CN107362844B - The micro-fluidic chip and its preparation method and application of 3D solid tumor model based on administration nano-drug administration system screening - Google Patents
The micro-fluidic chip and its preparation method and application of 3D solid tumor model based on administration nano-drug administration system screening Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- 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
- C12M23/12—Well or multiwell plates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses a kind of micro-fluidic chip of 3D solid tumor model based on administration nano-drug administration system screening and its applications.The chip is made of the substrate and cover plate being bonded together.The structure of cover plate is three parallel microchannels interconnected, the microchannel (1) formed including drug infusion and unilayered vessels, the microchannel (4) that there are many interface channel (2) and three-dimensional nodule the multicellular spheriods formation of extracellular matrix U-typed slot structure (3)；Substrate has the parallel channels (5) for being conducive to cell capture.The present invention can carry out the real-time monitoring of the targeting conveying of administration nano-drug administration system and its on-line evaluation of drug toxicity simultaneously, substantially increase the efficiency of drug evaluation.In addition, the present invention has better tumour biomimicking potential, accurate data are provided for evaluation before clinical drug.And micro-fluidic chip platform has reagent dosage small, low in cost, can analyze the advantages such as multiple samples simultaneously, has preferable drug screening application prospect.
Description
Technical field
The present invention relates to micro fluidic chip technical fields, more particularly to a kind of three-dimensional for administration nano-drug administration system screening
The micro-fluidic chip of solid tumor microenvironment.
Background technique
Malignant tumour is to seriously threaten the major disease of human life and health.Shown according to nearest data: the whole world is annual
There are 10,000,000 new cases, and dies of malignant tumour more than six million peoples.2015, China's cancer patient's number nearly 4,300,000
People occupies first of the whole world, and the death rate accounts for the 22.3% of the whole world, and is in sustainable growth trend [1].The appearance of administration nano-drug administration system is to evil
The treatment of property tumour provides new Gospel.Administration nano-drug administration system can effectively solve in itself water-soluble in transportational process in vivo
Difference, it is degradable the problems such as, the drug cycles time can be effectively improved, reduce toxic side effect, extend drug half-life period in vivo, mention
High curative effect provides unique advantage [2-5] in treatment human cancer.However, only a small number of administration nano-drug administration systems are applied to
Clinic, a large amount of systems only reside within experimental study stage [6].One of the main reasons therein is existing drug screening body
Be is not perfect, and the result difference for causing many administration nano-drug administration systems preclinical and clinical is very big, cannot finally put into final
In medicinal industry and antineoplaston [7].
In tumour medicine exploitation, for the drug effect and its toxicity for examining drug, the cell of two-dimensional surface is generally carried out first
The mode [8] of culture.However traditional cell experiment result can not embody it in the intracorporal all characteristics of people, because traditional
Cell local environment is more single in cell experiment, can not simulate complicated tumor microenvironment completely: between different cells
Metabolite exchange provides chemistry and body fluid and cell in the three-dimensional cell epimatrix structure of mechanical property, specific organization/organ
The conditions such as ratio and physiological fluid shearing force are all difficult to meet [9].
Under the premise of cell experiment proves that drug is effective, the research of next step will do it --- zoopery, animal are real
It tests for further verifying curative effect of medication, still, compared with drug effect in human body, the accuracy of results of animal is not high
[10].Main cause is as follows: (1) human body metabolism and animal body intracellular metabolite are not consistent；(2) tumour of zoopery is implantation
Rather than it is spontaneous；(3) usually zoopery uses nude mice, immunodeficiency；(4) zoopery is difficult to pair
The targeting transport of administration nano-drug administration system carries out real-time monitoring [11].Even if cell experiment and zoopery effect are very good, also not
It can ensure the success of clinical trial, the success rate of anti-tumor drug only has 5% [12] according to statistics.In addition, zoopery is often drawn
Hair moral controversy, especially results of animal can not predict drug under the premise of people's intracorporal pharmacodynamic characteristics well.It is good
In vitro models and animal metabolism model can preferably improve the efficiency of drug screening, save new drug development cost and
Time [13,14].Therefore, exploitation overcomes the shortcomings of the above conventional screening assays closer to the external model of internal microenvironment,
It is one of key scientific problems urgently to be resolved.
The microfluidic chip technology developed in recent years can efficiently solve problem above.Early 1990s micro-total analysis
The it is proposed of system (Miniaturized Total Analysis Systems, μ-TAS) concept is in analysis instrument and life science
Field produces significant impact, and guidance chemical analysis apparatus is towards micromation, automation, rapid, integrated with portability
Trend development [15,16].Based on micro electro mechanical processing technology (MicroElectromechanical Systems, MEMS),
The functional units such as Micropump, micro-valve, microchannel, microelectrode, microreactor, microflow sensor, micro- detector are made on chip
Very small chemical system is constituted, completeization such as the functions in entire laboratory, including sampling, dilution, reagent adding, reaction, separation, detection
It learns process integration to realize on the chip of heart size, meaning " laboratory on chip " (Lab on a Chip) can be referred to as
[17,18].A variety of monotechnicses during biology, chemistry, medical analysis can be flexibly integrated in one by micro-fluidic chip
On controllable small platform, have that integrated level is high, detection time is short, reagent consumption is few compared to traditional experiment, and can with compared with
More detection devices such as are used in combination at the advantages, occupy forward position leadership [19-21] in the fields such as analytical chemistry and life science.
Its related industry sharp increase has become undisputable fact.Such as it is engaged in the Illumina of gene sequencing sample preparation, it is engaged in collection
At flow path produce Fluidigm,.The Oxford of liver chip applied to toxicity trial, production figures PCR instrument
The companies such as the Bio-rad of Rain Dance and old brand, wherein some listings.U.S.'s Business magazine in 2004 is by lab-
On-a-chip is classified as " changing following one of the seven kinds of technologies ".BCC research report 2011 and microflow control technique market in 2012
Value is 5,100,000,000 dollars and 5,600,000,000 dollars respectively, from the point of view of the development of nearest 5 years 13% average growth rates, to its market in 2017
Value is estimated will be up to 10,300,000,000 dollars.
With the development of microfluidic chip technology, there is uniqueness relative to other biological chip in the development of cell field
Advantage: (1) chip micro-structure is suitable with human inner cell life organization structure size, and three-dimensional training altogether can be carried out with various kinds of cell
It supports, simulates the microenvironments such as intracorporal extracellular matrix, thus more can be close to intracorporal truth；(2) microenvironment of chip has
Accumulation conducive to growth factor etc. to the advantageous important factor of cell culture drains cell by the control of fluid, realization
Nutriment is replenished in time in waste scavenging, forms the microenvironment of more advantageous cell growth；(3) convenient for the transportational process to drug
Carry out effective real-time monitoring；(4) manipulation easy to accomplish to cell is easy to be combined the subsequent research of progress with detecting instrument
Deng these are all [22,23] that conventional method cannot achieve.Such as Prabhakarpandian designed on micro-fluidic chip and
The microchannel with internal similar blood vessel network is constructed, and forms three-dimensional tumour many cells using intermediate micro array structure
Microballoon, to realize in vitro to the real-time monitoring [24] of targeting drug delivery system traffic condition.Ingber seminar, Harvard University
It was constructed on micro-fluidic chip using pneumonocyte, permeable membrane and capillary cells such as rubber sassafras size for the first time in 2010
" lung chip ", the respiratory movement [25] of human lung can be simulated under the action of extraneous mechanical pump and regulating valve.It should " lung core
Piece " is capable of many functions of accurate simulation lungs, sucks the reaction etc. made after nanoparticle including lung, is expected to use
It is influenced in new drug evaluating drug effect and human lung's toxin.
In addition, the detection technique of micro-fluidic chip, is the key factor that can micro-fluidic chip play its important function.Mesh
Before, the Pharmaceutical Analysis on micro-fluidic chip based on cell detects the method for mostly using fluorescent marker, is filled by fluorescent microscopic imaging
Set acquisition data, it is difficult to its accurate quantitative analysis, and be unfavorable for the integrated and high-throughput drug screening [26-28] of system.It is more
Function microplate reader not only has higher analysis speed and sensitivity as one of detection means most important in drug screening,
And stability is good, and it is cheap, it is small to the stimulation of cell, it is living that micro-fluidic chip is carried out to online cell in conjunction with microplate reader
Journal of Sex Research, it will greatly accelerate the process of medicament research and development.In recent years, which also has on a small quantity for the research of drug screening
It reports [29-31].It is a large amount of studies have shown that exploitation is highly integrated, micro-fluidic chip external model of micromation, how by miniflow
Chip is controlled with microplate reader in conjunction with, carries out comprehensive administration nano-drug administration system research, development medicament transport, distribution and toxicity detection etc. into
Row evaluating drug effect will play very important facilitation to innovation drug research.
In conclusion the drug effect that the microfluidic chip technology with international forward position leading technology is introduced into nanometer administration is commented
In valence research, it can overcome in routine evaluations analysis method that model is simple, drug screening integration is poor, analysis result is accurately and reliable
The problems such as property is poor.Now it is micro- to cross the reasonable micro-fluidic chip of design for the quasi- novel administration nano-drug administration system with tumor-targeting of exploitation
Channel design is constructed containing Multicellular Tumor Spheroid, extracellular and peripheral vessels tumor microenvironment external model, is given to nanometer
The targeting conveying of medicine system and realizing controlled-release can be carried out research, while establish micro-fluidic chip and microplate reader combination platform, to nanometer
Drug delivery system carries out on-line evaluation, and utilizes conventional molecular biological means, explores its potential drug effect machine project
Research achievement provide novel technology platform for the in-vitro evaluation of administration nano-drug administration system, to drug early stage research and development and it is preclinical
Evaluation study has highly important academic significance and application value.
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Summary of the invention
The purpose of the present invention is to provide a kind of micro-fluidic chips based on 3D solid tumor model.
Another object of the present invention is to provide the researchs that said chip is screened in administration nano-drug administration system.
A kind of drug delivery system screening micro-fluidic chip of 3D solid tumor model, the chip is by the substrate that is bonded together
It is formed with cover plate；Three parallel microchannels being mutually communicated are provided on cover plate, respectively drug infusion and unilayered vessels is formed
Administration microchannel (1), the connection microchannel (2) comprising extracellular matrix layer, and with multiple U-typed slot structures (3) three
It ties up tumour many cells ball microchannel (4)；It is recessed that position on substrate corresponding to three-dimensional nodule many cells ball microchannel is provided with bar shaped
The parallel channels (5) of slot.
Extracellular matrix analogies, preferably basement membrane extract (BME) are provided in the connection microchannel.It is being administered
Umbilical vein vascular endothelial cells layer is provided between microchannel and connection microchannel.
Hydrophilic coating is set on the inner wall of administration microchannel and connection microchannel, preferably modification polyvinyl alcohol (PVA)
Or plunker PF-127 coating.
Each cell culture well aperture in the size and tissue culture plate of the micro-fluidic chip matches, and U-type groove
Position can be entirely accommodated in a cell culture well.
The interior diameter of the U-typed slot of the micro-fluidic chip is 150-250 μm, and preferably 200 μm, depth is 250-350 μ
M, preferably 300 μm, the width at U-typed channel opening are 100-200 μm, preferably 150 μm.Facilitate the capture of cell,
The Multicellular Tumor Spheroid of formation is 150-200 μm, meets the requirement of drug screening research cell spheroid size.
The position of the U-typed slot of the micro-fluidic chip is transpostion interval, and the depth that has on substrate is 10 μm parallel
Channel covers on this channel, allows cell to enter in slot to a greater degree on hydrodynamics, increases the efficiency of cell capture.
The depth of the connection microchannel of the micro-fluidic chip is more shallow than other microchannels, and connects the two sides setting of microchannel
There is the connector for penetrating through administration microchannel and three-dimensional nodule many cells ball microchannel, the width for connecting microchannel is connection
2-3 times of mouth width degree.Retain extracellular matrix largely in this channel.
The micro-fluidic chip needs to carry out hydrophily processing before cell is perfused, and such as modifies poly- 6,12,24,48 enol of second
(PVA) or plunker PF-127.
The substrate and cover plate of the micro-fluidic chip are made with biocompatible materials, preferably dimethyl silicone polymer
(PDMS) it is made.Its bio-compatibility is strong, and translucency is good, is suitable for cell culture and fluorescence monitoring.
A kind of tissue culture plate used that matches with aforementioned micro-fluidic chip, is provided with receiving on the tissue culture plate
The card slot of micro-fluidic chip, the position of card slot can make one of them in U-type groove and tissue culture plate on micro-fluidic chip
The position of cell culture well is corresponding.
The tissue culture plate is 6 orifice plates, 12 orifice plates, 24 orifice plates, 48 orifice plates or 96 orifice plates.
Application of the chip of the present invention in terms of administration nano-drug administration system screening, operating procedure are as follows: due to surface tension and
Basement membrane extract (BME) is perfused with slower flow velocity in connection microchannel (2) first, then by it in the hydrophobicity of material
Remaining (1), (4), (5) microchannel carries out hydrophily processing, by 1 × 106The tumour cell of a/mL flows into microchannel (4) and (5),
Make cell capture in U-typed slot, culture is for 24 hours.Channel (1) is passed through 1 × 105The huve cell of a/mL concentration, will
The Chip Vertical is placed, and makes endothelial cell with the cells of vascular wall layer of BME bracket formation single layer, by the chip water after 6h
Placing flat, and by extra cell clearance, the building of 3D solid tumor model is completed at this time.Administration nano-drug administration system is passed through (1),
Its traffic condition in solid tumor models is observed in real time using the photoluminescent property of nano-carrier.It then will be by machinery
96 orifice plates of processing load the chip, and the Activity determination for carrying out tumour cell can be directly combined with microplate reader.Finally, will be described
Cell in chip is collected, and the research of subsequent drug mechanism is carried out.
The production method of chip of the present invention:
1) chip designs: the chip chooses dimethyl silicone polymer as processing and fabricating material, using answering in recent years
The processing and fabricating of micro-fluidic chip is carried out with very universal soft lithography, can greatly simplify production process and reduces cost.
The micro-fluidic chip is mainly made of three parallel channels, is connected between parallel channels by interface channel.Wherein, three-dimensional nodule is more
Cell ball microchannel is made of upper and lower structures, and superstructure is made of many U-typed slots, and underlying basal is by shallower straight channel
It constitutes, facilitates the capture of tumour cell and the formation of three-dimensional nodule multicellular spheriods.
2) using PDMS as the chip manufacture method of material:
A. exposure mask is made in the micro-fluidic chip figure of design, graph area is clear area, and light-transmissive, non-graphic area is black
Color area, extinction and be unable to transmitted light.
B. a thin layer negativity SU-8 photoresist is spun coating on clean silicon wafer, using the SU-8 of different type, viscosity, glue-line
Thickness is can be controlled between 1-300 μm.Exposure mask is covered on silicon wafer, ultraviolet light exposes photoresist by photomask, does not expose
Light region developing solution dissolution.Remaining protrusion SU-8 structure is used as the mould formpiston of production PDMS substrate on silicon wafer and surface.
The production of c.PDMS substrate: PDMS monomer (dimethyl siloxane) and initiator is mixed with the weight ratio of 10:1
It closes, pours and cast from substrate force plate after degassing, polymerize 1.5 hours at 80 DEG C, be cooled to room temperature, lift-off stencil, obtain colorless and transparent
PDMS chip.
D. PDMS cover plate and substrate are cut into identical size.It is punched at the feeder connection position of cover plate, as core
Piece liquid storage tank.
E. glass substrate is put into togerther in oxygen plasma vacuum tube, is vacuumized 90 seconds, high frequency electric source is opened, after 90 seconds
Substrate and cover plate are taken out, is bonded immediately.
Beneficial effect
Cell culture method adopted by the present invention is compared with conventional cell culture processes (culture bottle, microwell plate etc.), energy
Enough modulating actions preferably controlled and reflect that between cell and extracellular matrix and cell is mutual, are a kind of closer bodies
Interior cultural method.
In addition, the present invention is applied to drug screening research can supervise drug in the dynamic of tumor microenvironment in real time
It surveys, this is that other training modes are difficult to realize.
To there is the present invention microfluidic chip technology of international forward position status to be introduced into the external of targeted nanometer drug delivery system
It in evaluation, and is combined with highly sensitive microplate reader, realizes accurate, quick, the high-throughput detection of drug toxicity.
The present invention can be simultaneously to the permeability of drug, and drug toxicity and mechanism of drug action are detected, greatly improved
The efficiency of drug screening.
It does not need in entire drug screening process using expensive instrument, and the consumption of reagent is very small, chip
Manufacturing cost is low, is easy to produce in batches, realizes micromation, the integrated and simplification of drug screening detection.The invention is medicine
Effect learns research and provides new technology platform, facilitates the early stage research and development etc. of the pre-clinical assessment and drug of drug.
Detailed description of the invention
Fig. 1 is the three-dimensional structure diagram a) and sectional view b) of the micro-fluidic chip of embodiment 1；1 is administration microchannel, and 2 be company
Microchannel is connect, 3 be U-shaped groove, and 4 be three-dimensional nodule multicellular spheriods microchannel, and 5 be parallel channels.
Fig. 2 is the cover plate interface channel mask dimensions design drawing that embodiment 2 makes.
Fig. 3 is the mask dimensions design drawing in the cover plate left and right side channel that embodiment 2 makes.
Fig. 4 is the mask dimensions design drawing for the substrate passageway that embodiment 2 makes.
Fig. 5 can be with the 96 orifice plate pictorial diagrams through over mechanical processing associated with microplate reader for the production of embodiment 4.
Fig. 6 is schematic perspective view of the invention.Wherein, 1-4 is the device being arranged on cover plate, and 5 be setting substrate
On device, 1 for administration microchannel, 2 for connection microchannel, 3 be U-shaped groove, 4 be three-dimensional nodule multicellular spheriods microchannel, 5
For parallel channels.
Specific embodiment
The structure of the chip of the present invention of embodiment 1
Chip of the present invention is made of the PDMS substrate and cover plate being bonded together.Its structure as shown in Figure 1, cover plate structure
For three parallel channels, it is connected between parallel channels by interface channel.Wherein, it is formed including drug infusion and unilayered vessels micro-
Channel (1), what the interface channel (2) and three-dimensional nodule multicellular spheriods of extracellular matrix were formed has 14 U-typed slot structures
Microchannel (3)；Substrate has the shallower parallel channels for being conducive to cell capture.The micro-assembly robot and internal reality that the present invention is formed
Body tumor microenvironment is quite similar, can observe drug in real time in the infiltration situation of micro-assembly robot, U-typed structure is every with 96 orifice plates
A hole is corresponding, and it is accurate to be conducive to, quick drug toxicity research.Cell by drug toxicity research can be from microchannel
Outflow carries out subsequent drug action mechanism research.
Embodiment 2 is using PDMS as the chip manufacturing of the present invention of substrate and cover plate
(1) silicon wafer is put into dense H2SO4/H2O2(3/1) solution boils slightly boiled 30 minutes, takes out use deionization after cooling
Water is rinsed to neutrality, and EtOH Sonicate heats 30 minutes on hot plate after five minutes.
(2) a thin layer SU-8 photoresist is spun coating on silicon wafer, makes bondline thickness be about 50 μm by controlling whirl coating speed.
Exposure mask with Fig. 2 structure is bonded with silicon wafer, ultraviolet light exposes photoresist by photomask.Carry out one again on the silicon wafer
Secondary whirl coating thickness is about 50 μm, and the exposure mask with Fig. 3 structure is bonded with silicon wafer, carries out re-expose using ultraviolet light, not
The part developing solution dissolution of exposure.Formpiston of the raised SU-8 structure as PDMS substrate on silicon chip surface.Then by silicon sun
Mould carries out silanization 1 hour.For the chip manufacturing of substrate also with SU-8 photoresist, bondline thickness is about 10 μm.There to be Fig. 4
The exposure mask of structure is bonded with silicon wafer, is developed after carrying out uv-exposure, silanization.
(3) PDMS performed polymer (monomer/curing agent=10/1 mixing) is cast on silicon formpiston simultaneously vacuum outgas, is then placed in
Solidify in 70 DEG C of baking ovens 2 hours or so, PDMS is removed from formpiston then, forms the substrate and cover plate of PDMS.It will be on PDMS
Structural unit cutting, cover plate punched.
(4) PDMS substrate is put into oxygen plasma vacuum tube together with calcium tablet cover plate, is vacuumized 90 seconds, open high frequency
Power supply takes out substrate and cover plate, is bonded immediately after 90 seconds.
Embodiment 3 forms 3D solid tumor micro-assembly robot using chip made from embodiment 2
(1) such as Fig. 1, channel (2) perfusion basement membrane extract (BME) gel of the resulting chip of the present invention is simulated into cell
Epimatrix then modifies channel (1) and (3) with 1% (w/v) polyvinyl alcohol (PVA), prevents cell from adhering on PDMS.
It (2) is 1 × 10 by concentration5A/mL tumour cell is fed into channel (3), keeps cell uniform, effectively captures
In U-typed slot, uniform tumour many cells microballoon is formed after cultivating 48h.
It (3) is 5 × 10 by concentration4A/mL human umbilical vein endothelial cell (HUVECs) flow channel (1), vertically puts
Chip is set, cell is affixed on BME glue and grows, place chip level after 2h, removes cell extra in channel.Due to BME
With cell adherent adhesion factor and growth factor is conducive to, cell is easy the adherency on BME and forms vascular cell layer.
Embodiment 4 carries out drug toxicity research using the three-dimensional breast cancer micro-assembly robot that embodiment 3 is formed
(1) drug of various concentration is passed through channel (1), passes it through vascular wall and ECM is reached locating for tumour cell
Position.Drug-treated for a period of time after, be added 10 μ L CCK-8 solution be incubated for 2h.
(2) as shown in figure 5, being machined to 96 conventional orifice plates, the micro-assembly robot core of the formation of embodiment 3 can be loaded
Piece, and every hole is made to cover U-typed slot structure all on every chip.
(3) being placed with CCK-8 treated chip on 96 orifice plates after processing, existed with multi-function microplate reader measurement
Light absorption value at 450nm.
Claims (12)
1. a kind of drug delivery system screening micro-fluidic chip of 3D solid tumor model, the chip by the substrate that is bonded together and
Cover plate composition；Three parallel microchannels being mutually communicated are provided on cover plate, respectively drug infusion and unilayered vessels is formed
It is administered microchannel (1), the connection microchannel (2) comprising extracellular matrix layer, and the three-dimensional with multiple U-typed slot structures (3)
Tumour many cells ball microchannel (4)；Position on substrate corresponding to three-dimensional nodule many cells ball microchannel is provided with strip groove
Parallel channels (5)；
Extracellular matrix analogies are provided in connection microchannel, extracellular matrix analogies are basement membrane extract (BME)；
Umbilical vein vascular endothelial cells layer is provided between administration microchannel and connection microchannel；
The depth of the connection microchannel of the micro-fluidic chip is more shallow than administration microchannel and cell ball microchannel, and connects micro- logical
The two sides in road are provided with the connector for penetrating through administration microchannel and three-dimensional nodule many cells ball microchannel, connect microchannel
Width is 2-3 times of connector width.
2. hydrophily is arranged on the inner wall of micro-fluidic chip according to claim 1, administration microchannel and connection microchannel
Coating.
3. micro-fluidic chip according to claim 2, hydrophilic coating is modification polyvinyl alcohol (PVA) or plunker
PF-127 coating.
4. micro-fluidic chip according to claim 1-3, the size and tissue culture plate of the micro-fluidic chip
On each cell culture well aperture match, and the position of U-type groove can be entirely accommodated in a cell culture well.
5. micro-fluidic chip according to claim 1-3, the interior diameter of the U-typed slot of micro-fluidic chip is 150-
250 μm, depth is 250-350 μm, and the width at U-typed channel opening is 100-200 μm.
6. micro-fluidic chip according to claim 1-3, the interior diameter of the U-typed slot of micro-fluidic chip is 200 μ
M, depth are 300 μm, and the width at U-typed channel opening is 150 μm.
7. micro-fluidic chip according to claim 1-3, the position of the U-typed slot of the micro-fluidic chip is to hand over
Fork interval, and the depth that has on substrate is 10 μm of parallel channels, the position of parallel channels and the position of U-type groove are corresponding.
The tissue culture plate used 8. a kind of and described in any item micro-fluidic chips of claim 1-3 match, the cell
The card slot for accommodating micro-fluidic chip is provided on culture plate, the position of card slot can make U-type groove and cell on micro-fluidic chip
The position of one of cell culture well on culture plate is corresponding.
The tissue culture plate used 9. a kind of and micro-fluidic chip according to any one of claims 8 matches, the tissue culture plate are 6
Orifice plate, 12 orifice plates, 24 orifice plates, 48 orifice plates or 96 orifice plates.
10. a kind of preparation method of the described in any item micro-fluidic chips of claim 1-7, is prepared micro-fluidic with soft lithography
Chip, material used are made of biocompatible materials.
11. a kind of preparation method of micro-fluidic chip described in any one of claim 10, biocompatible materials are polydimethylsiloxanes
Alkane (PDMS).
12. a kind of purposes that the described in any item micro-fluidic chips of claim 1-7 are screened as drug delivery system.
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