CN109517737A - A kind of micro-fluidic chip and metastasis models and model building method and application based on the chip - Google Patents
A kind of micro-fluidic chip and metastasis models and model building method and application based on the chip Download PDFInfo
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Abstract
The invention discloses a kind of micro-fluidic chip and the metastasis models based on the chip and model building method and application, the micro-fluidic chip include three layers of chip layer, two layers of perforated membrane and two layers of fixed plate；Every layer of chip layer is equipped with the cell culture chamber and width 0.1-10mm, high 10-500 μm of microfluidic channel of 1 0.1-10mm diameter；The aperture of the perforated membrane is 0.1-20 μm, can be used for that cell is adherent to be exchanged with mutual.The method for building up of metastasis models: using above-mentioned micro-fluidic chip, 1) collagen is fed into chip, it is coated with perforated membrane；2) vascular endothelial cell is fed into the chip second layer, constructs lumen of vessels model；3) tumour cell is fed into chip first layer, which can be used for the research of different Nasopharyngeal neoplasms, screening anti-tumor medicine, has the characteristics that stable, reliable and integrated level is high.
Description
Technical field
The invention belongs to the method for cell biology and organizational project, in particular to a kind of micro-fluidic chip and be based on the chip
Metastasis models and model building method and application.
Background technique
Tumour be to one of great disease of human health risk, have in tumor lethal patient according to statistics 90% all with tumour
It shifts related.But the physiological environment of humans and animals complexity, so that people is still known little about it the metastasis of tumour cell.Exploitation
Effective metastases real-time monitoring platform turns to Nasopharyngeal neoplasms process, mechanism and exploitation effective antitumour is fully understood
Shifting method is particularly important.Traditional animal model, due to people there are basic species variation so that anti-tumor drug
R&D cycle is long, it is high to spend.And existing traditional external model structure is simple, lacks physiological correlations, it is as a result often unreliable.
The new effective metastases research platform of exploitation becomes research hotspot.
In in the past few years, microfluidic chip technology is as the emerging technology developed rapidly in recent years, by its ruler
The features such as small, material and manufacture craft are various, flexible design is changeable is spent, field of biomedicine, conversion have been widely applied to
The related fields of the various clinicals such as medical domain medicine and basic research.Although many researchers are by micro-fluidic core
Chip technology carries out the research such as tumor cell migration, invasion, but rarely has the simulation to metastases overall process and antineoplastic
The research of object screening.We develop a kind of new micro-fluidic external model of three-dimensional nodule-blood vessel, and it is thin can not only to observe tumour
Proliferation, migration and the invasive procedure of born of the same parents, while can be used for the screening of anti-tumor drug.
Summary of the invention
The object of the present invention is to provide a kind of micro-fluidic chip and metastasis models and model construction based on the chip
Methods and applications.The present invention has the characteristics that easy to operate, integrated level is high, while simulating tumor vessel inner ring on a cellular level
Border has the metastasis models close to body physiological environment.
In order to achieve the above objectives, present invention employs the following technical solutions:
A kind of micro-fluidic chip, the micro-fluidic chip include three layers of chip layer and two layers of perforated membrane；Wherein, first layer is more
Pore membrane 6 is clipped between first layer chip layer 2 and second layer chip layer 3, and second layer perforated membrane 7 is clipped in second layer chip layer 3 and
Between three layers of chip layer 4；
Each layer of chip in three layers of chip layer is respectively provided with fluid inlet 8, microfluidic channel 9, cell culture chamber 10 and stream
Body outlet 11, wherein the both ends of microfluidic channel 9 are respectively fluid inlet 8 and fluid outlet 11, and the setting of cell culture chamber 10 exists
In microfluidic channel 9；Cell culture chamber in different layers is exchanged by perforated membrane.
Further, the micro-fluidic chip further includes two layers of fixed plate, and three layers of chip layer and two layers of perforated membrane are fixed on
Between two layers of fixed plate.
Preferably, the first layer chip layer 2 with a thickness of 0.5-10mm；Second layer chip layer 3 and third layer chip layer 4
With a thickness of 0.2-5mm, perforated membrane with a thickness of 0.1-30 μm, the aperture of perforated membrane is 0.1-20 μm.
Preferably, the height of the microfluidic channel 9 is 10-500 μm, width 0.1-10mm；Fluid inlet 8 and fluid
The diameter of outlet 11 is 0.1-5mm.
Preferably, the thickness of two layers of fixed plate is 0.5-10mm；First fixed plate 1 is inserted into equipped with several liquid-transport pipe-lines
Hole 13, the diameter that liquid-transport pipe-line is inserted into hole is 0.1-5mm.
Preferably, the material of chip layer is dimethyl silicone polymer (PDMS), and the material of perforated membrane is polycarbonate, fixed
The material of plate is PMMA.
The present invention also provides a kind of metastasis models based on micro-fluidic chip, the perforated membrane in micro-fluidic chip is adopted
It is coated with to form three-dimensional microenvironment with collagen；The microfluidic channel of second layer chip layer is perfused to be formed in vitro using vascular endothelial cell
Fine and close lumen of vessels；Tumour cell is injected in a manner of continuous perfusion in the microfluidic channel of first layer chip layer.
Invention also provides the construction method of above-mentioned metastasis models, the construction method the following steps are included:
Step 1: the perfusion of collagen in chip
The collagen for configuring final concentration of 0.1-10mg/ml, by collagen from every layer of chip layer in a manner of continuous pouring stream
Fluid inlet pours into microfluidic channel, and culture is incubated for collagen coating perforated membrane and forms three-dimensional microenvironment；
Step 2: lumen of vessels constructs on chip
Cultured vascular endothelial cell is digested for single cell suspension, by 0.1 × 106-10×106A/ml is unicellular outstanding
Liquid is injected in a manner of continuous perfusion in second layer chip layer microfluidic channel, is cultivated after filling single cell suspension, in the second layer
The lower surface of the cell culture chamber of chip layer forms fine and close cellular layer；Later by chip upside down, secondary pouring single cell suspension makes
The upper surface of second layer chip layer cell culture chamber forms fine and close cellular layer, and it is thin that driving single cell suspension continues culture blood vessel endothelium
Born of the same parents form external fine and close lumen of vessels；
Step 3: establishing metastasis models
Cultured tumour cell is digested for single cell suspension, by 0.1 × 106-10×106A/ml single cell suspension with
The mode of continuous perfusion is injected in first layer chip layer microfluidic channel, after filling tumor cell suspension, continues to cultivate, and is formed swollen
Tumor metastasis model.
Invention still further provides above-mentioned micro-fluidic chips or metastasis models to study in Nasopharyngeal neoplasms
With the application in screening anti-tumor medicine.
Preferably, Nasopharyngeal neoplasms research and screening anti-tumor medicine involved in tumour cell be breast cancer cell or
Liver cancer cells.
The present invention provides a kind of micro-fluidic chip, every layer of polydimethylsiloxanechip chip of the chip has independent microfluid
There are independent liquid inlet and outlet in channel, has independent cell culture chamber that can be exchanged by polycarbonate perforated membrane.
Two layers of fixed plate, three layers of chip layer and two layers of perforated membrane of the invention, which is combined, constitutes one group of chip system.
In the present invention, the fluid inlet and fluid outlet in pipeline infusion insertion hole and chip layer in the first fixed plate are distinguished
It corresponds, for liquid to be inputted microfluidic channel.It should be noted that when the pipeline infusion insertion hole in the first fixed plate
When needing to be connected to the fluid inlet of second layer chip layer and fluid outlet, need to beat in the corresponding position of first layer chip layer
Hole；Similarly, when the pipeline infusion insertion hole in the first fixed plate needs fluid inlet and fluid outlet with third layer chip layer
When connection, need to punch in the corresponding position of first layer chip layer and second layer chip layer.
The present invention provides the coated perforated membrane of collagen, collagen is 0.1-10mg/ml using concentration, forms cell culture chamber
Area be 0.0314-314mm2。
Preferably, upper layer fixed plate of the present invention has 12 liquid-transport pipe-line insertion holes；Two layers of fixed plate quadrangle has for spiral shell
The fixed fixation hole 14,15 of silk, diameter is 0.3-8mm.
Specifically, a kind of method for building up of the metastasis models based on micro-fluidic chip, using above-mentioned micro-fluidic chip,
It follows the steps below:
Step 1: the perfusion of collagen in chip
The collagen for configuring final concentration of 0.1-10mg/ml, with syringe pump in a manner of continuous pouring stream by collagen from every layer
Chip channel entrance pours into three-dimensional channel channel, and chip is placed in 37 DEG C of culture environments 2 hours, is incubated for gel and is coated with poly- carbon
Acid esters perforated membrane forms three-dimensional microenvironment；
Step 2: lumen of vessels constructs on chip
Cultured vascular endothelial cell (EAhy926) is digested for single cell suspension, with syringe pump by single cell suspension
(0.1-10*106A/ml) it is injected in a manner of continuous perfusion in the microfluidic channel of second layer chip layer, fill cell suspension
Afterwards, chip is placed in 37 DEG C of incubators 2 hours, the cell culture chamber lower surface of second layer chip layer forms fine and close cellular layer；
Later by chip upside down, secondary pouring cell suspension makes upper surface form fine and close cellular layer, then with syringe pump driving liquid after
Continuous culture vascular endothelial cell 3 days forms external fine and close lumen of vessels；
Step 3: establishing metastasis models
Cultured tumour cell is digested for single cell suspension, with syringe pump by single cell suspension (0.1-10*106A/
Ml) injected in a manner of continuous perfusion in the microfluidic channel of first layer chip layer, after filling cell suspension, in the incubator after
Continuous culture, forms metastasis models.
The application of metastasis models based on micro-fluidic chip can be used for the research of different Nasopharyngeal neoplasms features,
Specifically: microscopically observation is attached at the tumor cell proliferation ability of lumen of vessels upper surface, transfer ability, invades and invade bleeding
The ability of lumen, every 24 hours acquisition data, counts the cell quantity and moving distance of tumour cell.
The research of different Nasopharyngeal neoplasms features, the tumour cell studied have breast cancer cell (MDA-MB-231),
Liver cancer cells (HepG-2).
The screening of anti-tumor drug, specifically: the drug of various concentration is added in lumen of vessels flow fluid, medicine can be observed
Object transmits the inhibitory effect to tumor cell proliferation, migration and invasive ability by blood vessel.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of micro-fluidic chip of the present invention；
Fig. 2 is the structural schematic diagram of every layer of chip layer of the invention；
Fig. 3 is the floor map of perforated membrane of the present invention；
Fig. 4 is the structural schematic diagram of fixed plate of the present invention；
Fig. 5 is a kind of structure principle chart of the metastasis models based on micro-fluidic chip of the present invention；
Fig. 6 be a kind of metastasis models based on micro-fluidic chip of the present invention tumour cell MDA-MB-231 proliferation,
Migration, invasion overall process figure；
Fig. 7 is that a kind of tumor cell migration of the metastasis models based on micro-fluidic chip of the present invention designs and tumour is thin
Born of the same parents' HepG2 and MDA-MB-231 migration effect figure；
Fig. 8 is the present invention a kind of the tumour cell HepG2 and MDA-MB- of the metastasis models based on micro-fluidic chip
231 invasion effects and mode of invasion comparison diagram；
Fig. 9 be a kind of metastasis models based on micro-fluidic chip of the present invention 5Fu inhibit tumour cell HepG2 and
MDA-MB-231 proliferation migrates, the Statistical Comparison figure of invasion and conventional model；
Wherein, 1: the first fixed plate；2: first layer chip layer；3: second layer chip layer；4 third layer chip layers；5: the second
Fixed plate；6: first layer perforated membrane；7: second layer perforated membrane；8: fluid inlet；9: microfluidic channel；10: cell culture chamber；
11: fluid outlet；12: micropore；13: fluid line is inserted into hole；14: fixation hole；15: fixation hole.
Specific embodiment
Any feature is disclosed to obtain in this specification, it unless specifically stated, can be equivalent or with similar purpose by other
Alternative features are replaced.Unless specifically stated, each feature is an example in a series of equivalent or similar characteristics
?.It is described to understand the present invention just for the sake of help, it should not be considered as to concrete restriction of the invention.
Technical solution of the present invention is described in detail below with reference to embodiment.The reagent and biomaterial used below
It if not otherwise specified, is commercially produced product.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer suggests
Condition carry out.
1 micro-fluidic chip of embodiment
As shown in Figure 1, a kind of micro-fluidic chip, the micro-fluidic chip includes three layers of chip layer and two layers of perforated membrane；Its
In, first layer perforated membrane 6 is clipped between first layer chip layer 2 and second layer chip layer 3, and second layer perforated membrane 7 is clipped in the second layer
Between chip layer 3 and third layer chip layer 4；
As shown in Fig. 2, each layer of chip in three layers of chip layer is respectively provided with fluid inlet 8, microfluidic channel 9, cell training
Support room 10 and fluid outlet 11, wherein the both ends of microfluidic channel 9 are respectively fluid inlet 8 and fluid outlet 11, cell culture
Room 10 is arranged in microfluidic channel 9；Cell culture chamber in different layers is exchanged by perforated membrane.
The micro-fluidic chip further includes two layers of fixed plate (the first fixed plate 1 and the second fixed plate 5), three layers of chip layer and
Two layers of perforated membrane is fixed between two layers of fixed plate.
The first layer chip layer 2 with a thickness of 0.5-10mm；The thickness of second layer chip layer 3 and third layer chip layer 4
For 0.2-5mm, perforated membrane with a thickness of 0.1-30 μm, the aperture of the micropore 12 of perforated membrane is 0.1-20 μm.
The height of the microfluidic channel 9 is 10-500 μm, width 0.1-10mm；Fluid inlet 8 and fluid outlet 11
Diameter be 0.1-5mm.
As shown in figure 4, the thickness of two layers of fixed plate is 0.5-10mm；First fixed plate 1 is equipped with several liquid-transport pipe-lines
It is inserted into hole 13, there is fixation hole 14,15 for screw stays in two layers of fixed plate quadrangle, and the diameter in liquid-transport pipe-line insertion hole is
0.1-5mm。
The material of chip layer is dimethyl silicone polymer (PDMS), and the material of perforated membrane is polycarbonate, the material of fixed plate
Material is PMMA.
Establishing using the micro-fluidic chip of oneself design and production, there is the Vitro Tumor of physiological correlations to shift mould
Type does template construct PDMS chip with SU-8 glue using soft lithography, the chip made with punching pen get fluid inlet,
Fluid outlet and cell culture chamber, later by two layers of acrylic fixed plate clamping, fixed, one group of chip system of composition, picture 1-4
It is shown.
Metastasis models and its construction method of the embodiment 2 based on micro-fluidic chip
As shown in figure 5, a kind of metastasis models based on micro-fluidic chip, the perforated membrane in micro-fluidic chip uses glue
Primordial covering forms three-dimensional microenvironment；The microfluidic channel of second layer chip layer is perfused to form external densification using vascular endothelial cell
Lumen of vessels；Tumour cell is injected in a manner of continuous perfusion in the microfluidic channel of first layer chip layer.
The construction method of above-mentioned metastasis models, the construction method the following steps are included:
Step 1: the perfusion of collagen in chip
The collagen for configuring final concentration of 0.1-10mg/ml, with syringe pump in a manner of continuous pouring stream by collagen from every layer
Chip channel entrance pours into three-dimensional channel channel, and chip is placed in 37 DEG C of culture environments 2 hours, is incubated for gel and is coated with poly- carbon
Acid esters perforated membrane forms three-dimensional microenvironment；
Step 2: lumen of vessels constructs on chip
Cultured vascular endothelial cell (EAhy926) is digested for single cell suspension, with syringe pump by single cell suspension
(0.1-10*106A/ml) it is injected in a manner of continuous perfusion in the microfluidic channel of second layer chip layer, fill cell suspension
Afterwards, chip is placed in 37 DEG C of incubators 2 hours, the cell culture chamber lower surface of second layer chip layer forms fine and close cellular layer；
Later by chip upside down, secondary pouring cell suspension makes upper surface form fine and close cellular layer, then with syringe pump driving liquid after
Continuous culture vascular endothelial cell 3 days forms external fine and close lumen of vessels；
Step 3: establishing metastasis models
Cultured tumour cell is digested for single cell suspension, with syringe pump by single cell suspension (0.1-10*106A/
Ml) injected in a manner of continuous perfusion in the microfluidic channel of first layer chip layer, after filling cell suspension, in the incubator after
Continuous culture, forms metastasis models.
The real-time monitoring of 3 metastases overall process of embodiment
The metastasis models that micro-fluidic chip described in Application Example 1-2 is established are thin by cultured blood vessel endothelium
Born of the same parents (EAhy926) digestion is single cell suspension, with syringe pump by cell suspension (0.2*106A/ml) in a manner of continuous perfusion
It injects in second layer chip layer microfluidic channel, after filling cell suspension, chip is placed in 37 DEG C of incubators 2 hours, second
The cell culture chamber lower surface of layer chip layer forms fine and close cellular layer；Later by chip upside down, secondary pouring cell suspension makes
Surface forms fine and close cellular layer, then continues culture vascular endothelial cell 3 days with syringe pump driving liquid, forms external fine and close blood
Lumen.Cultured tumour cell is digested for single cell suspension, with syringe pump by cell suspension (0.1*106A/ml) with even
The mode of continuous perfusion injects first layer chip, after filling cell suspension in channel, stands 3h, and after cell is adherent, nothing is flowed on upper layer
Fetal calf serum (FBS) DMEM/F12 culture solution, lower layer flow the culture solution of DMEM/F12 containing 10%FBS, continue to train in the incubator
It supports 24 hours, tumor cell proliferation, migration, invasion overall process can be observed, as shown in Figure 6.
The different tumor cell migration features of embodiment 4
Cultured vascular endothelial cell (EAhy926) digestion is single by micro-fluidic chip described in Application Example 1
Cell suspension, with syringe pump by cell suspension (0.2*106A/ml) second layer chip, channel are injected in a manner of continuous perfusion
After inside filling cell suspension, chip is placed in 37 DEG C of incubators 2 hours, the cell culture chamber lower surface shape of second layer chip layer
At fine and close cellular layer；Later by chip upside down, secondary pouring cell suspension makes upper surface form fine and close cellular layer, then with injection
Pump driving liquid continues culture vascular endothelial cell 3 days, forms external fine and close lumen of vessels.Chip is split later, by another
PDMS chip (long 4mm, wide 4mm, high 1mm) is placed on 6 upper surface of upper layer perforated membrane, and it is 0.5mm that 6 diameters are equipped on the chip
Hole, as shown in Fig. 7 A (1) (2)；Tumor cell suspension (0.2*106/ml) is inoculated in the PDMS chip layer surface, stands 3h
Afterwards, another PDMS chip is removed, diameter is formed by 0.5mm cell mass (cell mass is formed based on the hole beaten), such as schemes
Shown in 7A (3) (4)；Last assembling chip simultaneously flows on 6 upper layer of perforated membrane without fetal calf serum (FBS) DMEM/F12 culture solution, under
The laminar flow culture solution of DMEM/F12 containing 10%FBS continues culture 12 hours in incubator, tumour cell can be observed along training
The direction of nutrient solution flowing migrates.As shown in Figure 7 B.
The different tumor cell invasion features of embodiment 5
Micro-fluidic chip described in Application Example 1, it is slender that cultured vascular endothelial cell (EAhy926), which is digested,
Born of the same parents' suspension, with syringe pump by cell suspension (0.2*106A/ml) second layer chip layer, channel are injected in a manner of continuous perfusion
After inside filling cell suspension, chip is placed in 37 DEG C of incubators 2 hours, the cell culture chamber lower surface shape of second layer chip layer
At fine and close cellular layer；Later by chip upside down, secondary pouring cell suspension makes upper surface form fine and close cellular layer.It will be cultured
Tumor cell of liver (HepG2) and breast cancer cell (MDA-MB-231) digestion are cell suspension, with syringe pump by cell suspension
(0.2*106A/ml) first layer chip layer is injected in a manner of continuous perfusion, it is quiet after filling cell suspension in microfluidic channel
3h is set, after cell is adherent, upper layer flowing flows DMEM/ containing 10%FBS without fetal calf serum (FBS) DMEM/F12 culture solution, lower layer
F12 culture solution continues culture 18 hours in the incubator, and HepG2 cell can be observed and directly destroy extracellular matrix across blood
Solencyte enters lumen of vessels, and MDA-MB-231 cytoclasis Intercellular protein invades lumen of vessels by space between cells, such as Fig. 8 A institute
Show, swept by three-dimension layer, can be further observed that this phenomenon, as shown in Figure 8 B, white is divided into tumour cell compared with highlights.
The evaluation of 6 5-Fu activity of resisting tumor metastasis of embodiment
The tumor proliferation of the foundation of micro-fluidic chip described in Application Example 1-5, migration, invasive model, in the second laminar flow
5-Fluorouracil (the 5-Fu of various concentration is added in dynamic culture solution (DMEM/F12+10%FBS)；0,0.1,1,10,100ug/
ML), continue culture 48 hours in the incubator, count the cell quantity of tumour cell, it is possible to find 5-Fu to HepG2 cell and
The proliferation of MDA-MB-231 cell has apparent inhibiting effect, as shown in Figure 9 A；It, can by counting tumor cell migration distance
It was found that 5-Fu has apparent inhibiting effect to the migration of HepG2 cell and MDA-MB-231 cell, as shown in Figure 9 B；Pass through statistics
The cell quantity of tumor cell invasion lumen of vessels, it is possible to find 5-Fu equally has apparent inhibiting effect to the invasion of two kinds of cells.
In addition, 5-Fu is to Nasopharyngeal neoplasms under the conditions of chip dynamic cultivation by comparing with staticizer Plate and transwell
There is more obvious inhibitory effect, as shown in Fig. 9 A, 9B, 9C.
Technological parameter (such as temperature, time) section bound value of the invention and interval value can realize this law,
Embodiment numerous to list herein.
Ordinary skill in the art knowledge can be used in the unspecified content of the present invention.
It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting.Although ginseng
It is described the invention in detail according to embodiment, it will be apparent to an ordinarily skilled person in the art that technical side of the invention
Case is modified or replaced equivalently, and without departure from the spirit and scope of technical solution of the present invention, should all be covered in the present invention
Scope of the claims in.
Claims (10)
1. a kind of micro-fluidic chip, which is characterized in that the micro-fluidic chip includes three layers of chip layer and two layers of perforated membrane；Its
In, first layer perforated membrane (6) is clipped between first layer chip layer (2) and second layer chip layer (3), second layer perforated membrane (7) folder
Between second layer chip layer (3) and third layer chip layer (4)；
Each layer of chip in three layers of chip layer be respectively provided with fluid inlet (8), microfluidic channel (9), cell culture chamber (10) and
Fluid outlet (11), wherein the both ends of microfluidic channel (9) are respectively fluid inlet (8) and fluid outlet (11), cell culture
Room (10) is arranged on microfluidic channel (9)；Cell culture chamber in different layers is exchanged by perforated membrane.
2. micro-fluidic chip according to claim 1, which is characterized in that the micro-fluidic chip further includes two layers of fixation
Plate, three layers of chip layer and two layers of perforated membrane are fixed between two layers of fixed plate.
3. micro-fluidic chip according to claim 1 or 2, which is characterized in that the first layer chip layer (2) with a thickness of
0.5-10mm；Second layer chip layer (3) and third layer chip layer (4) with a thickness of 0.2-5mm, perforated membrane with a thickness of 0.1-30
μm, the aperture of perforated membrane is 0.1-20 μm.
4. micro-fluidic chip according to claim 1-3, which is characterized in that the height of the microfluidic channel (9)
Degree is 10-500 μm, width 0.1-10mm；The diameter of fluid inlet (8) and fluid outlet (11) is 0.1-5mm.
5. micro-fluidic chip according to claim 1-4, which is characterized in that the thickness of two layers of fixed plate is
0.5-10mm；First fixed plate (1) is equipped with several liquid-transport pipe-lines insertion hole (13), and the diameter in liquid-transport pipe-line insertion hole is
0.1-5mm。
6. micro-fluidic chip according to claim 1-5, which is characterized in that the material of chip layer is poly dimethyl
Siloxanes, the material of perforated membrane are polycarbonate, and the material of fixed plate is PMMA.
7. the metastasis models based on any one of the claim 1-6 micro-fluidic chip, which is characterized in that micro-fluidic chip
In perforated membrane be coated with to form three-dimensional microenvironment using collagen；The cell culture chamber (10) of second layer chip layer uses blood vessel endothelium
Cell infusion forms external fine and close lumen of vessels；The microfluid that tumour cell injects first layer chip layer in a manner of continuous perfusion is logical
In road.
8. the construction method of metastasis models described in claim 7, the construction method the following steps are included:
Step 1: the perfusion of collagen in chip
The collagen for configuring final concentration of 0.1-10mg/ml, by collagen from the fluid of every layer of chip layer in a manner of continuous pouring stream
Entrance pours into microfluidic channel, and culture is incubated for collagen coating perforated membrane and forms three-dimensional microenvironment；
Step 2: lumen of vessels constructs on chip
Cultured vascular endothelial cell is digested for single cell suspension, by 0.1 × 106-10×106A/ml single cell suspension with
The mode of continuous perfusion is injected in second layer chip layer microfluidic channel, is cultivated after filling single cell suspension, in second layer chip
The lower surface of the cell culture chamber of layer forms fine and close cellular layer；Later by chip upside down, secondary pouring single cell suspension makes second
The upper surface of the cell culture chamber of layer chip layer forms fine and close cellular layer, and it is thin that driving single cell suspension continues culture blood vessel endothelium
Born of the same parents form external fine and close lumen of vessels；
Step 3: establishing metastasis models
Cultured tumour cell is digested for single cell suspension, by 0.1 × 106-10×106A/ml single cell suspension is with continuous
The mode of perfusion is injected in first layer chip layer microfluidic channel, after filling tumor cell suspension, continues to cultivate, and is formed tumour and is turned
Shifting formwork type.
9. micro-fluidic chip described in any one of claims 1-6 or metastasis models as claimed in claim 7 are thin in tumour
Application in born of the same parents' transfer research and screening anti-tumor medicine.
10. application according to claim 9, which is characterized in that Nasopharyngeal neoplasms research and screening anti-tumor medicine institute
The tumour cell being related to is breast cancer cell or liver cancer cells.
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