CN104017768B - Cells in vitro collection method and application, cell in vitro material and application thereof - Google Patents
Cells in vitro collection method and application, cell in vitro material and application thereof Download PDFInfo
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- CN104017768B CN104017768B CN201410280422.9A CN201410280422A CN104017768B CN 104017768 B CN104017768 B CN 104017768B CN 201410280422 A CN201410280422 A CN 201410280422A CN 104017768 B CN104017768 B CN 104017768B
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Abstract
The invention discloses a kind of cell collection method, cell material.For existing Transwell migration research, the scientific contents that experimental result is pointed out is utilized limited defect, present invention firstly provides a kind of cells in vitro collection method.This method is cultivated at Transwell cell migration and is terminated and after removing room on Transwell, lower indoor cultivation liquid, uses trypsinization and combines the method for mechanical oscillation and respectively the cell in upper after Transwell Cell migration assay room, lower room is digested, collected；In the operation of pancreatin cell dissociation, cleaning mixture is pH 7.35～7.45 phosphate buffer, and pancreatin cell dissociation buffer contains 0.25% pancreatin and 0.01%EDTA.Present invention simultaneously provides and separated, by the induction of identical chemotactic factor is lower, the high animal migration live cell material and low migratory activity cell material produced a kind of including.The present invention also provides for the application in cell behaviors analysis is tested of above-mentioned collection method and cell material.The present invention can make Transwell external migration laboratory exploration to the cell behaviors of cell migration and molecular biology research.
Description
Technical field
The present invention relates to a kind of cell collection method, cell material, particularly relate to a kind of cyton
Outer collection method, and cell in vitro material.Belong to biological, medical cell experiment in vitro technology neck
Territory.
Background technology
Cell migration is the Chemotaxis that cell produces under exogenous signal stimulus, it is common that cell
Move from low concentration king's area with high mercury after experiencing the Concentraton gradient of Cucumber.In physiological status
Under, cell migration is looked for food at small cell, wound healing, embryo's generation, tissue repair, immunity are anti-
Should play an important role during waiting；Under pathological state, cell migration has also promoted cancer to turn
The generation of the diseases such as shifting, atherosclerosis and arthritis and development.Thus, cell migration is to work as
One important research direction of front life sciences.
Transwell migrating technology is the experiment side that current most widely used research cell in vitro migrates
Method.The required Transwell of experiment migrates system and uses Transwell cell (Transwell insert)
Jointly build with common porous Tissue Culture Plate.Transwell cell profile is one and can place
Cup-formed device in ordinary cells culture plate orifice plate, bottom belt permeability film, with aperture on film
The micropore of size 0.1-12.0 μm.According to different experiment purposes in research, select different micro-
Bore dia.Cell migration research is generally selected with 8.0 μm or the Transwell of 12.0 μm micropores
System.Transwell cell is put in ordinary cells culture plate by experiment, the little indoor of Transwell
Deserve to be called room or interior room, in Tissue Culture Plate, claim lower room or mistress.In experiment, by adding in lower room
Adding various chemotactic factor, inducing cell migrates from down room, upper room.It is different according to production firm,
Transwell cell is also known as Boyden Chamber or Thincert cell.Due to general in field
Use the Transwell cell that Corning company of the U.S. produces, thus, current this kind of cell in vitro
Migration research method widely used " Transwell method " is as its conventional designation.Transwell migrates
Engineering reliability is strong, reproducible, is the most widest cell migration research method.But mesh
The analysis method in front Transwell migration research used experimental result is the most fairly simple, it is common that
Simple analytical calculation cell migration rate, utilizes limited to the scientific contents of experimental result prompting.
Cell In vivo study having revealed that, the various cells of the cell that cell migration activity is high and low are raw
Thing scholarship and moral conduct is, including cell adhesion, breed, break up, apoptosis etc. the most dramatically different, both at home and abroad
Scholars have been carried out further investigation to this.But limited by In vivo study, researcher is difficult to
Acquisition has the cell of different migratory activity and studies further.Experiment is migrated from Transwell
Design principle is analyzed, and it is strong that cell material can be divided into transfer ability by Transwell external migration experiment
The two group cells weak with transfer ability, create premise for completing above-mentioned cells in vitro behavioral study
Condition.But, cell migration in Transwell cell is an extremely complex process: thin
Protein and the ion in intracellular portion rearrange after the Concentraton gradient perceptually descending cell, and degeneration
Stretching out pole shape foot, form talin, tractive cell is forward, thus micro-through narrow and small Transwell
Hole, gradually moves to lower room from the upper room of Transwell film.Owing to the diameter of eukaryotic cell typically exists
Between 10 μm～100 μm, migrate experiment 8.0 μm～the micropore of 12.0 μm much larger than Transwell
Diameter.Thus in Transwell Cell migration assay, cell is except sticking to Transwell film
Two bottom surfaces up and down on, also have substantial amounts of cell meeting and " fall into " micropore on Transwell film
In, it is difficult to effectively collect for follow-up research.Especially Transwell cell area is less, often uses
The chemical digestion mode of rule cannot be carried out batched operation, and cell collecting amount is relatively low, is not enough to for rear
Continuous Celluar and Molecular Biology research.It addition, in conventional cell dissociation method cell
It is to cultivate from it to come off plane, has only to during cell dissociation destroy the connection between cell；
And in Transwell migration system, cell aperture to be passed is less than the narrow and small micropore of its diameter,
Digestion needs cell to be rapidly separated from its embedding attached micropore when collecting cell, this process very easy damaged
Cell, even results in cell cracking, fragmentation.Therefore, on the whole, although cell in vitro is studied
Technology has that intervention factor is simple, experiment condition is easily-controllable, economical quick, reproducible, and can
Carry out the advantages such as deep Mechanism Study, but restricted by prior art condition so that cell in vitro
Migrate experiment to be typically only capable to simply by analysis cell migration rate to understand the transfer ability of cell,
And desired by researcher by migrate experiment result cell further with, such as obtain height migrate
The cell of activity is studied further, compares migrating cell and non-migrating under cells in vitro experiment condition
The research of the biological behaviour of cell is difficult to carry out all the time.
Summary of the invention
The purpose of the present invention is aiming at the deficiencies in the prior art, it is provided that a kind of cells in vitro collection side
Method, the method specific aim combines with Cell migration assay, enters migrating the cell after experiment terminates
Row is effectively collected；The cell collected is to have different migratory activity under identical chemotactic factor is induced
Cell, can be applicable to relatively grinding of migrating cell and non-migrating cell under cells in vitro experiment condition
Study carefully；This cells in vitro collection method can be applicable to cell clone, propagation, apoptosis, gene and albumen
Expression, morphologic detection etc. are tested.
For achieving the above object, present invention firstly provides a kind of cells in vitro collection method, its technology
Scheme is as follows:
A kind of cells in vitro collection method, it is characterised in that: it is applied to Transwell cell migration real
Test；Transwell cell migration is cultivated after terminating and removing room on Transwell, lower indoor cultivation liquid,
Use trypsinization and combine the method for mechanical oscillation respectively to Transwell Cell migration assay after
Cell in upper room, lower room carries out digesting, collecting；In the operation of pancreatin cell dissociation, cleaning mixture is
PH 7.35～7.45 phosphate buffer, pancreatin cell dissociation buffer contains 0.25% pancreatin and 0.01%
EDTA。
Cells in vitro collection method technology basic technique principle of the present invention is to move external for Transwell
Move experiment to combine with external cell culture method.Transwell cell migration is cultivated and is terminated and remove
After the upper room of Transwell, lower indoor cultivation liquid, remain and be attached to Transwell permeability film upper strata
The cell on surface is non-migrating cell (transfer ability is low), and is attached to Transwell permeability film
Cell in underlying surfaces and the embedding Transwell of being attached to permeability membrane micropore is that migrating cell (migrates
Ability is high), by trypsinization and combine the method for mechanical oscillation and can will attach to permeability film surface
And the cell dissociation that is embedded in micropore collecting, thus obtain Transwell cell in vitro and migrate
Under experiment condition, the cell of non-migratory behavior carries out next step research with there being the cell of transfer behavior.
Under optimal way, above-mentioned cells in vitro collection method can be implemented according to following steps:
Step S1, initial wash
Transwell cell migration is cultivated and is terminated and remove room on Transwell, lower indoor cultivation
After liquid, upper room, lower room are separately added into phosphate buffer and wash culturing room, room in collection
It is stored in respectively in two centrifuge tubes with cleaning mixture in lower room, is respectively labeled as centrifuge tube A, centrifuge tube
B；
Step S2, digestion are collected
On Transwell, room, lower room are separately added into pancreatin cell dissociation buffer, treat that cell divides
Dissipate and suspend, collect Digestive system correspondence in upper room, lower room respectively and add centrifuge tube A, centrifuge tube B
In, in centrifuge tube A, centrifuge tube B, add Ox blood serum respectively terminate cell dissociation；
Step S3, again wash collection
Respectively on Transwell in room, lower room add phosphate buffer, horizontal oscillations,
Collect cleaning mixture correspondence in upper room, lower room respectively to add in centrifuge tube A, centrifuge tube B；
Step S4, centrifugal collecting cell
Centrifuge tube A, centrifuge tube B are centrifuged, remove supernatant, respectively to centrifuge tube A, from
Heart pipe B adds phosphate buffer, resuspended and collect cell mass bottom centrifuge tube, obtain receipts
Collection cell.
Above-mentioned cells in vitro collection method, needs to repeat step S3, again washing operation one when necessary
Secondary, i.e. after operation is collected in digestion, carry out twice washing collection.
Above-mentioned cells in vitro collection method carries out the collection of 2～3 attached cells altogether, only the 1st time,
I.e. in step S2, carrying out in trypsinization liquid, remaining all uses phosphate buffer washing to combine
The method of horizontal shaker mechanical oscillation is collected, and such operation can reduce cell and be exposed to pancreas
Time in enzyme, the collection efficiency of cell can be improved again.
Said method can at utmost improve collection effect by the cooperation between front and back's main operational steps
Rate.Specifically:
In step sl, room, lower room add on Transwell phosphate buffer washing,
Collect the most floating non-adherent cell.
In step s 2, on Transwell, room, lower room are separately added into pancreatin cell dissociation buffer
The cell being attached to the upper and lower both sides of Transwell film can be digested, to collection have phosphate buffer and
The centrifuge tube of trypsin solution adds Ox blood serum and terminates digestion reaction, and non-immediate on Transwell
Room, lower room add the cell culture medium containing Ox blood serum and terminates digestion reaction, can avoid in upper room
With generation bubble in lower room, both reduced the damage to cell, improved again cell collection rate.In routine
Trypsin digestion cell during, be to add the culture medium containing Ox blood serum pancreatin terminates cell
Digestion reaction, and if operating the most like this, due to the existence of serum composition, in piping and druming receipts
Being easy to produce bubble in collection cell processes, directly produce 3 aspects and endanger: first, bubble is broken
When splitting, mechanical stress is easy to damaging cells；Second, bubble once produces the collection that can affect liquid,
Cell is caused to be collected the most thorough；3rd, bubble once produces the definition affecting the visual field, is difficult to
The digestible degree of observation of cell under inverted microscope.And in the inventive method step S2, due to
Need not blow and beat cell, can thoroughly avoid bubble to produce.Simultaneously as be directly added into Ox blood serum
Neutralize digestion reaction rather than add the culture medium containing a small amount of Ox blood serum, it is also possible to reducing whole receipts
The volume of liquid collecting, thus reduce the time of follow-up centrifugal collection.
In step s3, by room on Transwell, lower room add phosphate-buffered again
Liquid washs and combines machinery horizontal oscillations, it is simple to the cell detachment of residual, it is possible to again collect residual
Cell in Transwell film both sides and be embedded in the cell in Transwell membrane micropore.In washing
Use horizontal mechanical vibration makes cell from Transwell permeability film, upper surface depart from and unconventional
Suction pipe piping and druming cell cause its depart from, can effectively reduce the damage to cell, improve collection rate,
And simplified operation, improve operating efficiency.
In step s 4, by centrifugation, resuspended operation, collect respectively and obtain target cell.
Under optimum condition, above-mentioned cells in vitro collection method can be done respectively and optimize as follows:
Optimize one: in method, use cleaning mixture is the phosphate buffer solution of pH 7.4.
Optimize two: in step S2, at pancreatin cell dissociation buffer containing 0.25% pancreatin and 0.01%EDTA
Under the conditions of, pancreatin cell dissociation buffer addition is to be covered each by Transwell bottom room and lower room being
Preferably.Trypsin is in close relations with the character of the type of cell and cell to the centrifugation of cell,
Different tissues or cell line are different to tryptic action-reaction, trypsin cell dispersion
Activity also relevant with its concentration, temperature and action time.Premenstruum, test determined, side of the present invention
In method, under conditions of combining with Transwell Cell migration assay, the upper room of Transwell, under
In room, the amount of pancreatin cell dissociation buffer is important cell decentralised control condition.For conventional
Transwell culturing room, it is proposed that pancreatin cell dissociation buffer addition be: 24 hole Transwell training
Above room adds 0.1ml, lower room adds 0.6ml to support room (Transwell cell diameter 6.5mm)；12 holes
Transwell culturing room (Transwell cell diameter 12mm) is upper, and room adds 0.5ml, lower room adds 1.5
ml；The upper room of 6 hole Transwell culturing room (Transwell cell diameter 24mm) add 1.5ml,
Lower room adds 2.6ml.
Optimize three: in step S2, use 100% Ox blood serum to terminate digestion reaction, its objective is to subtract
Total liquid volume in few centrifuge tube, reduces follow-up centrifugation time and improves cell collecting amount.Due to step
S2 uses have in the centrifuge tube of phosphate buffer and trypsin solution to collection and add Ox blood serum termination
Digestion reaction, trypsin solution is played peptizaiton by phosphate buffer that collect in advance in centrifuge tube,
Therefore use 100% Ox blood serum can terminate digestion reaction smoothly, and control centrifuge tube on this basis
Middle total liquid volume, improves collection efficiency.
Optimize four: in step S3, horizontal oscillations use horizontal shaker vibration, condition be 100r/min～
150r/min, vibration 5min.Cell dissociation operate in, use suction pipe piping and druming cell be make adherent
The scattered usual manner of cell detachment, but the present invention tests discovery, due to Transwell premenstruum
The particularity of permeability membrane structure bottom cell, uses suction pipe piping and druming to be difficult to make embedding be attached to permeability film
Cell in micropore completely falls off, and easily produces bubble, big to cell damage, cell
Collection efficiency is the highest.And the mild mechanical using horizontal shaker to realize vibrates and effectively controls vibration width
Degree, it is to avoid cell unbalance stress during piping and druming cell, avoids producing bubble simultaneously, is beneficial to be attached to micro-
In Porous materials, the cell with permeability film surface uniformly comes off.Can less cell injury, significantly
Improve collection rate.
Optimize five: in step S4, room, lower room centrifuge tube on Transwell are centrifuged, centrifugal bar
Part is: rotating speed 200RCF (xg)～250RCF (xg), time 10min.
Optimize six: in step S4, in centrifuge tube, add pH 7.35～7.45 phosphate buffer
0.5ml～1ml.
Cells in vitro collection method of the present invention can be applicable to cell behaviors analysis test, including
But it is not limited to cell clone, cell proliferation, the expression of apoptosis, cytogene and albumen test,
Morphocytology detection test etc..
The cell using cell in vitro collection method of the present invention to collect is to induce at identical chemotactic factor
Under show the cell of high and low two kinds of migratory activities, preferable cell material can be acted on and be applied to carefully
The cell behaviors of born of the same parents' migration and molecular biology research, including cell clone, propagation, wither
Die, gene and the expression of albumen, and morphologic detection etc..Based on this, the present invention provides a kind of
Cell in vitro material, its technical scheme is as follows:
A kind of cell in vitro material, it is characterised in that: include being separated by the induction of identical chemotactic factor is lower
The high animal migration live cell material produced and low migratory activity cell material；Described cell in vitro material
Preparation initially with Transwell migrate experiment make germinal cell material chemotactic factor induce under
Migrate and cultivate, after the cultivation of Transwell cell migration terminates, use above-mentioned cell in vitro collection method
Collecting cell, collecting the cell material obtained on Transwell in room is that described low migration is lived carefully
Born of the same parents' material, collecting the cell material obtained under Transwell in room is described high animal migration living cells
Material.
Above-mentioned cell in vitro material can be applicable to cell behaviors analysis test, including but do not limit
In cell clone, cell proliferation, apoptosis, cytogene and protein expression, morphocytology
Detection test etc..
Somatic cell collection method of the present invention can combine with various kinds of cell biological behaviour experimental technique,
Including cell clone experiment, cell proliferation experiment, the expression of cell apoptosis assay, gene and albumen
Experiment, and morphocytology detection etc..Its technical scheme is as follows:
A kind of cell behaviors analyzes method, is a kind of morphocytology detection method, and it is special
Levy and be: migrate experiment initially with Transwell and make germinal cell material under chemotactic factor is induced
Migrate and cultivate, after the cultivation of Transwell cell migration terminates, use above-mentioned cell in vitro collection method
Collect cell, respectively obtain high animal migration live cell material and low migratory activity cell material；Secondly
Respectively described high animal migration live cell material and low migratory activity cell material are seeded to cell cultivate
Base carries out cell injuring model；When Cell culture invitro terminates to detect high animal migration living cells material respectively
Material and low migratory activity cell material form；The culture medium selected in described cell injuring model and training
The condition of supporting determines according to germinal cell material behavior.
A kind of cell behaviors analyzes method, is a kind of cytoskeleton detection method,
It is characterized in that: migrate experiment initially with Transwell and make germinal cell material lure at chemotactic factor
Lead lower migration to cultivate, use above-mentioned cell in vitro to collect after the cultivation of Transwell cell migration terminates
Method collects cell, respectively obtains high animal migration live cell material and low migratory activity cell material；
Cytoskeleton to described high animal migration live cell material Yu low migratory activity cell material the most respectively
Dyeing；Detection cytoskeleton is observed in dyeing after terminating；Described cytoskeleton colouring method according to
Germinal cell material behavior determines.
A kind of cell behaviors analyzes method, is a kind of cell self-renewal ability detection method,
It is characterized in that: migrate experiment initially with Transwell and make germinal cell material lure at chemotactic factor
Lead lower migration to cultivate, use above-mentioned cell in vitro to collect after the cultivation of Transwell cell migration terminates
Method collects cell, respectively obtains high animal migration live cell material and low migratory activity cell material；
The highest animal migration live cell material and low migratory activity cell material are carried out cell clone training
Support；Clone cultivates and terminates poststaining detection cell self-renewal ability；Described cell clone culture bar
Part determines according to germinal cell material behavior.
A kind of cell behaviors analyzes method, is a kind of ability of cell proliferation detection method, its
It is characterised by: migrate experiment initially with Transwell and make germinal cell material induce at chemotactic factor
Lower migration is cultivated, and uses above-mentioned cell in vitro collection side after the cultivation of Transwell cell migration terminates
Method collects cell, respectively obtains high animal migration live cell material and low migratory activity cell material；Its
The secondary cell proliferation energy detecting high animal migration live cell material and low migratory activity cell material respectively
Power, detection method determines according to germinal cell material behavior.
Compared with prior art, the invention has the beneficial effects as follows: (1) cell in vitro of the present invention is collected
External for Transwell migration experiment is combined, to Transwell by method with external cell culture method
The later cell of external migration carries out digesting and collecting, and collects respectively and obtains high migratory activity and move with low
Move the cell of activity, make external migration of Transwell test from the simple transfer ability analyzing cell
With cell behaviors and the molecular biology research that transport efficiency is extended to cell migration；(2)
The outer cell collection method of the present invention in each operating procedure, by the different cell collection method of design (
In trypsinization liquid collect or in phosphate buffer collect), control pancreatin cell dissociation buffer concentration
With digestion time, select to add Ox blood serum and terminate the step react of cell dissociation and environment, combine machine
Tool vibration auxiliary cell comes off the optimization of essential condition in terms of scattered method etc. six from culturing room,
Ensure that on the whole Transwell migrate experiment fine under the conditions of can micro-to migration results
Amount cell is effectively collected, it is ensured that collect enough cells for follow-up research；(3) this
Bright providing is moved with low by the lower high animal migration live cell material separating generation of identical chemotactic factor induction
Moving living cellular material, this material is preferable cell migration material, it is possible to be applied to cell biological
Scholarship and moral conduct is and molecular biology research；(4) the invention provides and combine with somatic cell collection method
Various kinds of cell biological behaviour experimental technique；(5) the inventive method is cell in vitro migration mechanism
There is provided new technological means with behavioral study, and this technological means is set up at ripe Transwell
On external migration experiment, applied range.
Accompanying drawing explanation
Fig. 1-1: embodiment 1 cell inoculation-collecting amount curve.Result display cell collecting amount with
Inoculum concentration becomes positive correlation.
Fig. 1-2 a, Fig. 1-2 b show cell collection efficiency.Cell warp in Fig. 1-2 a display embodiment 1
After crossing Transwell cultivation 16h, migrate to the human marrow mesenchymal stem cell violet staining of lower room
Picture, it is seen that migrating cell well spreads over the film bottom surface of cell；In Fig. 1-2 b display embodiment 1
The cell dissociation method used by the present invention is taked to collect upper room non-migrating cell and lower room migrating cell,
Transwell film surface crystallization purple dyeing picture.Almost without more than cell on visible Transwell film
Stay, illustrate that the cell collection efficiency of the present invention is higher.
Inversion after cell material cultivates 24h respectively in Fig. 2 a, Fig. 2 b display embodiment 2 is micro-
Mirror photo.Fig. 2 a represents room non-migrating cell, and Fig. 2 b represents lower room migrating cell.From picture
In visible, different from upper room non-migrating cell, the cell of migration has a large amount of cell debris, it is seen that
Cell has a certain degree of mechanical damage in transition process.
Cell material skeleton coloration result in Fig. 3 a, Fig. 3 b display embodiment 3.Fig. 3 a represents
Room non-migrating cell, Fig. 3 b represents lower room migrating cell.Visible cell is cell shape in transition process
State changes so that upper room non-migrating cell and lower room migrating cell present visibly different cell
Matrix morphology feature.
In Fig. 4 a, Fig. 4 b display embodiment 4, cell material carries out the result of colony formation.
Fig. 4 a represents room non-migrating cell, and Fig. 4 b represents lower room migrating cell.Result shows, upper room
The plastidogenetic cell clone of non-migrating is less and sparse, and the clone that lower room migrating cell is formed is bigger
And fine and close, illustrate that the self-renewal capacity of the cell of different migratory activity differs.
Fig. 5 shows the growth curve of cell material in CKK-8 method detection embodiment 5.Can from figure
To find out, the multiplication capacity of the cell of different migratory activities is the most significantly different.
Detailed description of the invention
Below in conjunction with the accompanying drawings, the preferred embodiments of the present invention are further described.
Embodiment one
As shown in Fig. 1-1, Fig. 1-2, by the inventive method, external migration of Transwell is cultivated knot
The cell of bundle is collected.
The present embodiment uses mescenchymal stem cell (the human bone marrow of human bone marrow-derived
Mesenchymal stem cells, hereinafter referred to as hMSCs) as object of study, use people source
Restructuring PDGF-BB (platelet derived growth factor) as chemotactic factor induced cell migration.
1, experiment material and reagent
Transwell cell (i.e. room on Transwell): Coring company is applicable to 6 porocytes
The Transwell cell of culture plate, cell area 4.67cm2, diameter 24mm；Article No.: #3428,
#354576；Membrane material is polycarbonate (Polycarbonate, PC) or poly terephthalic acid second
Diol ester (Polyethylene Terephthalate, PET), pore size 8.0um.
Tissue Culture Plate (i.e. room under Transwell): 6 porocyte culture plates of Corning company,
Article No. CLS3516-50EA
The low sugar culture-medium of DMEM: Gibco Products, article No. 11885-076
Phosphate buffer: pH 7.35～7.45
Preparation chemotactic culture medium: by platelet derived growth factor (PDGF-BB) with 20ng/ml
Add DMEM solution to prepare, matching while using.
Platelet derived growth factor (PDGF-BB): R&D Products, article No. 220-BB-050
Pancreatin cell dissociation buffer: containing 0.25% pancreatin and the phosphate buffer of 0.01%EDTA,
Gibco Products, article No. R-001-100
Ox blood serum: Gibco Products, article No. 10082147
Crystal violet: Sigma Products, article No. HT90132
Centrifuge: Eppendorf Products, model 5810
Centrifuge tube: material is Merlon, 50ml, Corning Products
Blood cell calculator: sigma Products, article No. Z359629-1EA
Liquid-transfering gun: Eppendorf Products, specification 1ml
Leica inverted microscope (model DMI3000B)
2, experimental implementation
The external migration of 2.1Transwell cultivates (experiment)
1) cell pretreatment
The low sugar culture-medium of DMEM is changed by cell hunger 24h before carrying out Cell migration assay.
2) make cell suspension, prepare cell migration
The hungry cell cultivated discards culture fluid, washes 1 time with PBS, adds pancreatin cell dissociation buffer
1ml～2ml, the amount of addition is advisable bottom Tissue Culture Plate to cover, is added and contain after cell rounding
The low sugar DMEM culture medium of 10%FBS terminates digestion, uses blood cell calculator counting, centrifugal
Collect cell, rotating speed 200RCF (xg)～250RCF (xg), centrifugation time 5min, use DMEM
Low sugar culture-medium is resuspended, and adjusts cell density to 2.5 × 105/ ml obtains cell suspension, stand-by.
3) inoculating cell and cultivation
Transwell cell is added in Tissue Culture Plate, constitute the upper room of Transwell culturing room
With lower room；Room adds under Transwell preparation chemotactic culture medium, addition 2ml/ hole；
Obtained cell suspension 1ml adds room on Transwell, and during inoculation, attention action is slow, makes cell
Uniformly inoculation, and avoid producing bubble；
Cell is cultivated in 37 DEG C of 95% CO2 gas incubator 16h～24h.
The external migration of 2.2Transwell cultivates cell collection
Step S1, initial wash are collected
Transwell cell migration is cultivated and is terminated and remove room on Transwell, lower indoor cultivation liquid
After, be separately added in upper room, lower room phosphate buffer washing culturing room, addition be upper room,
The lower each 1ml in room；
In collection, room is stored in two 50ml centrifuge tubes respectively with cleaning mixture in lower room, is labeled as being centrifuged
Pipe A, centrifuge tube B.
Step S2, digestion are collected
Room, lower room are separately added on Transwell the pancreatin cell dissociation buffer of 37 DEG C of preheatings,
Addition is 1.5ml/ hole, upper room, 2.6ml/ hole, lower room；Transwell culturing room is placed in 95% 2
Carbonoxide incubator, cultivates 2min～5min for 37 DEG C；Period can be taken off Transwell culturing room in
Observe under inverted microscope, after and dispersion rounded until cell suspends, collect upper room, lower room respectively
Middle Digestive system correspondence adds in centrifuge tube A, centrifuge tube B；If finding, cell is under inverted microscope
The most rounded and dispersion suspends, and can put into and continue in carbon incubator to cultivate until cell is rounded and divides
Dissipate and suspend.
Adding 100% Ox blood serum respectively in centrifuge tube A, B and terminate cell dissociation, addition is pancreas
The 10%～20% of enzyme cell dissociation buffer.
Step S3, again wash collection
Room, lower room add on Transwell phosphate buffer respectively and washs culturing room, add
Amount is with step S1；
The Corning6 porocyte training of cell (is cultivated equipped with Transwell by Transwell culturing room
Support plate) it is placed in horizontal shaker, 100r/min～150r/min, time 5min,
Collect cleaning mixture correspondence in upper room, lower room respectively to add in centrifuge tube A, centrifuge tube B；
Repeat above operation.
Step S4, centrifugal collecting cell
Centrifuge tube A, centrifuge tube B are centrifuged, rotating speed 200RCF (xg)～250RCF (xg), time
Between 10min；
Remove supernatant respectively, in centrifuge tube A, centrifuge tube B, add phosphate buffer 0.5
Ml～1ml；Collect cell mass bottom lay equal stress on outstanding centrifuge tube A, B respectively, collect target cell.
Wherein, centrifuge tube A is separate with the restructuring PDGF-BB in people source for chemotactic factor induction is lower
The low migratory activity cell produced, is that the restructuring PDGF-BB with people source is for becoming in centrifuge tube B
Change the lower high migratory activity cell separating generation of factor induction.
Count with blood cell calculator, room in calculating, the cells ratio of lower room, cell migration rate, with
And cell collection efficiency (Fig. 1-1)；By violet staining analyze cell collection efficiency (Fig. 1-2 a,
Fig. 1-2 b).
In the present embodiment, violet staining concentration 0.1%, 10 minutes time；Leica is used to be inverted
Microscope DMI3000 B takes pictures.
Embodiment two
The induction of identical chemotactic factor is lower separates the high animal migration living cells and low migratory activity cell produced
Morphologic detection experiment.
Cell culture medium: the low sugar culture-medium of DMEM containing 10% hyclone
The low sugar culture-medium of DMEM: with embodiment one
Inverted microscope: with embodiment one
Collect cell in gained centrifuge tube A, centrifuge tube B with embodiment one, as material, to inoculate respectively
To cell culture medium, be placed in 37 DEG C, in 95% CO2 gas incubator, cultivate respectively after 24h
Cell morphology characteristic (Fig. 2 a, Fig. 2 b) is detected under inverted microscope.
Embodiment three
The induction of identical chemotactic factor is lower separates the high animal migration living cells and low migratory activity cell produced
Cytoskeleton test experience.
Phalloidin (the Phalloidin Tetramethylrhodamine B of tetramethylrhodamine labelling
Isothiocyanate:Sigma Products, article No. P1951
Inverted microscope: with embodiment one
Collect cell in gained centrifuge tube A, centrifuge tube B with embodiment one, as material, to be respectively adopted
The phalloidin of tetramethylrhodamine labelling, to cell dyeing, detects cytoskeleton under inverted microscope
Morphological characteristic (Fig. 3 a, Fig. 3 b).
In the present embodiment, phalloidin method operates with reference to Sigma product description.
Embodiment four
The induction of identical chemotactic factor is lower separates the high animal migration living cells and low migratory activity cell produced
Self-renewal capacity test experience.
Tissue Culture Plate, inverted microscope, crystal violet are all with embodiment one
Collect cell in gained centrifuge tube A, centrifuge tube B with embodiment one, as material, to inoculate respectively
To Tissue Culture Plate, 100/hole of inoculum density, incubation time 8d～10d；See clearly visible
Cell clone after terminate cultivate, use crystal violet cell clone is carried out dyeing use be inverted micro-
Mirror carries out observing (Fig. 4 a, Fig. 4 b).
In the present embodiment, cell culture condition and violet staining method are with embodiment one.
Embodiment five
The induction of identical chemotactic factor is lower separates the high animal migration living cells and low migratory activity cell produced
Multiplication capacity test experience.
CCK-8 reagent: Sigma Products, article No. 96992-500TESTS
Collect cell in gained centrifuge tube A, centrifuge tube B with embodiment one, as material, to be respectively adopted
CCK-8 method detection ability of cell proliferation (Fig. 5).
In the present embodiment, using 96 orifice plates to detect, cell inoculum concentration is 2000/hole, inspection
Surveying time point is 1,3,5,7,9 days.During detection, CCK-8 addition is 10ul/ hole, detection
Absorbance is 450nm.
Claims (4)
1. a cells in vitro collection method, it is characterised in that: it is applied to Transwell cell migration real
Test, implement according to following steps:
Step S1, initial wash
Transwell cell migration is cultivated and is terminated and remove room on Transwell, lower indoor cultivation
After liquid, upper room, lower room are separately added into the washing training of pH 7.35～7.45 phosphate buffer
Supporting room, in collections, in room and lower room, cleaning mixture is stored in two centrifuge tubes respectively, be respectively labeled as from
Heart pipe A, centrifuge tube B；
Step S2, digestion are collected
Room, lower room are separately added on Transwell pancreatin cell dissociation buffer, pancreatin cell
Digestive system addition for being covered each by Transwell bottom room and lower room, described pancreatin cell
Digestive system contains 0.25% pancreatin and 0.01%EDTA, treats that cell dispersion suspends, collects respectively
In room, lower room, Digestive system correspondence adds in centrifuge tube A, centrifuge tube B, respectively to centrifuge tube A,
Adding 100% Ox blood serum in centrifuge tube B and terminate cell dissociation, addition is pancreatin cell dissociation
The 10%～20% of liquid addition；
Step S3, again wash collection
Room, lower room add on Transwell pH 7.35～7.45 phosphate buffer respectively,
By Transwell culturing room horizontal oscillations, horizontal oscillations condition of culture 100r/min～
150r/min, time 5min, collect cleaning mixture correspondence in upper room, lower room respectively and add centrifuge tube
In A, centrifuge tube B；
Step S4, centrifugal collecting cell
Centrifuge tube A, centrifuge tube B are centrifuged, remove supernatant, respectively to centrifuge tube A, from
Heart pipe B adds pH 7.35～7.45 phosphate buffer, resuspended and collect bottom centrifuge tube
Cell mass, obtains collection cell.
Method the most according to claim 1, it is characterised in that: in described step S2, pancreatin is thin
Born of the same parents' Digestive system addition is one of following:
24 hole Transwell culturing room, upper room adds 0.1ml, lower room adds 0.6ml；
12 hole cell Transwell culturing room, upper room adds 0.5ml, lower room adds 1.5ml；
6 hole cell Transwell culturing room, upper room adds 1.5ml, lower room adds 2.6ml.
Cells in vitro collection method the most according to claim 1 and 2, it is characterised in that: it is applied to
Cell behaviors analysis is tested.
4. the cytobiology that the cells in vitro collection method that a kind utilizes described in claim 1 or 2 realizes
Behavior analysis method, is a kind of cell detection method, it is characterised in that: implement according to following steps:
Step S1, initially with Transwell migrate experiment make germinal cell material at chemotactic factor
Migrate cultivation under induction, when Transwell cell migration cultivate terminate after use above-mentioned external carefully
Born of the same parents' collection method collects cell, respectively obtains high animal migration live cell material thin with low migratory activity
Born of the same parents' material；
Step S2, implement one of following detection method
One of, morphocytology detection: respectively described high animal migration live cell material is moved with low
Shifting living cellular material is seeded to cell culture medium and carries out cell injuring model；When cell in vitro is trained
Foster end detects high animal migration live cell material and low migratory activity cell material form respectively；Institute
State the culture medium and condition of culture selected in cell injuring model according to germinal cell material behavior
Determine；
Two, cytoskeleton detection: respectively to described high animal migration live cell material with
The cytoskeleton dyeing of low migratory activity cell material；Detection cytoskeleton is observed in dyeing after terminating
Form；Described cytoskeleton colouring method determines according to germinal cell material behavior；
Three, cell self-renewal ability detection: respectively to high animal migration live cell material with low
Migratory activity cell material carries out cell clone culture；Clone cultivates and terminates poststaining detection cell
Self-renewal capacity；Described cell clone culture condition determines according to germinal cell material behavior；
Four, ability of cell proliferation detection: detect high animal migration live cell material respectively and move with low
Moving the ability of cell proliferation of living cellular material, detection method is true according to germinal cell material behavior
Fixed.
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