CN106047665B - It is a kind of for the method for high-flux cell migration research and cell migration research system - Google Patents
It is a kind of for the method for high-flux cell migration research and cell migration research system Download PDFInfo
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Abstract
The invention discloses a kind of for the method for high-flux cell migration research and cell migration research system, using the cell of the cell colony limitation chamber culture adherent growth in cell arranging devices, the cell mass of adherent growth is obtained.It is cultivated in the migration test chamber that the cell mass of adherent growth is placed in migration test device, obtains the picture in cell growth process, with the region that the growth by the cell mass of picture record is covered, calculate the migration distance of cell migration.Cell migration research is carried out using cell migration research system provided by the invention, chamber is limited by cell colony and obtains the cell mass of adherent growth, pass through the acquisition to the picture of cell growth in migration test chamber again, this research method has high repeatability, and can be by image procossing by the transfer ability quantification of cell.
Description
Technical field
The invention belongs to micro Process and technical field of cell biology, and in particular to one kind is ground for high-flux cell migration
The method and cell migration research system studied carefully.
Background technology
Cell migration and the occurrence and development of tumour are in close relations, and cell migration is by a variety of external environmental factors and interior
The joint effect of portion's factor.For the correlative study of cell migration, on the one hand can let us have more to cell migration behavior
Understanding, on the other hand, the treatment of tumour etc. He cell migration disease in close relations is explored, with important value.
Mainly there are two kinds of scratch experiment and invasion experiment in being tested in traditional biological method about cells in vitro migration
Experimental method, although scratch experiment is simple, repeatability is poor, and the process of cut can cause to damage to cell, experimental result
Also the effect of cell Proliferation and migration can not be distinguished；Invasion experiment, the environment of one side steady concentration gradient are difficult to tie up
It holds, on the other hand, the effect of cell Proliferation and migration can not be distinguished.However, for existing micro-fluidic chip, it is uncomfortable
It, also can not the proliferation of cell and the effect of migration for the high flux screening of sample.
Invention content
The technical problems to be solved by the invention are how to make a kind of high flux screening simple in structure, suitable for sample
System and a kind of cell migration test method for distinguishing the proliferation of cell and migration.
For the problem, the present invention provides a kind of cell migration research system, including：Cell arranging devices and migration are surveyed
Try device；
The cell arranging devices include at least one first through hole, and one end of each first through hole is both provided with
Chassis is provided at least one second through-hole on the chassis；
Second through-hole limits chamber as cell colony, for delimiting the growthform of attached cell；
The migration test device includes at least one third through-hole, and the third through-hole is used as migration test chamber,
For carrying out cell migration research to cell colony completely adherent in limiting chamber in cell colony.
Preferably, at least two the second identical through-holes are provided on the chassis.
Preferably, second through-hole is circle along the cross sectional shape of radial direction.
Preferably, the circle that second through-hole is diameter 0.20-0.30mm along the cross sectional shape of radial direction.
Preferably, the first through hole and the third through-hole along the cross sectional shape of radial direction for round, rectangle or
Person's polygon.
Preferably, the hole depth of the first through hole axially is 2-3mm, and the third through-hole is axially
Hole depth be more than or equal to 3mm.
Preferably, the cell arranging devices and migration test device are formed by PDMS.
In addition, the present invention also provides a kind of sides that cell migration research is carried out using above-mentioned cell migration research system
Method, including：
After the chassis of the cell arranging devices is adhered to clean culture dish, the cell arranging devices are carried out etc.
Gas ions processing, cell suspending liquid is added in into the first through hole；
After cell in the cell suspending liquid is sunk in the cell colony limitation chamber in the cell arranging devices,
Remaining cell suspending liquid in the first through hole is sucked out, so that the cell sunk in the cell colony limitation chamber is complete
It is adherent；
After cell in cell colony limitation chamber is completely adherent, the cell arranging devices are removed, are changed to
The migration test device adds in pre-prepd culture medium, so that completely adherent cell in the migration test device
Group is proliferated and migrates in the culture medium；
Every preset time period, take pictures to growth of the completely adherent cell in the migration test device, with
Migration distance of the cell in the culture medium is studied by the picture taken pictures to the cell in the migration test device.
Preferably, the picture by taking pictures to the cell in the migration test device studies cell described
Migrating data in culture medium, including：
It, will be in each completely adherent cell in the picture taken pictures to the cell in the migration test device
The region division that cell growth is covered is growth district and migration region；
The average value of distance between the every bit migrated in region and the center of circle of the growth district is obtained, calculates institute
Difference between the radius of circle where stating average value and the growth district, using as the migration distance；
Wherein, the growth district is the maximum connected region covered comprising the completely adherent cell growth,
And using the barycenter in the largest connected region as the circle in the center of circle in radius minimum circle, it is described migration region be by the complete patch
Do not include the region of the growth district in the region that the cell growth of wall is covered.
In method and cell migration research system provided by the present invention for high-flux cell migration research, using cell
The cell of cell colony limitation chamber culture adherent growth in arranging devices, obtains the cell mass of adherent growth.By adherent life
It is cultivated in the migration test chamber that long cell mass is placed in migration test device, 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.It is provided using the present invention
Cell migration research system carry out cell migration research, by cell colony limit chamber obtain the cell mass of adherent growth,
Again by the acquisition to the picture of cell growth in migration test chamber, this research method has high repeatability, and
It can be by image procossing by the transfer ability quantification of cell.
Description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is this hair
Some bright embodiments, for those of ordinary skill in the art, without creative efforts, can be with root
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the schematic diagram of the vertical view of cell arranging devices provided in an embodiment of the present invention；
Fig. 2 is the schematic diagram of the vertical view of migration test opinion provided in an embodiment of the present invention；
Fig. 3 is bowing for the structure of each first through hole and chassis in cell arranging devices provided in an embodiment of the present invention
View and side view；
Fig. 4 is vertical view and the side view of each third through-hole in migration test device provided in an embodiment of the present invention
Figure；
Fig. 5 is the form picture and image that MCF-7 cells provided in an embodiment of the present invention are grown in test chamber is migrated
Processing method schematic diagram, wherein, (a) is the form schematic diagram of the population of cells of zero moment, when (b) and (c) is 23h respectively
The form schematic diagram of population of cells under the conditions of 1% FBS and under the conditions of 10% FBS, (d) are that migration distance calculates signal
Figure；
Fig. 6 is MCF-7 cell Proliferations and the schematic diagram of migration under the conditions of various concentration FBS provided in an embodiment of the present invention,
Wherein, (e) is MCF-7 cell Proliferation change schematic diagrams under the conditions of various concentration FBS, and (f) is MCF- under the conditions of various concentration FBS
7 cell migrations are apart from schematic diagram.
Specific embodiment
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical solution in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
Part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
All other embodiments obtained without making creative work shall fall within the protection scope of the present invention.
A kind of cell migration research system is present embodiments provided, including：Cell arranging devices 010 as shown in Figure 1
With migration test device 020 as shown in Figure 2；
Cell arranging devices 010 include at least one first through hole 011, and one end of each first through hole is both provided with bottom
Disk is provided at least one second through-hole 012 on chassis；
Second through-hole 012 limits chamber as cell colony, for delimiting the growthform of attached cell；
Migration test device 020 includes at least one third through-hole 021, and third through-hole 021 is used as migration test chamber,
For carrying out cell migration research to cell colony completely adherent in limiting chamber in cell colony.
Further, at least two the second identical through-holes 012 are provided on chassis.
Cell colony limitation chamber can be one, certainly, can be on a chassis in order to improve the accuracy of experiment
The second identical through-hole of multiple size and shape is made, to carry out multigroup parallel laboratory test.As shown in fig. 1, on each chassis
It is both provided with 4 the second through-holes 012.
Further, the second through-hole is circle along the cross sectional shape of radial direction.
It will be appreciated that the second through-hole limits chamber as cell colony, it can be with 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 it is identical.Certainly, it is easier for circular second through-hole along the cross sectional shape of radial direction
In manufacture, and since there is no wedge angle, avoid influence of the wedge angle in the locular wall of cell colony limitation chamber to cell growth.
Further, the circle that the second through-hole is diameter 0.20-0.30mm along the cross sectional shape of radial direction.
Fig. 3 is bowing for the structure of each first through hole and chassis in cell arranging devices provided in an embodiment of the present invention
View and side view.Referring to Fig. 3, the second through-hole on the 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, for example, the second through-hole is diameter d=0.25mm along the cross sectional shape of radial direction
Circle, the through-hole of h=100 μm of the hole depth of the second through-hole.The distance between adjacent cell colony limitation chamber is not less than
0.8mm, to prevent the cell grown in test chamber is migrated from interfering with each other.
Further, the first through hole and the third through-hole are round, rectangle along the cross sectional shape of radial direction
Or polygon.
It will be appreciated that the cross sectional shape along radial direction is more easily made for circular first through hole and third through-hole
It makes and cleans.
Further, the hole depth of the first through hole axially is 2-3mm, and the third through-hole is axially square
To hole depth be more than or equal to 3mm.
As shown in figure 3, the circle that first through hole is a diameter of D along the cross sectional shape of radial direction, the hole of first through hole
Depth is H, for example, the circle that first through hole is diameter D=4mm along the cross sectional shape of radial direction, the hole depth H=of first through hole
The through-hole of 3mm.
Fig. 4 is vertical view and the side view of each third through-hole in migration test device provided in an embodiment of the present invention
Figure.Referring to Fig. 4, third through-hole is provided on migration test device, the third through-hole as migration test chamber, for pair
Completely adherent cell colony carries out cell migration research in cell colony limits chamber.
As shown in figure 4, a diameter of D ' of third through-hole, D '=5mm, third through-hole is more than the second through-hole, in cell
The cell of adherent growth provides enough migration spaces in group's limitation chamber.The hole depth of third through-hole is not less than 3mm.
Further, the cell arranging devices and migration test device are formed by PDMS (poly dimethyl oxygen alkane).
As a kind of specific embodiment, 4 independent cell colony restricted rooms are included in this cell arranging devices
Room, to be limited by extraneous chamber, to delimit the growthform of attached cell.Cell arranging devices rapidoprint is PDMS
(dimethyl silicone polymer) and Tissue Culture Dish.The shape of cell colony limitation chamber can be there are many form, such as square,
Rectangle, round, polygon.Diameter 4mm of first through hole of cell colony limitation chamber or so, thickness 3mm or so, each cell
Group limitation chamber a diameter of 0.25mm, thickness 0.1mm；The distance between flanking cell group limitation chamber is left for 0.8mm
The right side, certainly, the distance between flanking cell group limitation chamber can be adjusted according to specific experiment demand.
Diameter 5mm of migration test chamber as follow-up cultivation or so, more than thickness 3mm；It is formed and migrated in same
Multiple cell migration test chambers can be made by testing on the material of device, as long as being changed to carefully by cell colony limitation chamber
When born of the same parents migrate test chamber, the center of circle on culture dish of the third through-hole of each migration test chamber and first through hole are being trained
The center of circle position supported on ware corresponds to, on same migration test device, the distance between different migration test chamber
For 6mm, certainly, the distance between different migration test chamber can be adjusted according to specific experiment demand.
Present embodiments provide a kind of method that cell migration research is carried out using above-mentioned cell migration research system；
By the chassis of cell arranging devices with after clean culture dish adherency, being carried out at plasma to cell arranging devices
Reason, cell suspending liquid is added in into the first through hole；
After cell in the cell suspending liquid is sunk in the cell colony limitation chamber in the cell arranging devices,
Remaining cell suspending liquid in the first through hole is sucked out, so that the cell sunk in the cell colony limitation chamber is complete
It is adherent；
After cell in cell colony limitation chamber is completely adherent, the cell arranging devices are removed, are changed to institute
Migration test device is stated, pre-prepd culture medium is added in the migration test device, so that completely adherent cell mass
Body is proliferated and migrates in the culture medium；
Every preset time period, take pictures to growth of the completely adherent cell in the migration test device, with
Migration distance of the cell in the culture medium is studied by the picture taken pictures to the cell in the migration test device.
Further, the picture by taking pictures to the cell in the migration test device studies cell in institute
The migrating data in culture medium is stated, including：
It, will be in each completely adherent cell in the picture taken pictures to the cell in the migration test device
The region division that cell growth is covered is growth district and migration region；
The average value of distance between the every bit migrated in region and the center of circle of the growth district is obtained, calculates institute
Difference between the radius of circle where stating average value and the growth district, using as the migration distance；
Wherein, the growth district is the maximum connected region covered comprising the completely adherent cell growth,
And using the barycenter in the largest connected region as the circle in the center of circle in radius minimum circle, it is described migration region be by the complete patch
Do not include the region of the growth district in the region that the cell growth of wall is covered.
As more specifically embodiment, cell migration research is carried out including following using more than cell migration research system
Step：
(1) cell colony form is prepared by the method for soft lithographic and limits chamber, and naturally viscous with clean culture dish bottom
It is attached；
(2) the cell arranging devices that will be obtained in step (1) are placed in vacuum pump and vacuumize 1min or so and carry out 2-
10min corona treatments；
(3) cell suspension is added in into each cell colony form and limits chamber, sucked suspension after 30min, sink to 0.25mm
Cell in hole will not be sucked out；After cell is adherent, remove limitation chamber, use migration test device instead, and with it is adherent thin
Born of the same parents align one by one.
(4) cell culture fluid containing test substance is added in each migration observation chamber, to obtain test substance to thin
The influence of born of the same parents' migration.
(5) at regular intervals, it takes pictures, records cell expansion process, by image procossing, high-throughput quantification obtains
The proliferation of cell and migration.
For example, application cell arranging devices and migration test device are realized to MCF-7 breast cancer cells Bu Tong dense
Migration research detailed process under the conditions of degree fetal calf serum (FBS) is as follows：
(1) cell colony form limits.
Cell arranging devices are put into culture dish, are made after its bottom (chassis portion) is closely bonded with ware bottom, it will
Cell suspension is added in first through hole, so that cell suspension enters in each cell colony limitation chamber, each cell colony limit
Chamber processed can about accommodate the suspension of 0.02ml.In order to ensure that can limit cavity bottom in cell colony after cell is adherent forms tightly
The cell monolayer of solid matter row, the concentration of cell need to be controlled in ten thousand/ml of 50-100 in cell suspension, can also be carried out according to actual conditions
Adjustment.After adding in cell 30min, cell can sink to bottom hole, and suspension is sucked out at this time, change acellular culture medium, sink to
Cell in the second through-hole of 0.25mm will not be sucked out, and the cell in face can be inhaled mostly outside cell colony restricted room room
It walks.After adding in cell 2-4h, cell can start adherent.After adding in cell 8-12h, the cell in 0.25mm holes can complete adherent shape
Into irregular polygon.
(2) cell culture and migration test.
After treating that cell is completely adherent, by monoblock cell arranging devices (including upper and lower two layers, that is, first through hole and bottom
Disk) it takes off together, and change migration test device (the migration test chamber in 5mm holes), carefully to just (third through-hole is in culture dish
On hole center it is corresponding with control center of the first through hole on culture dish) and bottom is made to be bonded with ware bottom, later in each hole
Add in the culture medium of different condition.Each hole can about accommodate the culture medium of 0.06ml or so.
Since cell easily thirst during cell arranging devices to be changed into migration test chamber, so operation is non-
The training of 0.003ml or so bases is often added dropwise in the position in each hole with the volley of rifle fire as early as possible rapidly or after cell arranging devices are taken off
Base (serum-free influences very little to research later) is supported, then changes migration test chamber (the migration test chamber in 5mm holes).It changes
After migrating test chamber well and adding the culture medium of different condition, culture dish is put into carbon dioxide incubator, and will at this time
Zero moment is denoted as, long-term culture and observation are carried out to cell.In zero moment, cell is concentrated in the circle of diameter 0.25mm
In domain, a group is formed, it then can be due to being proliferated and migrating and slowly amplification outward.A pictures were clapped every several hours,
Record the process of cell colony amplification.
As shown in figure 5, (a) is the form schematic diagram of the population of cells of zero moment, 1% when (b) and (c) is 23h respectively
FBS under the conditions of and population of cells under the conditions of 10% FBS form schematic diagram, it can be seen that zero moment population of cells
Substantially it does not migrate, the population of cells after 23 hours under the conditions of FBS of the population of cells under the conditions of 1% FBS than 10% is more
Easily migrate.
(3) data processing quantitatively obtains cell migration data.
Shown in (d) in obtained experimental image such as Fig. 5, the area S-cell for having cell compartment (is removed in (d) of Fig. 5
Region except black region) be defined as cell proliferation it is horizontal.In the transfer ability for defining cell, first do a nothing and move
It moves it is assumed that assuming that cell does not migrate in whole process, but be mutually located next to outgrowth.Such case is equivalent to
All cells are gathered in a circle, that is, in the region of the circle (growth district) in (d) of Fig. 5, round radius is denoted as
Rgrowth, the center of circle is the barycenter for having largest connected domain in cell compartment.
Cell outside for circle (except the circle of (d) of Fig. 5, the region of non-black), it is believed that they are entirely migration
It goes out, this subregion is called migration region.The all pixels point in region is thus migrated to the distance R in the center of circletotalBe averaged
Value subtracts round radius Rgrowth, obtain a Length Quantity Dmigration, then this amount can serve as measure of cell migration water
Flat index may also be referred to as migration distance, i.e.,：
Dmigration=Mean (Rtotal)–Rgrowth
Mean(Rtotal) it is the average value for migrating each pixel in region to the distance in the center of circle, in (d) in Fig. 5
Rtotal(i) and Rtotal(j) represent migration region in ith pixel point and j-th of pixel to the center of circle distance.
It should be noted that proliferation water here gentle migration level represented is all that entire cell colony is put down in the visual field
Equal behavior.Furthermore, it is contemplated that the initial value of different experiments group is different, so needing that result is normalized.
(4) experimental result.
Part of test results is illustrated in Fig. 5.10%FBS control groups be regular culture conditions (DMEM basal mediums+
1% antibiotic+10%FBS), after 23h as shown in (c) in Fig. 5, cell is mutually located next to outgrowth, almost without any
Migration (continuous observation also provable population of cells entirely due to proliferation and migrate, but we do not use in analysis
The data of continuous observation)；1%FBS experimental groups are hungry condition of culture, and after 23h as shown in (b) in Fig. 5, cell is in addition to proliferation
Except, also significantly to the phenomenon that external migration；In addition there are one the control groups (being not shown in Fig. 5) of 0.1%FBS, approximate
In the control of serum-free, it is this under the conditions of cell activity it is very poor, migration and proliferation are all very faint.These qualitatively result and we
Be contemplated to be what is be consistent.
By that can obtain quantitative after image procossing as a result, (e) and (f) in as shown in Figure 6 is shown.0.1%FBS
Under the conditions of cell activity it is very poor, so either proliferation is horizontal or migrates and horizontal nearly all changes without what.For proliferation
For, FBS concentration is higher, and the proliferation of cell is also faster.And for migration, cell has under hungry condition of culture
Stronger transfer ability.The abscissa of (e) in Fig. 6 is Time (time), and unit is h (hour), and ordinate is
Normalized Area of Cells (cell normalized area), the proliferation for having reacted cell is horizontal, the horizontal stroke of (f) in Fig. 6
Coordinate is Time (time), and unit is h (hour), and ordinate is Normalized Migration Distance (cell normalizings
Change migration distance), the migration for having reacted cell is horizontal.
The third aspect, the present invention also provides a kind of method for making cell arranging devices, including：
Boss corresponding with the first through hole is formed on preset first silicon chip by patterning processes, using as formation
First mold of the first through hole；
Using first mold as forming tool, obtain that there is dent corresponding with the first through hole by molding process
The first unshaped device, the part in dent in the first unshaped device is removed, to obtain the first through hole；
Boss corresponding with second through-hole is formed on preset second silicon chip by patterning processes, using as formation
Second mold of second through-hole；
Using second mold as forming tool, by spin coating proceeding, the chassis with second through-hole is formed；
The chassis is fixed on to one end of the first through hole, to obtain that there is the thin of the cell colony limitation chamber
Born of the same parents' arranging devices.
Further, described one end that the chassis is fixed on to the first through hole, to obtain having the cell mass
Body limits the cell arranging devices of chamber, including：
After the chassis to be attached to one end of the first through hole, baking oven baking preset duration is put into, so that the chassis
It is bonded in one end of the first through hole.
Fourth aspect, the production method that the present embodiment has also carried migration test device, including：
Boss corresponding with the third through-hole is formed on preset third silicon chip by patterning processes, using as formation
The third mold of the third through-hole；
Using the third mold as forming tool, obtain that there is dent corresponding with the third through-hole by molding process
The second unshaped device, the part in dent in the second unshaped device is removed, to obtain the third through-hole.
Specifically, the production method of cell arranging devices and migration test device includes：
(1) preparation of mold.Drawn with L-Edit, by the shape of first through hole, the second through-hole be printed upon plastic film or
In optical mask, as exposed mask, for subsequent experimental.Three pieces of silicon chip molds are needed in total, and two pieces are used to make cell mass
Volume morphing limits chamber, and one piece is used to make cell culture and migration test chamber.Three pieces of mold preparation process are basically identical, only
It is that mask is variant.Using SU8-3050 glue, 10s is got rid of, then get rid of with the rotating speed of 1000rpm with the rotating speed of 500rpm in advance during spin coating
30s, photoresist thickness 0.2mm or so.It after drying 30min on 95 degree of electric hot plates, is put into exposure machine and is exposed, the time for exposure needs
Help document and actual light intensity with reference to exposure machine determine.30min is dried on 95 degree of electric hot plates again later, development can
To obtain mold.
(2) making of cell arranging devices and migration test device.First through hole and cell in cell arranging devices are moved
The third through-hole in test chamber is moved, is all obtained by PDMS injection moldings, the former needs A glue (matrix) and B glue (curing agent)
Proportioning be 9:1-10:1, the latter needs 7:1-10:1, their thickness is all in more than 3mm.Cell in cell arranging devices
Group's limitation chamber is obtained by PDMS spin coatings, and A, B glue proportioning are 9:1-10:1, turned in sol evenning machine with 1500rpm
Speed gets rid of 30s, can obtain the PDMS film that thickness is about 0.1mm.First three pieces of molds are put into 70 degree of baking ovens during curing and are dried
30min takes out two pieces of molds of cell colony form limitation chamber later, and PDMS has been cured at this time.It is being removed from mold
It is punched on the PDMS with dent corresponding with 4mm holes come with 4mm card punch, carefully by the cell cloth of only first through hole
It puts device aperture device to hole to be attached on chassis, is put into baking oven and dries overnight, PDMS will continue to cure, and first through hole and chassis can be sticked to
Together.
(3) surface of cell arranging devices is modified.It is thin being added in 4mm holes (first through hole on cell arranging devices)
Before born of the same parents, need to carry out the corona treatment vacuumized with 2-10min of 1min, cell arranging devices to be prevented to be put into dioxy
The internal gas meeting expanded by heating dissolved generates bubble, while can make hydrophobic PDMS surfaces after changing carbon incubator (37 degree)
Become hydrophilic, so as to successfully inject cell without generating bubble in bottom.Third through-hole (the migration test of 5mm
Device) it does not need to do such processing.It should be noted that after corona treatment PDMS can slowly revert to it is hydrophobic, so
This step needs to carry out in 30min before migration experiment starts.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, that is made any repaiies
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of cell migration research system, which is characterized in that including：Cell arranging devices and migration test device；
The cell arranging devices include at least one first through hole, and one end of each first through hole is both provided with bottom
Disk is provided at least one second through-hole on the chassis；
Second through-hole limits chamber as cell colony, for delimiting the growthform of attached cell；
The migration test device includes at least one third through-hole, and the third through-hole is used for as migration test chamber
Cell migration research is carried out to cell colony completely adherent in limiting chamber in cell colony；
Wherein, the method that cell migration research is carried out by the cell migration research system
Including：
By the chassis of the cell arranging devices with after clean culture dish adherency, plasma is carried out to the cell arranging devices
Body processing, cell suspending liquid is added in into the first through hole；
After cell in the cell suspending liquid is sunk in the cell colony limitation chamber in the cell arranging devices, it is sucked out
Remaining cell suspending liquid in the first through hole, so that the cell sunk in the cell colony limitation chamber pastes completely
Wall；
After cell in cell colony limitation chamber is completely adherent, the cell arranging devices are removed, are changed to described
Migration test device adds in pre-prepd culture medium, so that completely adherent cell colony in the migration test device
It is proliferated and migrates in the culture medium；
Every preset time period, take pictures to growth of the completely adherent cell in the migration test device, to pass through
The picture taken pictures to the cell in the migration test device studies migration distance of the cell in the culture medium.
2. according to the cell migration research system described in claim 1, which is characterized in that be provided at least two on the chassis
A the second identical through-hole.
3. according to the cell migration research system described in claim 2, which is characterized in that second through-hole is along radial direction side
To cross sectional shape for circle.
4. according to the cell migration research system described in claim 3, which is characterized in that second through-hole is along radial direction side
To cross sectional shape be diameter 0.20-0.30mm circle.
5. according to the cell migration research system described in claim 1, which is characterized in that the first through hole and the third
Through-hole is round, rectangle or polygon along the cross sectional shape of radial direction.
6. according to the cell migration research system described in claim 1, which is characterized in that the first through hole is axially square
To hole depth for 2-3mm, the hole depth of the third through-hole axially is more than or equal to 3mm.
7. according to the cell migration research system described in claim 1, which is characterized in that the cell arranging devices and migration
Test device is formed by PDMS.
8. a kind of cell migration research system using described in any one of claim 2-7 carries out the side of cell migration research
Method, which is characterized in that including：
By the chassis of the cell arranging devices with after clean culture dish adherency, plasma is carried out to the cell arranging devices
Body processing, cell suspending liquid is added in into the first through hole；
After cell in the cell suspending liquid is sunk in the cell colony limitation chamber in the cell arranging devices, it is sucked out
Remaining cell suspending liquid in the first through hole, so that the cell sunk in the cell colony limitation chamber pastes completely
Wall；
After cell in cell colony limitation chamber is completely adherent, the cell arranging devices are removed, are changed to described
Migration test device adds in pre-prepd culture medium, so that completely adherent cell colony in the migration test device
It is proliferated and migrates in the culture medium；
Every preset time period, take pictures to growth of the completely adherent cell in the migration test device, to pass through
The picture taken pictures to the cell in the migration test device studies migration distance of the cell in the culture medium.
9. according to the method described in claim 8, which is characterized in that described by the cell in the migration test device
Migrating data of the picture research cell taken pictures in the culture medium, including：
In the picture taken pictures to the cell in the migration test device, by the cell in each completely adherent cell
The covered region division of growth is growth district and migration region；
The average value of distance between the every bit migrated in region and the center of circle of the growth district is obtained, is calculated described flat
Difference between the radius of circle where mean value and the growth district, using as the migration distance；
Wherein, the growth district is the maximum connected region covered comprising the completely adherent cell growth, and with
Circle of the barycenter in the largest connected region for radius minimum in the circle in the center of circle, the migration region are by described completely adherent
Do not include the region of the growth district in the region that cell growth is covered.
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| 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 |
| CN103184263A (en) * | 2013-04-17 | 2013-07-03 | 孟文翔 | Tumour cell migration capability evaluation method and application |
| CN104774747A (en) * | 2015-04-14 | 2015-07-15 | 浙江大学 | Microfluidic liquid drop chip device for cell migration analysis experiment and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN103184263A (en) * | 2013-04-17 | 2013-07-03 | 孟文翔 | Tumour cell migration capability evaluation method and application |
| CN104774747A (en) * | 2015-04-14 | 2015-07-15 | 浙江大学 | Microfluidic liquid drop chip device for cell migration analysis experiment and method |
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