CN109929802A - The methods and applications of room adsorbing separation excretion body under a kind of orifice plate upper chamber culture cell based on Transwell - Google Patents
The methods and applications of room adsorbing separation excretion body under a kind of orifice plate upper chamber culture cell based on Transwell Download PDFInfo
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Abstract
The present invention relates to a kind of methods and applications of room adsorbing separation excretion body under orifice plate upper chamber culture cell based on Transwell, belong to field of biomedicine technology.The present invention prepares the water-soluble polymer solution containing a large amount of amino cations first, and room under Transwell orifice plate is transferred to after sterilizing；Then the cell with secretion excretion ability of immigrants is transferred to the Transwell orifice plate upper chamber equipped with complete medium and carries out cell culture；Since electrostatic and osmotic pressure act on while secreting excretion body in upper ventricular cell hatching process, excretion body passes through orifice plate and is clenched by lower indoor polymer, the small particle impurity for being mixed into lower room is removed by centrifugal treating again, to realize the spontaneous isolated purpose of excretion body, separative efficiency is high.In addition, the present invention can also can further be separated excretion body by deprotonation and centrifugal treating according to different experiment purposes from material, it is convenient for subsequent analysis of molecules and application.
Description
Technical field
The invention belongs to field of biomedicine technology, it is more particularly related to which a kind of be based on transwell orifice plate
The methods and applications of room adsorbing separation excretion body under upper chamber culture cell.
Background technique
Excretion body is found for the first time in nineteen eighty-three, is by cell active secretion into extracellular matrix, is in saucer shape or one
The bioactivity phospholipid bilayer vesicles of side recess, diameter carry the objects such as protein, lipid, nucleic acid in 30-15nm
Matter can reflect physiology, the pathologic condition of derived cell, play important physiopathology in a variety of biological aspects and disease
Function.Recent researches show that excretion body rises in cell-tocell transmitting, antigen presentation and the exchange of protein core acid substance etc.
To important role.Especially excretion body participates in the functions such as iuntercellular immune signal, angiogenesis, increment and differentiation, direct shadow
The generation of tumour, neurodegenerative disease etc. has been rung, thus excretion body is considered as analyzing the fingerprint of biological information feature.So
And the root problems such as mechanism of action about excretion body biology are not still solved effectively.Thus need in depth to study
The unique biological structure of excretion body and function inquire into the relevant biological mechanism of these structures.But to the excretion of separate sources
It is first of threshold for hindering the research of excretion body that body, which carries out separating-purifying,.Therefore the effective method of purification research of excretion body is compeled
The eyebrows and eyelashes.
Current existing excretion body separation method is mainly physics and Biological characteristics possessed by foundation excretion body itself,
1 is specifically included that, based on the different centrifugal method of excretion volume density, such as differential centrifugation, density-gradient centrifugation method, ultracentrifugation
Method etc.；2, the filter method based on excretion body size, such as ultrafiltration, molecular exclusion chromatography；3, affine in immunity crawl and PEG
The three classes method such as precipitation reagent box.Although these separation methods provide great convenience for research excretion body, also all deposit
In more obvious disadvantage.Supercentrifugation is the current separation most common method of excretion body, has easy to operate, and yield is suitable
In, the advantages such as it is widely used.The shortcomings that supercentrifugation is that centrifuge instrument is at high cost, and time-consuming, and repeated centrifugation may damage
Bad vesica membrane structure, and contain a large amount of protein contamination in obtained excretion body.Although secondly, filter membrane filtration method has had
A series of problems, such as product is released, but protein blocking fenestra can be generated when separation, and excretion body ruptures.Molecular-exclusion chromatography
Rule needs special instrument, and extremely time-consuming.Although the method for affine in immunity crawl can obtain the excretion of higher degree
Body, but use scope is small, while at high cost, low output is also not suitable for being widely applied.The PEG precipitation method are that current cost is minimum,
It is easy to operate, it can also be used to large capacity sample.But problem is also more: for example the sedimentation time is longer, in precipitating impurity protein compared with
More, purity and the rate of recovery are low, and granular size is uneven, and the polymer of introducing is difficult to the disadvantages of removing.
Based on the above reasons, the application is proposed.
Summary of the invention
In view of the problems of the existing technology, the purpose of the present invention is to provide one kind to be based on Transwell orifice plate upper chamber
Cultivate the methods and applications of room adsorbing separation excretion body under cell.The method of the present invention may be implemented in spontaneous during cell culture
Ground separates excretion body, and separative efficiency is high.
In order to realize above-mentioned first purpose of the invention, The technical solution adopted by the invention is as follows:
A method of based on room adsorbing separation excretion body under Transwell orifice plate upper chamber culture cell, prepared first containing big
The water-soluble polymer solution for measuring amino cation, is transferred to room under Transwell orifice plate after sterilizing；Then there will be secretion outer
The cell for secreting ability of immigrants is transferred to the Transwell orifice plate upper chamber equipped with complete medium, and carries out cell under low oxygen conditions
Culture；Since electrostatic interaction and osmotic pressure act on while secreting excretion body under hypoxemia stimulation in upper ventricular cell hatching process,
Excretion body passes through orifice plate and is clenched by lower indoor polymer；After cell culture, collects lower room polymer solution and carry out the
The processing of one low-speed centrifugal, obtains the polymer solution containing excretion body.
Further, above-mentioned technical proposal further includes that the polymer solution by gained containing excretion body successively carries out deprotonation
Change, the processing of the second low-speed centrifugal, it is final to collect the step of obtaining purifying excretion body.
Further, above-mentioned technical proposal, the Transwell orifice plate are to separate with upper and lower room and by certain pore size
A kind of device.The aperture of Transwell orifice plate of the present invention can be not construed as limiting, as long as not influencing adsorbing separation of the invention
Process, such as Transwell orifice plate can be Transwell-6 plate, Transwell-12 orifice plate or Transwell-24
Any one in orifice plate etc..
Further, above-mentioned technical proposal, the water-soluble polymer solution be water-solubility chitosan derivative solution,
Any one in gelatin solution, amination derivatives of hyaluronic acids solution, aminated alginic acid sodio-derivative solution etc.,
In: the chitosan derivatives are preferably carboxymethyl chitosan；The derivatives of hyaluronic acids can be de- for hyaluronic acid, part
Any one in acetyl group hyaluronic acid, hyaluronic acid methyl esters, acetylation hyaluronic acid etc., preferably hyaluronic acid；It is described
Sodium alginate derivative can be any one in sodium alginate, methyl-prop alkylene sodium alginate etc., preferably sodium alginate.
Further, above-mentioned technical proposal, the mass percent concentration of the water-soluble polymer solution are 1~5 %.
Further, above-mentioned technical proposal, the cell with secretion excretion ability of immigrants can be schwann cell, macrophage
Cell, fat stem cell, neural stem cell, mesenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma are dry thin
Any one in born of the same parents, urine Derived Stem Cells, endothelial progenitor cells or cardiac stem cells etc..
Further, above-mentioned technical proposal, inoculum density of the cell in complete medium be 0.5~2.0 ×
105/mL。
Further, the condition of above-mentioned technical proposal, the first low-speed centrifugal processing and the processing of the second low-speed centrifugal is equal
Are as follows: 500~1500rpm of centrifugal rotational speed, 5~10min of centrifugation time.The purpose of the first low-speed centrifugal processing is to remove
It is mixed into the small particle impurity (such as non-viable non-apoptotic cell film) under Transwell orifice plate in the polymer solution of room；Second low-speed centrifugal
The purpose of processing is to reach the mesh of separation excretion body to separate excretion body with polymer solution to obtain pure excretion body
's.
Further, above-mentioned technical proposal, it is described to be based under Transwell orifice plate upper chamber culture cell outside the adsorbing separation of room
The method for secreting body specifically comprises the following steps:
(1) 0.5~2% dual anti-, mixing, acquisition are added after mixing basal medium and serum according to the volume ratio of 5~15:1
Complete medium is added to Transwell orifice plate upper chamber, spare；
(2) water-soluble polymer is dissolved into and is configured to the polymer solution that mass percent concentration is 1~5% in deionized water,
Then the pH value of the polymer solution is reduced to 2~6, is made the protonated amino of polymer molecule, is obtained and contain a large amount of amino
The water-soluble polymer solution of cation is transferred to room under Transwell orifice plate, for use；
(3) there will be the cell of secretion excretion ability of immigrants with 0.5~2.0 × 105The inoculum density of/mL is transferred to equipped with training completely
The Transwell orifice plate upper chamber of base is supported, then Transwell orifice plate is put into progress hypoxemia culture, cell in cell incubator and is incubated
Excretion body is generated in Transwell orifice plate upper chamber under hypoxemia stimulation during change, lower room is spontaneously acted on by electrostatic, osmotic pressure
Collect excretion body；
(4) lower room solution, the centrifugal treating 5 under the conditions of 500~1500rpm are collected after cell culture, after upper chamber is removed
~10min removes mixed impurity, obtains the polymer solution containing excretion body, achievees the purpose that extract excretion body；Pass through again
The pH value of polymer solution of the gained containing excretion body is adjusted to 11~13 progress deprotonation processing, finally by polymer solution
Continue 5~10min of centrifugal treating under the conditions of 500~1500rpm, collection obtains purifying excretion body.
Further, above-mentioned technical proposal, step (2) pH value for reducing polymer solution, which specifically uses, to be polymerize
Proton release agent, acid flux material are added in object solution or in one or more of processing method of acid atmosphere.
Further, above-mentioned technical proposal, step (4) the deprotonation processing are specifically dripped in a polymer solution
Add lye, the lye is sodium hydroxide, potassium hydroxide or lithium hydroxide solution it is any one or several.
The principle of the method for the present invention is as follows:
The present invention utilizes amino-containing water-soluble polymer solution, is protonated by adjusting pH to the amino of polymer molecule
Processing.In general the unique texture of Transwell orifice plate is utilized, lower room loads water-soluble polymer solution, and upper chamber loads
Designated cell culture solution.Polymer in lower room be it is water-soluble, be dissolved in deionized water with a certain concentration, macromolecular chain is stretched
Exhibition and the amino for having protonation.Cell in upper chamber secretes the electronegative excretion body of exhibiting high surface under hypoxemia stimulation.In weight
Under power, osmotic pressure and electrostatic interaction, the excretion body of small volume diffuses through orifice plate to lower room, firmly " is caught " by polymer,
To realize the separation and Extraction to excretion body.Then lower room is further processed, other impurities can be removed.
The second object of the present invention is to provide the application of the isolated purifying excretion body of method described above, can apply
In preparation excretion body hydrogel.
A kind of temperature response type excretion body hydrogel includes the isolated purifying excretion of present invention method described above
Body.
A kind of Photosensitive excretion body hydrogel includes the isolated purifying excretion body of present invention method described above.
Compared with prior art, the invention has the following beneficial effects:
(1) the method for the present invention spontaneous grab excretion body from culture solution can be separated during cell culture,
Process is simple.At low cost simultaneously without expensive equipment, operation is simple.
(2) the method for the present invention is while separating excretion body, due to polymer solution amino under electrostatic interaction firmly
Excretion body " is caught ", ensure that the form of excretion body is complete, improve excretion body separation quality and purity, separative efficiency
It is high.
(3) polymer solution or mother cell used in the method for the present invention, which can according to need, is replaced, application range
More extensively.
(4) the method for the present invention allows further from obtaining isolating and purifying excretion in the polymer solution containing excretion body
Body, process is simple, to subsequent analysis of molecules, such as immune trace experiment, and the extraction of genome, identification, the offer condition such as measurement.
(5) the method for the present invention resulting polymers include excretion liquid solution, can be by design of material with light sensitivity or
The mixed solution of person's Thermo-sensitive forms the hydrogel containing excretion body under outside stimulus, in terms of hydrogel carries excretion body
Using providing new thinking.
Detailed description of the invention
Fig. 1 is the schematic illustration that the present invention carries out excretion body separation with Transwell orifice plate.
Fig. 2 is the TEM shape appearance figure for the excretion body that 1 method of the embodiment of the present invention is extracted；Wherein: scale is 100nm in figure.
Fig. 3 is the characteristic protein immunoblot experiment result of the isolated excretion body of 2 method of the embodiment of the present invention.
Specific embodiment
Below with reference to case study on implementation and attached drawing, invention is further described in detail.The implementation case is with skill of the present invention
Implemented under premised on art, provides detailed embodiment and specific operating process now to illustrate that the present invention has and create
Property, but protection scope of the present invention case study on implementation not limited to the following.
The information for including according to the application, to those skilled in the art can be easily to essence of the invention
Really description carries out various changes, without departing from spirit and scope of the appended claims.It should be understood that the scope of the present invention is not
Process, property defined by being confined to or component, because these embodiments and other descriptions are just for the sake of schematic
Illustrate certain aspects of the present disclosure.In fact, this field or those skilled in the relevant art obviously can be to embodiment party of the present invention
The various changes that formula is made all cover within the scope of the appended claims.
It is not intended to limit the scope of the invention for a better understanding of the present invention, expression dosage used in this application,
All numbers of percentage and other numerical value, are understood to be modified with word " about " in all cases.Therefore,
Unless stated otherwise, otherwise digital parameters listed in specification and appended book are all approximations, may
It can be changed according to the difference for the desirable properties for attempting to obtain.Each digital parameters at least should be considered as according to being reported
Effective digital and obtained by the conventional method of rounding up.
Fig. 1 is the schematic illustration that the present invention carries out excretion body separation with Transwell orifice plate (cell), and principle is as follows:
Excretion body is secreted by the cell in upper chamber, and diffuses through orifice plate to lower room from space between cells.Due to liquid level air pressure in lower room
In the presence of the culture solution of upper chamber can not be pressed into lower room；With the water-soluble polymer solution with amino cation in room at present molten
At stretched condition in agent, there is also active force between strand, it is also impossible to diffuse to upper chamber under the action of osmotic pressure.Therefore
The environment of upper chamber and lower room is relatively independent, under only excretion body can diffuse in the environment of gravity, osmotic pressure and electrostatic interaction
Room, thus by separating.The solution in lower room is diluted with deionized water again, convenient for being centrifuged off mixed impurity.Finally adjust
PH value of solution is amino deprotonation, and release electrostatic active force, centrifugal treating obtains purer excretion body again.
Embodiment 1
The method of room adsorbing separation excretion body under a kind of orifice plate upper chamber culture cell based on Transwell of the present embodiment, including
Following steps:
1, complete medium is prepared, DMEM culture medium and serum are mixed according to the volume ratio of 9:1,1% green strepto- is then added
Element is dual anti-, mixes, and obtains complete medium, the part complete medium is taken to be added to Transwell orifice plate upper chamber, remaining guarantor
It deposits spare.Microbiological contamination is prevented in the process.
2, accurate to weigh the carboxymethyl chitosan powder of 0.3g and sterilized, it is dissolved in the deionized water of 10mL, matches
The carboxymethyl chitosan solution that mass percent concentration processed is 0.3%.
3, hydrochloric acid is added dropwise into carboxymethyl chitosan solution described in step 2, adjusts the pH of the carboxymethyl chitosan solution
Value makes the protonated amino of carboxymethyl chitosan glycan molecule, externally shows positive electricity to 4.Microbiological contamination is prevented in the process.
4, it is transferred in the culture bottle containing complete medium after the mesenchymal stem cell in liquid nitrogen thawing, in temperature
Spend (37 °C), CO2(5%) it is cultivated under environment spare.Microbiological contamination is prevented in the process.
5, the carboxymethyl chitosan solution for preparing step 2 in ultra-clean station is transferred to room under Transwell orifice plate,
After cell in culture bottle is handled with pancreatin, it is transferred to Transwell orifice plate upper chamber, in temperature (37 °C), CO2(5%) environment
It is lower to cultivate 2 days.
6, it is taken out after cell covers in upper chamber, the polymer solution in lower room is transferred in centrifuge tube, with centrifugation
Machine is centrifuged 5 minutes at 1000rpm, and removal is mixed into the impurity of lower room, obtains the polymer solution containing excretion body.
7, the pH to 12 that the polymer solution containing excretion body is adjusted using sodium hydroxide solution, makes polymer molecule
In amino deprotonation, at this time due to the presence of carboxyl, solution externally shows negative electricity, release electrostatic active force, then in 1000rpm
Lower centrifugation 5 minutes, removes upper liquid, and deionized water is added, and obtains purifying excretion liquid solution.
8,0.1%SDS solution is added to centrifuge tube, extracts nucleic acid in solution after cracking, detected using qRT-PCR method
The amount of nucleic acid, as the result is shown after 20 circulations, fluorescence curve enters logarithmic phase, shows that the method for this separation excretion body can be with
Sufficient excretion body nucleic acid substances are obtained, and can be used for other subsequent experimental analyses.
Embodiment 2
The method of room adsorbing separation excretion body under a kind of orifice plate upper chamber culture cell based on Transwell of the present embodiment, including
Following steps:
1, complete medium is prepared, DMEM culture medium and serum are mixed according to the volume ratio of 9:1, add 1% mycillin
It is dual anti-, complete medium is prepared, takes the part complete medium to be added to Transwell orifice plate upper chamber, remaining is saved backup.
Microbiological contamination is prevented in the process.
2, the accurate gelatin powder for weighing 0.2g is dissolved in the deionized water of 10mL in ultraviolet lower sterilizing 10min, is prepared
The gelatin solution that mass percent concentration is 0.2%.
3, hydrochloric acid is added dropwise into step 2 gelatine solution, adjusts the pH value of institute's gelatine solution to 3, makes gelatin molecule
Protonated amino, externally show positive electricity.
4, it is transferred in the culture bottle containing complete medium after the neural stem cell cell in liquid nitrogen thawing, in temperature
(37 °C), CO2(5%) it is cultivated under environment spare.Microbiological contamination is prevented in the process.
5, gelatin solution is transferred to room under Transwell orifice plate in ultra-clean station, the cell in culture bottle is used
After pancreatin processing, it is transferred to Transwell orifice plate upper chamber, in temperature (37 °C), CO2(5%) it is cultivated 2 days under environment.
6, it is taken out after cell covers in upper chamber, the solution in lower room is transferred in centrifuge tube, is existed with centrifuge
It is centrifuged 5 minutes under 1000rpm, removal is mixed into the impurity of lower room, obtains the polymer solution containing excretion body.
7, the pH to 12 that the polymer solution containing excretion body is adjusted using sodium hydroxide solution, makes amino deprotonation
Change, at this time due to the presence of carboxyl, solution externally shows negative electricity, release electrostatic active force, then is centrifuged 5 minutes at 1000rpm, goes
Except upper liquid, deionized water is added, obtains purifying excretion liquid solution.
8, after using 0.1%SDS solution to the cracking of excretion body, protein is extracted.It is legal to egg using Coomassie brilliant blue
White matter is analyzed, and has the presence of protein as the result is shown.
Fig. 3 is the characteristic protein immunoblot experiment of the isolated excretion body of 2 method of the embodiment of the present invention as a result, saying
The excretion body that bright the method for the present invention obtains can be used for the experiments such as protein analysis detection.
Claims (10)
1. a kind of method of room adsorbing separation excretion body under orifice plate upper chamber culture cell based on Transwell, it is characterised in that: first
The water-soluble polymer solution for containing a large amount of amino cations is first prepared, room under Transwell orifice plate is transferred to after sterilizing；Then
To there is the cell of secretion excretion ability of immigrants to be transferred to the Transwell orifice plate upper chamber equipped with complete medium, and in hypoxemia item
Cell culture is carried out under part；While secreting excretion body under hypoxemia stimulation in upper ventricular cell hatching process due to electrostatic interaction and
Osmotic pressure effect, excretion body pass through orifice plate and are clenched by lower indoor polymer；After cell culture, lower room polymer is collected
Solution carries out the first low-speed centrifugal processing, obtains the polymer solution containing excretion body.
2. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: the method also includes the polymer solutions by gained containing excretion body successively to carry out deprotonation, second
Low-speed centrifugal processing, it is final to collect the step of obtaining purifying excretion body.
3. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: the Transwell orifice plate is Transwell-6 orifice plate, Transwell-12 orifice plate or Transwell-
Any one in 24 orifice plates.
4. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: the water-soluble polymer solution is water-solubility chitosan derivative solution, gelatin solution, amination
Any one in derivatives of hyaluronic acids solution, aminated alginic acid sodio-derivative solution.
5. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: the mass percent concentration of the water-soluble polymer solution is 1~5 %.
6. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: the cell with secretion excretion ability of immigrants is schwann cell, macrophage, fat stem cell, nerve
Stem cell, mesenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, urine Derived Stem Cells, endothelium
Any one in progenitor cells or cardiac stem cells.
7. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: inoculum density of the cell in complete medium is 0.5~2.0 × 105/mL。
8. the side of room adsorbing separation excretion body under the orifice plate upper chamber culture cell according to claim 1 based on Transwell
Method, it is characterised in that: the condition of the first low-speed centrifugal processing and the processing of the second low-speed centrifugal is equal are as follows: centrifugal rotational speed 500~
1500rpm, 5~10min of centrifugation time.
9. the isolated purifying excretion body of any one of claim 1~8 the method answering in preparation excretion body hydrogel
With.
10. a kind of temperature response type or Photosensitive excretion body hydrogel, it is characterised in that: include any one of claim 1~8 institute
State the isolated purifying excretion body of method.
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