CN102972351A - Method for capturing and cultivating caenorhabditis elegans single ovum based on micro-fluidic chip - Google Patents
Method for capturing and cultivating caenorhabditis elegans single ovum based on micro-fluidic chip Download PDFInfo
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- CN102972351A CN102972351A CN2012104394408A CN201210439440A CN102972351A CN 102972351 A CN102972351 A CN 102972351A CN 2012104394408 A CN2012104394408 A CN 2012104394408A CN 201210439440 A CN201210439440 A CN 201210439440A CN 102972351 A CN102972351 A CN 102972351A
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Abstract
Disclosed is a method for capturing and cultivating a caenorhabditis elegans single ovum based on a micro-fluidic chip. The micro-fluidic chip comprises two layers of polydimethylsiloxane (PDMS). The upper layer is a liquid way layer and is used for capturing, long-time cultivating and observing of the caenorhabditis elegans single ovum. The lower layer is blank PDMS and is connected with the upper PDMS in a sealed mode. The chip captures the caenorhabditis elegans single ovum by a micro-structure and is capable of cultivating the caenorhabditis elegans single ovum alone in a single cultivation room. The method for capturing the caenorhabditis elegans single ovum based on the micro-fluidic chip is easy to operate and the long-time cultivation of the caenorhabditis elegans during a whole life cycle of the caenorhabditis elegans is achieved easily. Exchange of nutrient substance and discharge of metabolic waste are achieved by the chip. The method for capturing the caenorhabditis elegans single ovum based on the micro-fluidic chip facilitates the long-time cultivation of the caenorhabditis elegans and can observe the behaviour and formation of the image of the single caenorhabditis elegans in a long time.
Description
Technical field
The present invention relates to micro-fluidic field, provide especially a kind of Caenorhabditis elegans list ovum based on channel microfluidic chip to catch and the method for long-term cultivation.
Background technology
Caenorhabditis elegans (Caenorhabditis elegans) is a kind of model organism of classics, and it is widely used in the fields such as modern Developmental Biology, genetics, genomics and Neurobiology.The at present cultivation of Caenorhabditis elegans mainly in the array microwell plate or agar plate carry out, be about to culture of nematodes in 96 orifice plates or agar plate, periodic replacement medium or nematode transferred on the new agar plate.Although culture of nematodes quantity is many, reagent and consumptive material consumption are large; Adopt manual carrying out for the operation of nematode, waste time and energy, flux is low; Operating procedure is comparatively loaded down with trivial details, complicated; Be difficult to realize the tracing study to the whole life course of single nematode.Generally speaking, along with the development of nematode correlative study, the development of current model organism technology far can not be satisfied the needs of modern biological medicine research and development.Develop a kind of new more high flux, the single line worm of automation is cultivated new platform, realizes that the microminiaturization of culture technique, integrated and with low costization are the inexorable trends of future development, also is the development need of drug evaluation research.And the micro-fluidic chip platform that grew up in recent years exactly can satisfy these requirements, and has become one of important single line worm research tool.Recently, micro-fluidic chip because of itself and Caenorhabditis elegans size coupling, environment is controlled, flux is high, easily be automated, the feature such as integrated becomes the nematode correlative study Important Platform, but still be the cultivation of the whole life process of nematode that realizes beginning from ovum.
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, flux is high, is a kind of based on the PDMS(dimethyl silicone polymer specifically) the Caenorhabditis elegans list ovum of channel microfluidic chip catches and long-period culture method.
The invention provides a kind of micro-fluidic chip, the material of this micro-fluidic chip is the PDMS polymer, adopt the irreversible sealing technology of the soft etching of PDMS and plasma to make up, by upper and lower two-layer the composition, the upper strata is for being used for the single ovum of nematode and catching and the liquid road floor of long-term cultivation, and lower floor is for being used for the blank PDMS layer of sealing-in;
Wherein, liquid road floor is comprised of 100～200 nematode analysis functional units, and all functions units in series connects; Each analytic function unit is comprised of line eggs and medicine entrance, culture of nematodes chamber, contains the single ovum grabber of a nematode in each culture of nematodes chamber;
Wherein, the single ovum grabber of nematode is positioned on the central shaft of culture of nematodes chamber; Line eggs and medicine entrance are connected with the culture of nematodes chamber by sample intake passage, and a plurality of culturing room are coupled together by a main channel, are connected at last waste liquid pool.
Micro-fluidic chip provided by the invention, described culture of nematodes chamber are oval, and major axis is 2mm, and minor axis is 1mm, highly is 80 μ m.
Micro-fluidic chip provided by the invention, the size of the single ovum grabber of described nematode is determined by size and the hydrodynamics characteristic of line eggs.The single ovum grabber of described nematode front end is wide to be 50 μ m, and the rear end is wide to be 30 μ m; Structural design is by being determined that by hydrodynamics angle of inclination, the single ovum grabber rear end of nematode is 110 °.
The present invention also provides a kind of Caenorhabditis elegans list ovum based on described micro-fluidic chip to catch and long-period culture method, and nematode sample introduction and cultural method are the mode of utilizing peristaltic pump to drive, and worm egg suspension is introduced nematode culturing room; Under hydromechanical effect, single ovum can be caught in each culture of nematodes chamber; Continuing to pour under the condition of culture fluid, ovum is hatched into larva and is grown up, and the size of culture of nematodes chamber can make it freely move about in whole life process.
The present invention has following advantage:
(1) the present invention adopts channels designs, and is simple to operate, and is easy to realize mass exchange, is conducive to the long-term cultivation of nematode.
(2) adopt the mode of passage perfusion to carry out food interpolation and waste liquid discharge, simple to operate, efficient is high, is conducive to the long-term cultivation of nematode.
(3) culturing room is equally as the single ovum grabber of nematode, and acquisition mode is to the worm's ovum not damaged, and method is simple, and flux is high.
The design of (4) cultivating single nematode in the single culturing room can be satisfied the needs of biological study so that the life-span of every worm, motor behavior and physiological characteristic reach long-term tracking in real time.
Description of drawings
Fig. 1 floor layout is followed successively by liquid road floor, blank PDMS floor from top to bottom; Wherein 1 is line eggs and medicine entrance, and 2 is the waste liquid mouth;
The single culture of nematodes of Fig. 2 chamber enlarged drawing 3 is the single ovum grabber of nematode in the dotted line frame wherein, arrow indicator solution flow path direction;
The single ovum of Fig. 3 nematode is caught the multiple-unit schematic diagram;
The single ovum of Fig. 4 nematode is caught enlarged drawing;
The growth (L1 phase) of nematode (wild type N2) on the 1st day chip after Fig. 5 catches;
The growth (L3 phase) of nematode (wild type N2) on the 3rd day chip after Fig. 6 catches;
The growth (L4 phase) of nematode (wild type N2) on the 5th day chip after Fig. 7 catches;
The growth of nematode (wild type N2) on the 7th day chip after Fig. 8 catches (the Adult phase, and lay eggs).
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
Laboratory designed, designed and the micro-fluidic chip of making: catch and the micro-fluidic chip material of long-term cultivation is the PDMS polymer for the preparation of Caenorhabditis elegans list ovum, the irreversible sealing-in of plasma, this chip is by two-layer, and the upper strata is liquid road floor, and lower floor is for being used for the blank PDMS of sealing-in.
Liquid road floor is comprised of 100～200 nematode analysis functional units, and all functions units in series connects; Each culture of nematodes chamber is oval, and major axis is 2mm, and minor axis is 1mm, highly be 80 μ m, (Fig. 2) can catch single ovum, wherein the single ovum grabber of nematode 3 (Fig. 2) are positioned on the central shaft of culture of nematodes chamber, and its size is determined by size and the hydrodynamics characteristic of line eggs; Line eggs and medicine entrance 1 (Fig. 1) are connected with the culture of nematodes chamber by sample intake passage, and a plurality of culturing room are coupled together by a main channel, are connected at last waste liquid pool 2 (Fig. 1).
Filled with in advance culture of nematodes liquid K Medium before the use.N2 is after agar plate is cultured to tiger to the strain of Caenorhabditis elegans wild type worm, carries out bleach operation a large amount of ovum are taken out, and make to join and form worm egg suspension among the KMedium.
Embodiment 1
The micro-fluidic chip system that utilizes the laboratory designed, designed and make, configuration as shown in Figure 1, 2, utilize peristaltic pump with the line eggs Spectroscopy With Suspension-injection in culturing room.Chip is totally 100～200 unit, and unit is separate, and sample introduction does not interfere with each other, and can reach single culturing room and all only have single ovum.
Embodiment 2
The micro-fluidic chip system that utilizes the laboratory designed, designed and make, the bleach operation is carried out in nematode wild type N2 match to the adult stage obtained a large amount of ovum, ovum is dispersed among the K Medium introduces in the chip, single line eggs is trapped in the single culturing room, as shown in Figure 3 and Figure 4.Caught rear the 1st day, ovum is hatched into L1 phase larva in K Medium medium, see Fig. 5.Through cultivating, long to the L3 phase at the 3rd day, the 5th day long to the L4 phase, grew up to adult on the 7th day and lay eggs.The nematode physiological behavior such as grow, move, lay eggs at chip is normal, shows that this chip microenvironment is suitable for the nematode long-term cultivation, has verified this platform feasibility.
Claims (6)
1. micro-fluidic chip, it is characterized in that: this micro-fluidic chip is by upper and lower two-layer the composition, and the upper strata is liquid road floor, and lower floor is blank PDMS layer;
Wherein, liquid road floor is comprised of 100～200 nematode analysis functional units, and all functions units in series connects; Each analytic function unit is comprised of line eggs and medicine entrance, culture of nematodes chamber, contains the single ovum grabber of a nematode in each culture of nematodes chamber;
Wherein, the single ovum grabber of nematode is positioned on the central shaft of culture of nematodes chamber; Line eggs and medicine entrance are connected with the culture of nematodes chamber by sample intake passage, and a plurality of culturing room are coupled together by a main channel, are connected at last waste liquid pool.
2. according to micro-fluidic chip claimed in claim 1, it is characterized in that: described culture of nematodes chamber is for oval, and major axis is 2mm, and minor axis is 1mm, highly is 80 μ m.
3. according to micro-fluidic chip claimed in claim 1, it is characterized in that: the size of the single ovum grabber of described nematode is determined by size and the hydrodynamics characteristic of line eggs.
4. according to micro-fluidic chip claimed in claim 3, it is characterized in that: the single ovum grabber of described nematode front end is wide to be 50 μ m, and the rear end is wide to be 30 μ m; Structural design is determined that by hydrodynamics angle of inclination, the single ovum grabber rear end of nematode is 110 °.
5. according to micro-fluidic chip claimed in claim 1, it is characterized in that: the material of described micro-fluidic chip is the PDMS polymer, adopts the irreversible sealing technology of the soft etching of PDMS and plasma to make up.
6. the Caenorhabditis elegans list ovum based on micro-fluidic chip claimed in claim 1 is caught and cultural method, it is characterized in that: nematode sample introduction and cultural method are the mode of utilizing peristaltic pump to drive, and worm egg suspension is introduced nematode culturing room; Under hydromechanical effect, single ovum can be caught in each culture of nematodes chamber; Continuing to pour under the condition of culture fluid, ovum is hatched into larva and is grown up, and the size of culture of nematodes chamber can make it freely move about in whole life process.
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| WO2016063199A1 (en) | 2014-10-20 | 2016-04-28 | Ecole Polytechnique Federale De Lausanne (Epfl) | Microfluidic device, system and method for the study of organisms |
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| WO2016063199A1 (en) | 2014-10-20 | 2016-04-28 | Ecole Polytechnique Federale De Lausanne (Epfl) | Microfluidic device, system and method for the study of organisms |
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