CN102206580A - Flow cytometry electrofusion apparatus - Google Patents
Flow cytometry electrofusion apparatus Download PDFInfo
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- CN102206580A CN102206580A CN2011100749628A CN201110074962A CN102206580A CN 102206580 A CN102206580 A CN 102206580A CN 2011100749628 A CN2011100749628 A CN 2011100749628A CN 201110074962 A CN201110074962 A CN 201110074962A CN 102206580 A CN102206580 A CN 102206580A
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Abstract
The invention discloses a flow cytometry electrofusion apparatus. The flow cytometry electrofusion apparatus comprises: a one-to-one cell fusion portion, a fusion cell detecting and sorting portion and a single cell collection portion. The one-to-one cell fusion portion is provided with a plurality of parallel cellular localization channels. Different cells can be adsorbed to ends of the channels, and two different cells can close to each other and be fused. The fused cells are placed until membranes of the different cells are completely fused, and then are put into the fusion cell detecting and sorting portion. The fusion cell detecting and sorting portion comprises a laser light source, a fluorescence detector and a deflector. The fused cells, i.e., red and green double fluorescent stained cells, are collected in a collecting tube. When collecting a fused cell, flow of droplet in the fluorescence detector is stopped. 200 [mu]l of cell culture medium is added to the collecting tube through a cell culture medium accumulator installed above the collecting tube to open the end of the collecting tube. The 200 [mu]l of the cell culture medium containing the fused cell flows into a well of a 96-well plate arranged blow the collecting tube. At this time, the flow of the liquid in a flow chamber is started, and the next fused cell is put into the next well of the 96-well plate. According to the present invention, one-time continuous accomplishment of one-to-one cell fusion can be realized, fusants can be detected correctly after fusing and each fusant can be cultured respectively.
Description
Technical field
The present invention relates to a kind of fluidic cell electricity fusion instrument, belong to field of biomedicine technology.
Background technology
The electricity integration technology is a kind of novel short integration technology of setting up the eighties in last century.Zimmerman had invented electric fusion instrument in 1981, and had proposed electric fusion notion first; 1987, Schweiger set up single to protoplastis electricity integration technology program.Its ultimate principle is that the mixed solution with two kinds of cells places the low-voltage alternating-current electric field, makes cell aggregation become beading, applies high electric field pulse then, to impel cytogamy.The main purpose of cytogamy is that the research heterogenous cell merges the influence factor of back gene recombination, produces hybridoma, and plant and microorganism cells cross-breeding etc.Development by decades, the electricity integration technology is ripe day by day, its many advantages are also more and more manifested, show following several aspect: the frequency that (1) inducing cell merges is the highest in all fusion methods, exceed 10-100 doubly than traditional method, particularly the fusion to marginal cell far away, the difficult cell that merges has effect preferably; (2) pair cell toxicological harmless effect is better than chemistry and merges and virus-mediated cytogamy; (3) scope of application is extensive, and animal, plant and microorganism can carry out cytogamy research with this technology.
Although also there are some problems in cell electricity integration technology having obtained successful Application aspect breeding, hybridization research and the cell clone, promptly cell freely contacts and effective fusion rate of causing is low.In traditional electric fusion process, cell forms the beading queuing in alternating-electric field be arbitrarily to take place, both two kinds of identical cells also can be lined up, multiple situation will take place in the fusion in ensuing DC electric field, allogenic cell merges and two above cytogamy all can take place, and directly to merge proportion just very little and test required heterogenous cell.To cause screening operation subsequently very complicated like this, seriously reduce test efficiency.The present invention can reach effectively not that allogenic cell merges one to one, has well solved problem on.
After the cytogamy, detecting of heterogenous cell fusant also is a crucial step, and this is directly connected to the efficient of follow-up test.Traditional screening method is to screen by selective medium, as screening fusant with the HAT substratum in the Monoclonal Antibody, the microorganism protoplastis merges with two kinds of drug-fast protoplastiss of difference and merges the back with containing two kinds of antibiotic substratum screenings, select medium therapy all very high, require to have the parental cell of special selection markers for the requirement of parental cell.In protoplast fusion breeding, have by nucleic acid and proteinic different properties and carry out parents' deactivation, one strain parent is carried out hot deactivation, ultraviolet inactivation is carried out in another strain, merges back active generation complementation by xenogenesis and screens, and the shortcoming of this method is not have the specificity false positive rate very high.The use of active fluoro dyestuff can realize extensive sorting after making cytogamy, this method is dyed two strain parents with red green two kinds of fluorescence dyes, merging the back detects two transfect cells and they is sorted out collection by the flow cytometer two channels, but this method has a large amount of false positive cell to be occurred, and dyestuff penetration takes place in the electric shock process can make in a large number not that fused cell is two states that dye.Therefore, existing cell-fusion techniques and equipment can't be realized a large amount of specific cytogamy.
Summary of the invention
Technology of the present invention is dealt with problems: the deficiency that overcomes conventional cell fusion instrument and fusion method, electrofusion chip and flow cytometer combine cleverly one to one with cell, can disposablely finish cell electricity fusion one to one continuously, merging the back fusant can correctly detect and each fusant be cultivated respectively.
Technical solution of the present invention: a kind of fluidic cell electricity fusion instrument comprises that cell merges part one to one, fused cell detects sorting part and unicellular collection part; Described cell merges part 1 one to one and comprises cytogamy chamber 2, many to parallel cellular localization passage 3 and cell suction passage 4, be provided with the airtight cytogamy chamber 2 of hollow in the middle of cell electrofusion chip 1,2 two ends, cytogamy chamber are respectively the first cell well 6 and the second cell well 7; The 2 liang of side bottoms in cytogamy chamber are distributed with many to parallel cellular localization passage 3, each cellular localization passage 3 is communicated with cell suction passage 4, and cell suction passage 4 is communicated with step by step and is amplified to end openings and is extended down to the up and down first cell suction port 5 and the second cell suction port 8 at two ends; The first cell well 6 is connected with two kinds of different enchylema by flexible pipe respectively with the second cell suction port 8 with the second cell well 7 and the first cell suction port 5, and the first cell suction port 5 is connected with syringe pump or electrode respectively with the second cell suction port 8; The enchylema that enters from the first cell well 6 and the second cell well 7 under the suction effect of syringe pump flows into to cytogamy chamber 2, and each cellular localization passage 3 place draws a cell in a side; The cellular localization passage 3 of opposite side is drawn another kind of cell, after cell is drawn and is finished, power up in the first cell suction port 5 of cell suction passage 4 ends and the electrode of the second cell suction port 8 respectively, make cell that man-to-man electricity take place and merge, the cell mixing liquid after the cytogamy is directly pumped into fluidic cell sorting part; Described fused cell detects sorting and partly comprises sample hose 9, sheath fluid tank 10, nozzle 12, laser apparatus 13, fluorimetric detector 14, optical splitter 15, optical collector 16, positive charge deflector plate 17, negative charge deflector plate 18, collector 19, fluid infusion mouth 26, computer 24, flow rate regulation pump 25 and drop charge device 21; The described unicellular part of collecting is made up of culture plate locating device 23 and 96 orifice plates 22 that are positioned on the culture plate locating device 23; Cell mixing liquid after the fusion is pumped in the sample hose 9, and enters exposure cell 11 after sheath fluid in the sheath fluid tank 10 mixes, and the irradiation back is regulated flow velocitys from nozzle 12 ejections by flow rate regulation pump 25; Laser apparatus 13 sends exciting light and makes that fluorescence dye inspires emission light in the cell, emission light is collected by optical collector 16, be divided into red green fluorescence by optical splitter 15, collect by photodetector 14 then, the cell signal that collection obtains is amplified, is sent to drop charge device 21 after the analog-to-digital conversion process by computer 24, is recharged; Charged drop carries cell and deflects by positive charge deflector plate 17, negative charge deflector plate 18, there is the cell of two kinds of fluorescence to fall into collector 19, the fluid infusion mouth 26 that be arranged in collector 19 upper ends this moment adds to collector 19 with cell culture fluid, and fall into 96 orifice plates 22 of collector 19 belows simultaneously, adjust culture plate locating device 23 afterwards, make next hole aim at cell harvestor 19, carry out the collection of next fusant.
The width of described cytogamy chamber 2 is 30-60 μ m, highly is 30-60 μ m, and length is 2cm.
What described a plurality of parallel cellular localization passage 3 was positioned the 2 liang of side bottoms in cytogamy chamber is spaced apart 80-100 μ m, and each placed channel cross section is the square of length of side 3-10 μ m, the long 100-200 μ of passage m.
Described cell suction passage 4 is communicated with step by step that to be amplified to open-ended diameter be 1-2mm.
The diameter of the described first cell well 6 and the second cell well 7 is 1-2mm.
Described fluid infusion mouth 26 is a 15ml test tube, and with hose connection and by peristaltic pump, the control flow velocity makes each unlatching can emit 200 μ l nutrient solutions in collector 19.
Described culture plate locating device 23 front and back and left and right sides adjustable distance are respectively 10-20mm.
The hole of described fluid infusion mouth 26, collector 19 and 96 orifice plates 22 should be aimed at.
The present invention's advantage compared with prior art is:
(1) the present invention combines electric one to one fusion part of cell and flow cytometer cleverly, can disposablely finish cell electricity fusion one to one continuously, and fusant can correctly detect and each fusant be cultivated respectively after the fusion.
(2) the cellular localization passage of both sides, cytogamy chamber of the present invention adsorbs different cells respectively, and cell can specificity match fully, and pairing rate height, and two heterogenous cells of each strict permission merge.
(3) convection type cell instrument of the present invention improves, and can finish individual cells analysis and collection work by regulating the enchylema flow velocity.
(4) the present invention is provided with the culture plate locating device, is convenient to each cell harvesting in a hole of 96 orifice plates, makes things convenient for follow-up work greatly.
Description of drawings
Fig. 1 is realization flow figure of the present invention;
Fig. 2 merges the structure iron of part one to one for cell of the present invention;
Fig. 3 detects sorting part and unicellular collection unit separation structure figure for fused cell of the present invention.
The 1-cell merges part one to one, 2-cytogamy chamber, 3-cellular localization passage, 4-cell suction passage, the 5-first cell suction port, the 6-first cell well, the 7-second cell well, the 8-second cell suction port, the 9-sample hose, the 10-sheath fluid tank, the 11-flow chamber, the 12-nozzle, the 13-laser apparatus, the 14-fluorimetric detector, the 15-spectroscope, the 16-optical collector, 17-positive charge deflector plate, 18-negative charge deflector plate, the 19-collector, 20-waste collection device, 21-drop charge device, the 22-96 orifice plate, 23-culture plate locating device, the 24-computer, 25-flow rate regulation pump, 26-fluid infusion mouth.
Embodiment
As shown in Figure 1, 2, 3, the present invention includes cell and merge part 1 one to one, cell merges part 1 one to one and is whole chip, is provided with the airtight cytogamy chamber 2 of hollow in whole chip.Cytogamy chamber 2 width are 30-60 μ m, highly are 30-60 μ m, and length is 2cm.2 two ends, cytogamy chamber are respectively the first cell well 6 and the second cell well 7.The 2 liang of side bottoms in cytogamy chamber are distributed with a plurality of parallel cellular localization passages 3.What a plurality of parallel cellular localization passages 3 were positioned the 2 liang of side bottoms in cytogamy chamber is spaced apart 80-100 μ m, and each placed channel cross section is the square of length of side 3-10 μ m, the long 100-200 μ of passage m.
Cellular localization passage 3 is communicated with cell suction passage 4, cell suction passage 4 ends are that the square of length of side 3-10 μ m connects with the cellular localization passage 3 of identical big or small cross section, another terminal and adjacent cell suction passage is terminal to be connected with the interconnection that cross-sectional area doubles jointly, for the overstriking first time of passage is amplified; This interconnection is linked to each other by the long-pending vertical passage of same cross-sectional, and two adjacent vertical passages connect by the interconnection that cross-sectional area doubles again, for the overstriking second time is amplified; By that analogy, after 5 amplifications, link to each other with the cell suction port of direct about 1mm.Cell suction passage 4 is communicated with step by step that to be amplified to open-ended diameter be 1-2mm.
The diameter of the first cell well 6 and the second cell well 7 is 1-2mm.The first cell well 6 and the second cell well 7 and the first cell suction port 5 and the second cell suction port 8 are hose connection with diameter 1-2mm respectively.The flexible pipe of the first cell well 6 and the second cell well 7 is connected with two kinds of different enchylema.The first cell suction port 5 is connected with syringe pump with the second cell suction port 8, but also connection electrode.Electricity leaves standstill 30min after merging, the first cell well 6 and 7 sucking-offs respectively of the second cell well, enter the sample hose 9 of flow cytometry part and mix the laggard flow chamber 11 of going into sheath fluid in the sheath liquid chamber 10, from nozzle 12 ejections, between flow chamber 11 and nozzle 12 flow rate regulation pump 25 is housed, purpose is the cell speed-controllable that makes the nozzle ejection.Laser apparatus 13 sends exciting light and makes that fluorescence dye inspires emission light in the cell, and emission light is collected by optical collector 16, is divided into red green fluorescence by optical splitter 15, is collected by fluorimetric detector 14 then.The cell signal that collection obtains is handled by computer 24, promptly at first the cell signal of collecting is amplified, and again through analog-digital converter, the cell signal behind the analog-digital converter is sent to carries out the cell drop charge in the drop charge device 21 again.Charged drop carries cell and deflects by positive charge deflector plate 17, negative charge deflector plate 18, has the cell of two kinds of fluorescence to fall into collector 19, and the cell that is not collected then falls into the adjacent waste collection device 20 of collector 19.At this moment, the fluid infusion mouth 26 that is arranged in collector 19 upper ends adds to collector 19 with 200 μ l cell culture fluids, fluid infusion mouth 26 is a 15ml test tube, and with hose connection and by peristaltic pump, the control flow velocity makes each unlatching can emit 200 μ l nutrient solutions in collector 19.Fluid infusion mouth 26 falls into 96 orifice plates 22 of collector below when adding to 200 μ l cell culture fluids in the collector 19, adjust culture plate locating device 23 afterwards, carries out the collection of next fusant.Culture plate locating device 23 front and back and left and right sides adjustable distance are respectively 10-20mm.The hole of fluid infusion mouth 26, collector 19 and 96 orifice plates 22 should be aimed at.
Principle of the present invention and using method are as follows: (with the pairing of K562 cell and Chinese hamster ovary celI and be fused to example)
(1) whole chip is just being placed on the testing table, the direct 1mm flexible pipe that the first cell well 6 connects is being put into the K562 cell suspension.The second cell well 7 is shut with the little flexible pipe that the second cell suction port 8 is connected.The hose connection syringe pump of the first cell suction port 5, open syringe pump K562 cell (red fluorescence) is sucked cytogamy chamber 2, continue for some time a large amount of cells in back and be adsorbed on cellular localization passage 3 ends that are connected with the first cell suction port 5 and keep bigger flow velocity to make cell reach stable absorption.
(2) flexible pipe of the first cell well 6 is put into acellular electricity and merged liquid, the second cell well, 7 hose connection syringe pumps wash out the cell that does not adsorb in the cytogamy chamber 2 with less flow velocity.Stop.
(3) the direct 1mm flexible pipe that the second cell well 7 is connected is put into Chinese hamster ovary celI (green fluorescence) suspension.The little flexible pipe that the first cell well 6 connects is shut.The hose connection syringe pump of the second cell suction port 8, open syringe pump Chinese hamster ovary celI is sucked cytogamy chamber 2, continue for some time a large amount of cells in back and be adsorbed on cellular localization passage 3 ends that are connected with the second cell suction port 8 and keep bigger flow velocity to make cell reach stable absorption.
(4) flexible pipe of the second cell well 7 is put into acellular electricity and merged liquid, the first cell well, 6 hose connection syringe pumps wash out the cell that does not adsorb in the cytogamy chamber 2 with less flow velocity.After stopping the first cell well 6 is shut.
(5) electrode and the power connection that the first cell suction port 5 is connected with the second cell suction port 8, the different cells that adsorb on the hecatomeral cells placed channel are more close making under the effect of alternating-current, and two cells are merged.
(6) from the first cell well 6 or 7 sucking-offs of the second cell well of both sides, carry out next step screening and analysis after fusion back cell leaves standstill 30min in cytogamy chamber 3.
(7) the cell mixing liquid after merging is pumped to the sample hose 9 of flow cytometry part, mixes the laggard flow chamber 11 of going into then with sheath fluid in the sheath liquid chamber 10, sprays from nozzle 12..
(8) laser apparatus 13 sends exciting light and makes that fluorescence dye inspires emission light in K562 cell and the Chinese hamster ovary celI, and emission light is collected by optical collector 16, is divided into red green fluorescence by optical splitter 15, is collected by photomultiplier detector 14 then.
(9) collect the cell signal obtain and amplify, be sent to drop charge device 21 after the analog-to-digital conversion process, be recharged by computer 24.Charged drop carries cell and deflects by positive and negative deflection electrode plate 17 and 18, has the cell of two kinds of fluorescence to fall into collector 19.
(10) the fluid infusion mouth 26 that is arranged in collector 19 upper ends adds to collector 19 with 200 μ l cell culture fluids, and falls into 96 orifice plates 22 of collector below simultaneously.
(11) adjust culture plate locating device 23, make next hole aim at cell harvestor 19, carry out the collection of next fusant.
The present invention is not limited only to above-mentioned two kinds of cells, can be applicable to the fusion between the big or small cell of difference by the size of revising cellular localization passage and cytogamy chamber.
The non-elaborated part of the present invention belongs to techniques well known.
Claims (8)
1. fluidic cell electricity fusion instrument, it is characterized in that comprising: cell merges part one to one, fused cell detects sorting part and unicellular collection part; Described cell merges part (1) one to one and comprises cytogamy chamber (2), many to parallel cellular localization passage (3) and cell suction passage (4), be provided with the airtight cytogamy chamber (2) of hollow in the middle of cell electrofusion chip (1), two ends, cytogamy chamber (2) are respectively the first cell well (6) and the second cell well (7); Cytogamy chamber (2) two side bottoms are distributed with many to parallel cellular localization passage (3), each cellular localization passage (3) is communicated with cell suction passage (4), and cell suction passage (4) is communicated with step by step and is amplified to end openings and is extended down to the up and down first cell suction port (5) and the second cell suction port (8) at two ends; The first cell well (6) is connected with two kinds of different enchylema by flexible pipe respectively with the second cell suction port (8) with the second cell well (7) and the first cell suction port (5), and the first cell suction port (5) is connected with syringe pump or electrode respectively with the second cell suction port (8); The enchylema that enters from the first cell well (6) and the second cell well (7) under the suction effect of syringe pump flows into to cytogamy chamber (2), locates to draw a cell at each cellular localization passage (3) of a side; The cellular localization passage (3) of opposite side is drawn another kind of cell, after cell is drawn and is finished, power up in the first cell suction port (5) of cell suction passage (4) end and the electrode of the second cell suction port (8) respectively, make cell that man-to-man electricity take place and merge, the cell mixing liquid after the cytogamy is directly pumped into fluidic cell sorting part; Described fused cell detects sorting and partly comprises sample hose (9), sheath fluid tank (10), nozzle (12), laser apparatus (13), fluorimetric detector (14), optical splitter (15), optical collector (16), positive charge deflector plate (17), negative charge deflector plate (18), collector (19), fluid infusion mouth (26), computer (24), flow rate regulation pump (25) and drop charge device (21); The described unicellular part of collecting is made up of culture plate locating device (23) and 96 orifice plates (22) that are positioned on the culture plate locating device (23); Cell mixing liquid after the fusion is pumped in the sample hose (9), and enters exposure cell (11) after sheath fluid in the sheath fluid tank (10) mixes, and the irradiation back is regulated flow velocity from nozzle (12) ejection by flow rate regulation pump (25); Laser apparatus (13) sends exciting light and makes that fluorescence dye inspires emission light in the cell, emission light is collected by optical collector (16), be divided into red green fluorescence by optical splitter (15), collect by photodetector (14) then, collect the cell signal that obtains and amplify, be sent in the drop charge device (21) after the analog-to-digital conversion process and be recharged by computer (24); Charged drop carries cell and deflects by positive charge deflector plate (17), negative charge deflector plate (18), there is the cell of two kinds of fluorescence to fall into collector (19), the fluid infusion mouth (26) that is arranged in collector (19) upper end this moment adds to collector (19) with cell culture fluid, and fall into 96 orifice plates (22) of collector (19) below simultaneously, adjust culture plate locating device (23) afterwards, make next hole aim at cell harvestor (19), carry out the collection of next fusant.
2. fluidic cell electricity fusion instrument according to claim 1, it is characterized in that: the width of described cytogamy chamber (2) is 30-60 μ m, highly is 30-60 μ m, length is 2cm.
3. fluidic cell electricity fusion instrument according to claim 1, it is characterized in that: what described a plurality of parallel cellular localization passages (3) were positioned cytogamy chamber (2) two side bottoms is spaced apart 80-100 μ m, each placed channel cross section is the square of length of side 3-10 μ m, the long 100-200 μ of passage m.
4. fluidic cell according to claim 1 electricity fusion instrument is characterized in that: described cell suction passage (4) is communicated with step by step that to be amplified to open-ended diameter be 1-2mm.
5. fluidic cell electricity fusion instrument according to claim 1, it is characterized in that: the diameter of the described first cell well (6) and the second cell well (7) is 1-2mm.
6. fluidic cell according to claim 1 electricity fusion instrument, it is characterized in that: described fluid infusion mouth (26) is a 15ml test tube, with hose connection and by peristaltic pump, the control flow velocity makes each unlatching can emit 200 μ l nutrient solutions in collector (19).
7. fluidic cell electricity fusion instrument according to claim 1, it is characterized in that: described culture plate locating device (23) front and back and left and right sides adjustable distance are respectively 10-20mm.
8. fluidic cell electricity fusion instrument according to claim 1, it is characterized in that: the hole of described fluid infusion mouth (26), collector (19) and 96 orifice plates (22) should be aimed at.
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| CN 201110074962 CN102206580B (en) | 2011-03-28 | 2011-03-28 | Flow cytometry electrofusion apparatus |
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| CN 201110074962 CN102206580B (en) | 2011-03-28 | 2011-03-28 | Flow cytometry electrofusion apparatus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105028200A (en) * | 2015-07-09 | 2015-11-11 | 武汉轻工大学 | Plant artificial seed production device and method |
| CN105143880A (en) * | 2013-04-26 | 2015-12-09 | 贝克顿·迪金森公司 | Methods and systems for the collection of light using total internal reflectance |
| CN112730408A (en) * | 2020-12-24 | 2021-04-30 | 贝克曼库尔特生物科技（苏州）有限公司 | Liquid flow detection system, liquid flow detection method, and sorting device |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9249385B1 (en) | 2014-12-18 | 2016-02-02 | City University Of Hong Kong | System and method for fusing cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101693874A (en) * | 2009-09-30 | 2010-04-14 | 重庆大学 | Cell electrofusion chip device based on micro-chamber array structure |
| CN101717717A (en) * | 2009-12-11 | 2010-06-02 | 江阴司特易生物技术有限公司 | Cell pairing and fusion chip |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101693874A (en) * | 2009-09-30 | 2010-04-14 | 重庆大学 | Cell electrofusion chip device based on micro-chamber array structure |
| CN101717717A (en) * | 2009-12-11 | 2010-06-02 | 江阴司特易生物技术有限公司 | Cell pairing and fusion chip |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143880A (en) * | 2013-04-26 | 2015-12-09 | 贝克顿·迪金森公司 | Methods and systems for the collection of light using total internal reflectance |
| CN105143880B (en) * | 2013-04-26 | 2018-03-09 | 贝克顿·迪金森公司 | For collecting the method and system of light using total internal reflection |
| CN105028200A (en) * | 2015-07-09 | 2015-11-11 | 武汉轻工大学 | Plant artificial seed production device and method |
| CN112730408A (en) * | 2020-12-24 | 2021-04-30 | 贝克曼库尔特生物科技（苏州）有限公司 | Liquid flow detection system, liquid flow detection method, and sorting device |
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