CN114591840A - Micro-fluidic chip for trapped suspension cell co-culture and cell culture method - Google Patents
Micro-fluidic chip for trapped suspension cell co-culture and cell culture method Download PDFInfo
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- 238000004113 cell culture Methods 0.000 title claims abstract description 34
- 238000003501 co-culture Methods 0.000 title claims abstract description 16
- 239000000725 suspension Substances 0.000 title claims description 15
- 238000004140 cleaning Methods 0.000 claims description 22
- 230000007246 mechanism Effects 0.000 claims description 18
- 238000007789 sealing Methods 0.000 claims description 11
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000006285 cell suspension Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000005096 rolling process Methods 0.000 claims description 7
- 238000012136 culture method Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 238000009417 prefabrication Methods 0.000 claims description 5
- 230000010405 clearance mechanism Effects 0.000 claims description 4
- 238000001125 extrusion Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 230000001464 adherent effect Effects 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000002503 metabolic effect Effects 0.000 claims description 3
- 230000001681 protective effect Effects 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 2
- 238000003780 insertion Methods 0.000 abstract description 2
- 230000037431 insertion Effects 0.000 abstract description 2
- 238000003825 pressing Methods 0.000 description 10
- 238000005452 bending Methods 0.000 description 3
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Abstract
The invention discloses a micro-fluidic chip for intercepting suspended cells for co-culture and a cell culture method, and relates to the technical field of cell culture. According to the invention, the connecting pin is inserted into the connecting sleeve, so that the chips can be connected, the elastic bent plate is pushed by the pushing spring to extrude the connecting pin, so that the chips are fixed, meanwhile, the top plate is extruded by the insertion of the connecting pin, so that the clamping plate is driven to extend out, the top block is pushed by the fastening spring to extend out from the side surface of the clamping plate, so that the chip is better fixed, and the use of the microfluidic chip is facilitated.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a micro-fluidic chip for intercepting suspension cells for co-culture and a cell culture method.
Background
Although the development of the technology is slow in recent years, the cell culture still provides great contribution to biological research in multiple fields for a long time.
The following problems exist in the prior art:
1. when the existing microfluidic chip for cell co-culture is used, the microfluidic chip needs to be connected and fixed through a fastening buckle or a connector, but the existing fastening buckle or the connector is inconvenient to use and has an insufficient fixing effect, so that the use of the microfluidic chip is influenced;
2. when the existing microfluidic chip for cell co-culture is used, oil stains and fingerprints are easily attached to an observation window on the microfluidic chip, and the observation window on the microfluidic chip is also inconvenient to clean, so that the observation effect is influenced when cells are observed;
3. when cells are cultured, the cell culture method is single, and the culture efficiency is low, so that the working cost and difficulty of the current equipment during cell culture are relatively high, and the requirement of actual working use is difficult to effectively meet.
Disclosure of Invention
The invention provides a micro-fluidic chip for intercepting suspension cells for co-culture and a cell culture method, which aim to solve the problems in the background technology.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a micro-fluidic chip for co-culture of trapped suspended cells, which comprises a cover plate layer, wherein a closed layer is arranged at the bottom end of the cover plate layer, a cell culture layer is arranged at the bottom end of the closed layer, an observation window is fixedly connected to the middle of the cover plate layer, a connecting mechanism is arranged at the top end of the cover plate layer, and a cleaning mechanism is fixedly connected to the side surface of the observation window.
The technical scheme of the invention is further improved as follows: the connecting mechanism comprises an upper pressing plate, the upper pressing plate is arranged at the top of the cover plate layer, the bottom fixedly connected with connecting pin of the upper pressing plate is movably connected with a connecting sleeve, the bottom fixedly connected with lower pressing plate of the connecting sleeve is fixedly connected with the inner side of the elastic bent plate, the inner side fixedly connected with telescopic ejector rod of the elastic bent plate is provided with a jacking spring.
The technical scheme of the invention is further improved as follows: the connecting pin comprises a connecting rod, the connecting rod is fixedly connected to the bottom end of the upper pressing plate, a top plate is movably connected to the bottom end inside the connecting rod, an extrusion spring is arranged at the top of the top plate, a clamping plate is movably connected to the side face of the top plate, a sliding block is fixedly connected to the side face of the clamping plate, a sliding groove is formed in the side face of the top plate, an ejecting block is movably connected to the two sides of the left end of the clamping plate, and a fastening spring is arranged on the side face of the ejecting block.
The technical scheme of the invention is further improved as follows: the clearance mechanism includes the protection frame, protection frame fixed connection is in the side of observation window, the inside fixedly connected with guide rod of protection frame, the side swing joint of guide rod has the sliding block, the left end swing joint of sliding block has the pulley, the right-hand member fixedly connected with clearance board of sliding block, the front end fixedly connected with drive plate of clearance board, the side fixedly connected with fixed block of clearance board, the fixed slot has been seted up to the inboard of protection frame.
The technical scheme of the invention is further improved as follows: the cleaning plate comprises a movable plate, the movable plate is fixedly connected to one side of the pulley, a scraper is fixedly connected to the bottom end of the movable plate, a worm is movably connected to the middle of the movable plate, the worm is fixedly connected to the inside of the protective frame, a worm wheel is meshed with the side face of the worm, the worm wheel is movably connected to the inside of the movable plate, a rolling roller is fixedly connected to the rear end of the worm wheel, and a brush is fixedly connected to the side face of the rolling roller.
In a second aspect, the present invention also provides a culture method for entrapping suspension cells, comprising the steps of:
the method comprises the following steps: preparing a chip;
step two: prefabrication of cells;
step three: culturing cells;
step four: observing the state of the cells;
step five: and (5) collecting metabolic liquid.
The technical scheme of the invention is further improved as follows: the first step further comprises the following steps:
a1: cleaning the prepared cover plate layer, the sealing layer and the cell culture layer by alcohol and sterile water, and drying after cleaning;
a2: the cover plate layer, the sealing layer and the cell culture layer are sent into an ultraviolet sterilization device for sterilization treatment, and the sterilization time is 5-10 minutes;
a3: and then the cover plate layer, the sealing layer and the cell culture layer are assembled together through a connecting mechanism.
The technical scheme of the invention is further improved as follows: the second step further comprises the following steps:
b1: digesting adherent cells into cell suspension by using trypsin, and centrifuging the cells by using a centrifuge;
b2: carrying out cell resuspension by using a culture medium, and diluting the cell suspension to obtain a seed solution;
b3: and standing the seed liquid prepared in the step B2 for 2-10 minutes.
The technical scheme of the invention is further improved as follows: the third step further comprises the following steps:
c1: sucking cells by using a pipette, injecting the cells into the cell injection channel through the cell injection port on the cover plate layer, and entering the cell culture layer for culturing the cells;
c2: the cells are injected into the microchannel without generating air bubbles, and the microfluidic chip is placed in a cell culture box.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
1. the invention provides a micro-fluidic chip for intercepting suspended cells for co-culture and a cell culture method.
2. The invention provides a micro-fluidic chip for intercepting suspended cells for co-culture and a cell culture method.
3. The invention provides a micro-fluidic chip for intercepting suspended cells for co-culture and a cell culture method, wherein when cells are cultured, each part of the micro-fluidic chip is disinfected and sterilized, so that the cells are prevented from being polluted during culture, the cells are conveniently connected and fixed through a connecting mechanism, the cells are conveniently cultured through prefabrication of cell suspension, and then the cells are sucked and injected by a liquid transfer gun, so that the cells can conveniently enter the micro-fluidic chip, the whole process is simple and quick, the culture efficiency is improved, and the culture cost is reduced.
Drawings
FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a schematic cross-sectional view of the connection mechanism of the present invention;
FIG. 3 is a cross-sectional view of the connecting pin of the present invention;
FIG. 4 is a schematic cross-sectional view of the cleaning mechanism of the present invention;
FIG. 5 is a schematic cross-sectional view of a cleaning plate according to the present invention;
FIG. 6 is a schematic flow chart of the present invention.
In the figure: 1. a cover plate layer; 11. an observation window; 2. a sealing layer; 3. a cell culture layer; 4. a connecting mechanism; 41. an upper pressure plate; 42. a connecting pin; 421. a connecting rod; 422. a top plate; 423. a compression spring; 424. clamping a plate; 425. a top block; 426. a fastening spring; 43. connecting sleeves; 44. a lower pressing plate; 45. an elastic bending plate; 46. a telescopic ejector rod; 47. the spring is tightly propped; 5. a cleaning mechanism; 51. a protective frame; 52. a guide rod; 53. a slider; 54. a pulley; 55. cleaning the plate; 551. moving the plate; 552. a squeegee; 553. a worm; 554. a worm gear; 555. a rolling roller; 556. a brush; 56. a drive plate; 57. a fixed block; 58. and fixing the grooves.
Detailed Description
The present invention will be described in further detail with reference to the following examples:
example 1
In a first aspect, as shown in fig. 1 to 6, the present invention provides a microfluidic chip for trapping suspended cells for co-culture, which comprises a cover plate layer 1, a sealing layer 2 is arranged at the bottom end of the cover plate layer 1, a cell culture layer 3 is arranged at the bottom end of the sealing layer 2, an observation window 11 is fixedly connected to the middle of the cover plate layer 1, a connecting mechanism 4 is arranged at the top end of the cover plate layer 1, a cleaning mechanism 5 is fixedly connected to the side surface of the observation window 11, the connecting mechanism 4 comprises an upper pressing plate 41, the upper pressing plate 41 is arranged at the top of the cover plate layer 1, a connecting pin 42 is fixedly connected to the bottom end of the upper pressing plate 41, a connecting sleeve 43 is movably connected to the bottom end of the connecting pin 42, a lower pressing plate 44 is fixedly connected to the bottom end of the connecting sleeve 43, an elastic bending plate 45 is fixedly connected to the inner side of the connecting sleeve 43, a telescopic ejecting rod 46 is fixedly connected to the inner side of the elastic bending plate 45, an ejecting spring 47 is arranged at the side surface of the telescopic ejecting rod 46, connecting pin 42 includes connecting rod 421, connecting rod 421 fixed connection is in the bottom of last clamp plate 41, the inside bottom swing joint of connecting rod 421 has roof 422, the top of roof 422 is provided with extrusion spring 423, the side swing joint of roof 422 has cardboard 424, the side fixedly connected with slider of cardboard 424, the spout has been seted up to the side of roof 422, the left end both sides swing joint of cardboard 424 has kicking block 425, the side of kicking block 425 is provided with fastening spring 426.
In this embodiment, through in inserting adapter sleeve 43 with connecting pin 42, thereby can be to apron layer 1, airtight layer 2, cell culture layer 3 connects, and move down in the top of top tight spring 47, make flexible ejector pin 46 push up elasticity bent plate 45, make elasticity bent plate 45 extrude connecting pin 42, thereby fix the chip, connecting pin 42's insertion can extrude roof 422 simultaneously, thereby make roof 422 extrude extrusion spring 423, and drive cardboard 424 and stretch out from the both sides of connecting rod 421, can ejecting with kicking block 425 from cardboard 424's side through fastening spring 426 simultaneously, thereby make the fixed effect to the chip better, during the use, still clear up observation window 11 through clearance mechanism 5, the use of micro-fluidic chip has been made things convenient for.
Example 2
As shown in fig. 1 to 6, on the basis of embodiment 1, the present invention provides a technical solution: preferably, the cleaning mechanism 5 comprises a protection frame 51, the protection frame 51 is fixedly connected to the side surface of the observation window 11, a guide rod 52 is fixedly connected inside the protection frame 51, a sliding block 53 is movably connected to the side surface of the guide rod 52, a pulley 54 is movably connected to the left end of the sliding block 53, a cleaning plate 55 is fixedly connected to the right end of the sliding block 53, a driving plate 56 is fixedly connected to the front end of the cleaning plate 55, a fixed block 57 is fixedly connected to the side surface of the cleaning plate 55, a fixed groove 58 is formed inside the protection frame 51, the cleaning plate 55 comprises a moving plate 551, the moving plate 551 is fixedly connected to one side of the pulley 54, a scraping plate 552 is fixedly connected to the bottom end of the moving plate 551, a worm 553 is movably connected to the middle of the moving plate 553, the worm 553 is fixedly connected to the inside of the protection frame 51, a worm wheel 551 is engaged with the side surface of the worm 554, the worm wheel 554 is movably connected to the inside of the moving plate 551, a rolling roller 555 is fixedly connected to the rear end of the worm wheel 554, a brush 556 is fixedly connected to a side surface of the rolling roller 555.
In this embodiment, when fingerprint and filth appear on the surface of observation window 11, through control drive plate 56, thereby can drive clearance board 55 and remove along guide rod 52, and through the meshing with between the worm 553, thereby can drive worm wheel 554 and rotate, and drive roll roller 555 and rotate, thereby can clear up the surface of observation window 11 through brush 556 and scraper blade 552, and through the setting of sliding block 53 and pulley 54, it is more convenient that messenger's clearance board 55 removes, it fixes clearance board 55 to have made things convenient for through fixed block 57 and fixed slot 58 simultaneously, avoided the direct contact with observation window 11, the cultivation to the cell has been made things convenient for and has been observed.
Example 3
In a second aspect, as shown in FIGS. 1-6, the present invention provides a method for culturing entrapped suspension cells, comprising the steps of:
the method comprises the following steps: preparing a chip;
step two: prefabrication of cells;
step three: culturing cells;
step four: observing the state of the cells;
step five: and (5) collecting metabolic liquid.
The first step also comprises the following steps:
a1: cleaning the prepared cover plate layer 1, the closed layer 2 and the cell culture layer 3 by alcohol and sterile water, and drying after cleaning;
a2: the cover plate layer 1, the sealing layer 2 and the cell culture layer 3 are sent into an ultraviolet sterilization device for sterilization treatment, and the sterilization time is 5-10 minutes;
a3: the cover plate layer 1, the sealing layer 2 and the cell culture layer 3 are assembled together by the connecting mechanism 4.
The second step also comprises the following steps:
b1: digesting adherent cells into cell suspension by using trypsin, and centrifuging the cells by using a centrifuge;
b2: carrying out cell resuspension by using a culture medium, and diluting the cell suspension to obtain a seed solution;
b3: and standing the seed liquid prepared in the step B2 for 2-10 minutes.
The third step also comprises the following steps:
c1: sucking cells by using a pipette, injecting the cells into the cell injection channel through a cell injection port on the cover plate layer 1, and entering the cell culture layer 3 for culturing the cells;
c2: the cells are injected into the microchannel without generating air bubbles, and the microfluidic chip is placed in a cell culture box.
In this embodiment, when cultivateing the cell, through the disinfection of each part of micro-fluidic chip to guaranteed that the cell can not receive the pollution when cultivateing, and through coupling mechanism 4, made things convenient for the connection of chip fixed, and through the prefabrication to the cell suspension, made things convenient for to cultivate the cell, absorb and pour into the cell by the pipette again, made things convenient for the cell to get into micro-fluidic chip's inside, whole process is simple quick, has improved the efficiency of cultivateing, has reduced the cost of cultivateing.
The present invention has been described in general terms in the foregoing, but it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Therefore, modifications or improvements are within the scope of the invention without departing from the spirit of the inventive concept.
Claims (9)
1. The utility model provides an intercept micro-fluidic chip of suspension cell coculture, includes apron layer (1), the bottom of apron layer (1) is provided with airtight layer (2), the bottom of airtight layer (2) is provided with cell culture layer (3), the middle part fixedly connected with observation window (11) of apron layer (1), its characterized in that: the top of apron layer (1) is provided with coupling mechanism (4), the side fixedly connected with clearance mechanism (5) of observation window (11).
2. The microfluidic chip for trapping suspended cells for co-culture according to claim 1, wherein: coupling mechanism (4) include top board (41), top board (41) set up the top at apron layer (1), the bottom fixedly connected with connecting pin (42) of top board (41), the bottom swing joint of connecting pin (42) has adapter sleeve (43), the bottom fixedly connected with holding down plate (44) of adapter sleeve (43), the inboard fixedly connected with elasticity bent plate (45) of adapter sleeve (43), the flexible ejector pin (46) of the inboard fixedly connected with of elasticity bent plate (45), the side of flexible ejector pin (46) is provided with top tight spring (47).
3. The microfluidic chip for trapping suspension cells for co-culture according to claim 2, wherein: connecting pin (42) includes connecting rod (421), connecting rod (421) fixed connection is in the bottom of last clamp plate (41), the inside bottom swing joint of connecting rod (421) has roof (422), the top of roof (422) is provided with extrusion spring (423), the side swing joint of roof (422) has cardboard (424), the side fixedly connected with slider of cardboard (424), the spout has been seted up to the side of roof (422), the left end both sides swing joint of cardboard (424) has kicking block (425), the side of kicking block (425) is provided with fastening spring (426).
4. The microfluidic chip for trapping suspension cells for co-culture according to claim 1, wherein: clearance mechanism (5) are including protection frame (51), protection frame (51) fixed connection is in the side of observation window (11), the inside fixedly connected with guide bar (52) of protection frame (51), the side swing joint of guide bar (52) has sliding block (53), the left end swing joint of sliding block (53) has pulley (54), the right-hand member fixedly connected with cleaning plate (55) of sliding block (53), the front end fixedly connected with drive plate (56) of cleaning plate (55), the side fixedly connected with fixed block (57) of cleaning plate (55), fixed slot (58) have been seted up to the inboard of protection frame (51).
5. The microfluidic chip and the cell culture method for the co-culture of the entrapped suspension cells according to claim 4, wherein: the cleaning plate (55) comprises a moving plate (551), the moving plate (551) is fixedly connected to one side of a pulley (54), a scraping plate (552) is fixedly connected to the bottom end of the moving plate (551), a worm (553) is movably connected to the middle of the moving plate (551), the worm (553) is fixedly connected to the inside of the protective frame (51), a worm wheel (554) is meshed with the side face of the worm (553), the worm wheel (554) is movably connected to the inside of the moving plate (551), a rolling roller (555) is fixedly connected to the rear end of the worm wheel (554), and a brush (556) is fixedly connected to the side face of the rolling roller (555).
6. A culture method for entrapping suspension cells is characterized in that: the culture method for entrapping suspension cells comprises the following steps:
the method comprises the following steps: preparing a chip;
step two: prefabrication of cells;
step three: culturing cells;
step four: observing the state of the cells;
step five: and (4) collecting metabolic liquid.
7. A culture method for entrapping suspension cells according to claim 6, wherein: the first step further comprises the following steps:
a1: cleaning the prepared cover plate layer (1), the sealing layer (2) and the cell culture layer (3) by alcohol and sterile water, and drying after cleaning;
a2: the cover plate layer (1), the sealing layer (2) and the cell culture layer (3) are sent into an ultraviolet sterilization device for sterilization treatment, and the sterilization time is 5-10 minutes;
a3: and then the cover plate layer (1), the sealing layer (2) and the cell culture layer (3) are assembled together through a connecting mechanism (4).
8. A culture method for entrapping suspension cells according to claim 6, wherein: the second step further comprises the following steps:
b1: digesting adherent cells into cell suspension by using trypsin, and centrifuging the cells by using a centrifuge;
b2: carrying out cell resuspension by using a culture medium, and diluting the cell suspension to obtain a seed solution;
b3: and standing the seed liquid prepared in the step B2 for 2-10 minutes.
9. A culture method for entrapping suspension cells according to claim 6, wherein: the third step further comprises the following steps:
c1: sucking cells by using a pipette, injecting the cells into the cell injection channel through a cell injection port on the cover plate layer (1), and entering the cell culture layer (3) for culturing the cells;
c2: the cells are injected into the microchannel without generating air bubbles, and the microfluidic chip is placed in a cell culture box.
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| WO2018079866A1 (en) * | 2016-10-25 | 2018-05-03 | 서울대학교산학협력단 | Microfluidic chip for co-culturing cells |
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| WO2018079866A1 (en) * | 2016-10-25 | 2018-05-03 | 서울대학교산학협력단 | Microfluidic chip for co-culturing cells |
| CN109171574A (en) * | 2018-10-27 | 2019-01-11 | 胡文保 | A kind of ceramic tile U.S. seam ceramic tile crevice cleaning device |
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