting density of the cell in the step (1) is 50-10000/cm2, use complete medium culture
Time be 1-10 days.
Preferably, the formula of serum free medium is to contain HT additives, 0.1-10mmol/L L- in the step (1)
Glutamine, 0.1-10g/L D- (+)-glucose, 0.1-100mL/L 100 × MEM nonessential amino acids, 0.1-
The CD-CHO culture mediums of 1000mL/L 100 × MEM vitamin solution.
Preferably, when cell density reaches 50-90% in the step (1), cleaned, added with phosphate balance liquid
Serum free medium, after 1-24 hours, new serum free medium being added, 12-48 collects serum free medium after hour, this
For the culture medium rich in extracellular vesica.
Preferably, the serum free medium obtained in the step (1) operates in Biohazard Safety Equipment, be sub-packed at a high speed from
Heart pipe.Centrifugal force is arranged to 0.1-169000 × G, a length of 0.1-100 minutes during centrifugation, collected after centrifugation supernatant, discards thin
The precipitation that born of the same parents and cell fragment are formed, collect serum free medium and be used for subsequent experimental.
Preferably, in the step (2) equilibrium liquid be containing 0.1-500mM phosphate, 0.1-500mM sodium chloride it is water-soluble
Liquid.It is highly preferred that equilibrium liquid is the aqueous solution containing 50mM phosphate, 50mM sodium chloride in the step (2).
Preferably, in the step (2) cleaning fluid be containing 0.1-500mM phosphate, 1-1000mM sodium chloride it is water-soluble
Liquid.It is highly preferred that cleaning fluid is the aqueous solution containing 50mM phosphate, 100mM sodium chloride in the step (2).Preferably, originally
It is ultra-pure water to invent the water that uses, the ultra-pure water for collect the water after evaporating and by 0.1-2 μm of filter membrane with reach it is sterile and
Without large particulate matter.
Preferably, equilibrium liquid cleaning chromatography pipe is used in the step (2)：Chromatography infratubal port is broken to short, hanging dropwise addition
0.1-100mL equilibrium liquids, treat that liquid drips off.
Preferably, chromatography pipe chromatographs pipe, high 0.1-100cm, 0.1-10*0.1- for Econo-Pac in the step (2)
The polypropylene post of 100cm sizes, bed volume about 0.1-200mL.
Preferably, before anion exchange resin being added into chromatography pipe in the step (2), resin 0.1-100% second
Alcohol dissolves, and is slowly rocked using preceding, interlaminar resin connection is avoided damage to, using resin suspension 0.1-10mL, wherein containing resin
0.1-10mL, treat that liquid flow is complete, it is seen that chromatography pipe lower floor forms resin bed.
Preferably, 0.1-1000mL equilibrium liquids balance resin is used in the step (2).
Preferably, 0.1-1000mL cleaning fluids cleaning resin is used in the step (2).
Preferably, eluent described in the step (2) is containing 0.1-500mM phosphate, 10-5000mM sodium chloride
The aqueous solution.Present inventor has found after further research, and the eluent described in the step (2) is contains 50mM phosphoric acid
Salt, 500mM sodium chloride the aqueous solution when, it is eluted, and effect is more preferable than other eluents in above-mentioned formula range, and what is obtained is outer
Secrete bulk concentration maximum, purity highest.
Preferably, the operation of the dropwise addition liquid formed in the step (2) after resin bed is, it is necessary to vacantly operate, and act
Softly, avoid influenceing resin bed.
Preferably, 0.1-100mL eluents are used in the step (2) every time, and in chromatography pipe lower end 0.2-50mL
Centrifuge tube collects eluent, and by 0.1-2 μm of filter membrane to reach sterile and without large particulate matter.
The invention provides the extracellular vesica obtained using method described above.
The invention provides extracellular vesica described above in treatment inflammation, the medicine or product that regulation is immunized is prepared
Purposes.Extracellular vesicle formation including the body including excretion can be applied to including but not limited to immunological regulation, tissue is repaiied
Multiple regeneration, beauty and skin care Related product or any acute or chronic inflammation and the damage relevant with being immunized and allergy etc..
According to an embodiment of the invention, the extracellular vesica protein quantification, can detect spy by detecting vesicle diameter
Determine surface antigen expression, and/or by the absence of particular surface antigen presentation identify.Such as the cell external capsule in the present invention
The detectable CD63 and CD81 antigen presentations of bubble, but CD9 antigens are not expressed on its surface.
The Econo-Pac chromatographies that anion chromatography column combination of the present invention includes belonging to polypropylene material are managed, 0.1-100cm,
0.1-10*0.1-100cm sizes, and the resin with positive charge.
The present invention can be greatly reduced in separation process due to the height using ultracentrifuge using anion chromatography method
High expense and operation difficulty.
Relative to prior art, beneficial effects of the present invention are：
The present invention based on anion exchange resin adsorbing separation cell to culture medium secrete including excretion body
The method of extracellular vesica is simple to operate, convenient, it is only necessary to can be grasped using anion chromatography pipe, post and simple experiment room articles for use
Make, it is not necessary to using ultra centrifugation techniques, save expense and time；The extracellular vesica purity of the cell secretion of acquisition is high, dense
Degree is big and can be mass-produced, and is easy to further clinical practice.
Brief description of the drawings
Fig. 1 is the side for the excretion body that anion exchange resin adsorbing separation mescenchymal stem cell of the present invention is secreted to culture medium
The technology path schematic diagram of one embodiment of method；
Fig. 2 is the basic principle schematic of anion chromatography method of the present invention；
Fig. 3 is the mescenchymal stem cell of the multipotency differentiation of stem cells in urothelial source in the embodiment of the present invention 1
Microphotograph, it is the mescenchymal stem cell of normal morphology；
Fig. 4 does nano-particle diameter detection by taking the 4th eluent in the embodiment of the present invention 1 after diluting 100 times
And Electronic Speculum qualification result (Nanosight).The visible separated excretion body particle diameter peaks of Fig. 4 A are 115nm, and unimodal
Distribution, meets excretion body expected results.Fig. 4 B are to amplify excretion body integrality under 46000 times of Electronic Speculum, under Electronic Speculum outside observable
Secrete somatocyst balloon-shaped structure, it is seen that in the visual field, size is about diameter 100nm for uniform vesica branch, meets excretion body electricity
Mirror outcome expectancy；
Fig. 5 is excretion body diameter branch, protein quantification and surface marker in the sample collected in the embodiment of the present invention 1
CD63 enzyme linked immunosorbent assay (ELISA) (ELISA) result displaying.The visible sample excretion body particle diameter branches of Fig. 5 A concentrate on
Near 120nm；The peak of visible protein content and CD63 expression quantity peak are in the 4th eluent in Fig. 5 B, in summary, I
Select excretion body included in the 4th eluent to complete follow-up functional experiment；
Fig. 6 is that the acute inflammation mouse model of ovalbumin (Ovalbumin, OVA) induction is utilized in the embodiment of the present invention 2
The function of preliminary identification excretion body.Fig. 6 A：The sample extracting method of the acute inflammation model mouse of OVA inductions.It is visible in Fig. 6 B
Excretion body can significantly reduce the leading acute inflammatory reactions of Th1.Visible excretion body can significantly reduce leading acute of Th2 in Fig. 6 C
Inflammatory reaction.EVs:Excretion body.
Embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to text in the art
Offer described technology or condition or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument,
For the conventional products of acquisition purchased in market can be passed through.
As shown in figure 1, it is divided for the present invention based on anion exchange resin adsorbing separation mescenchymal stem cell to culture medium
The schematic flow sheet of the method one embodiment for the extracellular vesica secreted, the basic principle schematic of anion chromatography method of the present invention
As shown in Fig. 2 this method comprises the following steps：
Step S1：Mescenchymal stem cell is planted according to certain cell density, with mescenchymal stem cell complete medium culture
To certain density；
Preferably, filled using mescenchymal stem cell complete medium in a manner of largely amplification property between a variety of sources of culture
Matter stem cell, planting density 50-10000/cm2, the time using mescenchymal stem cell complete medium culture is 4-6 days,
Cell density is observed until 60-70%；
Step S2：Serum free medium is changed, changes serum free medium after certain time again, then harvest is rich in outer
Secrete the culture medium of body；
Preferably, the formula of the step 1) serum free medium is added with HT additives (100 μM of hypoxanthine
Sodium, 16 μM of thymidines), 4-8mmol/L Glu, 2g/L D- (+)-glucose, 10mL 100 × MEM is non-must
Palpus amino acid, the CD-CHO culture mediums of 10mL 100 × MEM vitamin solution；Cell density under microscope reaches 60-70%
When, add 30-40mL/175cm2Serum free medium.After 6 hours, the serum free medium of harvest addition for the first time, add
New 40mL/175cm2Serum free medium, the serum free medium of the addition of harvest second after 42 hours, this is rich in outer
Secrete the culture medium of body.
Step S3：Chromatography pipe is cleaned using equilibrium liquid；
By optimization, the aqueous solution of the equilibrium liquid selection containing 50mM phosphate, 50mM sodium chloride, soft liquid feeding 4mL, avoid
Form bubble.
Step S4：Resin suspension is added into chromatography pipe, the beds of precipitation with positive charge is formed in bottom, ultimately forms tree
Lipid layer；
Preferably, soft liquid feeding 6mL is needed in step S4, resin bed is avoided damage to or forms bubble.
Step S5：Resin is balanced using equilibrium liquid.
By optimization, equilibrium liquid selects the aqueous solution containing 50mM phosphate, 50mM sodium chloride, is needed in step S5 soft
Liquid feeding 12mL, avoid damage to resin bed or form bubble.
Step S6：Chromatographic column is cleaned using cleaning fluid；
By optimization, cleaning fluid selects the aqueous solution containing 50mM phosphate, 100mM sodium chloride, is needed in step S6 light
Soft liquid feeding 40mL, avoid damage to resin bed or form bubble.
Step S7：The culture medium that will be enriched in excretion body adds chromatographic column；
Preferably, soft liquid feeding 160mL is needed in step S7, resin bed is avoided damage to or forms bubble.
Step S8：Eluent 1 and eluent 2 are gradually added, and eluent is collected respectively with centrifuge tube.
By optimization, the aqueous solution of the eluent selection containing 50mM phosphate, 500mM sodium chloride, 1mL elutions are gradually added
Liquid 1 (8 pipe) and eluent 2 (4 pipe), and eluent is collected respectively with 1.5mL centrifuge tubes.
Step S9：Eluate sample is dialysed
Preferably, dialysis tubing is cut into 10cm length, enough phosphate buffers (PBS) is connected to after sterilization treatment
Between sample, it is placed in 4 DEG C of agitator flat board overnight, reclaims sample.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
(1) method for the excretion body secreted based on anion exchange resin adsorbing separation mescenchymal stem cell to culture medium
Being secreted based on anion exchange resin adsorbing separation mescenchymal stem cell to culture medium for the present embodiment is extracellular
The method of vesica, comprises the following steps：
1. done using the mesenchyma in a variety of sources of mescenchymal stem cell complete medium culture in a manner of largely amplification property
Cell, planting density 50-10000/cm2, the time using mescenchymal stem cell complete medium culture is 4-6 days, observation
Cell density is until 60-70%；The mescenchymal stem cell of the multipotency differentiation of stem cells in urothelial source it is micro-
Mirror photo is as shown in Figure 3；
2. the cell density under microscope reaches 60-70%, 30-40mL/175cm is added2Serum free medium.6 is small
Shi Hou, the serum free medium of harvest addition for the first time, adds new 40mL/175cm2Serum free medium, after 42 hours
The serum free medium of second of addition of harvest, this is the culture medium rich in excretion body, collects (the name of 1mL serum free mediums
For IP)；
3. using 4mL equilibrium liquids cleaning chromatography pipe, soft liquid feeding, bubble is avoided the formation of；
4. 6mL resins suspension is added into chromatography pipe, the beds of precipitation with positive charge are formed in bottom, ultimately form 4mL
Resin bed；
5. use 12mL equilibrium liquids balance resin.
6. culture mediums of the 160mL rich in excretion body is added into chromatographic column, (the name of 1mL liquid is collected in instillation process midway
For FT)；
7. collect 1mL liquid (being named as W) in instillation process midway using 40mL cleaning fluids cleaning chromatographic column；
8. gradually adding 1mL eluents 1 (8 pipe) and eluent 2 (4 pipe), and elution is collected respectively with 1.5mL centrifuge tubes
Liquid.
9. the 4th eluent that will be required for next step functional experiment is dialysed.Dialysis tubing is cut into 10cm length,
Enough phosphate buffers (PBS) are connected to after sterilization treatment between sample, are placed in 4 DEG C of agitator flat board overnight,
It is 115nm to reclaim sample and Electronic Speculum identification (Fig. 4), the visible separated excretion body particle diameter peaks of Fig. 4 A, and Unimodal Distribution,
Meet excretion body expected results.Fig. 4 B are to amplify excretion body integrality under 46000 times of Electronic Speculum, observable excretion somatocyst under Electronic Speculum
Balloon-shaped structure, it is seen that in the visual field, size is about diameter 100nm for uniform vesica branch, meets excretion body Electronic Speculum result
It is expected that.
(2) protein quantification of excretion body and surface antigen CD63 ELISA are identified
The protein quantification of excretion body：
1) prepares working solution:According to standard items and sample size, add 1 volume BCA reagents B by 50 volume BCA reagent As
(50:1) appropriate BCA working solutions are prepared, are fully mixed.
2) dilution standards product：Take 10 microlitres of standard items to be diluted to 100 microlitres with PBS, make final concentration of 0.5mg/ml.Will
Standard items are added in the protein standard sample wells of 96 orifice plates by 0,1,2,4,8,12,16,20 microlitre, add PBS to supply to 20 microlitres.
3) adds proper volume sample to add PBS to 20 microlitres into the sample well of 96 orifice plates.
4) each holes of add 200 microlitres of BCA working solutions, and 37 DEG C are placed 30 minutes.
5) is cooled to room temperature, determines A562 with ELIASA, protein concentration is calculated according to standard curve.MSC surface
Marker CD63 ELISA authentication methods：
1) CD63 primary antibodies (kind is rabbit-anti mouse) are diluted 1000 times, add 200 μ L in each hole is incubated half at 37 DEG C
Hour；
2) blank well mark-on sheet or standard dilutions, mark-on sheet or various concentrations standard items (100 μ in remaining respective aperture
L/ holes), reacting hole is sealed with shrouding gummed paper, 37 DEG C are incubated 90 minutes；
3) board-washing 5 times.
4) blank well adds biotinylated antibody dilution, and remaining hole adds biotinylated antibody working solution (100 μ/l holes),
Reacting hole is sealed with shrouding gummed paper, 37 DEG C are incubated 60 minutes；
5) board-washing 5 times.
6) blank well adds enzyme combination diluent, and remaining hole adds enzyme conjugates working solution (100 μ/hole), with shrouding gummed paper
Reacting hole is sealed, 37 DEG C are incubated 30 minutes；
7) board-washing 5 times.
8) 100 μ of chromogenic substrate/hole is added, 37 DEG C of lucifuge is incubated 15 minutes；
9) 100 μ of terminate liquid/hole is added, OD450 values is detected after mixing at once, obtains result (Fig. 5), the visible samples of Fig. 5 A
Excretion body particle diameter branch is concentrated near 120nm；The peak of visible protein content and CD63 expression quantity peak exist in Fig. 5 B
In the 4th eluent, eluent now used is the aqueous solution containing 50mM phosphate, 500mM sodium chloride, in summary,
We select the excretion body included in the 4th eluent to complete follow-up functional experiment.
Embodiment 2
(1) macrophage function detects
1.1 experiment packets (totally 5 groups)：
A. Normal group：Complete medium (DMEM+10%FBS, 300 μ L/well, similarly hereinafter)
B. positive controls：Complete medium+10ng/mL Ovalbumin (OVA)
C. dexamethasone (DEX) treatment group：Complete medium+10ng/mL OVA+1 μM DEX
D. excretion body treatment group (5 × 108/ mL excretions body)：Complete medium+10ng/mL OVA+5 × 108/ mL excretion bodies
E. excretion body treatment group (2.5 × 109/ mL excretions body)：Complete medium+10ng/mL OVA+2.5 × 109Outside/mL
Secrete body
1.2 cell kind plates：The cells of recovery RAW 264.7, by cell according to 1 × 10548 well culture plates are planted in, every group sets
3 multiple holes, 15 holes are planted altogether, 300 μ L complete mediums are added per hole, it is overnight to be positioned over cell culture incubator culture.
1.3 excretion body anti-inflammatory properties are tested：With 1.5mL EP pipes according to step 2.1 each to assemble 1mL nutrient solutions processed, so
The complete medium in original each hole is absorbed afterwards, then nutrient solution configure assigned in every group of 3 multiple holes, often hole
300 μ L, cells and supernatant is collected after being positioned over cell culture incubator culture 4h, mouse IL-6 expression is detected with ELISA,
Excretion body can suppress macrophages secrete IL-6 in vitro.
(2) OVA inducing mouses systemic inflammatorome model
The sample extracting method of the acute inflammation model mouse of visible OVA inductions in Fig. 6 A.
2.1 Th1 dominate inflammatory model
Experiment packet (every group of 4-6 is only)：
A. Normal group：PBS sensitization+PBS atomizations excite (alternative)
B. positive controls：40 μ g OVA sensitization+0.5%OVA atomizations excite
C. excretion body treatment group (excretion body)：40 μ g OVA sensitization+0.5%OVA atomizations excite+200 μ L (10 X 109) outside
Secrete body treatment
Utilized in the 0th and the 7th day and OVA and not formula Freund's complete adjuvant sensitized mice is subcutaneously injected, and in the 18th, 19,20,21 day
Airway of mice inflammation is excited using the 0.5%OVA of atomization；Every mouse passes through tail vein injection in exciting the previous day (the 17th day)
200 μ L liquid solutions containing excretion, 4 hours anesthesia materials after last day excites.
A. mouse anesthesia method：1% yellow Jackets are injected intraperitoneally；
B. mouse materials sample：Bronchoalveolar lavage fluid (BALF) or other.
C. inflammatory cell quantity in BALF is detected with cell dyeing, and utilizes proinflammatory factor in ELISA method detection BALF
The expression of IFN-γ etc., visible excretion body can significantly reduce the leading acute inflammatory reactions of Th1 in Fig. 6 B.
2.2 Th2 dominate inflammatory model：
Experiment packet (every group of 4-6 is only)：
A. Normal group：PBS sensitization+PBS atomizations excite (alternative)
B. positive controls：40 μ g OVA sensitization+0.5%OVA atomizations excite
C. excretion body treatment group (excretion body)：40 μ g OVA sensitization+0.5%OVA atomizations excite+200 μ L (10 X 109) outside
Secrete body treatment
In the 0th, the 7th and the 14th day intraperitoneal injection OVA and aluminium hydroxide sensitized mice, and in the 21st, 22,23,24 day profit
Airway of mice inflammation is excited with the 0.5%OVA of atomization；Every mouse passes through tail vein injection in exciting the previous day (the 20th day)
200 μ L liquid solutions containing excretion, 4 hours anesthesia materials after last day excites.
A. mouse anesthesia method：1% yellow Jackets are injected intraperitoneally；
B. mouse materials sample：Bronchoalveolar lavage fluid (BALF) or other.
C. inflammatory cell quantity in BALF is detected with cell dyeing, and utilizes proinflammatory factor in ELISA method detection BALF
IL-4 etc. expression, visible excretion body can significantly reduce the leading acute inflammatory reactions of Th2 in Fig. 6 C.
Claims (10)
1. it is a kind of secreted based on anion exchange resin adsorbing separation cell to culture medium it is extracellular including excretion body
The method of vesica, it is characterised in that：Comprise the following steps：
1), will when cell density reaches 50-90% using the cell of the outer vesica of cell culture complete medium culture secretory cell
Culture medium is changed to serum free medium, collects the serum free medium rich in extracellular vesica；
2) using equilibrium liquid cleaning chromatography pipe, anion exchange resin is added into chromatography pipe, formed in bottom with positive charge
The beds of precipitation；Resin is balanced using equilibrium liquid；Resin is cleaned using cleaning fluid；The serum free medium of collection is added into chromatographic column,
Eluted using eluent, and collect eluent；
3) eluent of collection is dialysed, the extracellular vesica purified.
2. according to the method for claim 1, it is characterised in that：In the step (1) cell culture complete medium be added with
0.1-20% serum, 0.1-20mmol/L Glu and 50-1000000 unit/L penicillin and 50-1000000 are mono-
α-MEM the culture mediums of position/L streptomysins；The serum is fetal bovine serum.
3. according to the method for claim 1, it is characterised in that：The cell bag of the outer vesica of secretory cell in the step (1)
Include and filled between the multipotency induction mescenchymal stem cell of source of human stem cell, the mescenchymal stem cell in human embryo stem cell source, people's marrow
Matter stem cell and human umbilical cord mesenchymal stem cells, human adipose-derived stem cell, embryonic stem cell, inductive pluripotent stem cells and adult are thin
At least one of born of the same parents.
4. according to the method for claim 1, it is characterised in that：The planting density of cell in the step (1) is 50-
10000/cm2, the time using complete medium culture is 1-10 days.
5. according to the method for claim 1, it is characterised in that：In the step (1) formula of serum free medium be containing
Have a HT additives, 0.1-10mmol/L Glu, 0.1-10g/L D- (+)-glucose, 0.1-100mL 100 ×
MEM nonessential amino acids, the CD-CHO culture mediums of 0.1-1000mL 100 × MEM vitamin solution.
6. according to the method for claim 1, it is characterised in that：When cell density reaches 50-90%, put down with phosphate
The liquid that weighs cleans, and adds serum free medium, after 1-24 hours, adds new serum free medium, 12-48 collects nothing after hour
Blood serum medium, this is the culture medium rich in extracellular vesica.
7. according to the method for claim 1, it is characterised in that：Equilibrium liquid is to contain 0.1-500mM phosphorus in the step (2)
The aqueous solution of hydrochlorate, 0.1-500mM sodium chloride；Cleaning fluid is to contain 0.1-500mM phosphate, 1- in the step (2)
The aqueous solution of 1000mM sodium chloride.
8. according to the method for claim 1, it is characterised in that：Eluent described in the step (2) is to contain 0.1-
The aqueous solution of 500mM phosphate, 10-5000mM sodium chloride.
9. the extracellular vesica obtained using the method as described in claim 1-8 is any.
10. purposes of the extracellular utricule as claimed in claim 9 in the medicine for preparing treatment inflammation.
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| CN108918228A (en) * | 2018-06-04 | 2018-11-30 | 北京全式金生物技术有限公司 | Excretion body in serum or blood plasma prepares kit and excretion preparation |
| CN109825472A (en) * | 2019-03-01 | 2019-05-31 | 易春 | A kind of extracting method and kit of extracellular vesica |
| CN109929802A (en) * | 2019-04-02 | 2019-06-25 | 武汉理工大学 | The methods and applications of room adsorbing separation excretion body under a kind of orifice plate upper chamber culture cell based on Transwell |
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| CN113092642A (en) * | 2021-03-30 | 2021-07-09 | 苏州爱宝德生物科技有限公司 | A quick extraction element for extracellular vesicles |
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| CN108918228A (en) * | 2018-06-04 | 2018-11-30 | 北京全式金生物技术有限公司 | Excretion body in serum or blood plasma prepares kit and excretion preparation |
| CN109825472A (en) * | 2019-03-01 | 2019-05-31 | 易春 | A kind of extracting method and kit of extracellular vesica |
| WO2020191369A1 (en) * | 2019-03-21 | 2020-09-24 | Codiak Biosciences, Inc. | Process for preparing extracellular vesicles |
| CN109929802A (en) * | 2019-04-02 | 2019-06-25 | 武汉理工大学 | The methods and applications of room adsorbing separation excretion body under a kind of orifice plate upper chamber culture cell based on Transwell |
| CN111621471A (en) * | 2020-06-12 | 2020-09-04 | 成都世联康健生物科技有限公司 | Extraction method and application of soft tissue extracellular vesicles |
| CN112831457A (en) * | 2021-02-07 | 2021-05-25 | 辽宁润基生物科技有限公司 | Method for separating and concentrating exosome |
| CN113092642A (en) * | 2021-03-30 | 2021-07-09 | 苏州爱宝德生物科技有限公司 | A quick extraction element for extracellular vesicles |
| CN114591892A (en) * | 2021-08-30 | 2022-06-07 | 天九再生医学(天津)科技有限公司 | Exosome separation and purification method |
| CN113533589A (en) * | 2021-09-16 | 2021-10-22 | 天九再生医学(天津)科技有限公司 | Method for analyzing exosomal charge heterogeneity |
| CN114457004A (en) * | 2021-12-23 | 2022-05-10 | 江苏为真生物医药技术股份有限公司 | Exosome separation method in biological sample, kit and application thereof |
| CN114457004B (en) * | 2021-12-23 | 2024-04-16 | 江苏为真生物医药技术股份有限公司 | Method for separating exosomes in biological sample, kit and application thereof |
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