CN103074212B - Cell electrofusion device and method - Google Patents
Cell electrofusion device and method Download PDFInfo
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- CN103074212B CN103074212B CN201310025194.6A CN201310025194A CN103074212B CN 103074212 B CN103074212 B CN 103074212B CN 201310025194 A CN201310025194 A CN 201310025194A CN 103074212 B CN103074212 B CN 103074212B
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- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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Abstract
The invention discloses a cell electrofusion device. The cell electrofusion device is characterized in that a first rectangular groove which is sunk and used for mounting a sample carrying sheet and a second rectangular groove which is positioned in the first rectangular groove and further sunk are formed on a base; the second rectangular groove is a vacuum cavity; a joint is arranged on a side surface of the base; a flow passage allowing liquid to flow is arranged between the vacuum cavity and the joint; the joint is connected to a pressure loop; a rectangular clamping groove is formed on each of the two sides of the first rectangular groove on the base; through holes are formed on the clamping grooves; a first screw is mounted to fix a first electrode, and a second screw is mounted to fix a second electrode on the base; a square groove is formed on the sample carrying sheet, and a plurality of micropores are formed at the bottom of the groove, and assistant position system marks are arranged at the same side of each micropores; and the first electrode and the second electrode are clung to the bottom of the groove of the sample carrying sheet. The cell electrofusion device provides a new cell alignment method, can be fixed through cell adsorption and combined with operation of a robot, eliminates randomness of the traditional electrofusion method and greatly reduces the waste of cells.
Description
Technical field
The invention belongs to field of biomedicine technology, be specifically related to a kind of cell electricity fusing device and realize by this device the method that cell electricity merges.
Background technology
Cytogamy (cell fusion) is the cell engineering growing up the sixties in 20th century, also claim cell hybridization, refer to that two or more cells merge by biological chemistry or physics method, produce the hybrid cell that has both parent's inherited character, cell after fusion obtains the genetic material from two parental cells, there is new heredity or biological characteristics, through cultivating, can, for the preparation of cell engineering product, even produce special species or strain.Cytogamy becomes a core technology of cell engineering gradually, it is not only for the research in the fields such as caryoplasm mutual relationship, gene regulating genetic complementation, tumour generation, the assignment of genes gene mapping, old and feeble control provides powerful measure, and in fields such as genetics animals and plants distant hybridization breeding developmental biology immunology medicine food and agriculturals, thering is widespread use value, it has become a gordian technique in the modern biomedical researchs such as cross-breeding, drug screening, monoclonal antibody preparation, cloning of mammalian animal and anti-cancer vaccine research and development.
The induction of cytogamy can utilize the factors such as biological chemistry and physics.Therefore according to different induction modes, cell fusion method can be divided into virus fusion method, chemical fusion method, electro fusion method, laser fusion method etc.Due to cell electricity merge (cell electrofusion) method have controllability strong, easy and simple to handle, to advantages such as cell toxicological harmlesss, it is most widely used.
In early stage research, electric fusion instrument has been invented by the research group headed by Zimmermann, and people several times improve afterwards, have obviously improved the efficiency of cytogamy.At present, the commercialization instrument merging for cell electricity is a lot, as cell electricity fusion instrument and the related system of German Eppendorf, U.S. BTX, the exploitation of Hungarian BLSDeng company.Yet, in traditional electrical fusion method, too high operating voltage all has unsafe factor to experimenter and experimental cell, making to the piece electrical security of system and high-voltage power supply has proposed very high request, fusing device volume and cell sample consumption are large, fusion efficiencies and flux are lower, and experimental observation and analysis are inconvenient, and these have all limited the further popularization of electric fusion method.
In recent years, cell electro fusion and MEMS (micro electro mechanical system) (MEMS) in conjunction with and obtain new progress.Rely on micro fabrication, the microflow control technique that microchannel is feature of take has been realized the application of low voltage in electricity merges.Micro-fluidic chip is the main platform that microflow control technique is realized, and the development of microarray and micro fabrication has improved the quantity of microelectrode on chip greatly, makes cell electrofusion chip to high-throughput, high-level efficiency, high integration future development.The Zheng little Lin of University Of Chongqing etc. develops the multiple integrated micro-fluidic chip of microelectrode, and has obtained certain experiment effect.Micro-fluidic chip is portable easy of integration, but microelectrode particularly the cost of manufacture of high-density microelectrode array is high, to electric field controls require highly, the accuracy rate of cell pairing is also limited.
Therefore,, for above-mentioned technical problem, be necessary to provide a kind of structure improved cell electricity fusing device that has, to overcome above-mentioned defect.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of cell simple in structure electricity fusing device, to improve the accuracy rate of cell pairing.
For achieving the above object, the invention provides following technical scheme:
Cell electricity fusing device of the present invention, comprise substrate, load sample sheet, groove, the first electrode, the first screw, the second electrode, the second screw, joint, vacuum chamber, runner and micropore, substrate is provided with second rectangular tank in order to the first rectangular tank of load sample sheet to be installed and to be positioned at the first rectangular tank and further to cave in of depression, the second rectangular tank is vacuum chamber, joint is located at the side of substrate, between vacuum chamber and joint, be provided with the runner that makes liquid communication, joint is connected to pressure circuit, the both sides of suprabasil the first rectangular tank are respectively equipped with a rectangle draw-in groove, draw-in groove is provided with through hole, the first screw is installed respectively and is fixed the first electrode, the second screw is fixed the second electrode in substrate, load sample sheet is provided with square groove, bottom portion of groove is provided with some micropores, the first electrode and the second electrode are close to the bottom portion of groove of load sample sheet, the same side of each micropore is provided with auxiliary positioning mark.
Preferably, described substrate and load sample sheet all adopt light transmission bio-compatible material to make, and are convenient to examine under a microscope the experimentation of cytogamy.
Preferably, described some microwell arrays are arranged in bottom portion of groove, can fix a plurality of cells, improve the efficiency that electricity merges.
Preferably, described micro-pore diameter scope is 10～500 μ m, can operate for different cell or tissues.
Preferably, the first described electrode and the second electrode are made by copper or stainless steel.
In the same side of each micropore, be provided with auxiliary positioning mark, can demarcate, make the fixing cell position of this cell electricity fusing device clear and definite, the system of being convenient to catches and does not need to search for randomly.
The present invention also provides a kind of method that adopts above-mentioned cell electricity fusing device fused cell, comprises the steps:
1, will splash in the groove of load sample sheet containing needing the solution of fused cell A, trigger pressure loop, produces negative pressure cell A is wherein absorbed and fixed on the micropore of bottom portion of groove;
2, in glass capillary, suck all the other and treat fused cell B simultaneously, mobile kapillary and cell electricity fusing device, makes the position on the cell A side of capillary tip in being adsorbed, the kapillary cell B that spues at next-door neighbour cell A place, A, B two cells tightly stick together, and form a plurality of cell strings;
3, by cell electricity, merge power supply and send pulse signal to the first electrode and the second electrode, the cytolemma generation reversibility perforation of cell string, between A, B two cells, material is changed mutually, finally completes electricity and merges.
It is relatively low that cell electricity fusing device of the present invention substrate, load sample sheet and electricity merge electrode manufacture difficulty.From technique scheme, can find out, a kind of method that cell electricity fusing device of the present invention provides new cell to queue up, can adsorb fixing and robot manipulation by cell combines, realize the pairing of a plurality of designated cells, eliminate the randomness of traditional electrical fusion method, and greatly reduced the waste of cell; Whole experimentation can be observed under inverted microscope, or by associated picture acquisition system, image is presented on computer screen.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing relevant of the present invention in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the perspective view of cell electricity fusing device of the present invention;
Fig. 2 is the main body two dimensional structure schematic diagram of cell electricity fusing device of the present invention;
Fig. 3 is the part sectioned view of the joint of cell electricity fusing device of the present invention;
Fig. 4 is the load sample sheet micropore enlarged view of cell electricity fusing device of the present invention;
Fig. 5 is that the absorption of cell of the present invention electricity fusing device is fixed first and treated fused cell sample schematic diagram;
Fig. 6 is that cell electricity fusing device arrangement second batch of the present invention is treated fused cell sample schematic diagram, and wherein a is cell electricity fusing device of the present invention, and b is that second batch is treated fused cell, and c is kapillary;
Fig. 7 is that cell electricity fusing device of the present invention obtains cell string sample schematic diagram.
Embodiment
The invention discloses a kind of cell electricity fusing device, comprise substrate, load sample sheet, groove, the first electrode, the first screw, the second electrode, the second screw, joint, vacuum chamber, runner and micropore, substrate is provided with second rectangular tank in order to the first rectangular tank of load sample sheet to be installed and to be positioned at the first rectangular tank and further to cave in of depression, the second rectangular tank is vacuum chamber, joint is located at the side of substrate, between vacuum chamber and joint, be provided with the runner that makes liquid communication, joint is connected to pressure circuit, the both sides of suprabasil the first rectangular tank are respectively equipped with a rectangle draw-in groove, draw-in groove is provided with through hole, the first screw is installed respectively and is fixed the first electrode, the second screw is fixed the second electrode in substrate, load sample sheet is provided with square groove, bottom portion of groove is provided with some micropores, the first electrode and the second electrode are close to the bottom portion of groove of load sample sheet, the same side of each micropore is provided with auxiliary positioning mark.
Preferably, described substrate and load sample sheet all adopt light transmission bio-compatible material to make, and are convenient to examine under a microscope the experimentation of cytogamy.
Preferably, described some microwell arrays are arranged in bottom portion of groove, can fix a plurality of cells, improve the efficiency that electricity merges.
Preferably, described micro-pore diameter scope is 10～500 μ m, can operate for different cell or tissues.
Preferably, the first described electrode and the second electrode are made by copper or stainless steel.
In the same side of each micropore, be provided with auxiliary positioning mark, can demarcate, make the fixing cell position of this cell electricity fusing device clear and definite, the system of being convenient to catches and does not need to search for randomly.
The present invention also provides a kind of method that adopts above-mentioned cell electricity fusing device fused cell, comprises the steps:
1, will splash in the groove of load sample sheet containing needing the solution of fused cell A, trigger pressure loop, produces negative pressure cell A is wherein absorbed and fixed on the micropore of bottom portion of groove;
2, in glass capillary, suck all the other and treat fused cell B simultaneously, mobile kapillary and cell electricity fusing device, makes the position on the cell A side of capillary tip in being adsorbed, the kapillary cell B that spues at next-door neighbour cell A place, A, B two cells tightly stick together, and form a plurality of cell strings;
3, by cell electricity, merge power supply and send pulse signal to the first electrode and the second electrode, the cytolemma generation reversibility perforation of cell string, between A, B two cells, material is changed mutually, finally completes electricity and merges.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is described in detail, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, the every other embodiment that those of ordinary skills obtain under the prerequisite of not making creative work, belongs to the scope of protection of the invention.
As shown in Figures 1 to 4, cell electricity fusing device of the present invention, comprise substrate 1, load sample sheet 2, groove 3, the first electrode 4, the first screw 5, the second electrode 6, the second screw 7, joint 8, vacuum chamber 9, runner 10 and micropore 11, substrate 1 is provided with second rectangular tank in order to the first rectangular tank of load sample sheet 2 to be installed and to be positioned at the first rectangular tank and further to cave in of depression, the second rectangular tank is vacuum chamber 9, joint 8 is located at the side of substrate 1, between vacuum chamber 9 and joint 8, be provided with the runner 10 that makes liquid communication, joint 8 is connected to pressure circuit, the both sides of the first rectangular tank in substrate 1 are respectively equipped with a rectangle draw-in groove, draw-in groove is provided with through hole, the first screw 5 is installed respectively and is fixed the first electrode 4, the second screw 7 is fixed the second electrode 6 in substrate 1, load sample sheet 2 is provided with square groove 3, groove 3 bottoms are provided with some micropores 11.
Load sample sheet 2 and substrate 1 all adopt bio-compatible material preparation, and light transmission is good, is convenient to examine under a microscope experimentation.
On load sample sheet 2, the diameter range of micropore 11 is 10～500 μ m.Micropore 11 can break into laser-beam drilling machine, so arranges, and is convenient to process the through hole that diameter is different, and then can operates for different cell or tissues (as the fusion of zygote).Can be as required, do the quantity of portalling and be: 1,2 * 2 arrays, 3 * 3 arrays, 4 * 4 arrays, 5 * 5 arrays even more (size of pitch of holes, groove 3 all can change because of needs), to fix a plurality of cells, improve the efficiency that electricity merges.Between array hole, can demarcate by auxiliary positioning mark 12, make this install fixing cell position clear and definite, the system of being convenient to catches and does not need to search for randomly.
Electrode (the first electrode 4 and the second electrode 6) is fixed in substrate 1 with screw (the first screw 5 and the second screw 7), make its groove of being close to load sample sheet 23 bottoms, electrode replaceable, reusable, and be convenient to integrated with other functional modules and do not affect other operations.
Can as required, design different electrodes.Electrode materials can be selected copper or stainless steel, according to required electric field design electrode shape, and the distance between two-plate, realize thus different functions.
Cell of the present invention electricity fusing device automatically on fixing basis, merges electrode in conjunction with designed electricity in cell vacuum suck, to realize electricity, merges.The method of work that concrete cell electricity merges, comprise: will splash in the groove 3 of load sample sheet 2 containing needing the solution of fused cell (being designated as A), trigger pressure loop, produces negative pressure cell A is wherein absorbed and fixed on the micropore 11 of groove 3 bottoms (as shown in Figure 5); In glass capillary c, suck all the other and treat that fused cell b(is designated as B) simultaneously, movable glass kapillary c and cell electricity fusing device a(are as Fig. 6), make the position on the cell A side of glass capillary c tip in being adsorbed, glass capillary c is at the next-door neighbour cell B that spues of cell A place, viscosity due to cell itself, above-mentioned A, two kinds of (can be also same class) cells of B tightly stick together, and cell A, cell B are arranged in as shown in Figure 7 as far as possible, so far form a plurality of cell strings; By cell electricity, merge power supply and send pulse signal to two electrodes (the first electrode 4 and the second electrode 6), the cytolemma generation reversibility perforation of cell string, between two cells, material is changed mutually, finally completes electricity and merges.
In sum, it is relatively low that cell electricity fusing device of the present invention substrate, load sample sheet and electricity merge electrode manufacture difficulty.From technique scheme, can find out, a kind of method that cell electricity fusing device of the present invention provides new cell to queue up, can adsorb fixing and robot manipulation by cell combines, realize the pairing of a plurality of designated cells, eliminate the randomness of traditional electrical fusion method, and greatly reduced the waste of cell; Whole experimentation can be observed under inverted microscope, or by associated picture acquisition system, image is presented on computer screen.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and in the situation that not deviating from spirit of the present invention or essential characteristic, can realize the present invention with other specific form.Therefore, no matter from which point, all should regard embodiment as exemplary, and be nonrestrictive, scope of the present invention is limited by claims rather than above-mentioned explanation, is therefore intended to include in the present invention dropping on the implication that is equal to important document of claim and all changes in scope.Any Reference numeral in claim should be considered as limiting related claim.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should make specification sheets as a whole, and the technical scheme in each embodiment also can, through appropriately combined, form other embodiments that it will be appreciated by those skilled in the art that.
Claims (6)
1. cell electricity fusing device, it is characterized in that: comprise substrate (1), load sample sheet (2), groove (3), the first electrode (4), the first screw (5), the second electrode (6), the second screw (7), joint (8), vacuum chamber (9), runner (10) and micropore (11), substrate (1) is provided with second rectangular tank in order to the first rectangular tank of load sample sheet (2) to be installed and to be positioned at the first rectangular tank and further to cave in of depression, the second rectangular tank is vacuum chamber (9), joint (8) is located at the side of substrate (1), between vacuum chamber (9) and joint (8), be provided with the runner (10) that makes liquid communication, joint (8) is connected to pressure circuit, the both sides of the first rectangular tank in substrate (1) are respectively equipped with a rectangle draw-in groove, draw-in groove is provided with through hole, the first screw (5) is installed respectively and is fixed the first electrode (4), the second screw (7) is fixed the second electrode (6) in substrate (1), load sample sheet (2) is provided with square groove (3), groove (3) bottom is provided with some micropores (11), the first electrode (4) and the second electrode (6) are close to groove (3) bottom of load sample sheet (2), the same side of each micropore (11) is provided with auxiliary positioning mark (12).
2. cell electricity fusing device according to claim 1, is characterized in that: described substrate (1) and load sample sheet (2) all adopt light transmission bio-compatible material to make.
3. cell electricity fusing device according to claim 1, is characterized in that: described some micropores (11) array arrangement is in groove (3) bottom.
4. according to the cell electricity fusing device described in claim 1 or 3, it is characterized in that: described micropore (11) diameter range is 10～500 μ m.
5. cell electricity fusing device according to claim 1, is characterized in that: described the first electrode (4) and the second electrode (6) are made by copper or stainless steel.
6. a method that adopts cell electricity fusing device fused cell claimed in claim 1, is characterized in that, comprises the steps:
A, will splash in the groove (3) of load sample sheet (2) containing needing the solution of fused cell A, trigger pressure loop, produces negative pressure cell A is wherein absorbed and fixed on the micropore (11) of groove (3) bottom;
B, while suck all the other and treat fused cell B in glass capillary, mobile kapillary and cell electricity fusing device, makes the position on the cell A side of capillary tip in being adsorbed, the kapillary cell B that spues at next-door neighbour cell A place, A, B two cells tightly stick together, and form a plurality of cell strings;
C, by cell electricity, merge power supply and send pulse signal to the first electrode (4) and the second electrode (6), the cytolemma generation reversibility perforation of cell string, between A, B two cells, material is changed mutually, finally completes electricity fusion.
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| CN201310025194.6A CN103074212B (en) | 2013-01-23 | 2013-01-23 | Cell electrofusion device and method |
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| CN201310025194.6A CN103074212B (en) | 2013-01-23 | 2013-01-23 | Cell electrofusion device and method |
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| CN103382442B (en) * | 2013-07-11 | 2015-03-18 | 湖北省农业科学院畜牧兽医研究所 | Semi-fixed semi-mobile electrofusion electrode for mammal cloning |
| CN104789443B (en) * | 2014-06-23 | 2020-01-17 | 格子生物科技(上海)有限公司 | Cell positioning unit, cell positioning array, cell positioning device and forming method of cell positioning device |
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| CN101717717B (en) * | 2009-12-11 | 2012-07-18 | 江阴司特易生物技术有限公司 | Cell pairing and fusion chip |
| CN102174388B (en) * | 2011-01-25 | 2012-12-05 | 重庆大学 | Surface-electrode-technology-based high-throughput cell electrofusion chip device |
| CN102174387A (en) * | 2011-01-25 | 2011-09-07 | 重庆大学 | Low-voltage direct-current controlled continuous flow cell electrofusion chip |
| CN102321536B (en) * | 2011-09-30 | 2013-04-03 | 岭南大学校产学协力团 | High-flux cell electrofusion device based on microporous array film |
| CN203065488U (en) * | 2013-01-23 | 2013-07-17 | 苏州大学 | Cell electro-fusion device |
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