CN104830683B - A kind of bionical micro-fluidic chip for simulating interior tumor cell and its transfer microenvironment - Google Patents

A kind of bionical micro-fluidic chip for simulating interior tumor cell and its transfer microenvironment Download PDF

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CN104830683B
CN104830683B CN201510221851.3A CN201510221851A CN104830683B CN 104830683 B CN104830683 B CN 104830683B CN 201510221851 A CN201510221851 A CN 201510221851A CN 104830683 B CN104830683 B CN 104830683B
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pdms
micro
cell
interface channel
fluidic chip
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CN104830683A (en
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王琪
高占成
许志赟
郭哲
郝华龙
徐彤
徐一彤
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Second Hospital of Dalian Medical University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/02Form or structure of the vessel
    • C12M23/16Microfluidic devices; Capillary tubes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502707Containers 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M35/00Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Abstract

The invention discloses a kind of bionical micro-fluidic chip for simulating interior tumor cell and its transfer microenvironment.The micro-fluidic chip is formed by three layers of PDMS substrates and the interlaced irreversible sealing-in of the porous PDMS films of two-layer.Entrance and exit, the top half of vacuum passage that ground floor PDMS substrates are provided with passage for air circulation, pass in and out for liquid;Second layer PDMS substrates are provided with the passage for circulating for liquid, the entrance and exit for passing in and out for liquid, the top half of vacuum passage;Third layer PDMS substrates are provided with the cell culture chamber for cell culture;Passage on ground floor PDMS substrates is located at the upstream of second layer PDMS substrate upper channels, and the cell culture chamber on third layer PDMS substrates is located at the downstream of second layer PDMS substrate upper channels.Micro-fluidic chip of the invention can be used for the process of vitro detection Nasopharyngeal neoplasms, and formulating cancer immunotherapies for clinic provides basis.

Description

It is a kind of for simulate interior tumor cell and its transfer microenvironment it is bionical micro-fluidic Chip
Technical field
The invention belongs to clinical practice field, it is related to a kind of for simulating in-vivo tumour and its shifting the bionical micro- of microenvironment Fluidic chip.Micro-fluidic chip of the invention with the transfer process of dynamic monitoring tumour cell, can define the migration of tumour cell Pattern, for prevention and treatment metastases provide guidance.
Background technology
Cancer metastasis are the main causes including all cancer mortalities including lung cancer, belong to basic researchers and clinic One of ultimate challenge of scholar.Cancer metastasis are a physiology courses for complexity, generally include the status of a sovereign point neoplastic lesion propagation, Tumour cell disengaging, tumor cell migration, spilling simultaneously form metastatic tumour cell group at the second organ.With metastatic The tumour cell of phenotype shows following property:Cell mobility enhancing, degraded basement membrane composition ability enhancing, to thin around The ability enhancing of born of the same parents' migration, the ability enhancing, the ability enhancing in the second site autonomous proliferation that penetrate lymphatic vessel or blood vessel.So And, there is tumor cell migration height Organic selection, the process to be related to the interaction between tumour cell and host organ. Up to the present, the precise mechanism of the metastases of organ specificity is not studied also clear completely.
Lung cancer is the main factor of cancer mortality in the world, and the organ that cancer cell is transferred to distant place is lung cancer death Main cause.Clinical effectiveness shows that lung cancer is often transferred in brain tissue, bone tissue and liver organization.It is better understood by lung cancer Transfer mode is very crucial for the therapeutic strategy formulated for lung cancer patient.Therefore, developing one kind in vitro being capable of mould The in-vitro cell culture model for intending microenvironment in lung cancer metastasis body is problem demanding prompt solution.
Understanding the pathology of lung carcinoma cell transfer needs in the environment of the whole lung with physiologically active and distal organs The function of cancer cell and tissue of the research with physiologically active.However, presently, there are for evaluating normal physiological and disease The animal model of process is sufficiently expensive, and experimental period is very long, while also there are various dispute of ethic.Even more important It is that above-mentioned animal model can not well control the positioning and taxis of the lung carcinoma cell of transfer, so as to cause can not be correct The physiological status of lung carcinoma cell transfer in reaction mankind's body.In recent years, a kind of external 3D cell culture models have been obtained increasingly It is widely applied.Above-mentioned external 3D cell culture models have been used for the co-cultivation of kinds of tumor cells and interstitial cell.This A little models can be easily controlled condition and remove research paracrine signal to the influence between different type cell.Inventor is existing Upgraded and transformed on the basis of 3D cell culture models, developed and can be used for the bionical of lung cancer metastasis microenvironment in analogue body Micro-fluidic chip.
The content of the invention
In order to solve problems of the prior art, the invention provides one kind for simulate interior tumor cell and its Shift the bionical micro-fluidic chip of microenvironment.Different cells can be co-cultured in the micro-fluidic chip, in dynamic monitoring The process of trip tumour cell downstream target organ transfer, formulates cancer immunotherapies and provides basis for clinic.
Above-mentioned micro-fluidic chip of the invention employs following design:
The micro-fluidic chip is by three layers of PDMS substrates and the interlaced irreversible sealing-in of the porous PDMS films of two-layer Into a closed entirety;Ground floor PDMS substrates 1 be provided with air duct 11 for air circulation, pass in and out for liquid first Layer liquid inlet 12 and ground floor liquid outlet 13, the first vacuum passage 14 respectively positioned at the both sides of the air duct 11, the The top half of two vacuum passages 15;Second layer PDMS substrates 2 be provided with circulate for liquid fluid passage 21, pass in and out for liquid Second layer liquid inlet 22 and second layer liquid outlet 23, respectively positioned at the first vacuum passage of the both sides of the fluid passage 21 14th, the latter half of the second vacuum passage 15, the side of the fluid passage 21 is provided with three interface channels for stretching out, Respectively the first interface channel 211, the second interface channel 212, the 3rd interface channel 213, first interface channel 211, institute State the second interface channel 212, the end of the 3rd interface channel 213 and be respectively equipped with first entrance 214, second entrance 215, Three entrances 216;Third layer PDMS substrates 3 be provided with for the first cell culture chamber 31 of cell culture, the second cell culture chamber 32, 3rd cell culture chamber 33;First cell culture chamber 31, second cell culture chamber 32, the 3rd cell culture chamber 33 are located at first interface channel 211, second interface channel 212, the lower section of the 3rd interface channel 213 respectively, By the first porous PDMS films 5 and first interface channel 211, second interface channel 212, the 3rd interface channel 213 communicate;First vacuum passage 14, the top half of the second vacuum passage 15 and first vacuum passage 14, second Corresponding first vacuum passage 14 and the vacuum passage for forming structural integrity of the latter half structure of vacuum passage 15 15;The ground floor liquid inlet 12 and the ground floor liquid outlet 13 are respectively arranged at the upstream and downstream of the air duct 11 And homonymy;The second layer liquid inlet 22 and the second layer liquid outlet 23 are arranged at the upstream and downstream of the fluid passage 21 And homonymy, the air duct 11 is positioned at the surface of the fluid passage 21, the opening of the air duct 11 and the liquid The opening of body passage 21 is relative and is separated by the second porous PDMS films 4;The air duct 11 is covered in the fluid passage 21 upstream end, first interface channel 211, second interface channel 212, the 3rd interface channel 213 are located at institute State the downstream of fluid passage 21.
Further, the described first porous PDMS films 5, the second porous PDMS films 4 thickness for 10 μm, aperture be 10 μ m。
Further, in specific embodiments of the present invention, the air duct 11 and the fluid passage 21 it is transversal Face is rectangle, and size is:10mm long × 4mm wide, 35mm long × 4mm wide;First vacuum passage 14, the second vacuum passage 15 cross section is rectangle, and size is:7mm high, width 2mm;First cell culture chamber 31, second cell culture chamber 32nd, the shape of cross section of the 3rd cell culture chamber 33 is rectangle, and size is:1.5mm high, width 1.5mm.
Further, in specific embodiments of the present invention, the length of the air duct 11 is 10mm;The liquid leads to The length in road 21 is 35mm;First interface channel 211, second interface channel 212, the 3rd interface channel 213 Distance apart from one end of the fluid passage 21 is 2.2mm.
Further, in specific embodiments of the present invention, the ground floor liquid inlet 12 and the ground floor liquid The distance of outlet 13 is 15mm;The distance of the second layer liquid inlet 22 and the second layer liquid outlet 23 is 15mm.
Further, in specific embodiments of the present invention, first interface channel 211, second interface channel 212nd, the 3rd interface channel 213 is parallel to each other.
Further, in specific embodiments of the present invention, first vacuum passage 14 and the air duct 11 Distance is 2mm;Second vacuum passage 15 and the distance of the air duct 11 are 2mm.
All passages are disposed on the groove on substrate on micro-fluidic chip of the invention;On micro-fluidic chip of the invention The cell culture chamber groove that is disposed on substrate.
The material for constituting the upper and lower two-layer base material of micro-fluidic chip can be PDMS (dimethyl silicone polymer), PMMA (poly- first Base methyl acrylate), PC (makrolon), COC resins, ABS (acrylonitrile-butadiene-styrene copolymer), glass, quartz Or copper.In specific embodiments of the present invention, the base material 1 and base material 2 prepare material selection PDMS.
Present invention also offers the preparation method of above-mentioned micro-fluidic chip, the preparation method is comprised the following steps:
(1) the SU-8 formpistons with microchannel and micro-structural in above-mentioned micro-fluidic chip are prepared;
(2) the SU-8 formpistons with step (1) preparation, as template, are that raw material is replicated with PDMS, are prepared into ground floor PDMS bases Piece, second layer PDMS substrates, third layer PDMS substrates;
(3) the porous PDMS films in two above-mentioned micro-fluidic chips are prepared;
(4) the ground floor PDMS substrates, the second layer PDMS substrates, the third layer for preparing step (2) Two porous interlaced placement sealing-ins of PDMS films prepared by PDMS substrates and step (3) are formed.
Further, in specific embodiments of the present invention, the preparation method of micro-fluidic chip of the invention is as follows:
(1) microchannel, the micro-structural figure in above-mentioned micro-fluidic chip are drawn with computer aided design software CAD;Will figure It is printed upon on SU-8 films (Microchem, model 2075) as mask, mould, standard is made using standard photolithography process Photoetching process is known for those skilled in the art;
0007883528) and curing agent in mass ratio 10 (2) by PDMS, (Dow Corning, article No. is:1 mixes, true Empty drying box pours the die surface for being coated in step (1) preparation, 80 DEG C of baking 1h after vacuumizing;
(3) slowly PDMS is torn in template after cooling down, gets out entrance, outlet in PDMS substrates relevant position, then Cut into suitable size;
(4) porous PDMS film productions are PDMS and curing agent in mass ratio 15:1 mixes, sol evenning machine 3000rpm, 1 minute, The die surface for being coated in step (1) preparation is poured after vacuumizing, 65 DEG C are baked overnight;Slowly PDMS is torn in template after cooling Under, it is standby.
Su-8 photoetching techniques:New chemical amplification type negative-appearing image SU-8 photoresists overcome common photoresist using UV photoetching The not enough problem of depth-to-width ratio, is quite suitable for preparing high aspect ratio microstructures, therefore SU-8 glue is a kind of negativity, epoxy resin Type, near ultraviolet ray photoresist.Its absorbance in the range of black light (365nm-400nm) is very low, and whole photoresist layer The light exposure uniformity for being obtained, can obtain the thick film figure with vertical sidewall and high-aspect-ratio;It also has good Mechanical property, resistance to chemical corrosion and heat endurance;SU-8 is crosslinked after by ultraviolet radioactive, is that a kind of chemical amplification is born Property glue, can form the baroque figure such as step;And SU-8 glue is non-conductive, can make directly as insulator in plating With.Because it has more advantages, SU-8 glue is just gradually applied to the fields such as MFMS, chip package and micro Process.Directly adopt The new technology that depth-to-width ratio micro-structural high and micro- part are micro Process fields is prepared with SU-8 photoresists.
Present invention also offers application of the above-mentioned micro-fluidic chip during Nasopharyngeal neoplasms are monitored.Use the present invention Micro-fluidic chip monitoring interior tumor cell transfer process operating procedure it is as follows:
1st, essence simulation:Epithelial cell and tumour cell are co-cultured into the porous PDMS films 4 in micro-fluidic chip to contact The one side of air.
2nd, interstitial simulation:By vascular endothelial cell and interstitial cell (such as fibroblast, monocyte) co-culture in The one side of the porous contact liq of PDMS films 4 in micro-fluidic chip.
3rd, Nasopharyngeal neoplasms target organ simulation:By brain tissue cell, bone and its cells, hepatic tissue cell respectively in miniflow 3D cultures are carried out in cell culture chamber 31,32,33 in control chip.
4th, the validation checking of the metastases microenvironment of external structure is carried out
By detecting the compactness of cell viability, vascular endothelial cell and epithelial cell connection, the cancer of tumor cell induction Associated fibroblast cell, the expression of macrophage specific proteins utilize the simulation of micro-fluidic chip of the invention structure to evaluate The validity of the bionic model of Nasopharyngeal neoplasms microenvironment.
5th, the detection of Nasopharyngeal neoplasms process is carried out
(EMT) labelled protein expression evaluation metastatic cancer cell is converted by detecting that tumour cell occurs epithelial-mesenchymal Generation;Metastatic cancer cell growth pattern is compared by the observation of cellular morphology;There is mesenchymal-epithelial by tumour cell Conversion (MET) labelled protein expression evaluation metastatic cancer cell moves to the change after target organ;By detecting target organ The expression of labelled protein is damaged to evaluate whether metastatic cancer cell has been attacked in target organ.
In specific embodiments of the present invention, epithelial cell is bronchial epithelial cell;Tumour cell is lung carcinoma cell, A bionic model for simulated lung cancer metastasis microenvironment is constructed using micro-fluidic chip of the invention.
Principle:The present invention uses PDMS and with meshed permeable membrane material, according to internal cell and cell, cell and training Support between medium, tissue and tissue, the characteristic and fluid mechanics principle of organ and microenvironment interphase interaction, design and make one The individual high flux micro-fluidic chip for being capable of, multichannel integrated close to the multiple-unit of lung anatomical structure, simulated lung physiological function connection Laboratory.The key problem of the lab design is the anatomical structure for how rebuilding lung, including including bronchuses at different levels, alveolar Pulmonary parenchyma and interstitial lung and how the main Physiological Function of simulated lung, i.e. gas exchanges.With one layer with meshed permeable membrane The elastomer of the alveolar retraction of expansion can be promoted when substituting respiratory movement, by thin in film upper and lower surface difference bronchiolar epithelium Born of the same parents, vascular endothelial cell, macrophage, fibroblastic two dimension culture, simulate pulmonary parenchyma and interstitial, and constitute qi and blood screen Barrier, is then each leading into gas and liquid supply oxygen and nutriment, in two with film vertical direction with film parallel direction Side connects two vacuum passages, simulates the human body respiration rhythm and pace of moving things.When vacuum channel pump enters gas, permeable membrane retraction, gas passage Oxygen is pumped into, alveolar ectasia, simulates lungs air suction function;When vacuum channel pull gas, film stretching, gas passage oxygen are permeated Air pump goes out, and alveolar shrinks, and simulates lungs expiration function.So far, construct one close to lung anatomical structure, can simulated lung it is main The bionic lung chip physiological models of physiological function.On the basis of above-mentioned bionical chip lung physiological models, according to most lung cancer Originate from tunica mucosa bronchiorum epithelium, lung cancer is inoculated in bronchial epithelial cell area respectively, build bionical chip lung cancer pathology mould Type;According to lung cancer metastasis approach and predilection site, Deiter's cells culturing room, bone are built on above-mentioned lung cancer pathological model thin The target organ that the lung cancer such as brain, bone, liver are most often shifted is simulated in born of the same parents culturing room and hepatocyte cultures room respectively, builds different turns of lung cancer Shifting stage pathological model, reproduces invasion of lung cancer transfer overall process.
The advantages of the present invention are:
(1) micro-fluidic chip of the invention can realize the dimensional culture of cell, carry out biology work(in more preferable perfect aspect Can research;The reagent consumption that the micro-fluidic chip is used to detect is substantially less than traditional platform.
(2) cell is cultivated with good adaptability in micro-fluidic chip platform of the invention;The master of micro-fluidic chip It is PDMS to want material, and PDMS has a gas permeability of good biocompatibility and height, gas can with smooth by PDMS so that Ensure that the gas exchanges of the cell of culture and western medium in chip.
(4) the static macro environment of cell growth differs outstanding with the small solid space of internal cells survival on traditional platform Very, micro-fluidic chip system then compensate for traditional platform defect in this respect, and it has more preferable seal, therefore cell is given birth to The size for depositing space is even more like with the micro- space of physiology that human inner cell survives, the on-line checking in situ in this short space Also the confidence level of testing result is further increased.
(5) micro-fluidic chip platform operations of the invention are easy, time saving and energy saving.
Brief description of the drawings
Fig. 1 shows the exploded view of microfluidic chip structure of the invention;
Fig. 2 shows the exploded view of microfluidic chip structure of the invention;
Fig. 3 shows the integrally-built top view of micro-fluidic chip of the invention;
Fig. 4 shows the side view of the ground floor substrate structure of micro-fluidic chip of the invention;
Fig. 5 shows the side view of the second layer substrate structure of micro-fluidic chip of the invention;
Fig. 6 shows the side view of the third layer substrate structure of micro-fluidic chip of the invention;
Fig. 7 shows the side view of the porous PDMS films of micro-fluidic chip of the invention;
Fig. 8 displays detect bronchus using cell viability in H33342/PI dyeing detection chips and using immunostaining The connection of epithelial cell 16HBE and vascular endothelial cell HUVEC, wherein, A:Cellular morphology after 16HBE cells 24H;B: Cellular morphology after HUVEC cells 24H;C:Cellular morphology after 16HBE cells 48H;D:Cell shape after HUVEC cells 48H State;E:16HBE cells H33342/PI is dyeed:F:HUVEC cells H33342/PI is dyeed;G:E-cadherin in 16HBE cells Protein immunization is dyeed;H:E-cadherin protein immunizations dyeing in HUVEC cells;
Fig. 9 displays are thin using Immunofluorescence cell tracker expression assay detections lung cancer The common location situation of born of the same parents A549 and bronchial epithelial cell 16HBE and using immunostaining detection lung carcinoma cell, into fiber finer Born of the same parents, the expression of the specific proteins of macrophage, wherein, A:16HBE cellular morphologies, B:What 16HBE and A549 was co-cultured Cellular morphology;C:16HBE cell cell tracker are dyeed;D:16HBE and A549 co-cultures cell tracker dyeing;E: 16HBE cell CEA protein immunizations are dyeed;F:16HBE and A549 co-cultures the dyeing of CEA protein immunizations;G:α in fibroblast- SMA protein immunizations are dyeed;H:α-SMA protein immunizations dyeing in CAF (carcinoma-associated fibroblasts);I:CD206 in macrophage Protein immunization is dyeed;J:CD206 protein immunizations dyeing in CAM (cancer associated macrophages);
Figure 10 displays convert the expression of marker using epithelial cell interstitial in immunostaining detection lung carcinoma cell, its In, A:Cellular immunity colored graph;B:OD value statistical chart;
Figure 11 shows growth pattern of the lung cell A549 to the migration situation of remote organization and at remote organization, its In, A:The statistics of lung carcinoma cell migrating cell quantity;B:Cellular morphology of the lung carcinoma cell at remote organization;
Figure 12 shows using immunostaining to detect the expression of characteristic protein in remote organization, wherein, A:Feature Property protein immunization colored graph;B:OD value statistical chart;
Wherein, 1:Ground floor substrate;11:Air duct;12:Ground floor liquid inlet;13:Ground floor liquid outlet;14: First vacuum passage;15:Second vacuum passage;2:Second layer substrate;21:Fluid passage;22:Second layer liquid inlet;23:The Two layers of liquid outlet;211:First interface channel;212:Second interface channel;213:3rd interface channel;214:First entrance; 215:Second entrance;216:3rd entrance;3:Third layer substrate;31:First cell culture chamber;32:Second cell culture chamber 33: 3rd cell culture chamber;4:Second porous PDMS films;5:First porous PDMS films.
Specific embodiment
Present disclosure can more easily be understood by refering to following embodiments, these embodiments are for further The present invention is illustrated, is not meant to limit the scope of the present invention.
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
A kind of bionical micro-fluidic chip for lung cancer metastasis microenvironment in analogue body of embodiment 1
Micro-fluidic chip of the invention is by three layers of PDMS substrates and the interlaced irreversible envelope of the porous PDMS films of two-layer The closed entirety for connecing;Ground floor PDMS substrates 1 are provided with air duct 11 for air circulation, pass in and out for liquid Ground floor liquid inlet 12 and ground floor liquid outlet 13, the first vacuum passage 14, respectively positioned at the both sides of air duct 11 The top half of two vacuum passages 15;Second layer PDMS substrates 2 be provided with circulate for liquid fluid passage 21, pass in and out for liquid Second layer liquid inlet 22 and second layer liquid outlet 23, the first vacuum passage 14 respectively positioned at the both sides of fluid passage 21, The latter half of second layer liquid 15, the side of fluid passage 21 is provided with three interface channels for stretching out, and respectively A connection channel 211, the second interface channel 212, the 3rd interface channel 213, the first interface channel 211, the second interface channel 212nd, the end of the 3rd interface channel 213 is respectively equipped with first entrance 214, second entrance 215, the 3rd entrance 216;Third layer PDMS substrates 3 are provided with the first cell culture chamber 31, the second cell culture chamber 32, the 3rd cell culture chamber for cell culture 33;First cell culture chamber 31, the second cell culture chamber 32, the 3rd cell culture chamber 33 respectively be located at the first interface channel 211, Second interface channel 212, the lower section of the 3rd interface channel 213, by the interface channel 211 of the first porous PDMS films 5 and first, Two interface channels 212, the 3rd interface channel 213 are communicated;First vacuum passage 14, the top half of the second vacuum passage 15 and The corresponding and of the first vacuum passage 14 for forming structural integrity of the latter half structure of one vacuum passage 14, the second vacuum passage 15 Second vacuum passage 15;Ground floor liquid inlet 12 and second layer liquid outlet 13 are respectively arranged at the upstream and downstream of air duct 11 And homonymy;Second layer liquid inlet 22 and second layer liquid outlet 23 are arranged at the upstream and downstream and homonymy of fluid passage 21, air Passage 11 is located at the surface of fluid passage 21, and the opening of air duct 11 is relative with the opening of fluid passage 21 and by second Perforated membrane 4 separates;Air duct 11 is covered in the upstream end of fluid passage 21, the first interface channel 211, the second interface channel 212nd, the 3rd interface channel 213 is located at the downstream of fluid passage 21.
The thickness of the porous PDMS films 5 of the second porous PDMS films 4 and first is 10 μm, aperture is 10 μm.
The cross section of air duct 11 and fluid passage 21 is rectangle, and size is:10mm long × 4mm wide, 35mm long × 4mm wide;First vacuum passage 14, the cross section of the second vacuum passage 15 are rectangle, and size is:7mm high, width 2mm;First is thin Born of the same parents culturing room 31, the second cell culture chamber 32, the shape of cross section of the 3rd cell culture chamber 33 are rectangle, and size is:It is high 1.5mm, width 1.5mm.
The length of air duct 11 is 10mm;The length of fluid passage 21 is 35mm;First interface channel 211, second connects It is 2.2mm to connect the distance of road 212, the 3rd interface channel 213 apart from one end of fluid passage 21.
The distance of ground floor liquid inlet 12 and ground floor liquid outlet 13 is 15mm;Second layer liquid inlet 22 and second The distance of layer liquid outlet 23 is 15mm.
First interface channel 211, the second interface channel 212, the 3rd interface channel 213 are parallel to each other.
First vacuum passage 14 is 2mm with the distance of air duct 11;Second vacuum passage 15 and air duct 11 away from From being 2mm.
All passages are disposed on the groove on substrate on micro-fluidic chip of the invention;On micro-fluidic chip of the invention The cell culture chamber groove that is disposed on substrate.
The preparation of the micro-fluidic chip in the embodiment 1 of embodiment 2
The preparation method of the micro-fluidic chip in embodiment 1 is comprised the following steps:
(1) microchannel, the micro-structural figure in above-mentioned micro-fluidic chip are drawn with computer aided design software CAD;Will figure It is printed upon on SU-8 films (Microchem, model 2075) as mask, mould, standard is made using standard photolithography process Photoetching process is known for those skilled in the art;
(2) by PDMS (Dow Corning, article No.:0007883528) with curing agent in mass ratio 10:1 mixes, in vacuum Drying box pours the die surface for being coated in step (1) preparation, 80 DEG C of baking 1h after vacuumizing;
(3) slowly PDMS is torn in template after cooling down, gets out entrance, outlet in PDMS substrates relevant position, then Cut into suitable size;
(4) porous PDMS film productions are PDMS and curing agent in mass ratio 15:1 mixes, sol evenning machine 3000rpm, 1 minute, The die surface for being coated in step (1) preparation is poured after vacuumizing, 65 DEG C are baked overnight;Slowly PDMS is torn in template after cooling Under, it is standby.
The structure of the bionic model of lung carcinoma cell transfer microenvironment in the analogue body of embodiment 3
1st, the 2D of lung carcinoma cell is cultivated in micro-fluidic chip
(1) micro-fluidic chip uses ultraviolet irradiation-sterilize, and BME is coated with porous PDMS films, and specific coating process is: Chip is pre-processed, chip porous film surface is preferably attached to sharp cell.By 1:10 dilution proportion BME (Cultrex Basement membrane extract, R&D Systems, McKinley Place, MN, USA), with micro after being sufficiently mixed Sample injector injects the sample entrance port of micro-fluidic chip, and incubator overnight waits gelling solid.
(2) micro-fluidic chip is overturn, i.e., third layer PDMS substrates are upward.The monocyte of suspension is collected into centrifuge tube In, 1000rpm is centrifuged 5 minutes, abandons supernatant, adds fresh culture to prepare cell suspension.Mononuclear cell suspension is passed through into entrance 22 are injected into passage 21, make it with 103Individual/cm2Density plantation on perforated membrane PDMS films, afterwards using syringe pump with The volumetric flow rate of 24mm/h is by PMA culture mediums
(100ng/ml) is injected into micro-fluidic chip by entrance 22, and unnecessary culture medium is excluded by outlet 23.Will be micro- Fluidic chip inclines to allow signaling to one side of passage 21.Chip is tilted towards 30 degree of side, so that monocyte is heavy The side of central culture channel is dropped to, 37 DEG C, 5%CO is placed in2Incubator culture.Monocyte after 48h is stimulated as macrophage is thin Born of the same parents.
(3) after stimulating monocyte to become M0 macrophages, PMA culture mediums are replaced with normal incubation medium (1640 cultures Base).
(4) by human lung fibroblast's plantation with 104Individual/cm2Density plantation to M0 macrophage growths position (treat that cell is bred to exponential phase, use 0.25% Trypsin Induced, add fresh culture medium to blow and beat into cell suspension, 1000rpm is centrifuged 5 minutes, abandons supernatant, adds fresh culture, is made fibroblast suspension.By lung fibroblast WI38 With 104Individual cell/cm2Cell density be inoculated into the homonymy of macrophage, it is attached to porous film surface, be placed in 37 DEG C, 5%CO2Cultivated 4 hours under incubator static conditions.
(5) micro-fluidic chip is returned to horizontality, vascular endothelial cell HUVEC suspensions is injected into by entrance 22 In passage 21, make it with 104Individual/cm2Density plantation on porous PDMS films, stick to the another side of passage 21.
(6) after after the equal adherent growth of cell of chip perforated membrane downside, turning-over of chip is from upside by entrance 2 with 104It is individual thin Born of the same parents/cm2Cell density by bronchial epithelial cell 16HBE inject chip, it is small after static state makes it be attached to film surface 4 hours The heart lightly aspirates culture medium from outlet 2 from upper channel, continues through entrance and is continuously pumped into mixed culture medium, is placed in 37 DEG C, 5%CO2Incubator culture.Culture medium is excluded by outlet 13.
(7) after above-mentioned cell is covered with, lung carcinoma cell suspension is injected into passage 11 by entrance 12, makes it with 103 Individual/cm2Density plantation to chip upper strata bronchial epithelial cell area is inoculated on porous PDMS films, standing 4h attaches it. Culture medium is excluded by outlet 13.
2nd, the 3D cultures of micro-fluidic chip mesencephalic tissue cell, bone and its cells, hepatic tissue cell
(1) by astroglia, Gegenbaur's cell and liver cell (purchased from Shanghai Sheng Ke institutes of Chinese Academy of Sciences cell centre) Digested using pancreatin, it is resuspended in the cell basement membrane extract mixture of ice precooling (R&D supplements article No.), respectively will Cell suspension mixes in equal volume with BME.
(2) micro-fluidic chip is placed in into standing 30min in 37 DEG C of environment makes BME gels.Culture medium is noted by entrance 22 Enter in micro-fluidic chip, be continuously pumped into.Simultaneously, outlet 23 is sealed, makes unnecessary culture medium from entrance 214,215,216 Discharge.
(4) it is to cultivate in 37 DEG C of incubators that 95%, gas concentration lwevel is 5% micro-fluidic chip to be positioned over into humidity (vavuum pump parameter is physiological cyclic strain (10%at 0.2Hz).Incubation time 24h, carries out follow-up reality Test.
The validation checking of the bionic model of lung carcinoma cell transfer microenvironment in the analogue body of embodiment 4
Evaluate embodiment 3 build analogue body in lung carcinoma cell transfer microenvironment bionic model validity, by with It is lower to test to complete:
1st, the detection of cell viability
Detection method:Nutrient solution in sucking passage in chip system, PBS is injected in chip channel, and cleaning does not exist together The cell of reason group 2 times;H33342 (1 is pumped into afterwards:100) dye 15 minutes, PBS solution is cleaned 2 times;After pump into PI dyeing (1: 200) 5 minutes, PBS solution was cleaned 2 times;Under the microscope, fluorescence intensity and take a picture note of the observation under corresponding excitation Record.
2nd, the detection of lung carcinoma cell and bronchial epithelial cell common location
Cellular tracking agent C7000CM-DiL is by lung carcinoma cell labeled as red:Lung carcinoma cell is resuspended in before chip inoculation (1 μ g/ μ L) 37 DEG C of incubations 5min, 4 DEG C of placement 15min in C7000CM-DiL working solutions.
3rd, the observation of the growth pattern of human bronchial's epithelial cell and vascular endothelial cell
Detect that micro-fluidic chip mesobronchus epithelial cell and vascular endothelial cell connect using the immunostaining of E cadherins The compactness for connecing.E cadherin immunostaining processes are as follows:
A, bronchial epithelial cell and vascular endothelial cell that film both sides in micro-fluidic chip are rinsed using PBS;
B, cell 15min, 0.5%PBST solution rupture of membranes 10 minutes are fixed using 4% paraformaldehyde, PBS solution cleaning 2 It is secondary;
C, will closing serum dilution injection chip culture pond in, be put into wet box, 37 DEG C be incubated 1 hour;Matched somebody with somebody with fresh The 5%BSA dilution sheep closing serum stostes of system, according to 1:100 dilution proportion, is mixed with oscillator;
D, E cadherin antibodies (Abcam) dilution incubation 2h using final concentration of 2 μ g/mL;
E, the secondary antibody being coupled using Alexa 594 are incubated 1h;
F, the DAPI dyeing using final concentration of 10 μ g/mL, the time is 15min;Taken a picture using fluorescence microscope.
4th, lung carcinoma cell and carcinoma-associated fibroblasts, the feature of macrophage are detected
Using the method for immunostaining detect the expression of tumour cell labelled protein CEA, fibroblast labelled protein α- The expression of SMA, the expression of macrophage marker PROTEIN C D206, mark result are presented using immunofluorescence microscopy.
Immunostaining process is as follows:
A, use lung carcinoma cell, fibroblast, macrophage in PBS micro-fluidic chip;
B, cell 15min, 0.5%PBST solution rupture of membranes 10 minutes are fixed using 4% paraformaldehyde, PBS solution cleaning 2 It is secondary;
C, will closing serum dilution injection chip culture pond in, be put into wet box, 37 DEG C be incubated 1 hour.Matched somebody with somebody with fresh The 5%BSA dilution sheep closing serum stostes of system, according to 1:100 dilution proportion, is mixed with oscillator;
E, the antibody (1 using anti-human CEA albumen:100, abcam), the antibody (1 of anti-human α-SMA albumen:200,Santa Cruz), the antibody (1 of the albumen of anti-humen CD 20 6:50, abcam) it is incubated 2h;
F, the secondary antibody being coupled using Alexa 488 or 594 are incubated 1h;Dyeed using the DAPI of final concentration of 10 μ g/mL, Time is 15min;Taken a picture using fluorescence microscope.
2nd, experimental result:
The result of 2.1 cell viabilities:Cell viability more than 95% (Fig.8E-F).
2.2immunofluorescence cell tracker expression assay results show lung carcinoma cell A549 and bronchial epithelial cell 16HBE common locations (figure Fig.9C-D).
2.3E-cadherin protein immunizations are dyeed, test result indicate that bronchial epithelial cell 16HBE and blood vessel endothelium are thin Born of the same parents HUVEC is connected with each other closely (Fig.8G-H).
CEA expression (figure Fig.9E-F) in 2.4 lung carcinoma cells;Express alpha-SMA in fibroblast, expresses in macrophage CD206, it was demonstrated that lung carcinoma cell interacts with interstitial cell causes the interstitial cell to activate, it is related that fibroblast is converted into cancer Fibroblast α-SMA, macrophage is converted into cancer associated macrophages expression CD206 (figure Fig.9G, 9H, 9I, 9J).
Embodiment 5 monitors lung carcinoma cell transfer process using the bionic model of lung carcinoma cell transfer microenvironment in analogue body
1st, step
There is the detection of epithelial-mesenchymal conversion (EMT) in 1.1 lung carcinoma cells
By detecting that epithelial-mesenchymal converts (EMT) marker protein:E cadherins, N cadherins, Snail1, The expression of Snail2 come evaluate lung carcinoma cell whether there occurs epithelial-mesenchymal convert.
Immune labeled process is as follows:
A, use the lung carcinoma cell on PBS perforated membrane;
B, cell 15min, 0.5%PBST solution rupture of membranes 10 minutes are fixed using 4% paraformaldehyde, PBS solution cleaning 2 It is secondary;
C, will closing serum dilution injection chip culture pond in, be put into wet box, 37 DEG C be incubated 1 hour;Matched somebody with somebody with fresh The 5%BSA dilution sheep closing serum stostes of system, according to 1:100 dilution proportion, is mixed with oscillator;
D, use anti-human E cadherin antibodies (1:100, Proteintech), antibody (the 5 μ g/ of anti-human N cadherins Ml, abcam), the antibody (1 of anti-human Snail1 albumen:50, abcam), the antibody (1 of anti-human Snail2 albumen:400,Cell Signaling Technology), it is incubated 2h;
F, the secondary antibody being coupled using Alexa 488 or 594 are incubated 1h;Dyeed using the DAPI of final concentration of 10 μ g/mL, Time is 15min;Taken a picture using fluorescence microscope.
The identification of 1.2 pulmonary metastasis pattern of cell growth
Lung carcinoma cell is marked using C34557;C34557 working solutions (10 μm of ol/L) are added to 1640 cell culture mediums In, by cell incubation 20min;5min is incubated using fresh culture afterwards, is repeated 4 times.(in advance with Cellular tracking agent C34557 By lung carcinoma cell labeled as green:Lung carcinoma cell is resuspended in C34557 working solutions into 37 DEG C of (10 μm of ol/L) before chip inoculation to incubate Educate 20min, after be resuspended in 37 DEG C of incubation 5min in 4 times of volume mediums).
The invasion and attack of 1.3 detection pulmonary metastasis cells
Evaluate whether metastatic lung carcinoma cell can attack entrance by detecting the expression of tissue damage labelled protein In micro-fluidic chip multiple remote organ.Use the antibody (1 of anti-human brain tissue impairment labelled protein CXCR4:100,abcam)、 Anti-human bone tissue damage score albumen RANKL antibody (1:50, Santa Cruz Biotechnology), anti-human liver tissue injury Labelled protein AFP antibody carries out immunostaining.
Immunostaining process is as follows:
A, use PBS cell 2 times;
B, cell 15min, 0.5%PBST solution rupture of membranes 10 minutes are fixed using 4% paraformaldehyde, PBS solution cleaning 2 It is secondary;
C, will closing serum dilution injection chip culture pond in, be put into wet box, 37 DEG C be incubated 1 hour;Matched somebody with somebody with fresh The 5%BSA dilution sheep closing serum stostes of system, according to 1:100 dilution proportion, is mixed with oscillator;
E, use above-mentioned primary antibody, be incubated 2h;
F, the secondary antibody being coupled using Alexa 488 or 594 are incubated 1h;Dyeed using the DAPI of final concentration of 10 μ g/mL, Time is 15 minutes;Taken a picture using fluorescence microscope.
2nd, experimental result
2.1 lung carcinoma cell epithelial-mesenchymals are converted
After the lung carcinoma cell interstitial cell culture 4d related to cancer, lung carcinoma cell expression EMT, 3 kinds of EMT marker (N- Ca, Snail1, Snail2) show that lung carcinoma cell has occurred epithelial cell interstitial conversion (Fig.10A-B).
2.2 lung carcinoma cells are shifted at a distance
Cell count understands that it is thin that non-small cell lung cancer cell A549 adheres to brain tissue cell, bone and its cells, hepatic tissue Cell quantity on born of the same parents' upper porous film is respectively 148 ± 8,364 ± 16,299 ± 13;Small cell lung cancer cell H446 The cell quantity adhered on brain tissue cell, bone and its cells, hepatic tissue cell upper porous film is respectively 255 ± 16, 128 ± 8,278 ± 18 (Fig.11A).The above results show that the transcellular taxises of A549 are bone tissues>Hepatic tissue> Brain tissue;The transcellular taxises of H446 are hepatic tissues>Brain tissue>The clinic of bone tissue, these features and lung cancer patient is special Levy similar.
Growth pattern of the 2.3 metastatic lung carcinoma cells at multiple remote organs
With inverted fluorescence microscope observation of cell form.As shown in Fig.11B, the cellular morphology of distal end target organ is transferred to It is agglomerating.
The invasion and attack of 2.4 pulmonary metastasis cells
Result is expressed in CXCR4, Gegenbaur's cell Fob as shown in Fig.12A and Fig.12B, in star spongiocyte H1800 AFP is expressed in expression RANKL, liver cell L-02, shows that pulmonary metastasis cell has been attacked in target organ cell.
By above-described embodiment, find micro-fluidic chip of the invention can effectively in analogue body lung carcinoma cell transfer And invasive procedure, formulate therapeutic scheme for clinic and basis is provided.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that:Not Can these embodiments be carried out with various changes, modification, replacement and modification in the case of departing from principle of the invention and objective, this The scope of invention is limited by claim and its equivalent.

Claims (9)

1. it is a kind of for simulate interior tumor cell and its transfer microenvironment bionical micro-fluidic chip, it is characterised in that it is described Micro-fluidic chip is closed by one of three layers of PDMS substrates and the interlaced irreversible sealing-in of the porous PDMS films of two-layer It is overall;Ground floor PDMS substrates (1) is provided with the air duct (11) for supplying air circulation, the ground floor liquid for passing in and out for liquid and enters Mouthful (12) and ground floor liquid outlet (13), respectively positioned at the air duct (11) both sides the first vacuum passage (14) it is upper The top half of half part and the second vacuum passage (15);Second layer PDMS substrates (2) is provided with the liquid for circulating for liquid and leads to Road (21), the second layer liquid inlet (22) for passing in and out for liquid and second layer liquid outlet (23) are logical positioned at the liquid respectively The latter half of first vacuum passage (14) of road (21) both sides and the latter half of the first vacuum passage (15), the liquid lead to The side in road (21) is provided with three interface channels for stretching out, respectively the first interface channel (211), the second interface channel (212), the 3rd interface channel (213), first interface channel (211), second interface channel (212), the described 3rd The end of interface channel (213) is respectively equipped with first entrance (214), second entrance (215), the 3rd entrance (216);Third layer PDMS substrates (3) are provided with the first cell culture chamber (31), the second cell culture chamber (32), the training of the 3rd cell for cell culture Support room (33);First cell culture chamber (31), second cell culture chamber (32), the 3rd cell culture chamber (33) It is located under first interface channel (211), second interface channel (212), the 3rd interface channel (213) respectively Side, by the first porous PDMS films (5) and first interface channel (211), second interface channel (212), described the Three interface channels (213) are communicated;The top half of first vacuum passage (14), the upper half of second vacuum passage (15) Part is corresponding with the latter half structure of the latter half of first vacuum passage (14), second vacuum passage (15) Form first vacuum passage (14) and second vacuum passage (15) of structural integrity;The ground floor liquid inlet And the ground floor liquid outlet (13) is respectively arranged at the upstream and downstream and homonymy of the air duct (11) (12);Described second Layer liquid inlet (22) and the second layer liquid outlet (23) be respectively arranged at the fluid passage (21) upstream and downstream and together Side, the air duct (11) positioned at the surface of the fluid passage (21), the opening of the air duct (11) with it is described The opening of fluid passage (21) is relative and is separated by the second porous PDMS films (4);The air duct (11) is covered in described The upstream end of fluid passage (21), first interface channel (211), second interface channel (212), the 3rd connection Downstream of the passage (213) positioned at the fluid passage (21).
2. micro-fluidic chip according to claim 1, it is characterised in that the first porous PDMS films (5) and described The thickness of two porous PDMS films (4) is 10 μm, aperture is 10 μm.
3. micro-fluidic chip according to claim 1, it is characterised in that the air duct (11) and the fluid passage (21) cross section is rectangle, and size is:10mm long × 4mm wide, 35mm long × 4mm wide;First vacuum passage (14), The cross section of second vacuum passage (15) is rectangle, and size is:7mm long, width 2mm;First cell culture chamber (31), second cell culture chamber (32), the shape of cross section of the 3rd cell culture chamber (33) are rectangle, size For:1.5mm high, width 1.5mm.
4. micro-fluidic chip according to claim 1, it is characterised in that the length of the air duct (11) is 10mm; The length of the fluid passage (21) is 35mm;It is first interface channel (211), second interface channel (212), described Distance of 3rd interface channel (213) apart from the upstream end of the fluid passage (21) is 2.2mm.
5. micro-fluidic chip according to claim 1, it is characterised in that the ground floor liquid inlet (12) and described The distance of one layer of liquid outlet (13) is 15mm;The second layer liquid inlet (22) and the second layer liquid outlet (23) Distance is 15mm.
6. micro-fluidic chip according to claim 1, it is characterised in that first interface channel (211), described second Interface channel (212), the 3rd interface channel (213) are parallel to each other.
7. micro-fluidic chip according to claim 1, it is characterised in that first vacuum passage (14) and the air The distance of passage (11) is 2mm;Second vacuum passage (15) is 2mm with the distance of the air duct (11).
8. a kind of preparation method of the micro-fluidic chip any one of claim 1-7, it is characterised in that the preparation side Method is comprised the following steps:
(1) the SU-8 sun with the microchannel in the micro-fluidic chip any one of claim 1-7 and micro-structural is prepared Mould;
(2) the SU-8 formpistons for being prepared with step (1), as template, are raw material duplication with PDMS, are prepared into any in claim 1-7 Ground floor PDMS substrates, second layer PDMS substrates, third layer PDMS substrates described in;
(3) the porous PDMS films described in two claims 1 are prepared;
(4) the ground floor PDMS substrates, the second layer PDMS substrates, the third layer PDMS bases for preparing step (2) Two porous interlaced placement sealing-ins of PDMS films prepared by piece and step (3) are formed.
9. preparation method according to claim 8, it is characterised in that the preparation method is comprised the following steps:
(1) it is micro- logical in the micro-fluidic chip any one of use computer aided design software CAD drafting claims 1-7 Road and micro-structural;Drafting figure is printed upon on SU-8 films as mask, mould is made using standard photolithography process;
(2) by PDMS and curing agent in mass ratio 10:1 is mixed, and the die surface for being coated in step (1) preparation is poured after vacuumizing, 80 DEG C of baking 1h;
(3) slowly PDMS is torn in template after cooling down, entrance, outlet is got out in PDMS substrates relevant position, then cut Into suitable size;
(4) porous PDMS film productions are PDMS and curing agent in mass ratio 15:1 mixes, sol evenning machine 3000rpm, 1 minute, takes out true The die surface for being coated in step (1) preparation is poured after sky, 65 DEG C are baked overnight;Slowly PDMS is torn in template after cooling, It is standby.
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