CN104774747B - Microfluidic droplet chip apparatus for cell migration assay experiment and method - Google Patents
Microfluidic droplet chip apparatus for cell migration assay experiment and method Download PDFInfo
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Abstract
The invention discloses a kind of Microfluidic droplet chip apparatus for cell migration assay experiment, including：For carrying the perforated membrane of drop；For ensureing that perforated membrane is in the perforated membrane support component of the state of sprawling；It is enclosed in perforated membrane periphery for preventing the liquidproof evaporative component of droplet evaporation.The present invention discloses a kind of method carrying out cell migration using said apparatus.This device combines the advantage of micro-fluidic chip and drop technique, changed in the relative position of perforated membrane upper and lower surface using drop, build flexible minicell experimental provision, for the research of various kinds of cell migration model, such as competitive cell migration, cell chemotaxis migration, the coefficient cell migration of many cells etc..It is applied to high-flux medicaments sifting, the research of the pathophysiological mechanism of cell migration behavior, and research of the impact of peripheral cell or materials onto cells transfer behavior etc..
Description
Technical field
Field according to the present invention is microfluidic analysis field, particularly to a kind of micro- for cell migration assay experiment
Stream control drop chip apparatus and the method carrying out Cell migration assay using this device.
Background technology
Cell migration is closely bound up with multiple physiological and pathological phenomenons, former cancerous cell diffusion, the immunne response of immunocyte, embryo
Tire growth course etc. is directed to cell migration it is seen that cell migration is research pathology, the important content of physiological problem.Tradition
The method of research cell migration has the methods such as scarification, agarose plate method, Boyden cell, but traditional method reagent consumes
Amount is big, and flux is low, and cell counting is not accurately it tends to be difficult to Real Time Observation, and is difficult to simulation complex environment in order to study
Cell migration under the more complicated microenvironment of multi-mode.
Microflow control technique, so that its reagent consumption is low, flexible operation, structure are changeable, can simulate complicated microenvironment etc. excellent
Gesture, has obtained more application in recent years in terms of cell migration research.Passage as the employing of Kamm seminar adds the miniflow of gel
Control chip structure, forming material Concentraton gradient can be distributed, for cell migration in middle cell culture chamber and gel
Research.Using this seminar of this method have studied endotheliocyte migration (Advanced Materials, 2009,21,
4863), reaction after nerve synapse irriate (Lab on a Chip, 2011,11,497) etc., and in 2012 years (Nature
Protocols, 2012,7,1247) publish thesis the systemic concrete operations scheme that its method is described, is similar to the dress of this method
Put the also scholar by a lot of research cell migration behaviors to be used.Also have document report to study individual cells using narrow thin passage to move
The property moved, and find migration velocity and ratio (AngewandteChemie International relevant with cell density
Edition,2014,53,2344).By in chip channel process micro-pillar array, Wong etc. (Nature Materials,
2014,13,1063) have studied effect in malignant tumor migration for the epithelial cell interstitial conversion.Will be micro-fluidic integrated excellent
Gesture is used for complex environment many cells and co-cultures, and can preferably study cell migration behavior it might even be possible to simulate in internal organs environment
Cell behavior (Science, 2010,328,1662).
Numerous microfluidic system can form drug concentration gradient in microchannel, for studying cell migration mechanism, becoming
Change effect, drug screening of the factor etc..But its system, often with complicated liquid control device, needs loaded down with trivial details operation,
High-throughout experiment is difficult on chip piece.Also tend to presence using based on the micro-fluidic chip that enclosed type microchannel is designed
Passage easily blocks, the problems such as be difficult flexible operating.How to design the micro-fluidic chip of simple and flexible, and to possess above advantage be micro-
The important goal (Nature, 2014,507,181) that fluidic chip develops in cell biology.
Drop microflow control technique has micro, efficient, high-throughout feature, but its application in cytobiology at present
Also in the starting stage.
Content of the invention
The invention provides a kind of Microfluidic droplet chip apparatus for cell migration assay experiment, this device combines micro-
Fluidic chip and the advantage of drop technique, are changed in the relative position of perforated membrane upper and lower surface using drop, build flexibly micro-
How thin type cell experiment device is for the research of various kinds of cell migration model, such as competitive cell migration, cell chemotaxis migration,
Coefficient cell migration of born of the same parents etc..
The invention also discloses a kind of method carrying out cell migration assay experiment using said apparatus, the method combines micro-
Fluidic chip and the advantage of drop technique, are changed in the relative position of perforated membrane upper and lower surface using drop, build flexibly micro-
Type cell experiment is it is adaptable to the research of high-flux medicaments sifting, the pathophysiological mechanism of cell migration behavior, and surrounding is thin
Research of the impact of born of the same parents or materials onto cells transfer behavior etc..
The concrete technical scheme of the present invention is as follows：
A kind of Microfluidic droplet chip apparatus for cell migration assay experiment, including：
For carrying the perforated membrane of drop；
For ensureing that perforated membrane is in the perforated membrane support component of the state of sprawling；
It is enclosed in perforated membrane periphery for preventing the liquidproof evaporative component of droplet evaporation.
The Microfluidic droplet chip apparatus for Cell migration assay of the present invention, are mainly supported by perforated membrane, perforated membrane
Assembly, droplet position control assembly, liquidproof evaporative component composition, typically whole device are placed in transparent utensil during use.
The present invention can arrange droplet position control assembly in perforated membrane side, also can be respectively provided with droplet position control in both sides
Assembly processed, need to determine according to actual needs.Preferably, described perforated membrane side is covered with the first droplet position control assembly,
This first droplet position control assembly is provided with via-hole array.As further preferred, described perforated membrane opposite side is covered with
Two droplet position control assemblies, this second droplet position control assembly is provided with via-hole array.
As further preferred, the through hole in described first droplet position control assembly and the second droplet position control assembly
In projection section on perforated membrane at least one through hole or all overlapping, projection section or all overlapping through hole constitute one
Drop chain, multiple above-mentioned drop chains constitute drop chain array.
According to the present invention, described perforated membrane is fixed in perforated membrane support component, and perforated membrane is in sprawl state.More have
Profit, perforated membrane sprawls state in plane.Described droplet position control assembly is coordinated with perforated membrane, respectively in perforated membrane
Upper and lower surface forms aqueous phase droplets, partly or wholly passes through many between the drop of perforated membrane upper and lower surface
The fenestra of pore membrane is connected.Described liquidproof evaporative component be used to cover or wrap up perforated membrane and perforated membrane upper surface and under
The drop on surface, to prevent the evaporation of drop.
The material of perforated membrane support component of the present invention is macromolecule polymer material, such as polyethylene, polystyrene,
Politef, Merlon, polydimethylsiloxane, polymethyl methacrylate, silicone rubber etc., or inorganic and organic
Composite, or the inorganic material such as glass, quartz, silicon, metal.
Droplet position control assembly of the present invention has via-hole array, its from chip upper and lower surface fit perforated membrane,
Form similar " sandwich " type structure.Used in this droplet position control assembly, the material of chip is high molecular polymer material
Material, such as polyethylene, polystyrene, politef, Merlon, polydimethylsiloxane, polymethyl methacrylate, silicon rubber
Glue etc., or inorganic and organic composite material, or the inorganic material such as glass, quartz, silicon, metal.Described drop position
The thickness range putting the chip of control assembly is 1 micron to 5 centimetres, and on chip, the diameter range of through hole is 1 micron to 10 centimetres,
The cross sectional shape of through hole is circular, oval, rectangle or other polygons；For ensureing to pass through porous film surface between drop
Carry out mass transfer and cause cross-contamination or seepage, the region that chip is fitted with perforated membrane should non-leakage liquid.
Preferably, described liquidproof evaporative component is the drop on submergence perforated membrane and its surface, and with perforated membrane upper liquid
Drip immiscible liquid phase；Or, described liquidproof evaporative component is the wet box of the drop surrounding perforated membrane and its surface.Work as drop
During for aqueous phase, described liquidproof evaporative component adopts the liquid on the oil phase immiscible with aqueous phase droplets, submergence perforated membrane and its surface
Drip and droplet position control assembly；This oil phase has certain air permeability, to meet the requirement of cell culture.Described liquid-proof evacuator body
When assembly adopts the drop that wet box wraps up perforated membrane and its surface, keep higher humidity in wet box, reduce the evaporation of drop.Should
Wet box also can wrapping portion or whole perforated membrane support component and droplet position control assembly.
The material of perforated membrane of the present invention is macromolecule polymer material, such as polyethylene, polystyrene, polytetrafluoroethyl-ne
Alkene, Merlon, polydimethylsiloxane, polymethyl methacrylate, silicone rubber etc., or inorganic and organic composite material
Material, or the inorganic material such as glass, quartz, silicon, metal, commercially available commercialization perforated membrane product also can laboratory voluntarily add
Work；Perforated membrane thickness range is 1 micron to 5 millimeters, and on film, bore dia scope is 1 nanometer to 100 microns, and the cross sectional shape in hole can
Think circle, ellipse, rectangle or other polygons, the areal extent of perforated membrane is 10 square microns to 1000 square centimeters.Make
For preferred, the hole on perforated membrane is vertically independent through hole；Preferably, perforated membrane has transparent or semitransparent property, with convenient
Observe；Preferably, the upper and lower surface of perforated membrane has hydrophilic nmature with the part of drop contact.
The present invention also can be carried out at selectivity hydrophobe by the selection of the material to perforated membrane or to porous film surface
Reason, the hydrophilic and hydrophobic region being formed in porous film surface.Processed by hydrophilic and hydrophobicity, indirectly serve droplet position
The effect of control assembly.Preferably, the part that described perforated membrane carries drop has hydrophilic, remainder is hydrophobic region
Domain；Described perforated membrane side carries the region part or complete in the region of drop and at least one carrying drop of perforated membrane opposite side
Portion overlaps.
Droplet formation method of the present invention can be using a liquid device (as pipettor, capillary tube or spotting needle etc.) on perforated membrane
The hydrophilic region of surface and lower surface points out drop.Or the method that porous film surface crossed using liquid flow, in perforated membrane upper surface
Form drop with the hydrophilic region of lower surface.
Certainly the present invention also can not use any porous film surface processing method, using a liquid device (as pipettor, capillary
Pipe or spotting needle etc.) directly point out drop in the ad-hoc location of the upper and lower surface of perforated membrane.
According to the present invention, above-mentioned various structures droplet position control assembly or porous film surface are processed or perforated membrane table
Face, without technology such as any process, can be applied in combination in the same apparatus.Preferably, the method using via-hole array chip
Or the method that porous film surface selectivity is processed, be conducive to improving the accuracy that droplet position is controlled.
A kind of method carrying out Cell migration assay using any of the above-described technical scheme described device, including specifically grasping as follows
Make step：
A (), according to experiment needs, by the help of droplet position control assembly, determines in perforated membrane upper and lower surface
The required target location forming drop, that is, need to form the position of drop；
B (), by the help of droplet position control assembly, in the target location of the upper surface of perforated membrane, forms certain volume
Required drop.Droplet formation method is included using a liquid device (as pipettor, capillary tube or spotting needle etc.) in perforated membrane table
The hydrophilic region in face points out drop, or the method crossing porous film surface using liquid flow, in the hydrophilic region of porous film surface
Form drop.The species that drop includes solution includes：Cell suspension or culture fluid or stimulate liquid or induction liquid or diluent,
Or buffer or other functions solution.
(c) by the upper and lower surface reversion of perforated membrane so as to original lower surface upwards, by droplet position control assembly
Help, form one in the target location of this surface (i.e. the upper surface of now perforated membrane, namely the lower surface of original perforated membrane)
Determine the required drop of volume.Droplet formation method includes existing using a liquid device (as pipettor, capillary tube or spotting needle etc.)
The hydrophilic region of porous film surface points out drop, or the method crossing porous film surface using liquid flow, in porous film surface
Hydrophilic region forms drop.Drop includes solution type and includes：Cell suspension or culture fluid or stimulate liquid or induction liquid or
Diluent or buffer or other functions solution；
D () adopts (b), the operation order of (c) and method or (c), the operation order of (b) and method, in perforated membrane
Upper and lower surface forms multiple drops, constitutes and to be connected by perforated membrane fenestra, by multiple positioned at the upper and lower surface of perforated membrane
The drop chain of drop composition；Again with drop chain as elementary cell, constitute drop chain array, to carry out the cell experiment of complexity or to enter
Row high-flux cell is tested；
E () carries out follow-up cell experiment using the Microfluidic droplet chip apparatus being formed with drop, including：
I. this device is put into cell culture incubator, or in living cells work station, or other cell culture systems, carry out drop
The culture of inner cell and migration experiment；
Ii., in experimentation, according to experiment needs, add the liquid of certain volume in certain time to object droplet,
Carry out stimulation or dyeing or other operations of cell；According to experiment needs, take out one in object droplet in certain time
Divide liquid, carry out follow-up analysis, identification or experiment, or the replacing carrying out liquid in drop；Described liquid feeding and take liquid side
Method is using point liquid or liquid sucking device (as pipettor, capillary tube or spotting needle etc.), adds in the drop of porous film surface
The liquid of certain volume, or take out the liquid of certain volume from the drop of porous film surface；
Iii. microscope or other imaging observation equipment are adopted, to cultivating and migrating in experimentation, in perforated membrane
The state (position, form, quantity) of the cell in the drop on upper and lower surface and motion track etc. carry out optical imagery observation, bag
Include the cell being pointed to different layers in drop, the cell of perforated membrane upper surface, the cell of perforated membrane lower surface, in perforated membrane fenestra
In cell, carry out overall imaging or local tomography；Or remove the drop of perforated membrane one side, retain the liquid of perforated membrane another side
Drip, observe the state moving to this face cell, or the drop of removal perforated membrane one side, the cell of another side is selected
Property ground cell fix and the operation such as dye after, carry out imaging observation or preservation；When liquid-drop diameter is controlled in microscopic fields of view model
When enclosing interior, may be implemented in the absolute cell counting completing whole cells in drop under a visual field, cell counting is accurately, acceptable
Obtain the multinomial experimental datas such as the form of whole cells in drop, migration number, migration distance, migration velocity, migratory direction.
Iv. the experiment according to ii and iii, obtains the cell state information in different time and space for the drop inner cell, complete
Become cellular morphology mutation analysises, propagation, survival rate detection, migrate percentage test, the expression of functional protein and measure of spread, carefully
Born of the same parents classify, the research of cell-cell interaction, the influence research of stimulus object or inducer cell migration, the molecule of cell migration
Mechanism Study, drug screening etc. is tested.Preferably, on one chip, integrated complete above-mentioned various kinds of cell experiment.Described
Device can carry out individual processing to each cell drop on demand, such as realizes cellular morphology observation, cell position determines, cell is dead
Analysis alive etc..Meanwhile, integrated can complete multiple cell experiments in same chip block, such as cellular morphology change, proliferative conditions, deposit
Survival rate test, migration percentage test, drug screening etc..This is that general micro-fluidic chip is difficult to.
The using method of the Microfluidic droplet chip apparatus for Cell migration assay of the present invention, in perforated membrane
The position of the drop on upper and lower surface can accurately set, and upper and lower surface drop is part or all of with the contact surface of perforated membrane
Overlap.The drop on multiple upper and lower surfaces is connected by perforated membrane fenestra and forms the drop chain of various configuration.In drop chain,
Using material in a certain drop to the diffusion of other drops adjacent, the Concentraton gradient of this material can be formed in drop chain,
A series of cell experiments can be completed using this Concentraton gradient.Place different types of cell in the different drops in drop chain,
The characteristic being connected by perforated membrane fenestra using different drops, carries out the different intercellular experiments that interact, or simulation
The experiment (as using the cell construction drop chain from Different Organs or tissue) of biological living.By adjusting porous in drop chain
The relative position of the drop on upper and lower two surfaces of film, can be used for the Cell migration assay of various modes, and such as competition sexual cell moves
Shifting, cell chemotaxis migration, the coefficient cell migration of many cells etc. are tested.
The using method of the Microfluidic droplet chip apparatus for Cell migration assay of the present invention, in experimentation
In, the upper and lower surface of perforated membrane can as needed, repeatedly turning-over changed upper-lower position, is operated respectively with facilitating and processes
Drop positioned at the upper and lower surface of perforated membrane.This is also one of advantage of this device, can flexibly manipulate positioned at porous
The drop on the upper and lower surface of film.
The using method of the Microfluidic droplet chip apparatus for Cell migration assay of the present invention, in a drop
Interior can place single kind cell, also can place multiple different types of cells.State in drop for the cell is uniformly to divide
Cloth assumes dimensional culture state in a drop, or be affixed on porous film surface present monolayer two dimension cultivation conditions, or
Monolayer growth state presented on the boundary of drop and other solid phases or liquid phase, or concentrates on certain region of drop and assume group
Shape state.
It is an advantage of the invention that cell and reagent consumption are few, experiment flux is high；Can be with Real Time Observation cell state；Device
Structure is simply it is easy to build, simple to operate, flexible, convenient；Individually independent operation can be carried out to each drop, also can be to chip
On drop chain array carry out disposed of in its entirety；Can independently carry out repeating to test using multiple drop chain elements, it is to avoid due to logical
Road is connected the blocking causing or interfere；Chip can reuse；Experimentation does not need the liquid manipulation device of complexity；
When controlling liquid-drop diameter in the range of microscopic fields of view, can achieve the full drop imaging to this drop, under a visual field
Complete the absolute cell counting of whole cells in drop, cell counting accurately, can also obtain the form of whole cells in drop,
The multinomial experimental datas such as migration number, migration distance；Dosing or material incentive process are gentle, it is to avoid the impact of shearing force；Pass through
The design of drop chain, control and flexible combination can be completed with the Cell migration assay of various modes, and the cell phase of complexity
Interaction is tested, such as competitive cell migration, cell chemotaxis migration, the coefficient cell migration of many cells etc.；Cell is in liquid
Culture in dripping has various modes can select, including two and three dimensions condition, and mixing condition culture.
Brief description
Fig. 1 is that embodiment 1 is used for studying the Microfluidic droplet chip apparatus of competitive cell migration, the vertical section of its device
Schematic diagram.
Fig. 2 a is that embodiment 1 is used for studying the Microfluidic droplet chip apparatus of competitive cell migration, the vertical view of its device
Schematic diagram.
Fig. 2 b is partial enlarged drawing in Fig. 2 a.
Fig. 3 is that embodiment 1 Microfluidic droplet chip apparatus are used for studying the experimental result picture of competitive cell migration.
Fig. 4 a is that embodiment 2 is used for studying the Microfluidic droplet chip apparatus of cell chemotaxis migration, the vertical section of its device
Schematic diagram.
Fig. 4 b is the partial enlarged drawing of Fig. 4 a.
Fig. 5 a is that embodiment 2 is used for studying the Microfluidic droplet chip apparatus of cell chemotaxis migration, and the vertical view of its device is shown
It is intended to.
Fig. 5 b is the partial enlarged drawing of Fig. 5 a.
Fig. 6 be embodiment 2 Microfluidic droplet chip apparatus be used for study cell chemotaxis migration experimental result schematic diagram.
Fig. 7 be embodiment 2 Microfluidic droplet chip apparatus be used for study cell chemotaxis migration experimental result photo.
Fig. 8 a is that embodiment 3 is used for studying the Microfluidic droplet chip apparatus of cell migration under many cells collective effect, its
The vertical section schematic diagram of device.
Fig. 8 b is the partial enlarged drawing of Fig. 8 a.
Fig. 9 a is that embodiment 3 is used for studying the Microfluidic droplet chip apparatus of cell migration under many cells collective effect, its
The schematic top plan view of device.
Fig. 9 b is the partial enlarged drawing of Fig. 9 a.
Figure 10 is that embodiment 3 Microfluidic droplet chip apparatus are used for studying the experiment of cell migration under many cells collective effect
Result figure.
Figure 11 is that embodiment 4 is used for studying the Microfluidic droplet chip apparatus of macrophage Human Umbilical Vein Endothelial Cells invasion and attack impact,
The vertical section schematic diagram of its device.
Figure 12 a is that embodiment 4 is used for studying the Microfluidic droplet chip apparatus of macrophage Human Umbilical Vein Endothelial Cells invasion and attack impact,
The schematic top plan view of its device.
Figure 12 b is the partial enlarged drawing of Figure 12 a.
In figure：1- perforated membrane, 2- perforated membrane support component, the droplet position control group that 3- is combined with perforated membrane lower surface
Part, the droplet position control assembly that 4- is combined with perforated membrane upper surface, 5- briquetting, 6- liquidproof evaporative component, 7,8,9,10,
11st, 12,15,16,17,18,19,20,21, the 22, drop of the different composition of 23-, in 13- drop adherent with perforated membrane upper surface
Cell, 14- is moved to the cell of perforated membrane lower surface drop by perforated membrane fenestra by perforated membrane upper surface drop.
Specific embodiment
With specific embodiment, the present invention will be further described below, but protection scope of the present invention not limited to this.
Embodiment 1
The Microfluidic droplet chip apparatus of the competitive cell migration of research, referring to Fig. 1 and Fig. 2 a, Fig. 1 is device vertical section
Figure, Fig. 2 a is device top view, and Fig. 2 b is the partial enlarged drawing of Fig. 2 a.Whole chip is rounded, is placed in the transparent of 6 centimetres of diameter
In plastic culture dish, interior plus fluorocarbon oil (immiscible with aqueous solution, and have certain air permeability) submergence chip is as liquid-proof evacuator body
Assembly 6, being placed on chip using 7 millimeters of acrylic ring of thickness as briquetting 5 prevents chip from floating in oil phase.Perforated membrane 1 is
8 microns of aperture, the translucent polycarbonate membrane (PC) of 20 microns of thickness.Polydimethylsiloxane (PDMS) core containing array through-hole
Piece is fitted with the upper and lower surface of perforated membrane 1 respectively as droplet position control assembly 3, droplet position control assembly 4, and chip is thick
Spend 300 microns, contained through-hole diameter is 1.5 millimeters thereon, the relative position of two chip laminatings is by determining of designing in advance
Position hole determines.The PDMS annulus that perforated membrane support component 2 is 3 millimeters by thickness is constituted, and is fitted in PDMS via-hole array chip (liquid
Drip position control assembly 3, droplet position control assembly 4) upper it is ensured that chip level is stable, and avoid the drop of film lower surface with
Ware bottom contacts.
Make the lower surface of perforated membrane 1 upwards, the droplet position control assembly 3 now containing array through-hole is located on perforated membrane 1
Side.Add drop 7 and drop 8 in the hole of relevant position, all contain 1：The matrigel of 8 dilutions is dilute with cell optimum medium
Release.The difference of drop 7 and drop 8 is, drop 7 does not contain hyclone, and drop 8 contains 10% hyclone.Using above method,
Form the repeated droplet of drop 7 and drop 8 in the lower surface of perforated membrane 1.By this device be placed in cell culture incubator half an hour with
Upper it is ensured that matrigel cross-linked stable.Take out this device, acrylic ring briquetting 5 taken off, reversion perforated membrane 1 make its upper surface to
On, now the control assembly of droplet position containing array through-hole 3 is located at perforated membrane 1 lower surface, and the droplet position containing array through-hole controls
Assembly 4 is located at perforated membrane upper surface, then acrylic ring briquetting 5 is resetted.In corresponding hole midpoint drop 9, drop 9 is through starvation
Starve and process 12 hours, with the cell suspension that serum-free medium is resuspended.Can be formed in perforated membrane 1 upper surface using said method
The drop 9 of multiple same compositions.Above-mentioned single drop 7, drop 8, drop 9 constitute a drop chain, multiple above-mentioned drop chain structures
Become drop chain array.Above-mentioned middle drop 7, drop 8, drop 9 volume are 1000 nanoliters.Drop 7 and drop 9, drop 8 and drop
9 all have area on about 25% film overlapping.
Upwards, following table faces down final perforated membrane 1 upper surface, is placed in induced cell migration 24 hours in incubator.Drop 7
Cancer cell migration in the competitive induction drop 9 with drop 8.After induction in 24 hours terminates, microscope photographing drop 9 position film is upper and lower
Whole cells, count the whole cell number in this drop chain.Removal oil phase liquidproof evaporative component 6, PBS cleaning,
Paraformaldehyde is fixed, violet staining, tears and has the droplet position control assembly 4 of array through-hole, wipes unwanted cell, to every
Individual drop chain is imaged, and obtains moving to the cell number in drop 7 and drop 8.It is computed obtaining final product cell from drop 9 respectively
Move to drop 7, the ratio in drop 8.
Fig. 3 is competitive Cell migration assay result taking mouse colonic cell CT26 as a example.Cell counting simultaneously calculates
The percentage ratio of drop 7 migration to serum-free medium for the CT26 can be obtained and CT26 migrates to the drop 8 containing blood serum medium
Percentage ratio, the migration of two drops under perforated membrane of same tumor cell can be detected pole significant difference (* * * P ＜
0.001, t inspection).Different cells there is also difference to the reaction of competition migration.Cancerous cell can be analyzed using this device to not
Migration aptitude with environment.
Embodiment 2
With the Microfluidic droplet chip of research cell chemotaxis migration, referring to Fig. 4 a, Fig. 4 b and Fig. 5 a, Fig. 5 b, Fig. 4 a is use
The device of the good drop of point tested in cell three-dimensional migration, Fig. 4 b is the partial enlarged drawing of Fig. 4 a, and Fig. 5 a is for thin
The top view of the device of the good drop of point of born of the same parents' three-dimensional migration experiment, Fig. 5 b is the partial enlarged drawing of Fig. 5 a.Entirely chip is in
Circle, is placed in the transparent plastic culture dish of 6 centimetres of diameter, interior plus fluorocarbon oil submergence chip as vaporization prevention assembly 6, with thickness 7
The acrylic ring of millimeter is placed on chip as briquetting 5 and prevents chip from floating in oil phase.Perforated membrane 1 is 8 microns of aperture, thickness
20 microns of PC film.PDMS chip containing array through-hole is as droplet position control assembly 3, droplet position control assembly 4 respectively
Fit with the upper and lower surface of perforated membrane 1, PDMS chip thickness is 300 microns, contained through-hole diameter is 1.2 thereon
Millimeter, the relative position of two PDMS chip laminatings is determined by the location hole designing in advance.Perforated membrane support component 2 is by thickness 3
The PDMS annulus of millimeter is constituted, and is fitted in PDMS via-hole array chip (droplet position control assembly 3, droplet position control assembly
4) above it is ensured that chip level is stable, and the drop of film lower surface is avoided to contact with ware bottom.
Make perforated membrane 1 lower surface upwards, the droplet position control assembly 3 now containing array through-hole is located above perforated membrane 1.
Add drop 10 in the hole of relevant position, drop 10 contains 1：The matrigel of 8 dilutions, with serum-free medium dilution.Using
Above method can form multiple repeated droplet 10 in perforated membrane 1 lower surface.This device is placed in half an hour in cell culture incubator
Above it is ensured that matrigel cross-linked stable.Withdrawing device, acrylic ring briquetting 5 is taken off, reversion perforated membrane 1 make its upper surface to
On, the droplet position control assembly 3 now containing array through-hole is located at film lower surface, the droplet position control assembly containing array through-hole
4 are located at film upper surface, then acrylic ring briquetting 5 is resetted.In presetting location point drop 11, drop 11 is containing 50% tire
The culture medium of Ox blood serum, forms the drop 11 of multiple repetitions in film upper surface.In corresponding hole midpoint drop 12, drop 12 be through
Cross Nature enemy 12 hours, with the cell suspension that serum-free medium is resuspended, can be formed in film upper surface using said method
The drop 12 of multiple repetitions.Above-mentioned single drop 10, drop 11, drop 12 constitute a drop chain, multiple above-mentioned drop chain structures
Become drop chain array.Above-mentioned middle drop 10, drop 11, drop 12 volume are 800 nanoliters.Drop 10 and drop 11, drop 10
Overlapping with area on the film that drop 12 all has about 15%.
Upwards, following table faces down final perforated membrane 1 upper surface, is placed in induced cell migration 4 days in incubator.In drop 11
Serum can induce the part cell in drop 12 wear membrane orienting migration.People's mammary gland is added respectively in different drops 12
Cancerous cell MCF7, human colon cancer cell RKO, human breast cancer cell MDA-MB-231,4 days induction terminate after it can be observed that
MCF7 is almost acellular, and through perforated membrane, but RKO cell can pass through the directional migration in perforated membrane undirected drop 11 direction,
There is cell and pass through perforated membrane phenomenon in MDA-MB-231, and part cell migrates to drop 11 direction, that is, move to drop 10
The non-part overlapping with drop 12.
The three-dimensional migration result of MDA-MB-231 cell is cell 13 before migration referring to Fig. 6, wherein Fig. 6 a and Fig. 6 b
Position view with cell 14 after migration.Fig. 7 for MDA-MB-231 cell chemotaxis migrate 4 days after photograph via bright field and fluorogram
Piece is it can be seen that part cell has crossed drop 10 and the interface of drop 12.There is the cancerous cell MDA- of Interstitial cell property
MB-231 transfer ability is higher, and this device, method can be used to study Epithelial and stromal Cell transformation test it is also possible to for sentencing
The transfer ability of disconnected difference cancerous cell.
Embodiment 3
The Microfluidic droplet chip apparatus of cell migration under many cells collective effect, the structural representation of its device is referring to figure
8a, Fig. 8 b and Fig. 9 a and Fig. 9 b, Fig. 8 a are device profilograph, and Fig. 8 b is the partial enlarged drawing of Fig. 8 a, and Fig. 9 a overlooks for device
Figure, Fig. 9 b is the partial enlarged drawing of Fig. 9 a.Whole chip is rounded, is placed in the transparent plastic culture dish of 6 centimetres of diameter, interior plus
As vaporization prevention assembly 6, be placed on chip using 7 millimeters of acrylic ring of thickness as briquetting 5 prevents chip to fluorocarbon oil submergence chip
Oil phase floats.Perforated membrane 1 is 8 microns of aperture, the PC film of 20 microns of thickness.PDMS chip containing array through-hole is as drop
Position control assembly 3, droplet position control assembly 4 are fitted with the lower surface of perforated membrane 1 and upper surface respectively, PDMS chip thickness
For 300 microns, in droplet position control assembly 3, contained through-hole diameter is 1.2 millimeters, institute in droplet position control assembly 4
The through-hole diameter containing is 2.5 millimeters, and the relative position of two PDMS chip laminatings is determined by the location hole designing in advance.Porous
Film support component 2 is made up of the PDMS annulus of 3 millimeters of thickness, is fitted in PDMS via-hole array chip (droplet position control assembly
3rd, droplet position control assembly 4) above it is ensured that chip level is stable, and avoid the drop of film lower surface to contact with ware bottom.
Make perforated membrane 1 lower surface upwards, the droplet position control assembly 3 now containing array through-hole is located above film.In phase
Answer in the hole of position and add drop 16, drop 17, drop 18, drop 19, drop 20, except containing 1：The matrigels and complete of 8 dilutions
Outside full culture medium, contain respectively in each drop intentionally, liver, kidney, intestinal, the cells of organs such as lung, or not celliferous blank.Profit
The droplet array of petal-shaped distribution can be formed with above method in perforated membrane lower surface.Acrylic ring briquetting 5 is taken off, reversion
Perforated membrane 1 makes its upper surface upwards, and the droplet position control assembly 4 now containing array through-hole is located above film, acrylic ring pressure
Block 5 resets.This device is placed in cell culture incubator more than half an hour it is ensured that matrigel cross-linked stable.Crosslinking finish after from training
Withdrawing device in foster case, in presetting location point drop 15, drop 15 is through Nature enemy 12 hours, with serum-free culture
The cancer cell suspension that basic weight hangs, can form the drop 15 of multiple repetitions using said method in perforated membrane 1 upper surface.Above-mentioned list
Individual drop 15, drop 16, drop 17, drop 18, drop 19, drop 20 constitute a drop chain, and multiple above-mentioned drop chains are constituted
Drop chain array.Above-mentioned middle drop 16, drop 17, drop 18, drop 19, drop 20 volume are 800 nanoliters.Drop 15 volume
For 2000 nanoliters.Drop 16, drop 17, drop 18, drop 19, drop 20 respectively have area and drop 15 weight on about 25% film
Folded.
Upwards, following table faces down final chip upper surface, is placed in induced cell migration 1 day in incubator.Result reference picture
Shown in 10, Figure 10 left figure is the photo of a drop chain under 40 power microscopes in Induction Process, and Figure 10 right figure terminates for induction
Afterwards, the microphotograph after array chip 4 and drop thereon 15 is dyeed and removes in cell fixation.Each internal organs in the apparatus
Cell not only relatively independent but also interact, can preferably simulate the cancer cell migration under in vivo complicated cellular environment, use
To study the organ tendentiousness of former cancer cell migration.
Embodiment 4
Cell invasion is one kind of cell migration, and cell needs to secrete hydrolytic enzyme, and decomposing extracellular matrix glue could pass through
Perforated membrane.For studying the Microfluidic droplet chip apparatus of macrophage Human Umbilical Vein Endothelial Cells invasion and attack impact, the structure of its device is shown
It is intended to referring to Figure 11 and Figure 12 a, Figure 12 b, Figure 11 is device profilograph, and Figure 12 a is device top view for figure, Figure 12 b is figure
The partial enlarged drawing of 12a.Using wet box as vaporization prevention assembly 6, whole chip is square, and perforated membrane 1 is 100 microns of thickness
PDMS film, contains the through hole accounting for area 20% thereon, and through-hole diameter is 5 microns.Perforated membrane 1 is placed in perforated membrane by flattening and supports
In assembly 2, perforated membrane support component 2 is the square glass ware that two mirror images are placed.Two square glass wares guarantee perforated membrane 1 water
Steadily determine, and the upper and lower drop of protecting film is not damaged.
Make perforated membrane lower surface upwards, carefully take off the square glass ware being now placed in above perforated membrane, porous at this moment
Point out drop 21 with pipettor on the upper surface of film, contain with complete medium 1 in drop 21:The extracellular matrix glue of 5 dilutions,
The drop 21 of repetition is pointed out with pipettor on same surface.Square glass ware is resetted, whole chip apparatus upset, now many
Upwards, following table faces down pore membrane 1 upper surface, takes off the square glass ware of perforated membrane 1 top, uses pipettor many under stereoscope
Pore membrane 1 upper surface points out drop 22 and drop 23, contains macrophage RAW264.7, it is micro- that drop 23 includes 1 wherein in drop 22
1 rising:The endotheliocyte HUVEC suspension of 5 matrigels and 1 microlitre, first puts the 1 of 1 microlitre in drop 23:5 matrigels, after
The endotheliocyte suspension of 1 microlitre of same position point.Guarantee that the lower surface of drop 22 and drop 23 is all complete with the upper surface of drop 21
Full connected, drop 22 and drop 23 do not merge simultaneously.Using same method, in the perforated membrane upper surface forming drop 21 array
Form the array of drop 22 and drop 23.Above-mentioned single drop 21, drop 22, drop 23 constitute a drop chain, multiple above-mentioned
Drop chain constitutes drop chain array.Above-mentioned middle drop 22, drop 23 volume are 2 microlitres.Drop 21 volume is 8 microlitres.
Upwards, following table faces down final chip perforated membrane 1 upper surface, is fixed with square glass ware, is placed in (wet box in wet box
Have aperture breathe freely), after be put in incubator, induced cell migration 1 day.Final result shows that there is macrophage acts on relatively no
The matched group of macrophage, endotheliocyte deformation is bigger, and migration is more.
The substrate gum concentration adopting in this test is higher, and endotheliocyte passes through the hole on film to need to decompose first substrate
Glue, this device can study effect in the invasion and attack of endotheliocyte for the macrophage.Result is consistent with document report, that is, huge bite thin
The invasion and attack of born of the same parents' Human Umbilical Vein Endothelial Cells have facilitation, and this is likely due to the tumor necrosis factor-alpha (TNF-α) of macrophage generation
Cause the increase of endothelial cell migration.
Claims (6)
1. a kind of Microfluidic droplet chip apparatus for cell migration assay experiment are it is characterised in that include：
For carrying the perforated membrane (1) of drop；
For ensureing that perforated membrane (1) is in the perforated membrane support component (2) of the state of sprawling；
It is enclosed in perforated membrane (1) periphery for preventing the liquidproof evaporative component (6) of droplet evaporation；
Described perforated membrane (1) side is covered with the first droplet position control assembly (3), this first droplet position control assembly (3)
It is provided with via-hole array；
Described perforated membrane (1) opposite side is covered with the second droplet position control assembly (4), this second droplet position control assembly
(4) it is provided with via-hole array；
In through hole in described first droplet position control assembly (3) and the second droplet position control assembly (4), at least one leads to
Projection section or all overlapping on perforated membrane (1) for the hole, projection section or all overlapping through hole constitute a drop chain, many
Individual above-mentioned drop chain constitutes drop chain array；
Described perforated membrane (1) thickness range is 1 micron to 5 millimeters, and on film, bore dia scope is 1 nanometer to 100 microns；
Hole on described perforated membrane is vertically independent through hole.
2. the Microfluidic droplet chip apparatus for cell migration assay experiment according to claim 1 it is characterised in that
Described liquidproof evaporative component (6) is the drop on submergence perforated membrane (1) and its surface and to go up drop immiscible with perforated membrane (1)
Liquid phase；Or, described liquidproof evaporative component (6) is the wet box of the drop surrounding perforated membrane (1) and its surface.
3. the Microfluidic droplet chip apparatus for cell migration assay experiment according to claim 1 it is characterised in that
The part that described perforated membrane (1) both sides carry drop has hydrophilic, and remainder is hydrophobic region；Described perforated membrane (1)
At least one region carrying drop in region and perforated membrane (1) opposite side that side carries drop partly or entirely overlaps.
4. a kind of usage right requires 1-3 arbitrary claim described device to carry out the method for Cell migration assay it is characterised in that wrapping
Include：
A the target location of () side in perforated membrane (1) forms required drop, the solution in drop includes：Cell suspension, culture
One or more of liquid, stimulation liquid, induction liquid, diluent, buffer and other functions solution；
B the target location of () opposite side in perforated membrane (1) forms required drop；The solution of drop includes：Cell suspension, training
One or more of nutrient solution, stimulation liquid, induction liquid, diluent, buffer and other functions solution；
C () forms multiple drops by step (a) and step (b) in the both side surface of perforated membrane (1), constitute and pass through perforated membrane
(1) fenestra be connected, the drop chain that is made up of multiple drops positioned at the upper and lower surface of perforated membrane (1)；Again with drop chain as base
This unit, constitutes drop chain array, carries out cell experiment by these drop chain arrays.
5. the method carrying out Cell migration assay according to claim 4 is it is characterised in that under described cell experiment includes
Arrange one or more of experiment or operation：
The culture of drop inner cell and migration；
The stimulation of drop inner cell or dyeing；
Drop component analyses, identification；
The replacing of liquid in drop；
Optical imagery observation is carried out to the state and motion track of the cell in the drop of perforated membrane both side surface；
Remove the drop of perforated membrane side, retain the drop of perforated membrane opposite side, observe the drop inner cell migration of perforated membrane side
State to opposite side；
Remove the drop of perforated membrane side, optionally cell is carried out to the cell of opposite side and fixes and dye, carry out imaging and see
Survey or preserve；
Obtain drop inner cell in the cell state information in different time and space, complete cellular morphology mutation analysises, breed, deposit
Survival rate test；
Obtain drop inner cell migration percentage test；
Obtain the expression of functional protein and measure of spread in drop；
Cell divide, the research of cell-cell interaction；
The influence research that stimulus object or inducer migrate to drop inner cell；
The Study on Molecular Mechanism of cell migration, medicament screening experiment.
6. according to the method carrying out Cell migration assay described in claim 5 it is characterised in that cell experiment in step (c)
For：In drop chain, using material in a certain drop to the diffusion of other drops adjacent, form this material in drop chain
Concentraton gradient；Place different types of cell in the different drops in drop chain, pass through perforated membrane film using different drops
The characteristic that hole is connected, carries out the interaction experiment of different cells, or the experiment of simulation biological living；By adjusting drop
The relative position of the drop in perforated membrane both side surface in chain, carries out the Cell migration assay of various modes, including competitive thin
One or more of born of the same parents' migration, cell chemotaxis migration, the coefficient Cell migration assay of many cells.
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| CN105062879B (en) * | 2015-07-26 | 2017-06-27 | 江苏大学附属医院 | The multidirectional migration experiment device of cell |
| CN106635796B (en) * | 2015-10-30 | 2019-08-30 | 中国科学院微生物研究所 | Device and fixed cultural method for cell fixation culture |
| CN106047665B (en) * | 2016-07-29 | 2018-07-06 | 北京大学 | It is a kind of for the method for high-flux cell migration research and cell migration research system |
| CN110133180B (en) * | 2018-02-02 | 2020-05-22 | 厦门大学 | Substance detection system and method based on gated pressure threshold change |
| CN108837718A (en) * | 2018-06-11 | 2018-11-20 | 上海交通大学 | A kind of high throughput microlayer model gradient dilution device and method |
| CN110408536A (en) * | 2019-06-12 | 2019-11-05 | 上海大学 | Cell migration assay device and method |
| CN111242014A (en) * | 2020-01-10 | 2020-06-05 | 天津理工大学 | Liquid drop imaging method and device for urinary sediment cell microscopic image |
| CN112191288B (en) * | 2020-10-12 | 2021-12-24 | 云南省细胞工程中心有限公司 | Unicellular separator based on unicellular sequencing technology |
| CN113073029B (en) * | 2021-03-17 | 2023-03-21 | 长春长光辰英生物科学仪器有限公司 | Infiltration modified cell sorting chip for laser induced transfer and sorting method |
| WO2023137583A1 (en) * | 2022-01-18 | 2023-07-27 | 深圳华大生命科学研究院 | Method for determining effect relationship between various substances and cells, and microwell array chip |
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| CN102439717A (en) * | 2009-03-24 | 2012-05-02 | 芝加哥大学 | Slip chip device and method |
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| WO2013087888A1 (en) * | 2011-12-15 | 2013-06-20 | Commissariat à l'énergie atomique et aux énergies alternatives | 3d microfluidic system having nested areas and a built-in reservoir, method for the preparing same, and uses thereof |
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