CN203320000U - Cell chemotaxis analysis chip and device - Google Patents

Cell chemotaxis analysis chip and device Download PDF

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Publication number
CN203320000U
CN203320000U CN2013204117000U CN201320411700U CN203320000U CN 203320000 U CN203320000 U CN 203320000U CN 2013204117000 U CN2013204117000 U CN 2013204117000U CN 201320411700 U CN201320411700 U CN 201320411700U CN 203320000 U CN203320000 U CN 203320000U
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cell
passage
solution
chemotactic
chemotaxis
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CN2013204117000U
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罗春雄
杨薇
冉敏
欧阳颀
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Peking University
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Peking University
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Abstract

The utility model relates to the technical field of biology, in particular to a cell chemotaxis analysis chip. The cell chemotaxis analysis chip comprises a liquid inlet cavity, a chemotaxis channel, a fence channel and a liquid absorption cavity. The liquid inlet cavity is provided with a liquid inlet used for adding a cell solution, and is used for containing the cell solution. One end of the chemotaxis channel is communicated with the liquid inlet cavity, the other end of the chemotaxis channel is communicated with one end of the fence channel, and the inner diameter of the chemotaxis channel is larger than the diameter of cells contained inside the cell solution. The inner diameter of the fence channel is smaller than the diameter of the cells contained inside the cell solution, and the other end of the fence channel is communicated with the liquid absorption cavity. The liquid absorption cavity is used for absorbing a solution body in the cell solution. By utilizing the structure, the chemotaxis process of the cells under the specific concentration gradient can be observed in a rationing mode conveniently, the influence on the process from flowing is eliminated, and convenience is brought to study of cell chemotaxis.

Description

A kind of cell chemotaxis analysis chip and device
Technical field
The utility model relates to the biology techniques field, particularly a kind of cell chemotaxis analysis chip and device.
Background technology
The chemotaxis behavior is a basic vital process, its research not only is conducive to understand the biological signal conduction response mechanism, and the exploitation of relevant biosensor also has very high practical value.Yet research means is still relatively backward instantly, for the chemotactic research means of cell, the difficulty of current experimental implementation is larger, uses and inconvenience.
Microflow control technique is the front subject of develop rapidly in recent years, relies on its diversification, analyzes fast, the advantages such as consumption is few, high-throughput and real-time monitored, has now become a strong research means in the biomass cells analysis field.Recently, micro fluidic device (Erwin Berthier, Jill Surfus, James Verbsky, Anna Huttenlocher and David Beebe, An arrayed high-content chemotaxis assay for patient diagnosis, Integrative Biology, 2010,2,630-638.) also being widely used for zoologizeing the chemotaxis of cell, the experimental implementation difficulty is also larger on the whole, and more difficult control fluid flows on the impact of chemotaxis behavior itself.
In order to overcome the above problems, the utility model has been done useful improvement.
The utility model content
(1) technical problem that will solve
The purpose of this invention is to provide a kind of cell chemotaxis analysis chip that is easy to cell chemotaxis is carried out observation and analysis.
The technical problem that another will solve of the present utility model is to provide a kind of cell chemotaxis analytical equipment that includes a plurality of described cell chemotaxis analysis chips.
(2) technical scheme
For this technical theme of cell chemotaxis analysis chip, the utility model is achieved through the following technical solutions:
A kind of cell chemotaxis analysis chip, comprise feed liquor chamber, chemotactic passage, fence passage and imbibition chamber;
Described feed liquor chamber is provided with fluid inlet, and described fluid inlet is for adding cell solution; Described feed liquor chamber is for holding the described cell solution added;
One end of described chemotactic passage is connected with described feed liquor chamber, and the other end of described chemotactic passage is connected with an end of described fence passage, and the internal diameter of described chemotactic passage is greater than celliferous diameter in described cell solution;
The internal diameter of described fence passage is less than celliferous diameter in described cell solution; The other end of described fence passage is connected with described imbibition chamber;
Described imbibition chamber is for absorbing the solution of described cell solution.
Further, described chemotactic passage is many.
Further, the internal diameter of described every chemotactic passage is more than or equal to 10 microns and is less than or equal to 100 microns.
Further, described fence passage is many.
Further, the internal diameter of described every fence passage is more than or equal to 1 micron and is less than or equal to 10 microns.
For this technical theme of cell chemotaxis analytical equipment, the utility model is achieved through the following technical solutions:
A kind of cell chemotaxis analytical equipment, which is provided with a plurality of described cell chemotaxis analysis chips.
(3) beneficial effect
With prior art, with product, compare, the utility model has the following advantages:
Utilize structure of the present utility model, very easily quantitative observation to cell the chemotactic process under the certain concentration gradient, get rid of to flow on the impact of this process, for the research of cell chemotaxis behavior provides convenience.
The accompanying drawing explanation
Fig. 1 is schematic top plan view of the present utility model.
In accompanying drawing, the list of parts of each label representative is as follows:
1, feed liquor chamber, 1a, fluid inlet, 2, the chemotactic passage, 3, the fence passage, 4, the imbibition chamber.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is made a detailed explanation.
(1) introduce first the structure of cell chemotaxis analysis chip of the present invention below,
As shown in Figure 1, comprise feed liquor chamber 1, chemotactic passage 2, fence passage 3 and imbibition chamber 4; As can be seen from the figure, between feed liquor chamber 1, chemotactic passage 2, fence passage 3 and imbibition chamber 4, be communicated with successively;
Feed liquor chamber 1 is provided with for adding the fluid inlet 1a of cell solution; Feed liquor chamber 1 is for holding cell solution; That is to say, feed liquor chamber 1 is for to chemotactic passage 2, carrying liquid that a spatial accommodation is provided, to guarantee that mobile cell solution is endlessly to imbibition chamber 4 from feed liquor chamber 1;
One end of chemotactic passage 2 is connected with feed liquor chamber 1, and the other end of chemotactic passage 2 is connected with an end of fence passage 3, and the internal diameter of chemotactic passage 2 is greater than celliferous diameter in cell solution; That is to say, the cell in cell solution can enter in chemotactic passage 2.Chemotactic passage 2 can be one, here the situation below preferably: chemotactic passage 2 is many, these chemotactic passages are to be parallel to each other as shown in Figure 1 in a plane arranges, and the internal diameter of every chemotactic passage all is greater than celliferous diameter in described cell solution;
The internal diameter of fence passage 3 is less than celliferous diameter in cell solution; The other end of fence passage 3 is connected with imbibition chamber 4; That is to say, the cell in cell solution can be blocked by fence passage 3, only has the solution in cell solution and to be inhaled into imbibition chamber 4 by fence passage 3; Fence passage 3 can be one, here preferred following situation: fence passage 3 is many, and these fence passages are to be parallel to each other as shown in Figure 1 in a plane arranges, and the internal diameter of every fence passage is less than celliferous diameter in cell solution;
Imbibition chamber 4 is for the solution of absorptive cell solution.
The present invention is specially adapted to observation and the research of human body cell and zooblast, and the dimension of zooblast is generally 10 -4-10 -5M below provides one group of reference dimension as example:
The height of chemotactic passage 2 and imbibition chamber 4 can be chosen as 30 μ m, and because Fig. 1 is vertical view, the height of mentioning here is the outside height of vertical paper, and the height of the fence passage 3 between the two is 5 μ m.Chemotactic passage 2 is 4, and length is 600 μ m, and width is 30 μ m, and said length refers to the above-below direction in Fig. 1 here, and width refers to the left and right directions in Fig. 1.Fluid inlet 1a can select to make the hole that diameter is 5mm, and the cross-sectional sizes of imbibition chamber 4 can be made into the square of 600 * 600 μ m.Fence passage 3 is 20, and length is 200 μ m and width, highly is 5 μ m.
In general, the internal diameter of every chemotactic passage is more than or equal to 10 microns and is less than or equal to 100 microns; The internal diameter of every fence passage is more than or equal to 0.1 micron and is less than or equal to 10 microns.Certainly, those skilled in the art can also, according to the size of different cells to be observed, rationally further arrange and adjust the size of fluid inlet 1a, chemotactic passage 2, fence passage 3, the parameters such as spacing.
(2) next, the structure in conjunction with above-mentioned cell chemotaxis analysis chip provides its several using method;
Embodiment 1:
Step 1 runs through with the punch tool punching grade fluid inlet 1a obtained for feed liquor on the feed liquor chamber of circle in Fig. 1;
Step 2, be fixed in the cell chemotaxis analysis chip on cell culture apparatus, makes the cell chemotaxis analysis chip in vacuum state;
This area more often adopts the pre-plastomer of the PDMS (siloxanes of two kinds of different chemical functional groups, A:B=10:1) this material is made the cell chemotaxis analysis chip, major cause is to have light transmission and ventilation property preferably, light transmission makes the experimenter be convenient to its inner case of microscopic examination, ventilation property makes it be convenient to form vacuum state, be beneficial to the negative-pressure operation of back, for example can be by the whole vacuum pump that drops into of cell chemotaxis analysis chip, to utilize the ventilation property of himself, cell chemotaxis analysis chip inside is evacuated; Preferably being pumped into 5-100Pa gets final product.And PDMS is nontoxic, adopt this material to be tested also safer.Certainly in scope known to those skilled in the art, good material is made chip can certainly to adopt other ventilation properties and light transmission, if the ventilation property of selected other materials is poor, so also can select from fluid inlet 1a, cell chemotaxis analysis chip inside to be vacuumized.
The pre-plastomer of PDMS can be purchased from RTV615 (GE Toshiba Silicones Co.Ltd, Shizuoka, Japan);
Vacuum pump can adopt TS2-60 (Longer-Pump Company, Baoding, Hebei, China).
Step 3, after bleeding, at once by cell solution, the solution that contains cell adds from fluid inlet 1a, and due to the effect of negative pressure of vacuum, liquid can be sucked all chambers of cell chemotaxis analysis chip inside automatically;
Solution in cell solution sucks imbibition chamber 4 by chemotactic passage 2 and fence passage 3, and due to the Intercepting effects of fence passage 3, cell only is left on chemotactic passage 2.
Step 4, when chemotactic passage 2 and imbibition chamber 4 are all completed by liquid filling, whole flow velocity reduces to zero.After the solution that contains cell enters cell chemotaxis analysis chip inside, the outside residual solution that contains cell of cell chemotaxis analysis chip is siphoned away, for example, add again nutrient solution after can washing residual cell off with nutrient solution, for convenience, the nutrient solution here, can be used the solution part in archeocyte solution.
Step 5, cover upper transparent upper cover by cell culture apparatus, can prevent liquid evaporation, and carry out microscopic observation under cell culture environment.Under cell culture environment, treat that it is adherent.
Step 6, substitute the cell culture fluid for containing the identical or different chemokine of 30-100 μ L by the liquid in fluid inlet 1a, and chemokine is such as adopting protein micromolecular, organic molecule, ion etc.Liquid is without mobile the time in passage, and chemokine can complete the foundation of concentration gradient in chemotactic passage 2 by diffusion, then use microscopic observation.
Embodiment 2: the analysis allogenic cell is corresponding to different chemokine solution.
Step 1, MCF-7 MDA-MB-231 cell is with containing 10% serum (foetal calf serum, the DMEM nutrient solution of FBS) and 1% penicillin-Streptomycin sulphate is (containing the nutrient solution of each seed amino acid and glucose, Gibco), here mention 10% and 1% for volume fraction, lower same, at 37 ℃ and 5%CO 2In incubator, cultivate.Dissociated cell is used 0.25% Trypsin-EDTA (pancreatin cell dissociation buffer) solution-treated 3-4 minute, then adds the DMEM nutrient solution neutralization that contains 10%FBS, and 3000rpm is centrifugal, abandoning supernatant.Use 2% serum free culture system liquid washed cell, centrifugal rear resuspended, obtain the suspension liquid that contains cell.
Step 2, get two cell chemotaxis analysis chips, with vacuum pressure pump, (model can adopt Millipore-WP6122050, Beijing Min Taiyuan Science and Technology Ltd. of producer) after two cell chemotaxis analysis chips are bled 10 minutes, add cell suspension in two cell chemotaxis analysis chips, due to the effect of negative pressure, approximately in 1 minute, liquid can be inhaled into all chambers, because the size of fence passage is less than the size of cell, so cell is inhaled into and rests on outside the fence passage.
Step 3, remove unnecessary cell solution around these two cell chemotaxis analysis chips by liquid-transfering gun, washes remaining cell off, and add nutrient solution for these two cell chemotaxis analysis chips, and nutrient solution can adopt 2% serum free culture system liquid here.Cover the Tissue Culture Dish upper cover and prevent liquid evaporation, and carry out microscopic observation under cell culture environment.
Step 4, be placed on 37 ℃ and 5%CO by these two cell chemotaxis analysis chips 2Cultivate 2-3 hour in incubator, from fluid inlet, the liquid of one of them cell chemotaxis analysis chip is siphoned away and changes into chemokine solution after cell attachment, here chemokine solution can adopt 10% serum free culture system liquid, the concentration difference of the 2% serum free culture system liquid that utilizes 10% serum free culture system liquid and add before, set up chemotactic gradient; Again another cell chemotaxis analysis chip is added to another kind of chemokine solution from fluid inlet, for example contain 2% serum free culture system liquid of 1.5ug/mlEGF attractive substance, set up chemotactic gradient.Here the EGF attractive substance of using is the abbreviation of Epidermal Growth Factor, i.e. epithelical cell growth factor.
Step 5, be placed on 37 ℃ and 5%CO by cell 2In incubator, cultivate, and in real time Taking Pictures recording MDA-MB-231 chemotactic behavior to the EGF attractive substance to the chemotactic behavior of 10% serum free culture system liquid and MDA-MB-231, and contrasted.
Embodiment 3: analyze the response of different cells to chemokine solution of the same race.
Step 1, MCF-7 MDA-MB-231 cell and human breast carcinoma cell lines MCF-7 cell are respectively with containing 10% serum (foetal calf serum, the DMEM nutrient solution of FBS) and 1% penicillin-Streptomycin sulphate (containing the nutrient solution of each seed amino acid and glucose, Gibco), at 37 ℃ and 5%CO 2In incubator, cultivate.Dissociated cell is used 0.25% Trypsin-EDTA (pancreatin cell dissociation buffer) solution-treated 3-4 minute, then adds the DMEM nutrient solution neutralization that contains 10%FBS, and 3000rpm is centrifugal, abandoning supernatant.Use 2% serum free culture system liquid washed cell, centrifugal rear resuspended, obtain two kinds of suspension liquids that contain cell.
Step 2, get two cell chemotaxis analysis chips, and bleed after 10min with vacuum pressure pump, and two kinds of cell suspension are joined respectively in two cell chemotaxis analysis chips, and due to the effect of negative pressure, liquid can be inhaled into all chambers in 1 minute.The size of fence passage is less than the size of cell, so cell is inhaled into and rests on outside the fence passage.
Step 3, remove two cell chemotaxis analysis chips unnecessary cell solution on every side by liquid-transfering gun, and be that these two cell chemotaxis analysis chips add the nutrient solution that does not contain chemokine, 2% serum free culture system liquid for example, cover the Tissue Culture Dish upper cover and prevent liquid evaporation, and carry out microscopic observation under cell culture environment.
Step 4, be placed on 37 ℃ and 5%CO by two cell chemotaxis analysis chips 2Cultivate 2-3 hour in incubator, after cell attachment, the liquid in the feed liquor aperture is siphoned away respectively, and all change the 2% serum free culture system liquid that contains the 1.5ug/mlEGF attractive substance into.
Step 5, be placed on 37 ℃ and 5%CO by cell 2In incubator, cultivate, and in real time Taking Pictures recording MDA-MB-231 cell chemotactic behavior to 1.5ug/ml EGF attractive substance to the chemotactic behavior of 1.5ug/ml EGF attractive substance and MCF-7 cell, and contrasted.
(3) finally, provide a kind of preparation method of cell chemotaxis analysis chip;
Can adopt following method to scribe cell chemotaxis analysis chip of the present invention:
Step 1, optical mask or the plastic film of printing graphics chip as shown in Figure 1 cover on the silicon chip that scribbles photoresist material;
While making figure, can adopt the L-Edit instrument to draw, make the figure as Fig. 1, it is printed on exposure mask, exposure mask can adopt plastic film or optical mask; Because the optical mask cost is slightly high, can be printed on optical mask making the higher fence passage 3 of accuracy requirement, imbibition chamber 4, chemotactic passage 2 and feed liquor chamber 1 are printed on plastic film.
Step 2, adopt uviolizing, the transparent part of this optical mask or plastic film is accepted uviolizing generation crosslinking reaction and polymerization, and the part of non-irradiated with ultraviolet radiation can be dissolved by the developing, and the photoresist material of the remaining projection of silicon chip and surface thereof becomes the formpiston of making chip afterwards.That is to say, utilize the mask graph obtained, by multilayer contraposition cover, be engraved on silicon chip and prepare the photoresist material mould.Exposure energy is preferably 10mJ/cm 2-500mJ/cm 2, can in exposure intensity, be preferably for example 1mW/cm 2-10mW/cm 2Ultraviolet ray under irradiate for some time, for example 10-80 second, realize.
Step 3, cast in the pre-plastomer of PDMS on formpiston, is heating and curing, and then under ultraviolet lamp, irradiates and spend the night, and the purpose of uv irradiating is for sterilizing here.
For example, after prepared by the photoresist material mould of step 2, use the PDMS(A:B=10:1 of 5mm thickness) copy pattern, preferably at 20 ℃-120 ℃, be heating and curing 20 minutes to 72 hours, further preferably at 75 ℃, solidify 60 minutes, then cut the PDMS with figure, and bonding with substrate material, what substrate material was generally used is glass, so that microscopic examination for example adheres to naturally with culture dish, then be placed under ultraviolet lamp and irradiate and spend the night, the purpose of ultra violet lamp is to be the chip degerming, can obtain standby cell chemotaxis analysis chip.
Cell chemotaxis analysis chip of the present invention is the analysis of cells chemotaxis quickly and easily, imbibition chamber 4 can be prefabricated into to fixed size, can further quantitatively control like this volume of the liquid that sucks microchannel, in 4 imbibitions of imbibition chamber by cellular localization before stopping the fence passage that cell passes through, then add respectively the different small proteins with chemotactic effect at fluid inlet, liquid is without mobile the time in closed channel, protein micromolecular can complete by diffusion the foundation of concentration gradient in chemotactic passage 2, quantitative observation is to observing the chemotactic process of cell under the certain concentration gradient so very easily, and eliminating is flowed on the impact of this process.
Above-mentioned a plurality of described cell chemotaxis analysis chips are combined, can obtain the cell chemotaxis analytical equipment, for example, by 32 independently the cell chemotaxis analysis chip be divided into 4 rows, every row is parallel is arranged side by side 8, has formed a cell chemotaxis analytical equipment.Each cell chemotaxis analysis chip measure-alike, 8 cell chemotaxis analysis chips that can be so just a row with 8 volley of rifle fires add cell solution simultaneously, have improved conventional efficient; Certainly, can also in 8 chips of a row, add the signaling molecule of different concns, or different cell solutions, can facilitate like this response condition of paired observation allogenic cell to different chemokine solution, the embodiment 2 in the using method of front for example, or different cells are to identical chemokine solution response condition, the embodiment 3 in the using method of front for example, contrast be tested and be observed to above-mentioned each experiment can with a plurality of cell chemotaxis analysis chips, the error that may exist to reduce single experiment.That is to say, use this cell chemotaxis analytical equipment more to be convenient to carry out many group experiments and observe contrast simultaneously.Thereby for the research of cell chemotaxis sexual behaviour will provide a kind of more convenient instrument.
Above embodiment is only for illustrating the present invention; and be not limitation of the present invention; the those of ordinary skill in relevant technologies field; without departing from the spirit and scope of the present invention; can also make a variety of changes and modification; therefore all technical schemes that are equal to also belong to category of the present invention, and scope of patent protection of the present invention should be defined by the claims.

Claims (6)

1. a cell chemotaxis analysis chip, is characterized in that: comprise feed liquor chamber, chemotactic passage, fence passage and imbibition chamber;
Described feed liquor chamber is provided with fluid inlet, and described fluid inlet is for adding cell solution; Described feed liquor chamber is for holding the described cell solution added;
One end of described chemotactic passage is connected with described feed liquor chamber, and the other end of described chemotactic passage is connected with an end of described fence passage, and the internal diameter of described chemotactic passage is greater than celliferous diameter in described cell solution;
The internal diameter of described fence passage is less than celliferous diameter in described cell solution; The other end of described fence passage is connected with described imbibition chamber;
Described imbibition chamber is for absorbing the solution of described cell solution.
2. cell chemotaxis analysis chip according to claim 1, it is characterized in that: described chemotactic passage is many.
3. cell chemotaxis analysis chip according to claim 1 and 2, it is characterized in that: the internal diameter of described chemotactic passage is more than or equal to 10 microns and is less than or equal to 100 microns.
4. cell chemotaxis analysis chip according to claim 1, it is characterized in that: described fence passage is many.
5. according to the described cell chemotaxis analysis chip of claim 1 or 4, it is characterized in that: the internal diameter of described fence passage is more than or equal to 0.1 micron and is less than or equal to 10 microns.
6. a cell chemotaxis analytical equipment, is characterized in that: which is provided with a plurality of described cell chemotaxis analysis chips as arbitrary as claim 1 to 5.
CN2013204117000U 2013-07-11 2013-07-11 Cell chemotaxis analysis chip and device Withdrawn - After Issue CN203320000U (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361263A (en) * 2013-07-11 2013-10-23 北京大学 Cell chemotaxis analysis chip and device as well as use method and preparation method of cell chemotaxis analysis chip
CN113980794A (en) * 2021-09-22 2022-01-28 中国科学院合肥物质科学研究院 Multi-channel micro-fluidic chip suitable for cell migration analysis and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361263A (en) * 2013-07-11 2013-10-23 北京大学 Cell chemotaxis analysis chip and device as well as use method and preparation method of cell chemotaxis analysis chip
CN113980794A (en) * 2021-09-22 2022-01-28 中国科学院合肥物质科学研究院 Multi-channel micro-fluidic chip suitable for cell migration analysis and application thereof
CN113980794B (en) * 2021-09-22 2024-04-16 中国科学院合肥物质科学研究院 Multichannel microfluidic chip suitable for cell migration analysis and application thereof

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