CN102787071A - Study on in vivo fluid shearing force simulation cell behaviors on basis of microfluidic chip system - Google Patents
Study on in vivo fluid shearing force simulation cell behaviors on basis of microfluidic chip system Download PDFInfo
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Abstract
The invention relates to a study on in vivo fluid shearing force simulation cell behaviors on the basis of a microfluidic chip system. An external precise fluid injection pump is used as a motive power source, a microfluidic chip soft etching technology is used as the basis, and the fluid resistance principle is utilized for designing and manufacturing a microfluidic passage resistance network, so cells in a microfluidic chip cell culture microchamber can feel fluid shearing force with different intensities, and further, the cell behavior change is studied. Through the microfluidic chip system, the process of the inoculation and long-term culture of cells, the fluid shearing force stimulation on the cells and the cell dyeing and analysis and detection are integrated on a functionalization chip to be completed, and the cell behavior analysis and the in vivo ultra-low fluid shearing force simulation on a microfluidic chip platform are realized.
Description
Technical field
The invention belongs to the micro-fluidic chip system field, be specifically related to a kind of analogue body inner fluid shearing force cell behavior research based on micro-fluidic chip system.
Background technology
The micro-fluidic chip laboratory as this century an important science and technology represented its special advantages in a plurality of fields such as comprising biology, medical science, chemistry, materialogy, more because of its with cell size coupling, environment is close with physiological environment, heat and mass is fast, flux high can be integrated etc. characteristics become the important platform of cell research of new generation.Along with the continuous development of recent two decades, based on the existing very quantum jump of the cell research of micro-fluidic chip system, the relevant cell operation basically all can realize on chip like the cultivation of cell, sorting, evaluation etc.Further cytology research based on this; Particularly become the focus of concern day by day based on researchs such as the bionical microenvironment simulations of the cell of micro-fluidic chip system; Simulation even artificial cell microenvironment are used to study the related behavior of cell on micro-fluidic chip system; Its key point is exactly that the pair cell microenvironment is accurately simulated, and this also is simultaneously research cell and the interactional main contents of microenvironment.The analog cell microenvironment; What particularly simulation was relevant with the cell biological physical factor relies on prior art to exist the plant and instrument complicacy huge mostly such as microenvironments such as hydrodynamic shear, distraction force, matrix physical properties, Nanosurfaces; Complex operation; Simple to the microenvironment simulation, can not realize the problems such as real-time monitored of pair cell, and deepening continuously along with what micro-fluidic chip was studied; Micro-fluidic chip system maybe can become a kind of novel platform technology in order to the human simulation cell micro-environment, and is to study relevant tissue repair, reproduce the support that aspects such as transplanting and bionic provide technology and theory with cell micro-environment.
Summary of the invention
The purpose of this invention is to provide a kind of analogue body inner fluid shearing force cell behavior research based on micro-fluidic chip system; This micro-fluidic chip system is integrated on the functionalization chip by hydrodynamic shear stimulation, cell dyeing and analyte detection process inoculation, long-term cultivation, the cell of cell to accomplish, realized the simulation and the cell behavior analysis of extremely low hydrodynamic shear in the micro-fluidic chip platform upper.Cell and reagent consumption are low, and be easy and simple to handle, is easy to detect.
The present invention is a propulsion source with outside accurate fluid syringe pump; Be the basis with the soft lithographic technique of micro-fluidic chip; Utilize the fluid resistance principle design and make the microfluidic channel resistance network; Cause the cell in the micro-fluidic chip cell cultures microchamber can experience different big or small hydrodynamic shears; And then the research cell behavior changes, and this micro-fluidic chip system is integrated on the functionalization chip by hydrodynamic shear stimulation, cell dyeing and analyte detection process inoculation, long-term cultivation, the cell of cell to accomplish, realized the simulation and the cell behavior analysis of extremely low hydrodynamic shear in the micro-fluidic chip platform upper.Cell and reagent consumption are low, and be easy and simple to handle, is easy to detect.
The invention provides a kind of micro-fluidic chip system; This system is made up of two elementary cells: first elementary cell is the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear in the analogue body, second the cell behavior research micro-fluidic chip peripherals that elementary cell is an extremely low hydrodynamic shear in the analogue body.
Micro-fluidic chip system provided by the invention, the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear is the double-deck micro-fluidic chip that adopts soft etching of PDMS and irreversible sealing technology to make up in the described analogue body.
Micro-fluidic chip system provided by the invention, described micro-fluidic chip is divided into two portions: first part is a microchannel fluid resistance network, comprises a series of according to the different thicknesses of fluid resistance principle design, the microchannel of length; Second section is a plurality of cell cultures microchambers, and concrete structure is as shown in Figure 1, and the cell in the cell cultures microchamber will be experienced the different big or small extremely low hydrodynamic shear that the fluid resistance network forms, and then form cellular response and to be detected.
Micro-fluidic chip system provided by the invention; Microchannel and cell cultures microchamber in the fluid resistance network of described microchannel are of a size of: highly be 10 microns ~ 1 centimetre; Width is 30 microns ~ 1 centimetre, and the xsect pattern can be rectangle, square, circle, semicircle.
Micro-fluidic chip system provided by the invention, the number of described microchannel and cell cultures microchamber respectively are 1 ~ 1000.
Micro-fluidic chip system provided by the invention, said cell cultures microchamber is used for cell cultures can carry out finishing:
(1) can not carry out finishing for living state good cell in the cell cultures microchamber and handle the direct inoculation cell;
(2) can apply modification to channel inner surface for the cell that is difficult in the cell cultures microchamber being not in good state after attaching or the attaching; Applying the method for modifying is: directly in passage, inject the reagent that is used to increase the cell attaching; Treat that certain hour carries out later cell inoculation again, this kind reagent is one or more of gelatin, gelatinum, collagen type I, collagen type II, collagen VI type, Laminin ELISA;
(3) for simulation particular requirement is arranged in the microenvironment; Apply in the modification reagent and can add ESC, like one or more of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor (TGF).
Micro-fluidic chip system provided by the invention; Described micro-fluidic chip peripherals is the proofing unit that the outside is supported and cell behavior is analyzed that the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear in the analogue body is realized each function, mainly comprises precise injection pump, injector for medical purpose, medical Transfusion device, polystyrene tube and fluorescent microscope.
Micro-fluidic chip system provided by the invention, described precise injection pump can be realized the accurate control of the flow velocity direction of convection cell, and can go in the micro-fluidic chip passage through the peripheral tube pass, and then produces the hydrodynamic shear stimulation.
The present invention also provides the analogue body inner fluid shearing force cell behavior research based on described micro-fluidic chip system; Being the basis with the soft lithographic technique of micro-fluidic chip, is hydrodynamic source with outside accurate fluid syringe pump, accurately controls fluid flow rate; Utilize the fluid resistance principle design and make the microfluidic channel resistance network; Cause fluid in the cell cultures microchamber of flowing through to produce a certain size hydrodynamic shear, the cell in the cell cultures microchamber is experienced this hydrodynamic shear, and then the generation behavior changes; Via cell dyeing and analysis, the structure and the cell behavior analysis of hydrodynamic shear on the micro-fluidic chip platform have been realized.
Analogue body inner fluid shearing force cell behavior research based on micro-fluidic chip system provided by the invention, it is 1.0 * 10 that this micro-fluidic chip system can be used for simulating the hydrodynamic shear magnitude range
-9Dyne/cm
2~ 1dyne/cm
2Hydrodynamic shear.
The cell behavior research microfluidic system of extremely low hydrodynamic shear in the analogue body provided by the invention; Can realize the long-term attaching cultivation of mescenchymal stem cell; Utilize and extremely hang down the expression that hydrodynamic shear can impel mescenchymal stem cell Osteoblast Differentiation and motion associated protein in the mimic body, its result is like Fig. 2, shown in 3.In this system, mescenchymal stem cell is carried out the research of skeletonization differentiation mechanism, find the skelemin that the extremely low hydrodynamic shear of this kind can be through intervening cell and then promote its Osteoblast Differentiation.Shown in Fig. 4 difference.
The cell behavior research microfluidic system of extremely low hydrodynamic shear in the analogue body provided by the invention, the related detecting method of its mescenchymal stem cell is:
(1) Osteoblast Differentiation detection method: Ostase ALP dyeing, osteopontin OPN dyeing.
(2) cytoskeletal protein detection method: cytoskeleton scleroproein F-actin dyeing.
The cell behavior research microfluidic system that is used for extremely low hydrodynamic shear in the analogue body provided by the invention, easy and simple to handle, reagent and cell consumption amount are low, are easy to pair cell and observe detection, are a kind of novel platform technologys that is used for the human simulation cell micro-environment.
Description of drawings
The cell behavior research microfluidic chip structure figure of extremely low hydrodynamic shear in Fig. 1 analogue body, wherein 1 is microchannel fluid resistance network, 2 is the cell cultures microchamber;
Fig. 2 mescenchymal stem cell receives extremely low hydrodynamic shear to stimulate following Osteoblast Differentiation figure as a result;
Fig. 3 mescenchymal stem cell receives extremely low hydrodynamic shear to stimulate the real-time photo of motor behavior down;
The schematic diagram of mechanism that Fig. 4 mescenchymal stem cell stimulates through the extremely low hydrodynamic shear of ROCK approach impression;
Fig. 5 osteoprogenitor cells receives the cellular form under the extremely low hydrodynamic shear stimulation;
Fig. 6 chondrocyte receives the cellular form under the extremely low hydrodynamic shear stimulation.
Embodiment
Following embodiment will further explain the present invention, but therefore not limit the present invention.
The material of the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear is the PDMS polymkeric substance in the preparation analogue body, the irreversible sealing-in of plasma body.Chip channel after the preparation adds the water autoclaving, places in the aseptic worktable to dry again.With the mice embryonic mescenchymal stem cell is example, and chip liquid paths need not to apply, successively with 75% ethanol, aseptic secondary water, PBS, contain the α-MEM substratum rinse of foetal calf serum 15% and guarantee that the interior no bubble of passage is residual.Trysinization is used in the cultivation of in aseptic disposable culturing bottle, going down to posterity of mice embryonic mescenchymal stem cell during experiment, regulate proper density and be injected in the cell cultures microchamber with liquid-transfering gun from cell application of sample mouth, treats the agent of the complete adherent back replacement fresh culture of 24 hour cells.If carry out chemical inducer with the coefficient experiment of mechanical stimulation, then the differentiation culture agent is induced in replacement after 24 hours, and skeletonization adopts LG-DMEM+10%FBS+0.1 μ M DEXAMETHASONE BP98+50 μ g/ml xitix+100mM sodiums.Adjust the flow of accurate syringe pump; Make the scope that reaches extremely low hydrodynamic shear, mode is a continous perfusion, and perfusion can be investigated its motor behavior after 24 hours; Perfusion can be investigated into the secretion of bone alkaline phosphatase ALP after 7 days, perfusion can be investigated the situation of its osteopontin OPN after 14 days.Staining procedure is: with the substratum in the PBS replacement chip channel, and wash 3 times, with Paraformaldehyde 96 fixed cell 20 minutes, PBS washed 3 times.Skeletonization Ostase ALP dyeing is for washing with PBS after the cell fixation; Use the flushing of Tris damping fluid instead; In passage, add the ALP staining fluid of at present joining, take pictures for 3 times with the Tris flushing after 15 minutes, the light intensity of calculating Ostase (blueness) is in order to characterize the power of Osteoblast Differentiation.Blank control group is not for applying the situation of mechanical stimulation.Other dyeing use corresponding antibody to get final product for the dyeing of fluorescence immunoassay method.
The micro-fluidic chip system that utilizes the laboratory to design voluntarily and make; Configuration is as shown in Figure 1; The channel size degree of depth is 50 μ m, and width is that 50 μ m do not wait to 1mm, can produce the hydrodynamic shear of 4 different sizes altogether; At the syringe pump flow velocity is under the situation of 9 μ L/h, is respectively after the hydrodynamic shear Theoretical Calculation: 7.7 * 10
-3, 9.9 * 10
-4, 1.4 * 10
-4And2.9 * 10
-5Dyne/cm
2Chip inoculation mice embryonic mescenchymal stem cell, inoculum density 1 * 10
5Individual/ML, use the α-MEM substratum that contains foetal calf serum 15% instead after 24 hour cells are adherent, apply hydrodynamic shear, skeletonization ALP dyeing after 7 days, microscope is taken pictures, Image Pro software analysis.Its result is as shown in Figure 3, the Osteoblast Differentiation behavior of mescenchymal stem cell difference to some extent under the hydrodynamic shear effect of different sizes, and what can find out is 7.7 * 10
-3Dyne/cm
2The hydrodynamic shear effect under skeletonization the strongest, and 14 days after OPN be expressed as the positive.
The micro-fluidic chip system that utilizes the laboratory to design voluntarily and make; Configuration is as shown in Figure 1; The channel size degree of depth is 50 μ m, and width is that 50 μ m do not wait to 1mm, can produce the hydrodynamic shear of 4 different sizes altogether; At the syringe pump flow velocity is under the situation of 9 μ L/h, is respectively after the hydrodynamic shear Theoretical Calculation: 7.7 * 10
-3, 9.9 * 10
-4, 1.4 * 10
-4And2.9 * 10
-5Dyne/cm
2Chip inoculation mice embryonic mescenchymal stem cell, inoculum density 1 * 10
5Individual/ML; Use LG-DMEM+10%FBS+0.1 μ M DEXAMETHASONE BP98+50 μ g/ml xitix+100mM sodium osteogenic induction division culture medium after 24 hour cells are adherent instead; Apply hydrodynamic shear, skeletonization ALP dyeing after 7 days, skeletonization OPN dyeing after 14 days; Microscope is taken pictures, Image Pro software analysis.The Osteoblast Differentiation behavior of mescenchymal stem cell difference to some extent under the hydrodynamic shear effect of different sizes, what can find out is 7.7 * 10
-3Dyne/cm
2The hydrodynamic shear effect under skeletonization ALP and OPN express the strongest.
Embodiment 3
Osteoprogenitor cells: the micro-fluidic chip system that utilizes the laboratory to design voluntarily and make, its hydrodynamic shear scope is 7.7 * 10
-3, 9.9 * 10
-4, 1.4 * 10
-4And2.9 * 10
-5Dyne/cm
2Chip inoculation osteoprogenitor cells is cell 3T3-E1, inoculum density 1 * 10
5Individual/ML, after 24 hour cells are adherent, change liquid every day, and apply the hydrodynamic shear stimulation, mechanical stimulation is detected as bone characteristics property mark ALP after 7 days, find that hydrodynamic shear can promote osteoprogenitor cells to break up to the skeletonization direction.
Embodiment 4
The chondrocyte: the micro-fluidic chip system that utilizes the laboratory to design voluntarily and make, the channel size degree of depth are 100 μ m, and width is that 100 μ m do not wait to 2mm, and its hydrodynamic shear scope is 0.5,0.1,0.02,0.001dyne/cm
2Chip inoculation rat cartilage cell, inoculum density 1 * 10
5Individual/ML, after 24 hour cells are adherent, change liquid every day, and apply the hydrodynamic shear stimulation, mechanical stimulation can detect cartilage characteristic mark one Collagen Type VI, two Collagen Type VIs, protein-polysaccharide after 3 days, and the discovery hydrodynamic shear can be kept phenotype of chondrocytes.
Claims (9)
1. micro-fluidic chip system; It is characterized in that: this system is made up of two elementary cells: first elementary cell is the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear in the analogue body, second the cell behavior research micro-fluidic chip peripherals that elementary cell is an extremely low hydrodynamic shear in the analogue body.
2. according to the described micro-fluidic chip system of claim 1, it is characterized in that: the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear is the double-deck micro-fluidic chip that adopts soft etching of PDMS and irreversible sealing technology to make up in the described analogue body.
3. according to claim 1 or 2 described micro-fluidic chip systems, it is characterized in that: described micro-fluidic chip is divided into two portions: first part is a microchannel fluid resistance network, is made up of the microchannel; Second section is the cell cultures microchamber.
4. according to the described micro-fluidic chip system of claim 3; It is characterized in that: microchannel and cell cultures microchamber in the fluid resistance network of described microchannel are of a size of: highly be 10 microns ~ 1 centimetre; Width is 30 microns ~ 1 centimetre, and the xsect pattern can be rectangle, square, circle, semicircle.
5. according to the described micro-fluidic chip system of claim 3, it is characterized in that: the number of described microchannel and cell cultures microchamber respectively is 1 ~ 1000.
6. according to the described micro-fluidic chip system of claim 1; It is characterized in that: described micro-fluidic chip peripherals is the proofing unit that the outside is supported and cell behavior is analyzed that the cell behavior research micro-fluidic chip of extremely low hydrodynamic shear in the analogue body is realized each function, mainly comprises precise injection pump, injector for medical purpose, medical Transfusion device, polystyrene tube and fluorescent microscope.
7. according to the described micro-fluidic chip system of claim 6, it is characterized in that: described precise injection pump can be realized the accurate control of the flow velocity direction of convection cell, and can go in the micro-fluidic chip passage through the peripheral tube pass, and then produces the hydrodynamic shear stimulation.
8. study based on the analogue body inner fluid shearing force cell behavior of the described micro-fluidic chip system of claim 1; It is characterized in that: being the basis with the soft lithographic technique of micro-fluidic chip, is hydrodynamic source with outside accurate fluid syringe pump, accurately controls fluid flow rate; Utilize the fluid resistance principle design and make the microfluidic channel resistance network; Cause fluid in the cell cultures microchamber of flowing through to produce a certain size hydrodynamic shear, the cell in the cell cultures microchamber is experienced this hydrodynamic shear, and then the generation behavior changes; Via cell dyeing and analysis, the structure and the cell behavior analysis of hydrodynamic shear on the micro-fluidic chip platform have been realized.
9. according to the described analogue body inner fluid shearing force cell behavior research based on micro-fluidic chip system of claim 8, it is characterized in that: described hydrodynamic shear magnitude range is 1.0 * 10
-9Dyne/cm
2~ 1dyne/cm
2
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| CN103331097A (en) * | 2013-05-27 | 2013-10-02 | 陕西师范大学 | Application of polydimethylsiloxane micro fluidic chip in separating oligosaccharide and polysaccharide |
| CN104928147A (en) * | 2015-05-21 | 2015-09-23 | 重庆科技学院 | Double-layer cell screening culture chip and operation method |
| CN106318869A (en) * | 2016-11-27 | 2017-01-11 | 重庆科技学院 | Biological chip for cell fluid experiment |
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| CN106701573A (en) * | 2016-11-27 | 2017-05-24 | 重庆科技学院 | Use method of biological chip for cell fluid experiment |
| CN106544272B (en) * | 2016-11-27 | 2019-07-23 | 重庆科技学院 | A kind of application method of the biochip for shearing force experiment |
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| CN108339580B (en) * | 2018-03-20 | 2020-01-14 | 哈尔滨工业大学深圳研究生院 | Fluid shear force generation device and fluid shear force generation method |
| CN108977359B (en) * | 2018-07-27 | 2022-01-18 | 大连理工大学 | Micro-fluidic chip for cell culture and simulation of pulsating shear force environment after movement and detection method |
| CN108977359A (en) * | 2018-07-27 | 2018-12-11 | 大连理工大学 | A kind of micro-fluidic chip and detection method for dynamic shear force environment after cell culture and skimulated motion |
| CN109187318A (en) * | 2018-07-30 | 2019-01-11 | 西南交通大学 | Intravascular metal degradable high throughput based on micro-fluidic chip simulates monitoring system |
| CN113337451A (en) * | 2021-06-02 | 2021-09-03 | 吉林医药学院 | Accurate patterning method for single cells in multi-shear force microfluidic chip |
| CN113337451B (en) * | 2021-06-02 | 2023-03-07 | 吉林医药学院 | Accurate patterning method for single cells in multi-shear force microfluidic chip |
| CN114107056A (en) * | 2021-10-27 | 2022-03-01 | 中国科学院大学 | In-vitro vascular tissue-like model with fluid environment and application thereof |
| CN114107056B (en) * | 2021-10-27 | 2023-08-08 | 中国科学院大学 | In-vitro blood vessel-like tissue model with fluid environment and application thereof |
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