CN106701573A - Use method of biological chip for cell fluid experiment - Google Patents
Use method of biological chip for cell fluid experiment Download PDFInfo
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- CN106701573A CN106701573A CN201611058477.0A CN201611058477A CN106701573A CN 106701573 A CN106701573 A CN 106701573A CN 201611058477 A CN201611058477 A CN 201611058477A CN 106701573 A CN106701573 A CN 106701573A
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- 239000012530 fluid Substances 0.000 title claims abstract description 38
- 238000002474 experimental method Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 239000002699 waste material Substances 0.000 claims abstract description 10
- 230000004069 differentiation Effects 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 56
- 238000002347 injection Methods 0.000 claims description 27
- 239000007924 injection Substances 0.000 claims description 27
- 238000010008 shearing Methods 0.000 claims description 16
- 238000000018 DNA microarray Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 235000015097 nutrients Nutrition 0.000 claims description 12
- 210000000130 stem cell Anatomy 0.000 claims description 8
- 238000013461 design Methods 0.000 claims description 7
- 238000011160 research Methods 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 6
- 230000030570 cellular localization Effects 0.000 claims description 5
- 230000004936 stimulating effect Effects 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 4
- 230000002045 lasting effect Effects 0.000 claims description 4
- 239000002207 metabolite Substances 0.000 claims description 4
- 230000002503 metabolic effect Effects 0.000 claims description 3
- 238000012549 training Methods 0.000 claims description 3
- 230000008676 import Effects 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 238000011017 operating method Methods 0.000 claims description 2
- 230000007704 transition Effects 0.000 claims description 2
- 239000012780 transparent material Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000004113 cell culture Methods 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000010261 cell growth Effects 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 238000007599 discharging Methods 0.000 abstract 1
- 238000004088 simulation Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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Abstract
The invention provides a use method of a biological chip for a cell fluid experiment. The use method is characterized by comprising the following steps: 1, calculating a fluid flowing network of a cell culture area at an experimental fluid flowing speed according to shapes and specifications of selected chips; 2, classifying the selected chips into an experiment group and a contrast group, and culturing experimental cells under the same condition; 3, after the cells grow in an attachment manner, continuously injecting fluid which is required by the experiment and has a certain flowing speed; 4, detecting the differentiation situation of the cells under the acting force of the fluid through a high-speed high-definition camera; 5, at the end of the experiment, discharging waste liquid. The chip has the following beneficial effects that compared with a common chip, the chip is equipped with an extra culture detection unit under different mechanical experimental conditions of the fluid, and different shear forces are applied in a cell culture process, so as to realize instant and effective trace observation; real-time monitoring can be carried out within a period of time of detection, the fluid input rate can be controlled, and the detection reliability can be guaranteed; therefore, an experimental detection result can better reflect a real cell growth environment, and people can master the influence of the shear force on the cells.
Description
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.
Claims (3)
1. biochip application method is used in a kind of cell flow experiment, and the rectangle or the chip-side of circle for using have 2 rows totally 4
Individual injection orifice（6）, injection orifice（6）It is two-by-two a pair, 2 injection orifices of each pair（6）Totally 2 V passage is each connected respectively
（5）, 2 V passages（5）It is Y-shaped to be flowed to symmetrical 2 interface channels（4）；2 interface channels（4）Connection training respectively
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）By 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 concavo-convex lines
Shape, cultivation region two（7）Side it is mutually opposing by the circular arc wall opening of R=3mm-5mm, the two ends of arc are connected with each other series connection, connection
Into overlooking into the concavo-convex stripe shape of circular arc；Or, 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 formed objects, the both sides termination of pointed cone is connected with each other the shape being connected into, i.e. wedge angle is concavo-convex
Side shape；Cultivation region one（3）Sharp cone distal the length of side be 0.5mm-1mm, the convex-concave angle α between both sides1It is α1≤ 60 ° of wedge angle；Training
Support area two（7）Sharp cone distal the length of side be 2mm-5mm, the convex-concave angle α between both sides2It is α2≤ 90 ° of wedge angle；The cultivation region
One（3）With cultivation region two（7）L long it is equal, and L >=20mm, cultivation region one（3）With cultivation region two（7）Most width dimensions D
It is equal, and D sizes are interface channels（4）More than 3 times of width dimensions d, i.e. D >=3d；Characterized in that, using operating procedure
It is as follows：
The first step, tests, the chip cultivation region one of selection according to cell shearing power（3）With cultivation region two（7）Shape, and design
Flow velocity, the rectangle cultivation region of the chip that will be chosen needed for good experiment（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, the chip chosen more than is divided into experimental group and control group culture experiment cell；It is paired respectively from injection orifice 6
Injection stem cell and nutrient solution, periodically change nutrient solution, it is ensured that cell is normally metabolic by injection orifice 6；
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 6, stream
Body passes through cultivation region one（3）With cultivation region two（7）Produce the active force for stimulating cell；
4th step, cultivation region one is detected by high-definition camera or DVD（3）With cultivation region two（7）Cell under fluid force
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, experiment terminates, by waste liquid pool（1）The metabolite of cell and remaining nutrient solution in culture pond, by discharge
Passage（2）And outlet（8）Discharge.
2. biochip application method is used in a kind of cell flow experiment according to claim 1, is characterised by：Use chip
Cultivation region one（3）With cultivation region two（7）Floor height, with interface channel（4）The two of at the import for connecting and exit
The height dimension of individual cultivation region is all identical, i.e., when there is the gradient bottom surface, cultivation region one（3）With cultivation region two（7）Two bottom surfaces
The gradient is all consistent.
3. biochip application method is used in a kind of cell flow experiment 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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| CN201611058477.0A CN106701573B (en) | 2016-11-27 | 2016-11-27 | A kind of cell flow experiment biochip application method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904161A (en) * | 2017-12-15 | 2018-04-13 | 上海交通大学医学院附属仁济医院 | It is a kind of to visualize micro-fluidic chip of detection pathogen nucleic acid and preparation method thereof and detection method immediately |
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| CN106701573B (en) | 2018-10-23 |
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Effective date of registration: 20230418 Address after: Room 502, Building 1, No. 40, Xiayuangang East Street, Yuangang Village, Tianhe District, Guangzhou City, Guangdong Province, 510000 Patentee after: Guangzhou Huansheng Technology Co.,Ltd. Address before: No. 20, East Road, University City, Chongqing, Shapingba District, Chongqing Patentee before: Chongqing University of Science & Technology Effective date of registration: 20230418 Address after: B-102 #, New City State owned Assets Inspection and Testing Laboratory Building, No. 8 Shangju Road, Yunlong Economic Development Zone, Xuzhou City, Jiangsu Province, 221000 Patentee after: Xuzhou Longpeptide Medical Laboratory Co.,Ltd. Patentee after: Wei Bing Patentee after: Lu Jian Address before: Room 502, Building 1, No. 40, Xiayuangang East Street, Yuangang Village, Tianhe District, Guangzhou City, Guangdong Province, 510000 Patentee before: Guangzhou Huansheng Technology Co.,Ltd. |
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Denomination of invention: A method for using a biological chip for cell fluid experiments Granted publication date: 20181023 Pledgee: Bank of Jiangsu Co.,Ltd. Xuzhou Branch Pledgor: Xuzhou Longpeptide Medical Laboratory Co.,Ltd. Registration number: Y2024980038126 |