CN104830683A - Bionic micro-fluidic chip for simulating in vivo tumor cells and metastasis microenvironment - Google Patents

Bionic micro-fluidic chip for simulating in vivo tumor cells and metastasis microenvironment Download PDF

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CN104830683A
CN104830683A CN201510221851.3A CN201510221851A CN104830683A CN 104830683 A CN104830683 A CN 104830683A CN 201510221851 A CN201510221851 A CN 201510221851A CN 104830683 A CN104830683 A CN 104830683A
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passage
cell
micro
fluidic chip
pdms
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CN104830683B (en
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王琪
高占成
许志赟
郭哲
郝华龙
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 bionic micro-fluidic chip for simulating in vivo tumor cells and their metastasis microenvironment. The micro-fluidic chip is formed by interlacing and irreversible sealing of three layers of PDMS substrates and two layers of porous PDMS membranes. The first PDMS substrate layer is provided with a channel for air circulation, an inlet and an outlet for entry and exit of a liquid and an upper half part of a vacuum channel. The third PDMS substrate layer is provided with a cell culture room for cell culture. The channel of the first PDMS substrate layer is positioned up the channel of the second PDMS substrate layer. The cell culture room on the third PDMS substrate layer is positioned down the channel of the second PDMS substrate layer. The micro-fluidic chip provided by the invention can be used for in vitro detection of the tumor cell metastasis process and provides a basis for making clinical tumor treatment plans.

Description

A kind of for analogue body inner tumour cell and the bionical micro-fluidic chip shifting microenvironment thereof
Technical field
The invention belongs to clinical application field, relating to a kind of bionical micro-fluidic chip for simulating in-vivo tumour and transfer microenvironment thereof.Micro-fluidic chip of the present invention can the transfer process of dynamic monitoring tumour cell, and the migration model of definition tumour cell, for prevention and therapy metastases provides guidance.
Background technology
Cancer metastasis is the major cause of all cancer mortalities comprising lung cancer, belongs to one of ultimate challenge of basic researchers and clinical scholar.Cancer metastasis is a complicated physiological process, generally includes status of a sovereign point neoplastic lesion propagation, tumour cell disengaging, tumor cell migration, spilling form metastatic tumour cell group at the second organ place.The tumour cell with transitivity phenotype shows following character: the ability that cell mobility strengthens, the ability of degraded basement membrane composition strengthens, the ability of cell migration towards periphery strengthens, penetrates lymphatic vessel or blood vessel strengthens, strengthens in the ability of the second site autonomous proliferation.But tumor cell migration has height Organic selection, this process and relates to interaction between tumour cell and host organ.Up to the present, the precise mechanism of organ specific metastases is not also studied clear completely.
Lung cancer is the main factor of cancer mortality in the world, and cancer cell transfers to the main cause that organ is at a distance lung cancer death.Clinical effectiveness shows, and lung cancer is often transferred in cerebral tissue, osseous tissue and liver organization.Better understanding lung cancer metastasis pattern is very crucial for the therapeutic strategy formulated for lung cancer patient.Therefore, developing a kind of can the in-vitro cell culture model of microenvironment be problem demanding prompt solution in simulated lung metastasis of cancer body in vitro.
The pathology understanding lung carcinoma cell transfer need to have research under the lung of physiologically active and the environment of distal organs have the cancer cell of physiologically active and the function of tissue whole.But what exist at present is very expensive for evaluating the animal model of normal physiological and lysis, and experimental period is very long, also there is multiple dispute of ethic simultaneously.What is more important, above-mentioned animal model well can not control the location of lung carcinoma cell of shifting and taxis, thus cause can not be correct reaction mankind body in the physiological status of lung carcinoma cell transfer.In recent years, a kind of external 3D cell culture model obtains and applies more and more widely.Above-mentioned external 3D cell culture model has been used to the Dual culture of kinds of tumor cells and mesenchymal cell.These models can easily control condition go research paracrine signal on the impact between dissimilar cell.Contriver carries out upgrading and transforming on existing 3D cell culture model basis, develops the bionical micro-fluidic chip that may be used for lung cancer metastasis microenvironment in analogue body.
Summary of the invention
In order to solve problems of the prior art, the invention provides a kind of for analogue body inner tumour cell and the bionical micro-fluidic chip shifting microenvironment thereof.Different cell can be carried out Dual culture in this micro-fluidic chip, the process that dynamic monitoring upstream tumour cell shifts to downstream target organ, for clinical formulation cancer immunotherapies provides basis.
Above-mentioned micro-fluidic chip of the present invention have employed following design:
Described micro-fluidic chip is an airtight entirety by three layers of PDMS substrate and the interlaced irreversible sealing-in of two-layer porous PDMS film; The first layer PDMS substrate 1 is provided with for the passage 11 of air circulation, the entrance 12 passed in and out for liquid and outlet 13, lays respectively at upper part of the vacuum tunnel 14,15 of described passage 11 both sides; Second layer PDMS substrate 2 is provided with the passage 21 circulated for liquid, the entrance 22 passed in and out for liquid and outlet 23, lay respectively at lower part of the vacuum tunnel 14,15 of described passage 21 both sides, the side of described passage 21 is provided with three outward extending connecting passages 211,212,213, and the end of described connecting passage 211,212,213 is respectively equipped with entrance 214,215,216; Third layer PDMS substrate 3 is provided with the cell culture chamber 31,32,33 for cell cultures; Described cell culture chamber 31,32,33 lays respectively at the below of described connecting passage 211,212,213, is communicated with described connecting passage 211,212,213 by porous PDMS film 5; The described vacuum tunnel 14 of upper part of described vacuum tunnel 14,15 and lower part structure corresponding formation structural integrity of described vacuum tunnel 14,15 and described vacuum tunnel 15; Described entrance 12 and described outlet 13 are arranged at the upstream and downstream of passage 11 and homonymy respectively; Described entrance 22 and described outlet 23 are arranged at the upstream and downstream of described passage 21 and homonymy, and described passage 11 is positioned at directly over described passage 21, and the opening of described passage 11 is relative with the opening of described passage 21 and separated by described porous PDMS film 4; Described passage 11 covers the upstream extremity of described passage 21, and described connecting passage 211,212,213 is positioned at the downstream end of described passage 21.
Further, the thickness of described porous PDMS film is 10 μm, aperture is 10 μm.
Further, in specific embodiment of the invention scheme, the cross section of described passage 11 and described passage 21 is rectangle, is of a size of: long 10mm × wide 4mm, long 35mm × wide 4mm; The cross section of described passage 14,15 is rectangle, is of a size of: high 7mm, wide 2mm; The shape of cross section of described cell culture chamber is rectangle, is of a size of: high 1.5mm, wide 1.5mm.
Further, in specific embodiment of the invention scheme, the length of described passage 11 is 10mm; The length of described passage 21 is 35mm; Described connecting passage 211,212,213 is 2.2mm apart from the distance of one end of described passage 21.
Further, in specific embodiment of the invention scheme, the distance of described entrance 12 and described outlet 13 is 15mm; The distance of described entrance 22 and described outlet 23 is 15mm.
Further, in specific embodiment of the invention scheme, described connecting passage 211,212,213 is parallel to each other.
Further, in specific embodiment of the invention scheme, described passage 14 is 2mm with the distance of described passage 11; Described passage 15 is 2mm with the distance of described passage 11.
On micro-fluidic chip of the present invention, all passages are arranged at on-chip groove; Cell culture chamber on micro-fluidic chip of the present invention is arranged at on-chip groove.
The material forming the upper and lower two-layer base material of micro-fluidic chip can be PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), PC (polycarbonate), COC resin, ABS (acrylonitrile-butadiene-styrene copolymer), glass, quartz or copper.In specific embodiment of the invention scheme, described base material 1 and base material 2 prepare material selection PDMS.
Present invention also offers the preparation method of above-mentioned micro-fluidic chip, described preparation method comprises the following steps:
(1) preparation has the SU-8 formpiston of microchannel and microstructure in above-mentioned micro-fluidic chip;
(2) the SU-8 formpiston prepared with step (1), for template, is that raw material copies with PDMS, is prepared into the first layer PDMS substrate, second layer PDMS substrate, third layer PDMS substrate;
(3) the porous PDMS film in two above-mentioned micro-fluidic chips is prepared;
(4) two interlaced placement sealing-ins of described porous PDMS film prepared by described the first layer PDMS substrate step (2) prepared, described second layer PDMS substrate, described third layer PDMS substrate and step (3) form.
Further, in specific embodiment of the invention scheme, the preparation method of micro-fluidic chip of the present invention is as follows:
(1) microchannel in above-mentioned micro-fluidic chip, microstructure figure is drawn with computer aided design software CAD; Be printed on by figure as mask on SU-8 film (Microchem, model is 2075), adopt standard photolithography process to make mould, standard photolithography process is known for those skilled in the art;
(2) by PDMS (Dow Corning, article No. is 0007883528) and solidifying agent 10:1 mixing in mass ratio, after vacuum drying oven vacuumizes, the die surface being coated in step (1) and preparing is watered, 80 DEG C of baking 1h;
(3) cooling is slow afterwards tears PDMS in template, gets out entrance, outlet, then cut into suitable size in PDMS substrate corresponding position;
(4) porous PDMS film production is PDMS and solidifying agent 15:1 mixing in mass ratio, and sol evenning machine 3000rpm, 1 minute, waters the die surface being coated in step (1) and preparing, smoke for 65 DEG C and spend the night after vacuumizing; Cooling is slow afterwards tears PDMS in template, for subsequent use.
Su-8 photoetching technique: novel chemical amplification type negative-appearing image SU-8 photoresist material overcomes the problem that common photoresist material adopts UV photoetching depth-to-width ratio deficiency, extremely be suitable for preparing high aspect ratio microstructures, therefore SU-8 glue is a kind of negativity, epoxide resin type, near-ultraviolet ray photoetching glue.Its absorbance in near-ultraviolet light (365nm-400nm) scope is very low, and the exposure uniformity that whole photoresist layer obtains, the thick film figure with vertical side wall and high aspect ratio can be obtained; It also has good mechanical property, resistance to chemical corrosion and thermostability; SU-8 occurs crosslinked after being subject to uv-radiation, is a kind of chemical amplification negative photoresist, can forms the baroque figures such as step; And SU-8 glue is non-conductive, can directly use as isolator when electroplating.Because it has more advantages, SU-8 glue is just applied to the fields such as MFMS, Chip Packaging and micro Process gradually.The new technology that the high microstructure of depth-to-width ratio and micro-part are micro Process fields prepared by direct employing SU-8 photoresist material.
Present invention also offers the application of above-mentioned micro-fluidic chip in monitoring Nasopharyngeal neoplasms process.Use the operation steps of micro-fluidic chip of the present invention monitoring interior tumor cell transfer process as follows:
1, essence simulation: the one side of porous PDMS film 4 ingress of air in micro-fluidic chip by epithelial cell and tumour cell Dual culture.
2, interstitial simulation: the one side of porous PDMS film 4 contact liq in micro-fluidic chip by vascular endothelial cell and mesenchymal cell (such as inoblast, monocyte) Dual culture.
3, Nasopharyngeal neoplasms target organ simulation: carry out 3D cultivation by cell culture chamber 31,32,33 respectively in micro-fluidic chip of brain tissue cell, bone and its cells, hepatic tissue cell.
4, the validation checking of the metastases microenvironment of external structure is carried out
By detecting cell viability, the compactness that vascular endothelial cell is connected with epithelial cell, the carcinoma-associated fibroblasts of tumor cell induction, the expression of scavenger cell specific proteins carry out the validity of the bionic model of the simulation Nasopharyngeal neoplasms microenvironment that evaluation ideas micro-fluidic chip of the present invention builds.
5, the detection of Nasopharyngeal neoplasms process is carried out
The generation that (EMT) labelled protein expresses evaluation metastatic cancer cell is transformed by detecting tumour cell generation epithelial-mesenchymal; By the observation and comparison metastatic cancer cell growth pattern of cellular form; Transform (MET) labelled protein by tumour cell generation mesenchymal-epithelial and express the change evaluated after metastatic cancer cell moves to target organ; Evaluate metastatic cancer cell by the expression detecting target organ dysfunction labelled protein whether to have attacked in target organ.
In specific embodiment of the invention scheme, epithelial cell is bronchial epithelial cell; Tumour cell is lung carcinoma cell, namely uses micro-fluidic chip of the present invention to construct the bionic model of a simulated lung cancer metastasis microenvironment.
Principle: the present invention adopts PDMS and the meshed permeable membrane material of band, according to cells in vivo and cell, cell and developing medium, between tissue and tissue, the characteristic of organ and microenvironment interphase interaction and fluid mechanics principle, designing and making one can the high-throughput Microfluid based Lab on a chip of, hyperchannel connection integrated close to the multiple-unit of lung anatomical structure, simulated lung physiological function.The key problem of this lab design is the anatomical structure of how to rebuild lung, comprises the main Physiological Function of segmental bronchus at different levels, the pulmonary parenchyma of alveolar and interstitial lung and how simulated lung, i.e. gaseous interchange.The spandex fiber that the alveolar of expansion bounces back can be impelled when substituting respiratory movement with the meshed permeable membrane of one deck band, by at film upper and lower surface, bronchial epithelial cell, vascular endothelial cell, scavenger cell, fibroblastic two dimension are cultivated respectively, simulated lung essence and interstitial, and constitute ABB, then supply oxygen and divide and nutritive substance passing into gas and liquid respectively with film parallel direction, two vacuum tunnels are being connected, the simulation human body respiration rhythm and pace of moving things with the both sides of film vertical direction.When vacuum tunnel pumps into gas, permeable membrane bounces back, and gas passage oxygen pumps into, alveolar ectasia, simulation lungs air suction function; When vacuum channel pull gas, permeable membrane stretches, and gas passage oxygen pumps, and alveolar shrinks, simulation lungs expiration function.So far, construct one close to lung anatomical structure, can the bionic lung chip physiological models of simulated lung main Physiological Function.On above-mentioned bionical chip lung physiological models basis, originate from tunica mucosa bronchiorum epithelium according to most lung cancer, lung cancer is inoculated in bronchial epithelial cell district respectively, build bionical chip lung cancer pathological model; According to lung cancer metastasis approach and predilection site, above-mentioned lung cancer pathological model is built neurogliocyte culturing room, osteocyte culturing room and liver cell culture room and simulates the target organ that the lung cancer such as brain, bone, liver the most often shift respectively, build the different transition phase pathological model of lung cancer, reproduce invasion of lung cancer transfer whole process.
Advantage of the present invention and beneficial effect are:
(1) micro-fluidic chip of the present invention can realize the dimensional culture of cell, carrys out biological function research in better perfect aspect; This micro-fluidic chip for the reagent consumption that detects significantly lower than traditional platform.
(2) cell is cultivated and is had good adaptability in micro-fluidic chip platform of the present invention; The main raw of micro-fluidic chip is the ventilation property that PDMS, PDMS have good biocompatibility and height, and gas can be smooth and easy by PDMS, thus ensure that the gaseous interchange of cultured cells and western medium in chip.
(4) on traditional platform, the static macro environment of Growth of Cells and cells in vivo small solid space of surviving differs greatly, micro-fluidic chip system then compensate for traditional platform defect in this respect, it has better stopping property, therefore the size in cells survival space is more similar to the micro-space of the physiology that human inner cell survives, and the original position on-line checkingi in this short space also further increases the confidence level of detected result.
(5) micro-fluidic chip platform operations of the present invention is easy, time saving and energy saving.
Accompanying drawing explanation
Fig. 1 shows the exploded view of microfluidic chip structure of the present invention;
Fig. 2 shows the exploded view of microfluidic chip structure of the present invention;
Fig. 3 shows the integrally-built vertical view of micro-fluidic chip of the present invention;
Fig. 4 shows the side-view of the first layer substrate structure of micro-fluidic chip of the present invention;
Fig. 5 shows the side-view of the second layer substrate structure of micro-fluidic chip of the present invention;
Fig. 6 shows the side-view of the third layer substrate structure of micro-fluidic chip of the present invention;
Fig. 7 shows the side-view of the porous PDMS film of micro-fluidic chip of the present invention;
Fig. 8 display utilizes cell viability in H33342/PI staining examine chip and utilizes immunostaining to detect the connection of bronchial epithelial cell 16HBE and vascular endothelial cell HUVEC, wherein, and the cellular form after A:16HBE cell 24H; Cellular form after B:HUVEC cell 24H; Cellular form after C:16HBE cell 48H; Cellular form after D:HUVEC cell 48H; E:16HBE cell H33342/PI dyes: F:HUVEC cell H33342/PI dyes; E-cadherin protein immunization dyeing in G:16HBE cell; E-cadherin protein immunization dyeing in H:HUVEC cell;
Fig. 9 display utilizes the common positioning scenarios of Immunofluorescence cell tracker expression assay detection of lung cancer cell A549 and bronchial epithelial cell 16HBE and utilizes the expression of specific proteins of immunostaining detection of lung cancer cell, inoblast, scavenger cell, wherein, A:16HBE cellular form, the cellular form of B:16HBE and A549 Dual culture; C:16HBE cell cell tracker dyes; D:16HBE and A549 Dual culture cell tracker dyes; E:16HBE cell CEA protein immunization dyes; F:16HBE and A549 Dual culture CEA protein immunization dyes; G: α-SMA protein immunization dyeing in inoblast; α-SMA protein immunization dyeing in H:CAF (carcinoma-associated fibroblasts); I: CD206 protein immunization dyeing in scavenger cell; CD206 protein immunization dyeing in J:CAM (cancer associated macrophages);
Figure 10 display utilizes the expression of epithelial cell mesenchymal transformation marker in immunostaining detection of lung cancer cell, wherein, and A: cellular immunization colored graph; B: optical density value statistical graph;
Figure 11 shows lung cell A549 to the migration situation of remote organization and the growth pattern at place of remote organization, wherein, and A: lung carcinoma cell migrating cell number quantitative statistics; B: the cellular form that lung carcinoma cell is located in remote organization;
Figure 12 display utilizes immunostaining to detect the expression of characteristic protein in remote organization, wherein, and A: characteristic protein immunostaining figure; B: optical density value statistical graph;
Wherein, 1: the first layer substrate; 11: air passageways; 12: entrance; 13: outlet; 14,15: vacuum tunnel; 2: second layer substrate; 21: fluid passage; 22: entrance; 23: outlet; 211,212,213: connecting passage; 214,215,216: entrance; 3: third layer substrate; 31,32,33: cell culture chamber; 4,5: porous PDMS film.
Embodiment
Can more easily understand content of the present invention by consulting following embodiment, these embodiments just for further illustrating the present invention, and do not mean that restriction scope of the present invention.
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1 one kinds is for the bionical micro-fluidic chip of lung cancer metastasis microenvironment in analogue body
Micro-fluidic chip of the present invention is an airtight entirety by three layers of PDMS substrate and the interlaced irreversible sealing-in of two-layer porous PDMS film; The first layer PDMS substrate 1 is provided with for the passage 11 of air circulation, the entrance 12 passed in and out for liquid and outlet 13, lays respectively at upper part of the vacuum tunnel 14,15 of passage 11 both sides; Second layer PDMS substrate 2 is provided with the passage 21 circulated for liquid, the entrance 22 passed in and out for liquid and outlet 23, lay respectively at lower part of the vacuum tunnel 14,15 of passage 21 both sides, the side of passage 21 is provided with three outward extending connecting passages 211,212,213, and the end of connecting passage 211,212,213 is respectively equipped with entrance 214,215,216; Third layer PDMS substrate 3 is provided with the cell culture chamber 31,32,33 for cell cultures; Cell culture chamber 31,32,33 lays respectively at the below of connecting passage 211,212,213, is communicated with connecting passage 211,212,213 by porous PDMS film 5; The vacuum tunnel 14 of upper part formation structural integrity corresponding with lower part structure of vacuum tunnel 14,15 of vacuum tunnel 14,15 and vacuum tunnel 15; Entrance 12 and outlet 13 are arranged at the upstream and downstream of passage 11 and homonymy respectively; Entrance 22 and outlet 23 are arranged at the upstream and downstream of passage 21 and homonymy, and passage 11 is positioned at directly over passage 21, and the opening of passage 11 is relative with the opening of passage 21 and separated by porous-film 4; Passage 11 covers the upstream extremity of passage 21, and connecting passage 211,212,213 is positioned at the downstream end of passage 21.
The thickness of porous PDMS film 4 and porous PDMS film 5 is 10 μm, aperture is 10 μm.
The cross section of passage 11 and passage 21 is rectangle, is of a size of: long 10mm × wide 4mm, long 35mm × wide 4mm; The cross section of passage 14,15 is rectangle, is of a size of: high 7mm, wide 2mm; The shape of cross section of cell culture chamber is rectangle, is of a size of: high 1.5mm, wide 1.5mm.
The length of passage 11 is 10mm; The length of passage 21 is 35mm; The distance of one end of connecting passage 211,212,213 distance passage 21 is 2.2mm.
The distance of entrance 12 and outlet 13 is 15mm; The distance of entrance 22 and outlet 23 is 15mm.
Connecting passage 211,212,213 is parallel to each other.
Passage 14 is 2mm with the distance of passage 11; Passage 15 is 2mm with the distance of passage 11.
On micro-fluidic chip of the present invention, all passages are arranged at on-chip groove; Cell culture chamber on micro-fluidic chip of the present invention is arranged at on-chip groove.
The preparation of the micro-fluidic chip in embodiment 2 embodiment 1
The preparation method of the micro-fluidic chip in embodiment 1 comprises the following steps:
(1) microchannel in above-mentioned micro-fluidic chip, microstructure figure is drawn with computer aided design software CAD; Be printed on by figure as mask on SU-8 film (Microchem, model is 2075), adopt standard photolithography process to make mould, standard photolithography process is known for those skilled in the art;
(2) by PDMS (Dow Corning, article No.: 0007883528) and solidifying agent in mass ratio 10:1 mixing, after vacuum drying oven vacuumizes, water the die surface being coated in step (1) and preparing, 80 DEG C baking 1h;
(3) cooling is slow afterwards tears PDMS in template, gets out entrance, outlet, then cut into suitable size in PDMS substrate corresponding position;
(4) porous PDMS film production is PDMS and solidifying agent 15:1 mixing in mass ratio, and sol evenning machine 3000rpm, 1 minute, waters the die surface being coated in step (1) and preparing, smoke for 65 DEG C and spend the night after vacuumizing; Cooling is slow afterwards tears PDMS in template, for subsequent use.
The structure of the bionic model of lung carcinoma cell transfer microenvironment in embodiment 3 analogue body
1, in micro-fluidic chip, the 2D of lung carcinoma cell cultivates
(1) micro-fluidic chip uses uviolizing sterilization, and porous PDMS film is coated with BME, and concrete bag by process is: carry out pre-treatment to chip, be better attached to chip porous film surface in order to cell.By 1:10 dilution proportion BME (Cultrex basement membrane extract, R & D Systems, McKinley Place, MN, USA), fully inject the sample entrance port of micro-fluidic chip after mixing with micro sample adding appliance, incubator spends the night and waits for that gelling is solid.
(2) overturn by micro-fluidic chip, namely third layer PDMS substrate upward.Collect in centrifuge tube by the monocyte of suspension, centrifugal 5 minutes of 1000rpm, abandons supernatant, adds fresh culture and prepare cell suspension.Mononuclear cell suspension is injected in passage 21 by entrance 22, makes it with 10 3individual/cm 2density plant on porous-film PDMS film, afterwards use syringe pump with the volumetric flow rate of 24mm/h, PMA substratum (100ng/ml) is injected in micro-fluidic chip by entrance 22, unnecessary substratum is got rid of by outlet 23.Micro-fluidic chip is tilted to allow signaling to one side of passage 21.Chip is tilted to 30 degree, side, with the side making monocyte be deposited to central culture channel, is placed in 37 DEG C, 5%CO 2incubator is cultivated.Monocyte after 48h is stimulated as scavenger cell.
(3), after stimulating monocyte to become M0 scavenger cell, PMA substratum is replaced with normal incubation medium (1640 substratum).
(4) human lung fibroblast is planted with 10 4individual/cm 2the density position of planting M0 macrophage growth (treat that cell proliferation is to logarithmic phase, use 0.25% tryptic digestion, add fresh substratum and blow and beat into cell suspension, centrifugal 5 minutes of 1000rpm, abandon supernatant, add fresh culture, make inoblast suspension.By lung fibroblast WI38 with 10 4individual cell/cm 2cell density be inoculated into the homonymy of scavenger cell, make it be attached to porous film surface, be placed in 37 DEG C, 5%CO 2cultivate 4 hours under incubator static conditions.
(5) micro-fluidic chip is returned to horizontality, vascular endothelial cell HUVEC suspension is injected in passage 21 by entrance 22, makes it with 10 4individual/cm 2density plant on porous PDMS film, stick to the other side of passage 21.
(6) after the equal adherent growth of cell on the downside of chip porous-film, turning-over of chip passes through entrance 2 with 10 from upside 4individual cell/cm 2cell density by bronchial epithelial cell 16HBE inject chip, static state make its be attached to film surface 4 hours after, carefully from outlet 2 by substratum lightly from upper channel suction, continue through entrance and be continuously pumped into mixed culture medium, be placed in 37 DEG C, 5%CO 2incubator is cultivated.Substratum is got rid of by outlet 13.
(7) after above-mentioned cell covers with, lung carcinoma cell suspension is injected in passage 11 by entrance 12, makes it with 10 3individual/cm 2density plant on porous PDMS film and be inoculated into bronchial epithelial cell district, chip upper strata, standing 4h makes it attach.Substratum is got rid of by outlet 13.
2, the 3D of micro-fluidic chip mesencephalic tissue cell, bone and its cells, hepatic tissue cell cultivates
(1) by astroglia cell, scleroblast and liver cell (purchased from Shanghai Sheng Ke institute of Chinese Academy of Sciences cell centre) use trysinization, it is made to be resuspended in (R & D in the cell based counterdie extract mixtures of ice precooling, supplement article No.), respectively cell suspension is mixed with BME equal-volume.
(2) micro-fluidic chip is placed in the standing 30min of 37 DEG C of environment and makes BME gel.By entrance 22, substratum is injected micro-fluidic chip, be continuously pumped into.Simultaneously, outlet 23 is sealed, unnecessary substratum is discharged from entrance 214,215,216.
(4) micro-fluidic chip is positioned over humidity is 95%, gas concentration lwevel is cultivate in 37 DEG C of incubators of 5% that (vacuum pump parameter is physiological cyclic strain (10%at 0.2Hz).Incubation time 24h, carries out subsequent experimental.
The validation checking of the bionic model of lung carcinoma cell transfer microenvironment in embodiment 4 analogue body
The validity of the bionic model of lung carcinoma cell transfer microenvironment in the analogue body that Evaluation operation example 3 builds, come by following experiment:
1, the detection of cell viability
Detection method: suck nutrient solution in passage in chip system, injects PBS in chip channel, the cell of cleaning different treatment group 2 times; Pump into H33342 (1:100) afterwards to dye 15 minutes, PBS solution cleans 2 times; After pump into PI dyeing (1:200) 5 minutes, PBS solution cleans 2 times; Under the microscope, the fluorescence intensity under corresponding excitation is observed and photographic recording.
2, lung carcinoma cell and the bronchial epithelial cell detection of locating altogether
Lung carcinoma cell is labeled as redness by Cellular tracking agent C7000CM-DiL: before chip inoculation, lung carcinoma cell is resuspended in (1 μ g/ μ L) 37 DEG C in C7000CM-DiL working fluid and hatches 5min, places 15min for 4 DEG C.
3, the observation of the growth pattern of human bronchial's epithelial cell and vascular endothelial cell
Use the compactness that the immunostaining of E cadherin detection micro-fluidic chip mesobronchus epithelial cell is connected with vascular endothelial cell.E cadherin immunostaining process is as follows:
A, the bronchial epithelial cell using film both sides in PBS flushing micro-fluidic chip and vascular endothelial cell;
B, use 4% paraformaldehyde fixed cell 15min, 0.5%PBST solution rupture of membranes 10 minutes, PBS solution cleans 2 times;
C, serum dilution will be closed inject chip cultivation pool, and put into wet box, hatch 1 hour for 37 DEG C; Dilute sheep with freshly prepared 5%BSA and close serum stoste, according to the dilution proportion of 1:100, mix with vibrator;
D, E cadherin antibody (Abcam) diluent using final concentration to be 2 μ g/mL hatch 2h;
E, two of Alexa 594 coupling are used anti-ly to hatch 1h;
F, the DAPI dyeing using final concentration to be 10 μ g/mL, the time is 15min; Use fluorescent microscope is taken a picture.
4, detection of lung cancer cell, with carcinoma-associated fibroblasts, scavenger cell feature
Use the expression of the method detection tumour cell labelled protein CEA of immunostaining, the expression of inoblast labelled protein α-SMA, the expression of macrophage marker PROTEIN C D206, mark result uses immunofluorescence microscopy to present.
Immunostaining process is as follows:
A, use PBS clean lung carcinoma cell, inoblast, scavenger cell in micro-fluidic chip;
B, use 4% paraformaldehyde fixed cell 15min, 0.5%PBST solution rupture of membranes 10 minutes, PBS solution cleans 2 times;
C, serum dilution will be closed inject chip cultivation pool, and put into wet box, hatch 1 hour for 37 DEG C.Dilute sheep with freshly prepared 5%BSA and close serum stoste, according to the dilution proportion of 1:100, mix with vibrator;
E, use the antibody (1:100, abcam) of anti-human CEA albumen, the antibody (1:200, Santa Cruz) of anti-human α-SMA albumen, the antibody (1:50, abcam) of anti-humen CD 20 6 albumen hatch 2h;
F, two of Alexa 488 or 594 coupling are used anti-ly to hatch 1h; Use final concentration is the DAPI dyeing of 10 μ g/mL, and the time is 15min; Use fluorescent microscope is taken a picture.
2, experimental result:
The result of 2.1 cell viabilities: cell viability more than 95% (Fig.8E-F).
2.2immunofluorescence cell tracker expression assay result shows that lung cell A549 and bronchial epithelial cell 16HBE locate altogether (figure Fig.9C-D).
2.3E-cadherin protein immunization dyes, and experimental result shows that bronchial epithelial cell 16HBE and vascular endothelial cell HUVEC is interconnected closely (Fig.8G-H).
In 2.4 lung carcinoma cells, CEA expresses (figure Fig.9E-F); Express alpha-SMA in inoblast, CD206 is expressed in scavenger cell, demonstrate lung carcinoma cell and mesenchymal cell to interact and cause mesenchymal cell to activate, inoblast is converted into carcinoma-associated fibroblasts α-SMA, and scavenger cell is converted into cancer associated macrophages and expresses CD206 (figure Fig.9G, 9H, 9I, 9J).
Embodiment 5 utilizes the bionic model monitoring lung carcinoma cell transfer process of lung carcinoma cell transfer microenvironment in analogue body
1, step
1.1 lung carcinoma cell generation epithelial-mesenchymal transform the detection of (EMT)
(EMT) marker protein is transformed: the expression of E cadherin, N cadherin, Snail1, Snail2 is evaluated lung carcinoma cell and whether be there occurs epithelial-mesenchymal conversion by detecting epithelial-mesenchymal.
Immune labeled process is as follows:
A, the lung carcinoma cell used on PBS cleaning porous-film;
B, use 4% paraformaldehyde fixed cell 15min, 0.5%PBST solution rupture of membranes 10 minutes, PBS solution cleans 2 times;
C, serum dilution will be closed inject chip cultivation pool, and put into wet box, hatch 1 hour for 37 DEG C; Dilute sheep with freshly prepared 5%BSA and close serum stoste, according to the dilution proportion of 1:100, mix with vibrator;
D, use anti-human E cadherin antibody (1:100, Proteintech), antibody (the 5 μ g/ml of anti-human N cadherin, abcam), the antibody (1:50 of anti-human Snail1 albumen, abcam), the antibody (1:400 of anti-human Snail2 albumen, Cell Signaling Technology), hatch 2h;
F, two of Alexa 488 or 594 coupling are used anti-ly to hatch 1h; Use final concentration is the DAPI dyeing of 10 μ g/mL, and the time is 15min; Use fluorescent microscope is taken a picture.
The qualification of 1.2 pulmonary metastasis pattern of cell growths
C34557 is used to mark lung carcinoma cell; C34557 working fluid (10 μm of ol/L) is joined in 1640 cell culture mediums, by cell incubation 20min; Use fresh culture to hatch 5min afterwards, repeat 4 times.(with Cellular tracking agent C34557, lung carcinoma cell is labeled as green in advance: lung carcinoma cell is resuspended in (10 μm of ol/L) 37 DEG C in C34557 working fluid hatches 20min before chip inoculation, then to be resuspended in 4 times of volume medium 37 DEG C hatch 5min).
The invasion and attack of 1.3 detection pulmonary metastasis cells
Organize the expression of damage score albumen to evaluate metastatic lung carcinoma cell whether can to attack and enter in the multiple remote organ of micro-fluidic chip by detecting.Use the antibody (1:100 of anti-human brain tissue impairment labelled protein CXCR4, abcam), anti-human osseous tissue damage score albumen RANKL antibody (1:50, Santa Cruz Biotechnology), anti-human liver tissue injury labelled protein AFP antibody carry out immunostaining.
Immunostaining process is as follows:
A, use PBS clean cell 2 times;
B, use 4% paraformaldehyde fixed cell 15min, 0.5%PBST solution rupture of membranes 10 minutes, PBS solution cleans 2 times;
C, serum dilution will be closed inject chip cultivation pool, and put into wet box, hatch 1 hour for 37 DEG C; Dilute sheep with freshly prepared 5%BSA and close serum stoste, according to the dilution proportion of 1:100, mix with vibrator;
E, use above-mentioned primary antibodie, hatch 2h;
F, two of Alexa 488 or 594 coupling are used anti-ly to hatch 1h; Use final concentration is the DAPI dyeing of 10 μ g/mL, and the time is 15 minutes; Use fluorescent microscope is taken a picture.
2, experimental result
2.1 lung carcinoma cell epithelial-mesenchymal transform
After the mesenchymal cell that lung carcinoma cell is relevant with cancer cultivates 4d, lung carcinoma cell expresses EMT, and 3 kinds of EMT marker (N-Ca, Snail1, Snail2) show that epithelial cell mesenchymal transformation (Fig.10A-B) has occurred lung carcinoma cell.
2.2 lung carcinoma cells shift at a distance
Cell counting is known, and the cell quantity that non-small cell lung cancer cell A549 adheres on brain tissue cell, bone and its cells, hepatic tissue cell upper porous film is 148 ± 8 respectively, 364 ± 16,299 ± 13; The cell quantity that small cell lung cancer cell H446 adheres on brain tissue cell, bone and its cells, hepatic tissue cell upper porous film is 255 ± 16 respectively, 128 ± 8,278 ± 18 (Fig.11A).The above results shows that the transcellular taxis of A549 is osseous tissue > hepatic tissue > cerebral tissue; The transcellular taxis of H446 is hepatic tissue > cerebral tissue > osseous tissue, and the Clinical symptoms of these characteristic sum lung cancer patients is similar.
2.3 metastatic lung carcinoma cells are at the growth pattern at multiple remote organ place
By inverted fluorescence microscope observation of cell form.As shown in Fig.11B, the cellular form transferring to far-end target organ is agglomerating.
The invasion and attack of 2.4 pulmonary metastasis cells
Result, as shown in Fig.12A and Fig.12B, is expressed in star spongiocyte H1800 in CXCR4, scleroblast Fob to express in RANKL, liver cell L-02 and is expressed AFP, show that pulmonary metastasis cell has been attacked in target organ cell.
By above-described embodiment, find that micro-fluidic chip of the present invention can the transfer of lung carcinoma cell and invasive procedure in analogue body effectively, for clinical formulation treatment plan provides basis.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.

Claims (10)

1., for analogue body inner tumour cell and the bionical micro-fluidic chip shifting microenvironment thereof, it is characterized in that, described micro-fluidic chip is an airtight entirety by three layers of PDMS substrate and the interlaced irreversible sealing-in of two-layer porous PDMS film; The first layer PDMS substrate 1 is provided with for the passage 11 of air circulation, the entrance 12 passed in and out for liquid and outlet 13, lays respectively at upper part of vacuum tunnel 14 and upper part of vacuum tunnel 15 of described passage 11 both sides; Second layer PDMS substrate 2 is provided with the passage 21 circulated for liquid, the entrance 22 passed in and out for liquid and outlet 23, lay respectively at lower part of vacuum tunnel 14 and lower part of vacuum tunnel 15 of described passage 21 both sides, the side of described passage 21 is provided with three outward extending connecting passages 211, connecting passage 212, connecting passages 213, and the end of described connecting passage 211, connecting passage 212, connecting passage 213 is respectively equipped with entrance 214, entrance 215, entrance 216; Third layer PDMS substrate 3 is provided with cell culture chamber 31, cell culture chamber 32, cell culture chamber 33 for cell cultures; Described cell culture chamber 31, cell culture chamber 32, cell culture chamber 33 lay respectively at the below of described connecting passage 211, connecting passage 212, connecting passage 213, are communicated with described connecting passage 211, connecting passage 212, connecting passage 213 by porous PDMS film 5; The described vacuum tunnel 14 of upper part of described vacuum tunnel 14, upper part of described vacuum tunnel 15 and lower part of described vacuum tunnel 14, lower part structure corresponding formation structural integrity of described vacuum tunnel 15 and described vacuum tunnel 15; Described entrance 12 and described outlet 13 are arranged at the upstream and downstream of passage 11 and homonymy respectively; Described entrance 22 and described outlet 23 are arranged at the upstream and downstream of described passage 21 and homonymy respectively, and described passage 11 is positioned at directly over described passage 21, and the opening of described passage 11 is relative with the opening of described passage 21 and separated by described porous PDMS film 4; Described passage 11 covers the upstream extremity of described passage 21, and described connecting passage 211, connecting passage 212, connecting passage 213 are positioned at the downstream end of described passage 21.
2. micro-fluidic chip according to claim 1, is characterized in that, the thickness of described porous PDMS film is 10 μm, aperture is 10 μm.
3. micro-fluidic chip according to claim 1, is characterized in that, the cross section of described passage 11 and described passage 21 is rectangle, is of a size of: long 10mm × wide 4mm, long 35mm × wide 4mm; The cross section of described passage 14, described passage 15 is rectangle, is of a size of: long 7mm, wide 2mm; The shape of cross section of described cell culture chamber is rectangle, is of a size of: high 1.5mm, wide 1.5mm.
4. micro-fluidic chip according to claim 1, is characterized in that, the length of described passage 11 is 10mm; The length of described passage 21 is 35mm; Described connecting passage 211, connecting passage 212, connecting passage 213 are 2.2mm apart from the distance of the upstream extremity of described passage 21.
5. micro-fluidic chip according to claim 1, is characterized in that, the distance of described entrance 12 and described outlet 13 is 15mm; The distance of described entrance 22 and described outlet 23 is 15mm.
6. micro-fluidic chip according to claim 1, is characterized in that, described connecting passage 211, connecting passage 212, connecting passage 213 are parallel to each other.
7. micro-fluidic chip according to claim 1, is characterized in that, described passage 14 is 2mm with the distance of described passage 11; Described passage 15 is 2mm with the distance of described passage 11.
8. a preparation method for the micro-fluidic chip according to any one of claim 1-7, is characterized in that, described preparation method comprises the following steps:
(1) microchannel during preparation has according to any one of claim 1-7 a micro-fluidic chip and the SU-8 formpiston of microstructure;
(2) the SU-8 formpiston prepared with step (1), for template, is that raw material copies with PDMS, is prepared into the first layer PDMS substrate according to any one of claim 1-7, second layer PDMS substrate, third layer PDMS substrate;
(3) two porous PDMS films according to claim 1 are prepared;
(4) two interlaced placement sealing-ins of described porous PDMS film prepared by described the first layer PDMS substrate step (2) prepared, described second layer PDMS substrate, described third layer PDMS substrate and step (3) form.
9. preparation method according to claim 8, is characterized in that, described preparation method comprises the following steps:
(1) microchannel in the micro-fluidic chip of computer aided design software CAD drafting according to any one of claim 1-7 and microstructure is used; Drafting figure is printed on as mask on SU-8 film, adopts standard photolithography process to make mould;
(2) by PDMS and solidifying agent 10:1 mixing in mass ratio, water the die surface being coated in step (1) and preparing after vacuumizing, smoke 1h for 80 DEG C;
(3) cooling is slow afterwards tears PDMS in template, gets out entrance, outlet, then cut into suitable size in PDMS substrate corresponding position;
(4) porous PDMS film production is PDMS and solidifying agent 15:1 mixing in mass ratio, and sol evenning machine 3000rpm, 1 minute, waters the die surface being coated in step (1) and preparing, smoke for 65 DEG C and spend the night after vacuumizing; Cooling is slow afterwards tears PDMS in template, for subsequent use.
10. the micro-fluidic chip according to any one of claim 1-7 is detecting the application in Nasopharyngeal neoplasms process.
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