Biochip application method is used in a kind of cell flow experiment
Technical field
The present invention relates to a kind of application method of the biochip for testing special cell culture, from using being one
Plant the application method of the cell culture biochip for fluid experiment being used in cell culture experiments.
Background technology
Traditional biochip technology there are problems that it is many insoluble, be mainly manifested in conventional bio-chip use
Be in vitro gene and protein molecular, experience from intracellular environment extracting and developing, purification, surface adhesion, molecule with reference to etc.
After a series for the treatment of, the cell after treatment changes, and can not play observation, the effect of analysis in time.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 Real Time Observation was contrasted
Chip.In the differentiation and proliferation research to cell, cyto-mechanics experiment is often used, fluid in analogue body, and cell is probed into right
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.
Accordingly, it would be desirable to invent that a kind of biochip and its application method for cell flow experiment is provided, with solution
State problem.
The content of the invention
Technology contents of the invention are a kind of cell flow experiment biochip application methods, it is characterised in that:In length
Square or circular chip-side has 2 rows totally 4 injection orifices, and injection orifice is two-by-two a pair, and 2 injection orifices of each pair each connect respectively
Connect totally 2 V passage, 2 V passages are Y-shaped to be flowed to symmetrical 2 interface channels.2 interface channel connects 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 connected 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 two ends of arc are connected with each other series connection, connect into and overlook into concavo-convex line
Strip, is similar to wave-like.The side of cultivation region two is, the two ends phase of arc mutually opposing by the circular arc wall opening of R=3mm-5mm
Connect series connection, connects into and overlooks into concavo-convex stripe shape, is similar to wave-like.This design, mainly increases fluid to thin
The active force of born of the same parents, while avoid producing vortex again as far as possible.Because 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 the stem cell in the presence of different shearing forces
Differentiation situation.Or, the shape on the cultivation region one of the chip and the side of cultivation region two has another rule that wave is sharp cone distal
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 formed objects, bores both sides
Termination is connected with each other the shape being connected into.The length of side of the sharp cone distal of cultivation region one 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 the wedge angle rabbet shape of circular arc rabbet shape and acute angle, this 2 kinds of shape specifications.This design
It is, for contrast experiment, to increase turbulization, forms vortex, compares the influence of the change to cell of mechanical stimulation.The culture
Area one is equal with the L long 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 more than 3 times of interface channel width dimensions d, i.e. D >=3d.This design is the optimization for meeting experiment condition.And can
Calculated according to hydrodynamics formula according to requirement of experiment, the shear stress under the different width of research on adjustment, studied identical or not
Influence of the shear stress change produced under identical flow velocity to cell is characterized in that, as follows using operating procedure.
The first step, tests, the chip cultivation region one of selection and the shape of cultivation region two, and design according to cell shearing power
Flow velocity needed for experiment, the rectangle cultivation region of the chip that will be chosen and the shape and size of taper cultivation region, and whole passage chis
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, the chip chosen more than is divided into experimental group and control group culture experiment cell;From injection orifice respectively into
To injection stem cell and nutrient solution, nutrient solution is periodically changed by injection orifice, it is ensured that cell is normally metabolic.
3rd step, after after cell attachment growth, from two pairs of fluids of the certain flow rate of the lasting injection experimentses requirement of injection orifice,
Fluid produces the active force for stimulating cell by cultivation region one and cultivation region two.
4th step, by cell under high-definition camera or DVD detection cultivation regions 1 and the fluid force of cultivation region 27
Differentiation situation, detects cell signal, collects data;According to cellular localization, and fluid flow network positioning, to the cell collected
Experimental data is analyzed, influence of the research different fluid shearing force to cell.
5th step, by the metabolite of cell in waste liquid pool culture pond and remaining nutrient solution, by passing away and row
Outlet discharge.
Preferably, the floor height of the cultivation region one and cultivation region two, at the import connected 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 bottom surface, two bottom surfaces of cultivation region one and cultivation region two
The gradient it is all consistent.So it is easy to ensure the contrast of experimental data.
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 with region.So ensure that chip can be detected under microscope or CCD.The chip is in rectangle culture
Grid groove is all machined with the region of area and taper cultivation region, to cell shearing power during in order to cellular localization and fluid simulation
Positioning.
The advantage of chip of the present invention:Under good cell culture environment, by the experimental implementation on chip, Neng Gouqi
To the experimental observation contrast of simultaneous quantitative orientation, so that influence experiment of the research shearing force to cell more facilitates, reduce
Immediately the experimental period and error observed.Compared with existing design, chip of the present invention has following beneficial effect:Compare common core
Piece is provided with the culture systems of different shearing forces, and different shearing forces are applied in stem cell incubation, reach immediately, have
Effect, micro observation.Real-time monitoring can be carried out within the time period of detection, the input rate of nutriment is controlled, it is ensured that detection
Reliability so that result can more react real stem cell growth environment, grasp the influence that shearing force is broken up to stem cell, be
Follow-up work is prepared.
Brief 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;5.V passages;6. injection orifice;7. train
Support area two;8. outlet, 9. grid groove.
Specific embodiment
1-2 is further illustrated to the present invention below in conjunction with the accompanying drawings.A kind of cell flow experiment is used with biochip
Method, chooses cultivation region 1 and the shape specification identical chip of cultivation region 27, is divided into experimental group and control group, carries out cell training
Support the experiment for stimulating cell with flow of fluid.
Embodiment one
The first step, the chip form specification of selection is:The side of cultivation region 1 is by the mutually opposing series connection of circular arc wall of R=0.8mm
Composition, the side of cultivation region 27 are by the mutually opposing circular arc rabbet shape chip being composed in series of circular arc wall of R=4mm.Cultivation region
The most width dimensions D=10mm of the one 3 and L=25mm long of cultivation region 27, cultivation region 1 and cultivation region 27, interface channel width
Size d=1mm.Experimental Flowing Object injection flow velocity 15 μ L/S, 30 μ L/S.The rectangle cultivation region 3 of the chip that will be chosen and taper culture
In the shape and size in area 7, and whole channel size input computers, charted with CAD software;Then cartographic data is imported
In fluid simulation software, the fluid flow network of the cultivation region 1 and cultivation region 27 under Experimental Flowing Object flow velocity is calculated.
Second step, with more than 20 chips chosen, is divided into experimental group 10 and control tissue culture 10, under the same conditions
Culture experiment cell.Stem cell and nutrient solution are injected in pairs respectively from injection orifice 6, nutrient solution is periodically changed by injection orifice 6, protect
Card cell is normally metabolic.
3rd step, after after cell attachment growth, from two pairs of streams of the certain flow rate of the lasting injection experimentses requirement of injection orifice 6
Body, fluid produces the active force for stimulating cell by cultivation region 1 and cultivation region 27.
4th step, by under high speed high-definition camera machine testing cultivation region 1 and the fluid force of cultivation region 27 cell point
Change situation, detects cell signal, collects data.According to cellular localization, and fluid flow network positioning, to the cell reality collected
Test data to be analyzed, influence of the research different fluid shearing force to cell.
5th step, experiment terminates, by the metabolite and remaining nutrient solution of cell in the culture pond of waste liquid pool 1, by row
Go out passage 2 and outlet 8 is discharged.
Embodiment two
The first step, the chip form specification of selection is:The length of side of the sharp cone distal of cultivation region 1 is 0.5mm, the convex-concave angle between both sides
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.Cultivation region
The most width dimensions D=30mm of the one 3 and L=40mm long of cultivation region 27, cultivation region 1 and cultivation region 27, interface channel width
Size d=2.5mm.Experimental Flowing Object injection flow velocity 25 μ L/S, 50 μ L/S.The rectangle cultivation region 3 of the chip that will be chosen and taper are trained
Support in the shape and size in area 7, and whole channel size input computers, charted with CAD software;Then cartographic data is led
Enter in fluid simulation softward, calculate the fluid flow network of the cultivation region 1 and cultivation region 27 under Experimental Flowing Object flow velocity.
Second step, with 12 chips chosen, is divided into experimental group 6 and control group 6, under the same conditions culture experiment
Cell.Stem cell and nutrient solution are injected in pairs respectively from injection orifice 6, nutrient solution is periodically changed by injection orifice 6, it is ensured that cell is just
Normal metabolism.
3rd step, after after cell attachment growth, from two pairs of streams of the certain flow rate of the lasting injection experimentses requirement of injection orifice 6
Body, fluid produces the active force for stimulating cell by cultivation region 1 and cultivation region 27.
4th step, the differentiation situation of cell under cultivation region 1 and the fluid force of cultivation region 27 is detected by high definition DVD,
Detection cell signal, collects data;According to cellular localization, and fluid flow network positioning, to the cell experiment data collected
It is analyzed, influence of the research different fluid shearing force to cell.
5th step, experiment terminates, by the metabolite and remaining nutrient solution of cell in the culture pond of waste liquid pool 1, by row
Go out passage 2 and outlet 8 is discharged.