CN102816682B - Micro-fluidic chip and method for studying exosmosis of tumor cell cluster - Google Patents
Micro-fluidic chip and method for studying exosmosis of tumor cell cluster Download PDFInfo
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- CN102816682B CN102816682B CN201110151866.9A CN201110151866A CN102816682B CN 102816682 B CN102816682 B CN 102816682B CN 201110151866 A CN201110151866 A CN 201110151866A CN 102816682 B CN102816682 B CN 102816682B
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Abstract
The invention provides a micro-fluidic chip and a method for studying exosmosis of a tumor cell cluster. The micro-fluidic chip is composed of one to three vascular units which are connected successively in an end-to-end manner to form a microvascular system, wherein two ends of the microvascular system are connected with a nutrient solution injection port and a waste reservoir; each vascular unit comprises a main channel and five side channels; the side channels are disposed on one side of the main channel and are parallel mutually; the side channels are vertically connected with the main channel; the height of each side channel is lower than that of the main channel; and the side channels of each vascular unit are separately connected with independent matrix injection ports. The micro-fluidic chip can be used for studying the exosmosis link of the tumor metastasis process.
Description
Technical field
The present invention relates to micro-fluidic chip technology to be applied to biomedical research field a kind of method that provides especially micro-fluidic chip and study tumor cell group thereof to exosmose.
Background technology
Several key links of tumour hematogenous metastasis are: cell free goes out original site, through organizing barrier, intravasation, in circulation under survival, stick and the blood vessel of the target organ of overflowing, in the target organ step such as survive and grow.Tumour cell sticks and this process of target organ blood vessel of overflowing is called and exosmoses, and is the rate-limiting step in metastases process.This process of research at present adopts the method for setting up animal model, but experimentation on animals need to consume a large amount of time, manpower, fund, also relates to the problems such as animal ethics.Therefore, will provide an Important Platform as research tumour hematogenous metastasis taking external model simulation capillary blood vessel.Micro-fluidic chip technology, rapidly to biomedical sector infiltration, had shown wide application prospect in recent years, and increasing sign shows that micro-fluidic chip technology will become an epochmaking platform of biomedical research.The present invention can simulate and build the capillary blood vessel that comprises tube chamber, vascular endothelial cell and pipe Zhou Jizhi on chip, and study tumor cell group enters the process of exosmosing of pipe Zhou Jizhi through vascular endothelial cell, for research metastases provides an effective instrument.
Summary of the invention
A kind of method that the object of the present invention is to provide micro-fluidic chip and study tumor cell group thereof to exosmose, builds in vitro a microenvironment that more approaches Ti Nei vascular tissue structure to realize, thereby studies metastases.
The invention provides a kind of micro-fluidic chip, it is characterized in that: this micro-fluidic chip is made up of 1 ~ 3 blood vessel unit that structure is identical, blood vessel unit successively head and the tail connects and composes Microvasculature, and the two ends of Microvasculature are connected with waste liquid pool 2 with nutrient solution injection port 1 respectively; Each blood vessel unit comprises 3 and 5 of 1 main channels wing passage being parallel to each other 4, and wing passage 4 is positioned at a side of main channel 3 and is connected with main channel 3 is vertical; The wing passage 4 of each blood vessel unit is connected with matrix injection port 5 independently; Wherein, described wing passage 4 is positioned at a side of main channel 3, and its height is lower than main channel 3.
In micro-fluidic chip provided by the invention, each blood vessel unit comprises main channel and wing passage; Wing passage is concentrated a side that is positioned at main channel, and wing passage height, lower than main channel, therefore forms fluid blocking-up and connects between main channel and wing passage, prevents that the matrix of matrix sample introduction process medium fluid state from entering in main channel.
Wherein, the upper layer of material of described micro-fluidic chip is preferably PDMS polymkeric substance, and lower floor is glass, the irreversible sealing-in of PDMS and glass material after Cement Composite Treated by Plasma, and make PDMS surface be converted into hydrophily by hydrophobic state.
The present invention also provides a kind of method of exosmosing based on micro-fluidic chip study tumor cell group, and its detailed process is as follows:
---the extracellular matrix surrogate of fluid state is added in matrix injection port, and under the effect of capillary force, in this matrix inflow side passage, described surrogate is basement membrane-like material;
---after the extracellular matrix surrogate in wing passage forms gel, in nutrient solution injection port, add cell culture fluid, under the effect of capillary force, nutrient solution is full of main channel;
---vascular endothelial cell is added in main channel by nutrient solution injection port, chip is holded up, matrix injection port, leaves standstill 20 to 30 minutes down, makes vascular endothelial cell be attached at the surface of extracellular matrix surrogate, then chip is kept flat;
---the cell mass of tumour cell is added in main channel by nutrient solution injection port;
Wherein, described extracellular matrix surrogate room temperature leaves standstill and can be transformed into colloidal state by liquid state in 15 to 30 minutes.
The method that micro-fluidic chip provided by the invention and study tumor cell group thereof exosmose, can on the chip of more than square centimeters, simulate capillary blood vessel, build in vitro a microenvironment that more approaches Ti Nei vascular tissue structure, the link of exosmosing in simulation tumour cell transfer process, has important biomedical research value and economic worth.
Brief description of the drawings
Fig. 1 micro-fluidic chip schematic diagram of the present invention, wherein (1) nutrient solution injection port, (2) waste liquid pool, (3) main channel, (4) wing passage, (5) matrix injection port;
Fig. 2 shows the enlarged diagram of 1 blood vessel unit, wherein (3) main channel, (4) wing passage;
Fig. 3 micro-fluidic chip photo in kind, is full of dyestuff in chip;
Finding under the microscope of Fig. 4 main channel and wing passage junction, wherein (3) main channel, it acts on similar lumen of vessels, (6) vascular endothelial cell HUVEC, (7) chip material PDMS, plays a supportive role, (8) extracellular matrix surrogate, and it acts on similar blood vessel Zhou Jizhi;
Fig. 5 shows cell mass and the interactional situation of extracellular matrix surrogate of different time tumour cell ACC-M, wherein (1) 30 minute, (2) 24 hours, (3) 48 hours, (4) 72 hours;
Fig. 6 show different time ACC-M cell mass under Chemokine CXCL12 effect with the interactional situation of extracellular matrix surrogate, wherein (1) 30 minute, (2) 24 hours, (3) 48 hours, (4) 72 hours;
Fig. 7 shows cell mass and vascular endothelial cell HUVEC and the interactional situation of extracellular matrix surrogate of different time tumour cell ACC-M, wherein (1) 30 minute, (2) 24 hours, (3) 48 hours, (4) 72 hours, (5) HUVEC cell, (6) ACC-M cell mass.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
A kind of micro-fluidic chip, concrete structure is shown in Fig. 1 and Fig. 3, this micro-fluidic chip is made up of 3 blood vessel units, and the formation Microvasculature that joins end to end successively, and the two ends of Microvasculature are connected with waste liquid pool 2 with injection port 1; Fig. 2 specifically sees in blood vessel unit, and each blood vessel unit comprises 3 and 5 of 1 main channels wing passage being parallel to each other 4, and wing passage 4 is positioned at a side of main channel 3 and is connected with main channel 3 is vertical, and its height is lower than the height of main channel 3; The wing passage 4 of each blood vessel unit is connected with matrix injection port 5 independently respectively, therefore between main channel 3 and wing passage 4, forming fluid blocking-up connects, prevent from flowing into main channel in the matrix of extracellular matrix surrogate sample introduction process medium fluid state from wing passage, described chip upper layer of material is PDMS polymkeric substance, is packaged on lower floor's glass surface by irreversible sealing technology.
Use the micro-fluidic chip study tumor cell group in embodiment 1 to exosmose, in wing passage, add extracellular matrix surrogate (Basement Membrane Extract on ice by matrix injection port, BME), chip room temperature is placed 20 minutes, after BME forms gel, add cell culture fluid by nutrient solution injection port, then add the cell mass of tumour cell ACC-M by nutrient solution injection port, chip is placed in to the CO of 37 DEG C
2in incubator, cultivate, took pictures and determine cell relative position (see figure 5) respectively at 30 minutes, 24 hours, 48 hours, 72 hours.The position of ACC-M cell mass is substantially unchanged.
As described in Example 2, micro-fluidic chip used is to design in embodiment 1, in wing passage, add matrix BME on ice by matrix injection port, chip room temperature is placed 20 minutes, after BME forms gel, add cell culture fluid by nutrient solution injection port, then add the cell mass of tumour cell ACC-M by nutrient solution injection port, cell adds Chemokine CXCL12 (100 ng/ml) at matrix injection port after leaving standstill, and is placed in the CO of 37 DEG C
2in incubator, cultivate, took pictures and determine cell relative position (see figure 6) respectively at 30 minutes, 24 hours, 48 hours, 72 hours.Result demonstration, under the effect of CXCL12, ACC-M cell mass is attacked gradually and is entered in BME matrix.
As described in Example 2, micro-fluidic chip used is to design in embodiment 1, in wing passage, add BME on ice by matrix injection port, chip room temperature is placed 20 minutes, after BME forms gel, add cell culture fluid by nutrient solution injection port, then vascular endothelial cell HUVEC is added in main channel by nutrient solution injection port, chip is holded up, matrix injection port down, leave standstill 30 minutes, make HUVEC be attached at BME stromal surface, then chip is kept flat, add afterwards the cell mass of tumour cell ACC-M by nutrient solution injection port, chip is placed in to the CO of 37 DEG C
2in incubator, cultivate, took pictures and determine cell relative position (see figure 7) respectively at 30 minutes, 24 hours, 48 hours, 72 hours.The position of ACC-M cell mass is substantially unchanged.
Claims (3)
1. a micro-fluidic chip, it is characterized in that: this micro-fluidic chip is made up of 1～3 blood vessel unit that structure is identical, blood vessel unit successively head and the tail connects and composes Microvasculature, and the two ends of Microvasculature are connected with waste liquid pool (2) with nutrient solution injection port (1) respectively; Each blood vessel unit comprises 1 main channel (3) and 5 wing passages that are parallel to each other (4), wing passage (4) be positioned at a side of main channel (3) and with vertical being connected of main channel (3), its height lower than main channel (3); The wing passage (4) of each blood vessel unit is connected with matrix injection port (5) independently.
2. according to micro-fluidic chip claimed in claim 1, it is characterized in that: the upper layer of material of described micro-fluidic chip is PDMS polymkeric substance, lower floor is glass.
3. the method based on micro-fluidic chip study tumor cell group exosmoses described in claim 1, its detailed process is as follows:
---by the extracellular matrix surrogate of fluid state, by matrix injection port joining side passage, wherein, described surrogate is basement membrane-like material;
---after the extracellular matrix surrogate in wing passage forms gel, in nutrient solution injection port, add cell culture fluid;
---vascular endothelial cell is added in main channel by nutrient solution injection port, chip is holded up, matrix injection port down, leaves standstill and sets level after 20 to 30 minutes;
---after vascular endothelial cell is attached at extracellular matrix surrogate, the cell mass of tumour cell is added in main channel by nutrient solution injection port;
Described extracellular matrix surrogate leaves standstill and changes colloidal state into by liquid state in 15 to 30 minutes in room temperature.
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| CN101745121A (en) * | 2008-12-08 | 2010-06-23 | 中国科学院大连化学物理研究所 | Genetic therapy breast cancer drug preparation method based on microfluidic chip |
| CN101782569A (en) * | 2009-01-21 | 2010-07-21 | 中国科学院大连化学物理研究所 | Microfluidic chip group used for screening formyl peptide receptor agonist and screening method |
| CN102021116A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院大连化学物理研究所 | Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same |
| CN102071138A (en) * | 2009-11-23 | 2011-05-25 | 中国科学院大连化学物理研究所 | Microfluidic chip and application thereof |
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| CN101745121A (en) * | 2008-12-08 | 2010-06-23 | 中国科学院大连化学物理研究所 | Genetic therapy breast cancer drug preparation method based on microfluidic chip |
| CN101782569A (en) * | 2009-01-21 | 2010-07-21 | 中国科学院大连化学物理研究所 | Microfluidic chip group used for screening formyl peptide receptor agonist and screening method |
| CN102021116A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院大连化学物理研究所 | Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same |
| CN102071138A (en) * | 2009-11-23 | 2011-05-25 | 中国科学院大连化学物理研究所 | Microfluidic chip and application thereof |
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