CN106047665A - Method for high-throughput cell migration research and cell migration research system - Google Patents
Method for high-throughput cell migration research and cell migration research system Download PDFInfo
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Abstract
The invention discloses a method for high-throughput cell migration research and a cell migration research system. Anchorage-dependent growing cells are cultured by adopting a cell population limiting chamber in a cell arrangement device, so as to obtain an anchorage-dependent growing cell population. The anchorage-dependent growing cell population is put in a migration testing chamber in a migration tester for culturing, pictures in the growth processes of the cells are obtained, and a migration distance that the cells migrate is calculated with an area which covered by the growth, which is recorded through the pictures, of the cell population. The cell migration research is carried out by adopting the cell migration research system provided by the invention; the anchorage-dependent growing cell population is obtained through the cell population limiting chamber; afterwards, through the obtaining of the growth pictures of the cells in the migration testing chamber, such a research method has extremely high repeatability; the migration capability of the cells can be quantified through image processing.
Description
Technical field
The invention belongs to micro Process and technical field of cell biology, be specifically related to a kind of migration for high-flux cell and grind
The method studied carefully and cell migration research system.
Background technology
The generation of cell migration and tumor and development relationship are close, and cell migration is by multiple external environmental factor and interior
The joint effect of portion's factor.For the correlational study of cell migration, on the one hand can let us cell migration behavior have more
Understanding, on the other hand, the treatment for tumor etc. and cell migration disease in close relations is explored, and has important value.
Traditional biological method migrates about cells in vitro experiment mainly has scratch experiment and invasion and attack test two kinds
Experimental technique, although scratch experiment is simple, but repeatability is poor, and cell can be caused damage, experimental result by the process of cut
Also the effect of cell proliferation and migration cannot be made a distinction；Invasion and attack test, on the one hand the environment of steady concentration gradient is difficult to tie up
Hold, on the other hand, it is impossible to the effect of cell proliferation and migration is made a distinction.But, for existing micro-fluidic chip, uncomfortable
For the high flux screening of sample, also cannot the propagation of cell and the effect of migration.
Summary of the invention
The technical problem to be solved is how to make a kind of simple in construction, be suitable to the high flux screening of sample
System, and the cell migration method of testing that a kind of propagation by cell and migration make a distinction.
For this problem, the invention provides a kind of cell migration research system, including: cell arranging devices and migration are surveyed
Examination device；
Described cell arranging devices includes that at least one first through hole, one end of each described first through hole are provided with
Chassis, described chassis is provided with at least one second through hole；
Described second through hole limits chamber as cell colony, for delimiting the growthform of attached cell；
Described migrate test device include at least one third through-hole, described third through-hole as migrate test chamber,
For the most adherent cell colony in limiting chamber at cell colony is carried out cell migration research.
Preferably, described chassis is provided with the second through hole that at least two is identical.
Preferably, described second through hole is circular along the cross sectional shape of radial direction.
Preferably, described second through hole is along the circle that cross sectional shape is diameter 0.20-0.30mm of radial direction.
Preferably, described first through hole and described third through-hole along the cross sectional shape of radial direction be circular, rectangle or
Person's polygon.
Preferably, described first through hole hole depth axially is 2-3mm, and described third through-hole is axially
Hole depth more than or equal to 3mm.
Preferably, described cell arranging devices and migration test device are formed by PDMS.
It addition, present invention also offers a kind of side using above-mentioned cell migration research system to carry out cell migration research
Method, including:
After the chassis of described cell arranging devices is adhered to clean culture dish, described cell arranging devices is carried out
Gas ions processes, and adds cell suspending liquid in described first through hole；
After cell in described cell suspending liquid sinks in the cell colony restriction chamber in described cell arranging devices,
Remaining cell suspending liquid in first through hole described in sucking-off, so that the cell sunk in described cell colony restriction chamber is complete
Adherent；
Described cell colony limit the cell in chamber the most adherent after, described cell arranging devices is removed, is replaced by
Described migration tests device, adds pre-prepd culture medium described migration in test device, so that the most adherent cell
Colony breeds in described culture medium and migrates；
Every preset time period, the most adherent cell is taken pictures in the described growth migrated in test device, with
By the picture research cell migration distance in described culture medium obtained that the described cell migrated in test device is taken pictures.
Preferably, described by the described cell tested in device that migrates is taken pictures the picture research cell obtained described
Migration data in culture medium, including:
The described cell migrated in test device is being taken pictures in the picture obtained, by the most adherent each cell
The region that cell growth is covered is divided into growth district and migrates region；
Obtain the meansigma methods of the every bit in described migration region and the spacing in the center of circle of described growth district, calculate institute
State the difference between the radius of meansigma methods and described growth district place circle, using as described migration distance；
Wherein, described growth district is to comprise the maximum connected region that the most adherent described cell growth is covered,
And the circle that in the circle with the barycenter in described largest connected region as the center of circle, radius is minimum, described migration region is to be pasted completely by described
The region that the cell growth of wall is covered does not includes the region of described growth district.
In the method for high-flux cell migration research of present invention offer and cell migration research system, use cell
Cell colony in arranging devices limits chamber and cultivates the cell of adherent growth, obtains the cell mass of adherent growth.By adherent life
Long cell mass is placed in the migration test chamber migrated in test device and cultivates, and obtains the picture in cell growth process, with
The region covered by the growth of the cell mass of picture record, calculates the migration distance of cell migration.The employing present invention provides
Cell migration research system carry out cell migration research, limit chamber by cell colony and obtain the cell mass of adherent growth,
Again by migrating the acquisition of the picture of cell growth in test chamber, this research method has high repeatability, and
Can be by image procossing by the transfer ability quantification of cell.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is this
Some bright embodiments, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to root
Other accompanying drawing is obtained according to these accompanying drawings.
Fig. 1 is the schematic diagram of the top view of the cell arranging devices that the embodiment of the present invention provides；
Fig. 2 is the schematic diagram of the top view migrating test suggestion that the embodiment of the present invention provides；
Fig. 3 is bowing of the structure on each first through hole in the cell arranging devices that the embodiment of the present invention provides and chassis
View and side view；
Fig. 4 is top view and the side-looking of each third through-hole migrated in test device that the embodiment of the present invention provides
Figure；
Fig. 5 is form picture and the image of MCF-7 cell growth in migrating test chamber that the embodiment of the present invention provides
Processing method schematic diagram, wherein, (a) is the form schematic diagram of the population of cells in zero moment, when (b) and (c) are 23h respectively
The form schematic diagram of the population of cells under the conditions of the FBS of 1% and under the conditions of the FBS of 10%, (d) is that migration distance calculates signal
Figure；
Fig. 6 is MCF-7 cell proliferation and the schematic diagram of migration under the conditions of the variable concentrations FBS that the embodiment of the present invention provides,
Wherein, (e) is MCF-7 cell proliferation change schematic diagram under the conditions of variable concentrations FBS, and (f) is MCF-under the conditions of variable concentrations FBS
7 cell migration distance schematic diagrams.
Detailed description of the invention
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
The a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art
The every other embodiment obtained under not making creative work premise, broadly falls into the scope of protection of the invention.
Present embodiments provide a kind of cell migration research system, including: cell arranging devices 010 as shown in Figure 1
Device 020 is tested with migrating as shown in Figure 2；
Cell arranging devices 010 includes at least one first through hole 011, and one end of each first through hole is provided with the end
Dish, chassis is provided with at least one second through hole 012；
Second through hole 012 limits chamber as cell colony, for delimiting the growthform of attached cell；
Migrate test device 020 include at least one third through-hole 021, third through-hole 021 as migrate test chamber,
For the most adherent cell colony in limiting chamber at cell colony is carried out cell migration research.
Further, chassis is provided with the second through hole 012 that at least two is identical.
It can be one that cell colony limits chamber, certainly, in order to improve the accuracy of experiment, and can be on a chassis
Make the second through hole that multiple size and dimension is identical, to carry out organizing parallel laboratory test more.As shown in fig. 1, on each chassis
It is provided with 4 the second through holes 012.
Further, the second through hole is circular along the cross sectional shape of radial direction.
It will be appreciated that the second through hole limits chamber as cell colony, it is permissible along the cross sectional shape of radial direction
It is rectangle, polygon, circle etc., as long as ensureing to limit chamber along radial direction side as the cell colony in same group of parallel laboratory test
To section shape and size identical.Certainly, the second through hole that the cross sectional shape along radial direction is circular is easier
In manufacture, and owing to there is not wedge angle, it is to avoid cell colony limits the impact of the wedge angle cell growth in the locular wall of chamber.
Further, the second through hole is along the circle that cross sectional shape is diameter 0.20-0.30mm of radial direction.
Fig. 3 is bowing of the structure on each first through hole in the cell arranging devices that the embodiment of the present invention provides and chassis
View and side view.Seeing Fig. 3, the second through hole on this cell arranging devices is a diameter of along the cross sectional shape of radial direction
The circle of d, the hole depth of the second through hole is h, and such as, the second through hole is diameter d=0.25mm along the cross sectional shape of radial direction
Circle, the through hole of the hole depth h=100 μm of the second through hole.The distance that adjacent cell colony limits between chamber is not less than
0.8mm, to prevent the cell of growth in migrating test chamber from interfering with each other.
Further, described first through hole and described third through-hole are circular, rectangle along the cross sectional shape of radial direction
Or polygon.
It will be appreciated that the first through hole that cross sectional shape is circle and third through-hole along radial direction are more easily made
Make and clean.
Further, described first through hole hole depth axially is 2-3mm, described third through-hole axially side
To hole depth more than or equal to 3mm.
As it is shown on figure 3, the first through hole is along the circle that cross sectional shape is a diameter of D of radial direction, the hole of the first through hole
Deep is H, and such as, the first through hole is along the circle that cross sectional shape is diameter D=4mm of radial direction, hole depth H=of the first through hole
The through hole of 3mm.
Fig. 4 is top view and the side-looking of each third through-hole migrated in test device that the embodiment of the present invention provides
Figure.Seeing Fig. 4, this migration test device is provided with third through-hole, described third through-hole is as migrating test chamber, for right
In cell colony limits chamber, the most adherent cell colony carries out cell migration research.
As shown in Figure 4, a diameter of D ', D ' of third through-hole=5mm, third through-hole is more than the second through hole, thinks at cell
Colony limits the cell of adherent growth in chamber and provides enough migration spaces.The hole depth of third through-hole is not less than 3mm.
Further, described cell arranging devices and migration test device are formed by PDMS (poly dimethyl oxygen alkane).
As a kind of specific embodiment, this cell arranging devices includes 4 independent cell colony restricted room
Room, to be limited by extraneous chamber, delimit the growthform of attached cell.Cell arranging devices rapidoprint is PDMS
(polydimethylsiloxane) and Tissue Culture Dish.Cell colony limits the shape of chamber can various ways, such as square,
Rectangle, circular, polygon.Cell colony limits diameter about the 4mm of the first through hole of chamber, thickness about 3mm, each cell
Colony limits a diameter of 0.25mm of chamber, and thickness is 0.1mm；It is that 0.8mm is left that flanking cell colony limits the distance between chamber
The right side, certainly, the distance that flanking cell colony limits between chamber can adjust according to specific experiment demand.
Diameter about the 5mm, more than thickness 3mm that migrate test chamber as follow-up cultivation；Formed at same and migrate
Multiple cell migration test chamber can be made, as long as being replaced by carefully cell colony is limited chamber on the material of test device
When born of the same parents migrate test chamber, the center of circle on culture dish and first through hole of the third through-hole of each migration test chamber are being trained
Support position, the center of circle correspondence on ware, testing on device with migration, the different distances migrated between test chamber
For 6mm, certainly, the different distances migrated between test chamber can adjust according to specific experiment demand.
Present embodiments provide a kind of method using above-mentioned cell migration research system to carry out cell migration research；
After the chassis of cell arranging devices is adhered to clean culture dish, cell arranging devices is carried out at plasma
Reason, adds cell suspending liquid in described first through hole；
After cell in described cell suspending liquid sinks in the cell colony restriction chamber in described cell arranging devices,
Remaining cell suspending liquid in first through hole described in sucking-off, so that the cell sunk in described cell colony restriction chamber is complete
Adherent；
Described cell colony limit the cell in chamber the most adherent after, described cell arranging devices is removed, is changed to institute
State migration test device, add pre-prepd culture medium described migration in test device, so that the most adherent cell mass
Body is bred in described culture medium and migrates；
Every preset time period, the most adherent cell is taken pictures in the described growth migrated in test device, with
By the picture research cell migration distance in described culture medium obtained that the described cell migrated in test device is taken pictures.
Further, described by the described cell tested in device that migrates is taken pictures the picture research cell obtained in institute
State the migration data in culture medium, including:
The described cell migrated in test device is being taken pictures in the picture obtained, by the most adherent each cell
The region that cell growth is covered is divided into growth district and migrates region；
Obtain the meansigma methods of the every bit in described migration region and the spacing in the center of circle of described growth district, calculate institute
State the difference between the radius of meansigma methods and described growth district place circle, using as described migration distance；
Wherein, described growth district is to comprise the maximum connected region that the most adherent described cell growth is covered,
And the circle that in the circle with the barycenter in described largest connected region as the center of circle, radius is minimum, described migration region is to be pasted completely by described
The region that the cell growth of wall is covered does not includes the region of described growth district.
As more specifically embodiment, use above cell migration research system to carry out cell migration research and include following
Step:
(1) prepare cell colony form by the method for soft lithographic and limit chamber, and naturally glue at the bottom of clean culture dish
Attached；
(2) the cell arranging devices that will obtain in step (1), is placed in vacuum pump evacuation about 1min and carries out 2-
10min Cement Composite Treated by Plasma；
(3) cell suspension is added each cell colony form and limits chamber, suck suspension after 30min, sink to 0.25mm
Cell in hole will not be sucked out；After cell attachment, remove restriction chamber, use instead migration test device, and with the most adherent carefully
Born of the same parents' para-position one by one.
(4) cell culture fluid containing test substance is added, to obtain test substance to carefully at each observation chamber that migrates
The impact that born of the same parents migrate.
(5) at set intervals, taking pictures, record cell expansion process, by image procossing, high-throughput quantification obtains
The propagation of cell and migration.
For example, application cell arranging devices realizes MCF-7 breast cancer cell the denseest with migrating test device
Migration research detailed process under the conditions of degree hyclone (FBS) is as follows:
(1) cell colony form limits.
Cell arranging devices is put in culture dish so that it is at the bottom of bottom (chassis portion) and ware closely after laminating, will
Cell suspension adds in the first through hole, so that cell suspension enters each cell colony and limits in chamber, each cell colony limits
Chamber processed about can accommodate the suspension of 0.02ml.Formed tightly in order to cavity bottom can be limited at cell colony after ensureing cell attachment
The cell monolayer of solid matter row, in cell suspension, the concentration of cell need to control at 50-100 ten thousand/ml, it is possible to carries out according to practical situation
Adjust.After adding cell 30min, cell can sink at the bottom of hole, now by suspension sucking-off, changes acellular culture medium, sinks to
Cell in second through hole of 0.25mm will not be sucked out, and the cell in face, cell colony restricted room outdoor can be inhaled mostly
Walk.After adding cell 2-4h, cell can start adherent.After adding cell 8-12h, the cell in 0.25mm hole can complete adherent shape
Become irregular polygon.
(2) cell is cultivated and migrates test.
After treating that cell is the most adherent, monoblock cell arranging devices (is included upper and lower two-layer, namely the first through hole and the end
Dish) take off together, and change migration test device (the migration test chamber in 5mm hole), carefully align that (third through-hole is at culture dish
Shang Kong center is corresponding at culture dish Shang Kong center with the first through hole) and make to fit, afterwards in each hole at the bottom of bottom and ware
Add the culture medium of different condition.Each hole about can accommodate the culture medium of about 0.06ml.
Owing to during cell arranging devices changes into migration test chamber, cell easily thirst, so operation is non-
The most rapidly, or the training of about 0.003ml basis is dripped with the volley of rifle fire in the position in each hole as early as possible after taking cell arranging devices off
Support base (research afterwards is affected the least by serum-free), then change migration test chamber (the migration test chamber in 5mm hole).Change
Migrate well test chamber and add different condition culture medium after, culture dish is put into CO2 gas incubator, and will now
It was designated as zero moment, cell is carried out long-term cultivation and observation.In zero moment, cell is concentrated in the circle of diameter 0.25mm
In territory, form a colony, the most outwards can expand due to propagation and migration subsequently.A pictures is clapped every several hours,
The process of record cell colony amplification.
As it is shown in figure 5, (a) is the form schematic diagram of the population of cells in zero moment, 1% when (b) and (c) is 23h respectively
FBS under the conditions of and the form schematic diagram of population of cells under the conditions of the FBS of 10%, it can be seen that zero the moment population of cells
The most not migrating, after 23 hours, the population of cells under the conditions of the FBS of 1% is than the population of cells under the conditions of the FBS of 10% more
It is susceptible to migrate.
(3) data process, and quantitatively obtain cell migration data.
The area S-cell having cell compartment, as shown in (d) in Fig. 5, (is removed in (d) of Fig. 5 by the experimental image obtained
Region outside black region) it is defined as the propagation level of cell.When defining the transfer ability of cell, first do a nothing and move
Move it is assumed that i.e. assume that cell does not migrate during whole, but be mutually close to outgrowth.This situation is equivalent to
All cells is gathered in a circle, and namely in the region of the circle in (d) of Fig. 5 (growth district), the radius of circle is designated as
Rgrowth, the center of circle is to have the barycenter in largest connected territory in cell compartment.
Cell (outside the circle of (d) of Fig. 5, the region of non-black) outside for circle, it is believed that they are entirely migration
Going out, this subregion is called migration region.Thus migrate all pixels distance R to the center of circle in regiontotalAverage
Value deducts round radius Rgrowth, obtain a Length Quantity Dmigration, then this amount can serve as measure of cell migration water
Flat index, it is also possible to be referred to as migration distance, it may be assumed that
Dmigration=Mean (Rtotal)–Rgrowth
Mean(Rtotal) it is the meansigma methods migrating each pixel in region to the distance in the center of circle, in (d) in Fig. 5
Rtotal(i) and RtotalJ () represents the ith pixel point migrated in region and the jth pixel distance to the center of circle.
It should be noted that the gentle migration of proliferation water here level represents is all the flat of whole cell colony in the visual field
All behaviors.Furthermore, it is contemplated that the initial value of different experiments group is different, so needing result is normalized.
(4) experimental result.
Fig. 5 illustrates part of test results.10%FBS matched group be regular culture conditions (DMEM basal medium+
1% antibiotic+10%FBS), after 23h as shown in (c) in Fig. 5, cell is mutually close to outgrowth, almost without any
(continuous print observes the most provable population of cells entirely due to breed and migrate, but we do not use when analyzing in migration
The data of Continuous Observation)；1%FBS experimental group is hungry condition of culture, and after 23h as shown in (b) in Fig. 5, cell is except propagation
Outside, also have significantly to the phenomenon of external migration；Additionally also have the matched group (not shown in Fig. 5) of a 0.1%FBS, approximation
In the comparison of serum-free, this under the conditions of cytoactive very poor, migrate and breed the faintest.These qualitatively result and we
Expection be consistent.
Can obtain quantitative result after image procossing, as shown in Figure 6 in (e) and (f) shown in.0.1%FBS
Under the conditions of cytoactive very poor, so either propagation level or migration level nearly all do not have anything to change.For propagation
For, FBS concentration is the highest, and the propagation of cell is the fastest.And for migrating, cell has under hungry condition of culture
Higher transfer ability.The abscissa of (e) in Fig. 6 is Time (time), and unit is h (hour), and vertical coordinate is
Normalized Area of Cells (cell normalized area), has reacted the propagation level of cell, the horizontal stroke of (f) in Fig. 6
Coordinate is Time (time), and unit is h (hour), and vertical coordinate is Normalized Migration Distance (cell normalizing
Change migration distance), react the migration level of cell.
The third aspect, present invention also offers a kind of method making cell arranging devices, including:
On the first default silicon chip, the boss corresponding with described first through hole is formed, using as formation by patterning processes
First mould of described first through hole；
With described first mould as forming tool, obtain that there is the indenture corresponding with described first through hole by molding process
The first unshaped device, remove the part in indenture in described first unshaped device, to obtain described first through hole；
On the second default silicon chip, the boss corresponding with described second through hole is formed, using as formation by patterning processes
Second mould of described second through hole；
With described second mould as forming tool, by spin coating proceeding, form the chassis with described second through hole；
Described chassis is fixed on one end of described first through hole, to obtain there is the thin of described cell colony restriction chamber
Born of the same parents' arranging devices.
Further, described one end that described chassis is fixed on described first through hole, to obtain having described cell mass
Body limits the cell arranging devices of chamber, including:
After described chassis is attached to one end of described first through hole, put into baking oven baking preset duration, so that described chassis
It is bonded in one end of described first through hole.
Fourth aspect, the present embodiment has also put forward the manufacture method migrating test device, including:
On the 3rd default silicon chip, the boss corresponding with described third through-hole is formed, using as formation by patterning processes
3rd mould of described third through-hole；
With described 3rd mould as forming tool, obtain that there is the indenture corresponding with described third through-hole by molding process
The second unshaped device, remove the part in indenture in described second unshaped device, to obtain described third through-hole.
Specifically, the manufacture method of cell arranging devices and migration test device includes:
(1) preparation of mould.With L-Edit draw, the shape of the first through hole, the second through hole is printed upon plastic film or
In optical mask, as exposed mask, for subsequent experimental.Altogether needing three pieces of silicon chip moulds, two pieces are used for making cell mass
Volume morphing limits chamber, and one piece is used for making cell and cultivates and migrate test chamber.Three pieces of mould preparation process are basically identical, only
It is that mask is variant.Use SU8-3050 glue, get rid of 10s in advance with the rotating speed of 500rpm during spin coating, then get rid of with the rotating speed of 1000rpm
30s, photoresist thickness about 0.2mm.After drying 30min on 95 degree of electric hot plates, putting into exposure machine and be exposed, time of exposure needs
Help document and actual light intensity with reference to exposure machine determine.Drying 30min the most again on 95 degree of electric hot plates, development just may be used
To obtain mould.
(2) cell arranging devices and the making of migration test device.The first through hole and cell in cell arranging devices move
Moving the third through-hole in test chamber, all obtained by PDMS injection molding, the former needs A glue (substrate) and B glue (firming agent)
Proportioning be 9:1-10:1, the latter needs 7:1-10:1, and their thickness is all at more than 3mm.Cell in cell arranging devices
Colony is limited chamber and is obtained by PDMS spin coating, and A, B glue proportioning is 9:1-10:1, turning with 1500rpm in sol evenning machine
Speed gets rid of 30s, can obtain the PDMS film that thickness is about 0.1mm.First three pieces of moulds are put into during solidification in 70 degree of baking ovens and dry
30min, takes out cell colony form afterwards and limits two pieces of moulds of chamber, and now PDMS has cured.Taking off on mould
Punch, carefully by the cell cloth of the only first through hole with 4mm card punch on the PDMS with the indenture corresponding with 4mm hole come
Putting device aperture to be attached to hole on chassis, put into and dry overnight in baking oven, PDMS may proceed to solidification, and the first through hole and chassis can be bonded at
Together.
(3) surface modification of cell arranging devices.Thin adding in 4mm hole (the first through hole on cell arranging devices)
Before born of the same parents, need the Cement Composite Treated by Plasma of evacuation and the 2-10min carrying out 1min, to prevent cell arranging devices from putting into dioxy
After changing carbon incubator (37 degree), the internal gas dissolved can produce bubble by expanded by heating, can make hydrophobic PDMS surface simultaneously
Become hydrophilic such that it is able to successfully injected by cell without producing bubble in bottom.The third through-hole of 5mm (migrates test
Device) need not do such process.After it should be noted that Cement Composite Treated by Plasma, PDMS can slowly revert to hydrophobic, so
This step needs to carry out in 30min before migrating experiment and starting.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (9)
1. a cell migration research system, it is characterised in that including: cell arranging devices and migration test device；
Described cell arranging devices includes that at least one first through hole, one end of each described first through hole are provided with the end
Dish, described chassis is provided with at least one second through hole；
Described second through hole limits chamber as cell colony, for delimiting the growthform of attached cell；
The described test device that migrates includes at least one third through-hole, and described third through-hole, as migrating test chamber, is used for
The most adherent cell colony in limiting chamber at cell colony is carried out cell migration research.
2. according to the cell migration research system described in claim 1, it is characterised in that be provided with at least two on described chassis
Individual the second identical through hole.
3. according to the cell migration research system described in claim 2, it is characterised in that described second through hole is along radial direction side
To cross sectional shape be circular.
4. according to the cell migration research system described in claim 3, it is characterised in that described second through hole is along radial direction side
To the circle that cross sectional shape is diameter 0.20-0.30mm.
5. according to the cell migration research system described in claim 1, it is characterised in that described first through hole and the described 3rd
Through hole is circular, rectangle or polygon along the cross sectional shape of radial direction.
6. according to the cell migration research system described in claim 1, it is characterised in that described first through hole axially side
To hole depth be 2-3mm, described third through-hole hole depth axially be more than or equal to 3mm.
7. according to the cell migration research system described in claim 1, it is characterised in that described cell arranging devices and migration
Test device is formed by PDMS.
8. the cell migration research system that a kind uses according to any one of claim 1-7 carries out the side of cell migration research
Method, it is characterised in that including:
After being adhered to clean culture dish on the chassis of described cell arranging devices, described cell arranging devices is carried out plasma
Body processes, and adds cell suspending liquid in described first through hole；
After cell in described cell suspending liquid sinks in the cell colony restriction chamber in described cell arranging devices, sucking-off
Remaining cell suspending liquid in described first through hole, so that the cell sunk in described cell colony restriction chamber pastes completely
Wall；
Described cell colony limit the cell in chamber the most adherent after, described cell arranging devices is removed, is replaced by described
Migrate test device, add pre-prepd culture medium described migration in test device, so that the most adherent cell colony
Described culture medium is bred and migrates；
Every preset time period, the most adherent cell is taken pictures in the described growth migrated in test device, to pass through
Cell migration distance in described culture medium studied by the picture obtained of taking pictures the described cell migrated in test device.
Method described in the most according to Claim 8, it is characterised in that described by the described cell migrated in test device
Cell migration data in described culture medium studied by the picture obtained of taking pictures, including:
The described cell migrated in test device is being taken pictures in the picture obtained, by the cell in the most adherent each cell
The region that growth is covered is divided into growth district and migrates region；
Obtain the meansigma methods of the every bit in described migration region and the spacing in the center of circle of described growth district, calculate described flat
Difference between the radius of average and described growth district place circle, using as described migration distance；
Wherein, described growth district is to comprise the maximum connected region that the most adherent described cell growth is covered, and with
The circle that in the circle that barycenter is the center of circle in described largest connected region, radius is minimum, described migration region is by described the most adherent
The region that cell growth is covered does not includes the region of described growth district.
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| CN110408536A (en) * | 2019-06-12 | 2019-11-05 | 上海大学 | Cell migration assay device and method |
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| US20030040031A1 (en) * | 2000-11-08 | 2003-02-27 | Enoch Kim | System for monitoring cell motility in real-time |
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| US20030040031A1 (en) * | 2000-11-08 | 2003-02-27 | Enoch Kim | System for monitoring cell motility in real-time |
| CN101508954A (en) * | 2009-03-18 | 2009-08-19 | 南开大学 | Scuffing experimental device for cell migration research and high throughput medicament sifting motion |
| CN102796659A (en) * | 2012-08-21 | 2012-11-28 | 北京大学 | Porous single cell observation plate and use thereof |
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