CN101748059A - Micro-fluidic chip and method for research on oriented movement of cell in three-dimensional medium - Google Patents
Micro-fluidic chip and method for research on oriented movement of cell in three-dimensional medium Download PDFInfo
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- CN101748059A CN101748059A CN200810229391A CN200810229391A CN101748059A CN 101748059 A CN101748059 A CN 101748059A CN 200810229391 A CN200810229391 A CN 200810229391A CN 200810229391 A CN200810229391 A CN 200810229391A CN 101748059 A CN101748059 A CN 101748059A
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Abstract
The invention provides a micro-fluidic chip and a method for research on oriented movement of cell in three-dimensional medium. The micro-fluidic chip comprises a cell injection port, a cell culture pool, two parallel perfusion channels and a waste liquid pool; the lower ends of the perfusion channels are connected with the waste liquid pool, the cell culture pool is connected with the two parallel perfusion channels, the upper end of the waste liquid pool is connected with the cell injection port; and the micro-fluidic chip is used to provide three-dimensional space support for growth and migration of the cell, simultaneously stable concentration gradient is formed in the three-dimensional space, and with the inducement of the chemoattractant, the cell moves in orientation in the three-dimensional medium. The invention has the advantages of simple and fast operation, low sample use level and the like.
Description
Technical field
The present invention relates to the micro-fluidic chip technology is applied to the biomedical research field, a kind of micro-fluidic chip and the research cell directed method that moves in three-dimensional medium is provided, utilize micro-fluidic chip to grow and move the three-dimensional space support is provided for cell, and in the stable concentration gradient of this three-dimensional space generation, under the inducing of chemoattractant, directed moving takes place in cell in three-dimensional medium, and can investigate two kinds of different cells moving under the same conditions simultaneously.
Background technology
The orientation of cell moves in processes such as fetal development, wound healing, tumor-infiltrated and transfer and plays a significant role.The model that the migration of tradition research cell in vitro is adopted often can only be observed cell the creeping of two-dimensional surface, and with the residing three-dimensional space of cells in vivo very big-difference is arranged.The micro-fluidic chip technology is a kind of new technology that grows up on the capillary electrophoresis basis early 1990s, quilt " nature " magazine was classified one of most important cutting edge technology of 21 century as in 2006, the micro-fluidic chip technology was permeated to biomedical sector rapidly in recent years, shown wide application prospect, increasing sign shows that the micro-fluidic chip technology will become an epochmaking platform of biomedical research.The present invention can growth and the migration for cell provide the three-dimensional medium support on chip, and utilizes lamina flow principle to generate stable concentration gradient, and inducing cell moves at the three-dimensional medium interior orientation.With the conventional two-dimensional research model relatively, the three-dimensional microenvironment that this micro-fluidic chip is provided for cell growth and migration is more near internal milieu.And the present invention is simple to operate, quick, is easy to the biomedical researcher and accepts.
Summary of the invention
The purpose of this invention is to provide a kind of micro-fluidic chip and the research cell directed method that moves in three-dimensional medium.
The invention provides a kind of micro-fluidic chip, this micro-fluidic chip is made up of syringe pump, two parallel perfusion passages, cell injection port, cell cultures pond, waste liquid pools; The upper end of perfusion passage connects syringe pump, and the lower end connects waste liquid pool; The cell cultures pond perfusion passage parallel with two links to each other, and the upper end in cell cultures pond links to each other with the cell injection port.
The material of micro-fluidic chip provided by the invention is the PDMS polymkeric substance, with the irreversible envelope of glass material.
Micro-fluidic chip provided by the invention, described cell cultures pond are oval.
The present invention also provides with micro-fluidic chip research cell directed method that moves in three-dimensional medium, and procedure is as follows: with the growth conditions good cell through trysinization, centrifugal, then with the cell that obtains with concentration 10
5/ ml and Cultrex Basement Membrane Extract (BME, R﹠amp; D Systems MN) at mixing on ice, adds in the cell cultures pond by the mixture of cell injection port with cell and BME, is positioned over the about 30min of room temperature and is frozen into glue to BME; After treating that BME forms gel, the upper end of both sides perfusion passage connects syringe pump, wherein pour into the cell culture fluid that contains chemokine in the lateral irrigation circulation road, opposite side pours into the cell culture fluid that does not contain chemokine as blank, flow velocity is 0.5 μ l/min, perfusion was cultivated 24 hours continuously, formed stable concentration gradient in the cell cultures pond; Characterize cell growth conditions and the activity BME three-dimensional space in of cultivation after 24 hours with rhodamine 123 (Rodamin 123) and propidium iodide (PI).
Micro-fluidic chip provided by the invention research cell directed method that moves in three-dimensional medium, described BME is a basement membrane-like material, in the cell cultures pond,, cell growth and migration support for providing three-dimensional, and during low temperature liquid state, be frozen into colloidal state during room temperature.
Micro-fluidic chip research cell provided by the invention directed method that moves in three-dimensional medium, the concentration gradient in the described cell cultures pond characterizes with the dna molecular (molecular weight is about 6000D) of fluorescence dye (FAM) mark.
Micro-fluidic chip research cell provided by the invention directed method that moves in three-dimensional medium, the flow velocity of keeping syringe pump in the entire method process is 0.5 μ l/min.
Micro-fluidic chip research cell provided by the invention directed method that moves in three-dimensional medium is carried out the detection of two kinds of cells simultaneously on chip; This micro-fluidic chip has two cell cultures ponds, can add different types of cell respectively, detects the mobility of different cells under the same conditions.
In a word, the present invention can be on more than one square centimeters chip, for the growth and the migration of cell provides the three-dimensional medium support, and generates stable concentration gradient in medium, and inducing cell takes place by directed moving under the effect of chemokine.With respect to traditional research method, for the growth of cell with move more approaching intravital microenvironment is provided.And that the present invention has is simple to operate, fast and characteristics such as amount of samples is few.Have important biomedical researching value and economic worth.
Description of drawings
Fig. 1 micro-fluidic chip synoptic diagram of the present invention, wherein (1) cell injection port, (2) cell cultures pond, (3) perfusion passage, (4) waste liquid pool;
Fig. 2 shows the fluorescent signal in the different time cell cultures pond: liquid flow velocity is 0.5 μ l/min in the perfusion passage;
Fig. 3 generates stable linear fluorescence concentration gradient in the cell cultures pond;
Fig. 4 breast cancer cell MCF7 is growth conditions and active the detection in three-dimensional medium;
Fig. 5 breast cancer cell MCF7 induces at EGF directional migration takes place down;
Fig. 6 breast cancer cell MCF7 induces at EGF mobility takes place down.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Embodiment
Used micro-fluidic chip designs voluntarily for this laboratory and prepares.Chip material is the PDMS polymkeric substance, is packaged on glass surface by irreversible sealing technology.As shown in Figure 1, two parallel perfusion channel sizes are long 5mm, wide 400 μ m, and the elliptical erythrocyte cultivation pool is of a size of major diameter 1mm, minor axis 200 μ m.The mixture that in the cell cultures pond, adds breast cancer cell MCF7 and Basement Membrane Extract (BME) by the cell injection port at low temperatures, after thing to be mixed forms gel, the last termination syringe pump of both sides perfusion passage, in the perfusion passage, pour into cell culture fluid, place 37 ℃ CO
2Culturing cell is 24 hours in the incubator.Adopt the MCF7 cytoactive of Rodamin-123 (Rh-123) and PI joint-detection BME embedding, 24 hours surviving rate that the result shows the MCF7 cell in BME is greater than 90% (Fig. 4).
The nutrient solution of Urogastron (EGF) that perfusion contains 0～200ng/ml different concns in a wing passage is as experimental side, and opposite side pours into the nutrient solution side in contrast that does not contain chemoattractant in contrast, and the perfusion culturing cell is 24 hours continuously.Under the inducing of EGF, most of MCF7 cell is to the higher direction migration of EGF concentration in 24 hours, then be not moved (Fig. 5) at the MCF7 cell near control sides perfusion passage, the cell migration rate of experimental side is significantly greater than control sides (Fig. 6) under the EGF of 200ng/ml concentration induces.
Claims (7)
1. micro-fluidic chip, it is characterized in that: this micro-fluidic chip is made up of cell injection port (1), cell cultures pond (2), two parallel perfusion passages (3), waste liquid pools (4);
The lower end of perfusion passage connects waste liquid pool, and the cell cultures pond perfusion passage parallel with two links to each other, and the upper end in cell cultures pond links to each other with the cell injection port.
2. according to the described micro-fluidic chip of claim 1, it is characterized in that: the material of described micro-fluidic chip is the PDMS polymkeric substance, with the irreversible envelope of glass material.
3. according to the described micro-fluidic chip of claim 1, it is characterized in that: described cell cultures pond is for oval.
4. the described micro-fluidic chip of claim 1 is studied cell directed method that moves in three-dimensional medium, and it is characterized in that: procedure is as follows:
Add in the cell cultures pond by the mixture of cell injection port, be positioned over room temperature and be frozen into glue to BME with cell and BME;
After treating that BME forms gel, the upper end of both sides perfusion passage connects syringe pump, wherein pour into the cell culture fluid that contains chemokine in the lateral irrigation circulation road, opposite side pours into the cell culture fluid that does not contain chemokine as blank, flow velocity is 0.5 μ l/min, perfusion was cultivated 24 hours continuously, and chemokine forms stable concentration gradient in the cell cultures pond;
Characterize cell growth conditions and the activity BME three-dimensional space in of cultivation after 24 hours with rhodamine 123 and propidium iodide.
5. according to the described micro-fluidic chip research of claim 4 cell directed method that moves in three-dimensional medium, it is characterized in that: described BME is a basement membrane-like material, is liquid during low temperature, is frozen into colloidal state during room temperature.
6. according to the described micro-fluidic chip research of claim 4 cell directed method that moves in three-dimensional medium, it is characterized in that: the concentration gradient in the described cell cultures pond characterizes with the dna molecular of fluorochrome label.
7. according to the described micro-fluidic chip research of claim 6 cell directed method that moves in three-dimensional medium, it is characterized in that: the molecular weight of described dna molecular is 6000D.
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| CN102746986A (en) * | 2012-07-13 | 2012-10-24 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102816695A (en) * | 2011-06-08 | 2012-12-12 | 大连医科大学 | Micro-fluidic chip and method for studying effect of fluid shearing force on cell with the micro-fluidic chip |
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
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- 2008-12-08 CN CN200810229391A patent/CN101748059A/en active Pending
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Application publication date: 20100623 |