CN104745445A - Three-dimensional micro-fluidic chip for building cellular network and preparation method of three-dimensional micro-fluidic chip - Google Patents
Three-dimensional micro-fluidic chip for building cellular network and preparation method of three-dimensional micro-fluidic chip Download PDFInfo
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- CN104745445A CN104745445A CN201310746728.4A CN201310746728A CN104745445A CN 104745445 A CN104745445 A CN 104745445A CN 201310746728 A CN201310746728 A CN 201310746728A CN 104745445 A CN104745445 A CN 104745445A
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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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- 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
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
Abstract
The invention relates to the technical field of micro-fluidic chips, in particular to a three-dimensional micro-fluidic chip for building a cellular network and a preparation method of the three-dimensional micro-fluidic chip. The micro-fluidic chip comprises a first film layer and a second structural layer; the first film layer is provided with a cellular network culturing pond and longitudinal through pores, and the cellular network culturing pond is communicated with the pores. The second structural layer is provided with a runner structure; and a plurality of branch pipes for communicating the outside and the runner structure, outlet holes and inlet holes by which a cell culture medium circulates are also formed in the runner structure. The second structural layer covers the surface of the first film layer, and the branch pipes correspond to the pores. The invention further provides the preparation method of the three-dimensional micro-fluidic chip. The problem that cells which are not sensitive to PLL coating difficultly form a cellular network is solved, and a favorable application platform is provided for scientific research on the cellular network.
Description
Technical field
The present invention relates to micro fluidic chip technical field, especially a kind of micro-fluidic chip being applied to life science and preparation method thereof.
Background technology
Inside current life science, the conclusion that most scientific researches is all experiment based on cell and draws.When studying the interaction between cell and cell, if can build a complete cellular network structure, that is very useful for such research.Building a complete cellular network structure, if use general traditional method, will be a very difficult task.If application microfluidic chip technology, its operating process will simplify greatly.
Microfluidic chip technology is the new technology grown up the nineties in 20th century.Microflow control technique has far-reaching influence power at present, 2004, micro-fluidic chip is classified as wherein one of the technology in " the change world " by U.S. Business2.0 journal surface article, and within 2006, Nature magazine releases special edition with regard to this may become " this centurial technology ".Through the development of more than ten years, micro-fluidic chip is widely applied at biochemical analysis field.At present, have many research reports and utilize the network structure bag of micro-fluidic chip to be formed the neuronal cell network of 8*8 by poly-l-lysine (PLL), this is mainly realized by regional compare sensitivity PLL bag based on neuronal cell.But have a lot of synapse cell in the later stage of neuronal cell network growth and grow into grid structure PLL bag by outside region, therefore such bag, by the cell of mode for other kind a lot, is not especially had effect to PLL bag by insensitive cell.
Summary of the invention
For solving the problem, the invention provides a kind of three-dimensional microflow control chip building cellular network and preparation method thereof, the requirement to specific cells kind can be avoided, make cell can form complete, stable cellular network structure in this micro-fluidic chip.
The preparation method of this micro-fluidic chip, it comprises the steps:
Step one: the network graphic providing to be provided with to be interconnected and the first template of column, described first template is formed thickness and is not less than described network graphic height and the first film layer being less than described stem height, described network graphic and pillar construction form cellular network cultivation pool and longitudinally through duct for being transferred on described the first film layer;
Step 2: form the second structural sheet being provided with flow passage structure in the second template, described flow passage structure is arranged with several and is communicated with arm that is extraneous and described flow passage structure; Wherein, described arm is corresponding with described column; The height of described arm is greater than described stem height;
Step 3: described second structural sheet is covered on described the first film layer surface, makes described arm corresponding with described column, then the first film layer, the second structural sheet described in thermofixation process;
Step 4: the described the first film layer after step 3 process, the second structural sheet are peeled off from described first template, and on described flow passage structure, making is used for outlet opening, the ingate of cell culture medium circulation, obtains described micro-fluidic chip.
Further, described the first film layer, the second structural sheet are cast in respectively in the first template, the second template by the mixed solution of prepolymer and cure-crosslinking agent, formed after thermofixation process; Described prepolymer and cure-crosslinking agent mass ratio are 10 ~ 20:1.
Further, control that described network graphic height is 5 ~ 10 microns, described stem height is 40 ~ 50 microns.
Further, described the first film layer thickness is 10 ~ 20 microns.
Further, the height of described arm is 50 ~ 70 microns.
Further, described thermofixation is treated to and toasts 30 ~ 120 minutes at 80 ~ 100 DEG C.
The present invention also provides the microfluidic chip structure adopting aforesaid method to obtain, and it comprises:
The first film layer, it is provided with cellular network cultivation pool and longitudinally through duct, described cellular network cultivation pool and described hole link; And
Be provided with the second structural sheet of flow passage structure; Described flow passage structure is arranged with several and is communicated with arm that is extraneous and described flow passage structure, and for the outlet opening of cell culture medium circulation, ingate;
Described second structural sheet is covered on described the first film layer surface; Described arm is corresponding with described duct.
Further, described the first film layer, the second structural sheet are poured into a mould by the mixed solution of prepolymer and cure-crosslinking agent, formed after thermofixation process; Described prepolymer and cure-crosslinking agent mass ratio are 10 ~ 20:1.
Further, described the first film layer thickness is 10 ~ 20 microns.
Further, the height controlling described cellular network cultivation pool is 5 ~ 10 microns.
Further, the height of described arm is 50 ~ 70 microns.
Beneficial effect:
Making method of the present invention is simple, repeated and workable.By building the micro-fluidic chip of a three-dimensional through hole structure, on the one hand for Growth of Cells provides the environment of a relative closure, thus can source of pollution be effectively avoided to invade; On the other hand, utilize cell in the medium from drop characteristic, make it grow at specific micro-fluidic chip cellular network structural region; Not only can provide a complicated and diversified network structure for eurypalynous cell, also solve the problem being difficult to be formed cellular network to PLL bag by insensitive cell, various types of cell all can be grown in the network structure preset, and the region do not had outside Growth of Cells to network structure, the scientific research for cellular network provides a favourable application platform.
Accompanying drawing explanation
Fig. 1 is microchannel chip structural representation of the present invention.
The Making programme figure that Fig. 2 (a) (b) (c) (d) is microchannel chip of the present invention.
Fig. 3 is the second structural sheet sectional view of Fig. 2 (c) of the present invention along AA ' line.
Embodiment
Below, by reference to the accompanying drawings the embodiment of the present invention is described in detail.
The invention provides a kind of three-dimensional microflow control chip building cellular network, as shown in Figure 1, this micro-fluidic chip is carried on a slide glass 40, and it comprises the first film layer 20 and second structural sheet 30 bilayer structure of bonding mutually; Wherein:
The first film layer 20 is provided with cellular network cultivation pool 21 and longitudinally through duct 22, and described cellular network cultivation pool 21 is communicated with described duct 22.
Second structural sheet 30 is provided with flow passage structure 31; Described flow passage structure 31 is arranged with several and is communicated with arm 32 that is extraneous and described flow passage structure, and for the outlet opening 34 of cell and substratum circulation thereof, ingate 33.Described second structural sheet 30 is covered on described the first film layer 20 surface; Described arm 32 is corresponding with described duct 22.
Introduce the preparation method of this micro-fluidic chip below, comprise the steps:
Cure-crosslinking agent (SYLGARD, 184, Silicone Elastomer Curing Agent is provided; Lot number 0007450507) for subsequent use; And prepolymer (SYLGARD, 184, Silicone Elastomer Base; Lot number 0007450507) for subsequent use.
Get prepolymer and cure-crosslinking agent is stirred into mixed solution, discharge bubble.The mass ratio preparing prepolymer and cure-crosslinking agent is respectively two portions of mixed solutions of 10:1,20:1, for subsequent use.The present embodiment utilizes the product of prepolymer and cure-crosslinking agent polymerization reaction take place (main component is polydimethylsiloxane (PDMS)) as the material of micro-fluidic chip.
There is provided photoresist material SU8-2010, SU82050 for subsequent use.
Step one: etch the network graphic 11 and column 12 structure that are interconnected at photoresist material SU8-2010, then carries out silanization treatment and forms the first template 10 in 2 minutes, as shown in Figure 2 (a) shows.Wherein, the network graphic 11 of described first template 10 is highly 5 ~ 10 microns, and the height of described column 12 is 40 ~ 50 microns.
Then described first template 10 is placed on spin coating instrument (not shown) center, the mixed solution (mass ratio is 20:1) getting prepolymer described in 1mL and cure-crosslinking agent is cast in described first template 10.The mixed solution being positioned at column 12 top blows off by recycling nitrogen, and after horizontal positioned for some time, liquid to be mixed covers flat again, is placed in 80 DEG C, baking oven baking 20 minutes, forms transparent the first film layer 20, as Fig. 2 (b).The thickness of the first film layer 20 is not less than described network graphic 11 height and is less than described column 12 height, ensures that the network graphic 11 in the first template 10 can be transferred on described the first film layer 20 completely.Therefore, the first film layer 20 thickness is preferably 10 ~ 20 microns.Described network graphic 11 and column 12 are formed after being transferred to described the first film layer 20 shown in cellular network cultivation pool 21 and longitudinally through duct 22(composition graphs 1 respectively).
Step 2: the flow passage structure etching convex at photoresist material SU8-2050, then carries out silanization treatment and forms the second template (not shown) in 2 minutes.Then the mixed solution (mass ratio is 10:1) of described prepolymer and cure-crosslinking agent is cast in described second template, bubble is got rid of 10 minutes under vacuum environment, finally to move in baking oven 80 DEG C of bakings 30 minutes, obtain the second transparent structural sheet 30, as Fig. 2 (c).
The second structural sheet 30 formed thus is provided with flow passage structure 31, and particularly, this flow passage structure 31 can be such as some the fluid channel be arranged side by side (see Fig. 3).Flow passage structure 31 arranges several and be communicated with arm 32 that is extraneous and flow passage structure 31, the cell culture medium carried from flow passage structure 31 just can flow into duct 22, then arrive cellular network cultivation pool 21 from each arm 32.For achieving the above object, arm 32 offer the duct 22 being equivalent to the first film layer 20 with described column 12() position is corresponding, and in order to ensure the planarization of the first film layer 20, second structural sheet 30, the height of arm 32 is greater than described column 12 height, and arm 32 is highly preferably 50 ~ 70 microns.For the relative width of arm 32 with duct 22, be not strict with, within general 10 microns.But in order to allow cell and substratum thereof flow into duct 22 smoothly, preferred arm 32 wants wider compared with duct 22.
Step 3: described second structural sheet 30 is peeled off from the second template, second structural sheet 30 is covered on described the first film layer 20 surface under Stereo microscope is auxiliary, as shown in Figure 2 (d) shows, make described arm 32 corresponding with the column 12 of the first template 10, the part column 12 protruding from the first film layer 20 surface just stretches in arm 32.Then described the first film layer 20, second structural sheet 30 to be sent in the lump in baking oven 80 DEG C of bakings 120 minutes, described the first film layer 20, second structural sheet 30 is bonded together.
Step 4: described the first film layer 20, second structural sheet 30 after step 3 thermofixation process is peeled off from described first template 10, and making is used for outlet opening 34, the ingate 34 of cell culture medium circulation on the flow passage structure 31 of the second structural sheet 30, now obtain micro-fluidic chip (shown in composition graphs 1).
Step 5: in order to ensure the steadiness of microfluidic chip structure further, is placed in described micro-fluidic chip on a clean slide 40 and carries out plasma treatment (800W, 10s), then sends into 80 ~ 100 DEG C of bakings in baking oven and spend the night.
For attached cell, its growth pattern can be limited to the two dimensional structure of two dimension.According to the growth characteristics of this cell, the bilayer structure utilizing above-mentioned micro-fluidic chip to design can obtain perfect cellular network.Wherein, be positioned at second structural sheet on upper strata, completed the conveying of cell and substratum thereof by its flow passage structure, be positioned at lower floor's the first film layer and then received the cell and substratum thereof that flow down from the arm of flow passage structure by longitudinally through duct.Meanwhile, because the density ratio substratum of cell is large, can sinks to bottom duct gradually in the process of inoculation, the three-dimensional through hole structure of a through levels can be utilized like this to realize the inoculation position of fixed cell.After inoculating cell, the cell in flow passage structure can wash away with substratum conveying, and the cell sunk in duct can continue to flow in cellular network cultivation pool along predetermined network structure to grow, and finally forms specific cellular network.
Above-mentioned micro-fluidic chip embodies excellent repeatability and ease for operation in actual applications.The invention provides the cellular network growth experiment of different types of cell at this micro-fluidic chip.The cell type tested comprise to PLL bag by the Primary rat hippocampus neuronal cell of sensitivity (numbering: No. 1) and to PLL bag by insensitive PC-12 neural-like cells (numbering: No. 2).No. 1 cell and No. 2 cells effectively can be formed predetermined cellular network structure by the results show micro-fluidic chip of the present invention.And in the later stage of continuing to cultivate, the network structure of two kinds of cells is more obvious and ripe, but can not grow to outside cellular network cultivation pool.
Micro-fluidic chip of the present invention, on the one hand for Growth of Cells provides the environment of a relative closure, thus can effectively avoid source of pollution to invade; On the other hand, a complicated and diversified network structure can be provided for eurypalynous cell, solve the problem being difficult to be formed cellular network to PLL bag by insensitive cell, various types of cell all can be grown in the network structure preset, and the region do not had outside Growth of Cells to network structure, the scientific research for cellular network provides a favourable application platform.
Claims (11)
1. build a preparation method for the three-dimensional microflow control chip of cellular network, it is characterized in that, comprise the steps:
Step one: the network graphic providing to be provided with to be interconnected and the first template of column, described first template is formed thickness and is not less than described network graphic height and the first film layer being less than described stem height, described network graphic and pillar construction form cellular network cultivation pool and longitudinally through duct for being transferred on described the first film layer;
Step 2: form the second structural sheet being provided with flow passage structure in the second template, described flow passage structure is arranged with several and is communicated with arm that is extraneous and described flow passage structure; Wherein, described arm is corresponding with described column; The height of described arm is greater than described stem height;
Step 3: described second structural sheet is covered on described the first film layer surface, makes described arm corresponding with described column, then the first film layer, the second structural sheet described in thermofixation process;
Step 4: the described the first film layer after step 3 process, the second structural sheet are peeled off from described first template, and on described flow passage structure, making is used for outlet opening, the ingate of cell culture medium circulation, obtains described micro-fluidic chip.
2. preparation method according to claim 1, is characterized in that, described the first film layer, the second structural sheet are cast in respectively in the first template, the second template by the mixed solution of prepolymer and cure-crosslinking agent, formed after thermofixation process; Described prepolymer and cure-crosslinking agent mass ratio are 10 ~ 20:1.
3. preparation method according to claim 1, is characterized in that, controls that described network graphic height is 5 ~ 10 microns, described stem height is 40 ~ 50 microns.
4. the preparation method according to claim 1 or 3, is characterized in that, described the first film layer thickness is 10 ~ 20 microns.
5. the preparation method according to claim 1 or 3, is characterized in that, the height of described arm is 50 ~ 70 microns.
6. preparation method according to claim 1, is characterized in that, described thermofixation is treated to toasts 30 ~ 120 minutes at 80 ~ 100 DEG C.
7. build a three-dimensional microflow control chip for cellular network, it is characterized in that, comprising:
The first film layer, it is provided with cellular network cultivation pool and longitudinally through duct, described cellular network cultivation pool and described hole link; And
Be provided with the second structural sheet of flow passage structure; Described flow passage structure is arranged with several and is communicated with arm that is extraneous and described flow passage structure, and for the outlet opening of cell culture medium circulation, ingate;
Described second structural sheet is covered on described the first film layer surface; Described arm is corresponding with described duct.
8. micro-fluidic chip according to claim 7, it is characterized in that, described the first film layer, the second structural sheet are poured into a mould by the mixed solution of prepolymer and cure-crosslinking agent, formed after thermofixation process; Described prepolymer and cure-crosslinking agent mass ratio are 10 ~ 20:1.
9. micro-fluidic chip according to claim 7, it is characterized in that, described the first film layer thickness is 10 ~ 20 microns.
10. micro-fluidic chip according to claim 7 or 9, is characterized in that, the height controlling described cellular network cultivation pool is 5 ~ 10 microns.
11. micro-fluidic chips according to claim 7, it is characterized in that, the height of described arm is 50 ~ 70 microns.
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| CN108473929A (en) * | 2016-01-20 | 2018-08-31 | 综研化学株式会社 | Cell culture substrate and its manufacturing method |
| CN112452367A (en) * | 2020-12-10 | 2021-03-09 | 深圳先进技术研究院 | Double-layer micropore chip, preparation method of double-layer micropore chip and biological device |
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