CN106701573B - A kind of cell flow experiment biochip application method - Google Patents
A kind of cell flow experiment biochip application method Download PDFInfo
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- CN106701573B CN106701573B CN201611058477.0A CN201611058477A CN106701573B CN 106701573 B CN106701573 B CN 106701573B CN 201611058477 A CN201611058477 A CN 201611058477A CN 106701573 B CN106701573 B CN 106701573B
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Abstract
Invention provides a kind of cell flow experiment biochip application method, it is characterised in that includes：The chip form specification that the first step is chosen, calculates the fluid flow network in the cell culture area under Experimental Flowing Object flow velocity.The second step chip chosen, is divided into experimental group and control group, under the same conditions culture experiment cell.Third walks, and after cell adherent growth, continues the fluid for the certain flow rate that injection experiments require.4th step detects the differentiation situation of cell under fluid force by high speed high-definition camera.5th step, experiment terminate, and waste liquid is discharged.Chip of the present invention has following advantageous effect, and the culture detection unit being provided with than common chip under the conditions of different fluid experiment of machanics applies different shearing forces in cell cultivation process, reaches instant, effective, micro observation more.It can in real time be monitored within the period of detection, control fluid input rate, ensure the reliability of detection so that laboratory test results can more react true cell growth environment, grasp influence of the shearing force to cell.
Description
Technical field
It is one that the present invention relates to a kind of application methods of the biochip of the dedicated cell culture of experiment from using
Kind is used in the application method of the cell culture biochip for fluid experiment in cell culture experiments.
Background technology
There is many insoluble problems for traditional biochip technology, are mainly manifested in conventional bio-chip use
Be in vitro gene and protein molecular, experience is combined from intracellular environment extracting and developing, purification, surface adhesion, molecule etc.
After a series of processing, treated, and cell changes, and can not play the role of timely observation, analysis.So to change
This situation, it is necessary to fundamentally thoroughly change the mentality of designing of biochip, design is a kind of to form what observation in real time compared
Chip.In the differentiation and proliferation research to cell, cyto-mechanics experiment is often used, and fluid in analogue body, cell is probed into pair
The influence of different shearing forces is one therein, but lacks the cell culture biochip for shearing force experiment at present, has been reached
To the cell that can be obtained under the conditions of simulation hydrodynamics to the real-time reflection situation of shearing force.
Therefore, it is necessary to invent to provide a kind of biochip and its application method for cell flow experiment, in solution
State problem.
Invention content
The technology contents of the present invention are a kind of cell flow experiment biochip application methods, it is characterised in that：In length
It is a pair two-by-two that rectangular or circular chip-side, which has 2 rows totally 4 injection orifices, injection orifice, and each pair of 2 injection orifices respectively connect
Totally 2 channel V is connect, 2 channels V are Y-shaped to be flowed to symmetrical 2 interface channels.2 interface channels connect respectively
Cultivation region one and cultivation region two are connect, two cultivation regions are symmetrical.2 cultivation region other ends, are respectively connected with interface channel, 2
Interface channel is finally flowed to 1 waste liquid pool.Waste liquid pool is connect by passing away with outlet.The cultivation region one and culture
The both sides and interface channel both sides of the inlet and outlet in area two are all arc transitions, turbulent flow occur to reduce.The side of cultivation region one
It is mutually opposing by the circular arc wall opening of R=0.5mm-1mm, the both ends of arc are connected with each other series connection, connect into and overlook into concave-convex line
Strip, similar wave-like.The side of cultivation region two is, the both ends phase of arc mutually opposing by the circular arc wall opening of R=3mm-5mm
Connect series connection, connects into and overlooks into concave-convex stripe shape, similar wave-like.This design mainly increases fluid to thin
The active force of born of the same parents, while avoiding generating vortex as possible again.Since cultivation region one is different with the width side of cultivation region two, twoth area are caused
Internal shearing force is different.Cell is cultivated in twoth area, and comparative analysis goes out stem cell under the action of different shearing forces
Break up situation.Alternatively, it is another rule of sharp cone distal that the shape on the side of the cultivation region one and cultivation region two of the chip, which has wave,
Lattice.The shape on the side of cultivation region one and cultivation region two is to overlook the mutually opposing opening of pointed cone for being rendered as same size, bores both sides
End is connected with each other the shape being connected into.The length of side of one sharp cone distal of cultivation region is 0.5mm-1mm, the convex-concave angle α between both sides1It is
α1≤ 60 ° of wedge angle.The length of side of the sharp cone distal of cultivation region two is 2mm-5mm, the convex-concave angle α between both sides2It is α2≤ 90 ° of point
Angle.Namely chip of the invention has circular arc rabbet shape and the wedge angle rabbet shape of acute angle, this 2 kinds of shape specifications.This design
It is to increase for contrast experiment and generate turbulent flow, forms vortex, compare influence of the variation to cell of mechanical stimulation.The culture
Area one is equal with the long L of cultivation region two, and L >=20mm, and cultivation region one is equal with the most width dimensions D of cultivation region two, and D
Size is 3 times or more of interface channel width dimensions d, i.e. D >=3d.This design is the optimization for meeting experiment condition.And it can
It is calculated according to hydrodynamics formula according to requirement of experiment, the shear stress under the different width of research on adjustment, research is identical or not
The shear stress generated under identical flow velocity changes the influence to cell it is characterized in that, as follows using operating procedure.
The first step is tested, the shape of the chip cultivation region one and cultivation region two of selection according to cell shearing power, and is designed
Flow velocity needed for experiment, by the shape and size of the rectangle cultivation region and taper cultivation region of the chip chosen, and whole channels ruler
In very little input computer, charted with CAD software；Then cartographic data is imported in fluid simulation software, calculates Experimental Flowing Object
The fluid flow network of cultivation region one and cultivation region two under flow velocity.
Second step is divided into experimental group and control group culture experiment cell with the chip chosen above；From injection orifice respectively at
To injection stem cell and culture solution, culture solution is periodically replaced by injection orifice, ensures that cell is normally metabolic.
Third walks, and after cell adherent growth, continues the fluid for the certain flow rate that injection experiments require from two pairs of injection orifices,
Fluid generates the active force of stimulation cell by cultivation region one and cultivation region two.
4th step detects cell under cultivation region 1 and 27 fluid force of cultivation region by high-definition camera or DVD
Break up situation, detect cell signal, collects data；According to the positioning of cellular localization and fluid flow network, to the cell of collection
Experimental data is analyzed, influence of the research different fluid shearing force to cell.
The metabolite of cell in waste liquid pool culture pond and remaining culture solution are passed through passing away and row by the 5th step
Outlet discharge.
Preferably, the floor height of the cultivation region one and cultivation region two in the entrance to connect with interface channel and goes out
The height dimension of two cultivation regions at mouthful is all identical, i.e., when there is the gradient in bottom surface, two bottom surfaces of cultivation region one and cultivation region two
The gradient it is all consistent.It is convenient for ensureing the comparison of experimental data in this way.
Preferably, the making material of the chip is all transparent material；The chip is in cultivation region one and cultivation region two
Grid groove is all machined on region.Ensure that chip can detect under microscope or CCD in this way.The chip is in rectangle culture
All be machined with grid groove on the region of area and taper cultivation region, in order to when cellular localization and fluid simulation to cell shearing power
Positioning.
The advantages of chip of the present invention：Under good cell culture environment, pass through the experimental implementation on chip, Neng Gouqi
The experimental observation comparison oriented to simultaneous quantitative reduces to keep influence experiment of the research shearing force to cell more convenient
Immediately the experimental period and error observed.Compared with existing design, chip of the present invention has following advantageous effect：Compare common core
Piece is provided with the culture systems of different shearing forces, applies different shearing forces in stem cell incubation, reaches instant, has
Effect, micro observation.It can in real time be monitored within the period of detection, control the input rate of nutriment, ensure detection
Reliability so that result can more react true stem cell growth environment, grasp the influence that shearing force breaks up stem cell, be
Follow-up work is prepared.
Description of the drawings
A kind of schematic top plan view with circular arc rabbet shape cultivation region of Fig. 1 inventions.
A kind of schematic top plan view with wedge angle rabbet shape cultivation region of Fig. 2 inventions.
Wherein：1. waste liquid pool；2. passing away；3. cultivation region one；4. interface channel；The channels 5.V；6. injection orifice；7. training
Support area two；8. outlet, 9. grid grooves.
Specific implementation mode
1-2 is further illustrated the present invention below in conjunction with the accompanying drawings.A kind of cell flow experiment is used with biochip
Method chooses the chip identical with 27 shape specification of cultivation region of cultivation region 1, is divided into experimental group and control group, carries out cell training
It supports and fluid flows the experiment for stimulating cell.
Embodiment one
The chip form specification of the first step, selection is：The side of cultivation region 1 is mutually opposing by the circular arc wall of R=0.8mm
It is composed in series, the side of cultivation region 27 is the mutually opposing circular arc rabbet shape chip being composed in series of circular arc wall by R=4mm.Training
Support the long L=25mm in area 1 and cultivation region 27, most width dimensions D=10mm of cultivation region 1 and cultivation region 27, interface channel
Width dimensions d=1mm.Experimental Flowing Object injects 15 μ L/S of flow velocity, 30 μ L/S.By the rectangle cultivation region 3 for the chip chosen and taper
In the shape and size of cultivation region 7, and whole channel size input computers, charted with CAD software；Then by cartographic data
It imports in fluid simulation software, calculates the fluid flow network of the cultivation region 1 and cultivation region 27 under Experimental Flowing Object flow velocity.
Second step, the chip chosen with 20 or more are divided into experimental group 10 and control tissue culture 10, under the same conditions
Culture experiment cell.From injection orifice 6, injection stem cell and culture solution are protected periodically by the replacement culture solution of injection orifice 6 in pairs respectively
It is normally metabolic to demonstrate,prove cell.
Third walks, and after cell adherent growth, continues the stream for the certain flow rate that injection experiments require from two pairs of injection orifices 6
Body, fluid generate the active force of stimulation cell by cultivation region 1 and cultivation region 27.
4th step passes through point of cell under high speed high-definition camera machine testing cultivation region 1 and 27 fluid force of cultivation region
Change situation, detect cell signal, collects data.It is real to the cell of collection according to the positioning of cellular localization and fluid flow network
It tests data to be analyzed, influence of the research different fluid shearing force to cell.
5th step, experiment terminate, and by the metabolite of cell and remaining culture solution in 1 culture pond of waste liquid pool, pass through row
Go out channel 2 and outlet 8 is discharged.
Embodiment two
The chip form specification of the first step, selection is：The length of side of one 3 sharp cone distal of cultivation region is 0.5mm, convex between both sides
Re-entrant angle α1=30 ° of wedge angle.The length of side of the sharp cone distal of cultivation region 27 is 4mm, the convex-concave angle α between both sides2=80 ° of wedge angle.Training
Support the long L=40mm in area 1 and cultivation region 27, most width dimensions D=30mm of cultivation region 1 and cultivation region 27, interface channel
Width dimensions d=2.5mm.Experimental Flowing Object injects 25 μ L/S of flow velocity, 50 μ L/S.By the rectangle cultivation region 3 for the chip chosen and cone
In the shape and size of shape cultivation region 7, and whole channel size input computers, charted with CAD software；Then by the number that charts
According to importing in fluid simulation software, the fluid flowing net of the cultivation region 1 and cultivation region 27 under Experimental Flowing Object flow velocity is calculated
Network.
Second step, the chip chosen with 12 are divided into experimental group 6 and control group 6, under the same conditions culture experiment
Cell.From injection orifice 6, injection stem cell and culture solution are ensureing cell just periodically by the replacement culture solution of injection orifice 6 in pairs respectively
Normal metabolism.
Third walks, and after cell adherent growth, continues the stream for the certain flow rate that injection experiments require from two pairs of injection orifices 6
Body, fluid generate the active force of stimulation cell by cultivation region 1 and cultivation region 27.
4th step detects the differentiation situation of cell under cultivation region 1 and 27 fluid force of cultivation region by high definition DVD,
Cell signal is detected, data are collected；According to the positioning of cellular localization and fluid flow network, to the cell experiment data of collection
It is analyzed, influence of the research different fluid shearing force to cell.
5th step, experiment terminate, and by the metabolite of cell and remaining culture solution in 1 culture pond of waste liquid pool, pass through row
Go out channel 2 and outlet 8 is discharged.
Claims (3)
1. a kind of cell flow experiment biochip application method, the rectangle used or circular chip-side have 2 rows totally 4
A injection orifice（6）, injection orifice（6）It is a pair, each pair of 2 injection orifices two-by-two（6）Respectively connect totally 2 channel V
（5）, 2 channels V（5）It is Y-shaped to be flowed to symmetrical 2 interface channels（4）；2 interface channels（4）It is separately connected training
Support area one（3）With cultivation region two（7）, two cultivation regions are symmetrical；Two cultivation region other ends, are respectively connected with interface channel
（4）, 2 interface channels（4）Finally it is flowed to 1 waste liquid pool（1）, waste liquid pool（1）Pass through passing away（2）With outlet（8）
Connection；The cultivation region one（3）With cultivation region two（7）Inlet and outlet both sides and interface channel（4）Both sides are all circular arcs
Transition；Cultivation region one（3）Side by the mutually opposing series connection of circular arc wall of R=0.5mm-1mm, connect into and overlook into concave-convex lines
Shape, cultivation region two（7）Side it is mutually opposing by the circular arc wall opening of R=3mm-5mm, the both ends of arc are connected with each other series connection, connection
At the stripe shape overlooked into circular arc bumps；Alternatively, the cultivation region one（3）With cultivation region two（7）The shape on side is to overlook to be in
It is now the mutually opposing opening of pointed cone of same size, the both sides end of pointed cone is connected with each other the shape being connected into, i.e. wedge angle is concave-convex
Side shape；Cultivation region one（3）The length of side of sharp cone distal be 0.5mm-1mm, the convex-concave angle α between both sides1It is α1≤ 60 ° of wedge angle；Training
Support area two（7）The length of side of sharp cone distal be 2mm-5mm, the convex-concave angle α between both sides2It is α2≤ 90 ° of wedge angle；Above-mentioned two is simultaneously
Row technical solution, the cultivation region one（3）With cultivation region two（7）Long L it is equal, and L >=20mm, cultivation region one（3）And training
Support area two（7）Most width dimensions D it is equal, and D sizes are interface channels（4）3 times or more of width dimensions d, i.e. D >=3d；
It is characterized in that, as follows using operating procedure：
The first step is tested, the chip cultivation region one of selection according to cell shearing power（3）With cultivation region two（7）Shape, and design
Flow velocity needed for good experiment, by the rectangle cultivation region for the chip chosen（3）With taper cultivation region（7）Shape and size, Yi Jiquan
In portion's channel size input computer, charted with CAD software；Then cartographic data is imported in fluid simulation software, is calculated
Cultivation region one under Experimental Flowing Object flow velocity（3）With cultivation region two（7）Fluid flow network；
Second step is divided into experimental group and control group culture experiment cell with the chip chosen above；It is pairs of respectively from injection orifice 6
Stem cell and culture solution are injected, culture solution is periodically replaced by injection orifice 6, ensures that cell is normally metabolic；
Third walks, and after cell adherent growth, continues the fluid for the certain flow rate that injection experiments require, stream from two pairs of injection orifices 6
Body passes through cultivation region one（3）With cultivation region two（7）Generate the active force of stimulation cell；
4th step detects cultivation region one by high-definition camera or DVD（3）With cultivation region two（7）Cell under fluid force
Break up situation, detect cell signal, collects data；According to the positioning of cellular localization and fluid flow network, to the cell of collection
Experimental data is analyzed, influence of the research different fluid shearing force to cell；
5th step, experiment terminate, by waste liquid pool（1）The metabolite of cell and remaining culture solution, pass through discharge in culture pond
Channel（2）And outlet（8）Discharge.
2. a kind of cell flow experiment biochip application method according to claim 1, is characterized in that：Use chip
Cultivation region one（3）With cultivation region two（7）Floor height, with interface channel（4）The two of the entrance and exit that connect
The height dimension of a cultivation region is all identical, i.e., when there is the gradient in bottom surface, cultivation region one（3）With cultivation region two（7）Two bottom surfaces
The gradient is all consistent.
3. a kind of cell flow experiment biochip application method according to claim 1, it is characterised in that：Use core
The making material of piece is all transparent material；The chip is in cultivation region one（3）With cultivation region two（7）Region on be all machined with net
Lattice groove（9）.
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