WO2017155170A1 - Élément microfluidique et procédé de traitement de cellule unique l'utilisant - Google Patents

Élément microfluidique et procédé de traitement de cellule unique l'utilisant Download PDF

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Publication number
WO2017155170A1
WO2017155170A1 PCT/KR2016/009420 KR2016009420W WO2017155170A1 WO 2017155170 A1 WO2017155170 A1 WO 2017155170A1 KR 2016009420 W KR2016009420 W KR 2016009420W WO 2017155170 A1 WO2017155170 A1 WO 2017155170A1
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well
single cell
solution
microfluidic device
microchannel
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PCT/KR2016/009420
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English (en)
Korean (ko)
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문희성
김연정
유창은
한경연
박웅양
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삼성전자 주식회사
사회복지법인 삼성생명공익재단
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Publication of WO2017155170A1 publication Critical patent/WO2017155170A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers

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  • a microfluidic device and a single cell treatment method using the same.
  • CTCs circulating tumor cells
  • the CTC collection methods that have been published so far include a gene reading method using a polymerase chain reaction (PCR), a centrifugation method, a reading method using magnetophoresis, and a method using fluorescence staining or a filter.
  • PCR polymerase chain reaction
  • centrifugation method a centrifugation method
  • magnetophoresis a reading method using magnetophoresis
  • fluorescence staining or a filter a method using fluorescence staining or a filter.
  • the conventional methods may result in the loss of a single cell while undergoing a process of removing a large number of blood cells contained in blood for CTC detection.
  • a treatment such as staining by injecting the single cells from which the blood cells are removed again into a separate experimental device, there is a possibility that a single cell loss occurs.
  • microfluidic device capable of minimizing the loss of a single cell during various processing of a single cell.
  • the present invention also provides a single cell processing method capable of continuously processing a single cell through a microfluidic device.
  • a target solution containing a single cell as a target cell, or a reagent containing a first well, a second well disposed spaced apart from the first well, and a second from the lower surface of the first well A microfluidic device including two or more microchannels extending to the bottom surface of the well and connecting the first well and the second well, the width of the microchannel being smaller than the size of the single cell.
  • the ratio of the area of the lower surface of the first well to the width direction cross-sectional area of the microchannel may be 1000: 1 to 400000000: 1.
  • the first reservoir may further include a first reservoir formed between the two or more microchannels and the second well, and the two or more microchannels and the second well may be connected to the first reservoir.
  • the two or more microchannels may connect the first well and the second well in parallel.
  • the width of the fine flow path may be 0.5 ⁇ m to 15 ⁇ m.
  • the bottom surface of the first well and the bottom surface of the second well may be coplanar.
  • the lower surface of the first well as viewed from the top may have a circular shape.
  • the bottom surface diameter of the first well may be 1 mm to 50 mm.
  • the microfluidic device may include two or more single cell processing units including the first well, the second well, and the two or more microchannels.
  • the two or more single cell processing units may be arranged to have a matrix form.
  • a single cell treatment method using the microfluidic device injecting a sample solution containing a single cell into the first well, injecting a fixed solution into the first well to fix the single cell
  • a single cell treatment method comprising the steps of: injecting a permeate solution into the first well to pretreat the fixed single cell; and injecting a dye solution into the first well to stain the pretreated single cell.
  • a negative force may be applied to the second well to remove the sample solution, the fixative solution, the penetrating solution, and the dye solution from the first well.
  • the sample solution, the fixer, the penetrant, and the dye solution removed from the first well may be accommodated in the second well through the microchannel.
  • the flow rate of the sample solution, the fixed solution, the penetrating solution, and the dye solution passing through the microchannel by the negative pressure applied may be greater than the moving speed of the single cell induced by the negative pressure.
  • the single cell treatment method may further include the step of washing the first well by injecting a washing solution into the first well.
  • the washing liquid may be removed from the first well by applying a negative force to the second well.
  • microfluidic device capable of minimizing the loss of a single cell during various processes of single cell.
  • FIG. 1 is a perspective view of a microfluidic device according to one embodiment
  • FIG. 2 is a plan view from above of the microfluidic device of FIG. 1;
  • FIG. 3 is a cross-sectional view taken along line III-III of FIG.
  • FIG. 7 is a perspective view showing a microfluidic device including two or more single cell processing units according to another embodiment
  • FIG 8 to 18 are views sequentially showing a single cell treatment method according to one embodiment.
  • a "single-cell” which is a target cell, is a very rare cell, for example with only a few to several dozens present in a sample, for example, a drug in the blood. It refers to a circulating tumor cell (CTC), etc. present in one of the billion cells of blood cells.
  • CTC circulating tumor cell
  • "reagent” means various kinds of drugs for biochemical treatment of the single cell, and includes, for example, washing solution such as buffer solution, fixed solution, penetrating solution, dye solution, and the like. to be.
  • sample liquid means that the "single cell" is contained in a reagent such as a buffer solution.
  • FIG. 1 is a perspective view illustrating a microfluidic device according to an embodiment
  • FIG. 2 is a plan view of the microfluidic device of FIG. 1 as viewed from above.
  • the microfluidic device 100 is disposed on the substrate 10 and the substrate 10, and is a single cell processor configured to receive and process a single cell as a target cell. And 20.
  • the substrate 10 may be formed of a material that does not chemically react with various reagents.
  • the substrate 10 may be made of an optically transparent material to confirm the presence of a single cell in the sample.
  • the substrate 10 may be made of a material such as glass or plastic. For example, a slide glass may be used as the substrate 10.
  • the single cell processing unit 20 is disposed directly on the substrate 10.
  • the single cell processing unit 20 includes a body 21, a first well 22, a second well 23, and two or more microchannels 24.
  • the body 21 is formed on the substrate 10 and may have a cross-sectional area corresponding to the cross-sectional area of the substrate 10.
  • the body 21 may be made of an optically transparent material so as to confirm the presence of a single cell in the sample.
  • the body 21 may be made of a material such as glass or plastic.
  • the body 21 may be formed of a material having hydrophobicity, for example, polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the body 21 is formed of a hydrophobic material, so that a strong fluid resistance is applied to the inside of the microchannel 24 so that a first external force is not applied. Reagents in the well 22 can be prevented from flowing into the microchannel 24 and the second well 23.
  • one embodiment is not necessarily limited thereto, and may be formed of a material having hydrophilicity, and at least, the surface forming the sidewalls of the first well 22 and the second well 23 and the fine flow path 24 will be described.
  • the surfaces to be formed may be controlled to have hydrophobicity through surface modification or the like.
  • the first well 22 is a semi-closed space in which the body 21 is opened up and down, but the upper surface formed by contact with the upper surface of the substrate 10 is opened. That is, the sidewall of the first well 22 is the body 21, but the lower surface of the first well 22 is the upper surface of the substrate 10.
  • a sample or reagent may be injected through the first well 22.
  • the second well 23 is also a semi-closed space with an open top surface formed by the same method as the first well 22.
  • the second well 23 may receive and remove a sample or a reagent contained in the first well 22 through the microchannel 24.
  • the first well 22 and the second well 23 may be formed to have a circular shape.
  • the shape of the first well 22 and the second well 23 may have a polygonal shape such as a triangular shape, a square shape, a hexagonal shape, an octagonal shape, an elliptic shape, or the like.
  • the microchannel 24 extends from the bottom surface of the first well 22 to the bottom surface of the second well 23 to connect the first well 22 and the second well 23 to each other.
  • the micro-channel 24 according to the exemplary embodiment may be a space formed between the substrate 10 and the body 21 by contacting the substrate 10 after pattern etching the lower surface of the body 21.
  • two or more micro-channels 24 may extend from the first well 22 toward the second well 23.
  • two or more microchannels 24 extend from the first well 22 toward the second well 23 in parallel, respectively, and then the first well. At one point between the 22 and the second well 23 may be formed in a shape that is combined into one path and connected to the second well 23.
  • the microchannel 24 serves as a passage for supplying the sample liquid or the reagent contained in the first well 22 to the second well 23. That is, the microchannel 24 may serve as a drainage way for discharging the reagent contained in the first well 22 to the second well 23.
  • the width of the microchannel 24 is smaller than the size of a single cell.
  • a single cell may not pass through the microchannel 24 and remain inside the first well 22. That is, the micro channel 24 may discharge only the sample liquid or the reagent except the single cell inside the first well 22 to the second well 23.
  • the sample liquid or reagent contained in the first well 22 has a predetermined pressure or more. Only to be discharged to the second well 23 through the micro-channel 24.
  • the microfluidic device 100 may be formed of a sample liquid including a single cell injected into the first well 22, or a sample fluid containing only a single cell except for a single cell. Can be removed by moving to two wells (23).
  • the sample liquid or reagent contained in the first well 22 may have a predetermined positive pressure to overcome the high hydraulic resistance of the microchannel 24. 22), or a predetermined negative pressure must be applied to the second well 23.
  • Equation 1 The hydraulic resistance applied to the fine flow path 24 is expressed by Equation 1 below.
  • Equation 1 R h denotes a hydraulic resistance applied to the fine passage 24, ⁇ P denotes a pressure change amount to be applied, and Q denotes a flow rate through the fine passage 24, respectively.
  • a first pressure may be applied to the second well 23 by applying a negative pressure greater than the product of the hydraulic resistance of the microchannel 24 and the flow rate through the microchannel 24 using a pipette or the like.
  • the sample liquid contained in 22) or the sample can be moved to the second well 23.
  • the pressure capable of overcoming the hydraulic resistance of the micro-channel 24 (for example, a negative pressure greater than or equal to a threshold pressure due to the hydraulic resistance of the micro-channel 24) is equal to the first well 22 or the second well 23. )
  • the sample liquid or reagent contained in the first well 22 remains entirely in the first well 22 due to the high hydraulic resistance of the microchannel 24. That is, the micro flow path 24 may serve as a kind of check valve.
  • the microfluidic device 100 selectively removes only the sample solution including the single cells injected into the first well 22 or only the remainder except the single cells in the sample from the first well 22. It is possible to easily control the flow of the sample liquid or reagent moving from the first well 22 to the microchannel 24 without providing a separate valve in the microchannel.
  • FIG. 3 is a cross-sectional view taken along line III-III of FIG. 2.
  • the microchannel 24 has a predetermined width D2 and a predetermined length H2.
  • the width of the micro-channel 24 may have various values depending on the type of single cell to be a target cell, for example, 0.5 ⁇ m to 15 ⁇ m, for example 1 ⁇ m to 15 ⁇ m, eg For example, 1 ⁇ m to 12 ⁇ m, for example 1 ⁇ m to 10 ⁇ m.
  • the width of the micro-channel 24 is less than 0.5 ⁇ m, the removal time of the sample liquid or reagent for treating single cells is excessively increased, thereby reducing the treatment efficiency, and the width of the micro-channel 24 exceeds 15 ⁇ m.
  • the size of the microchannel 24 is about the same as that of the single cell into which the microchannel 24 is inserted, and the single cell is sucked into the microchannel 24 by the negative pressure applied to the second well 23. By closing or moving to the second well 23 to be removed, a single cell loss can occur.
  • the cross section in the width direction of the micro-channel 24 may be variously designed in consideration of the ease of processing of the body 21, for example, may be formed in a semicircle, an ellipse, a square shape, or the like.
  • the length of the micro-channel 24 is the interval between the first well 22 and the second well 23, the amount of sample liquid or sample injected into the first well 22, the second well 23 It can be variously designed according to the sound pressure applied to, for example, 5 ⁇ m to 1 mm, for example 5 ⁇ m to 800 ⁇ m, for example 5 ⁇ m to 600 ⁇ m, for example 5 ⁇ m to 500 ⁇ m have.
  • the length of the micro-channel 24 When the length of the micro-channel 24 is less than 5 ⁇ m, the flow rate of the fluid passing through the micro-channel 24 is excessively increased due to the negative pressure applied to the second well 23, so that the sample liquid in the first well 22, or It is difficult to precisely control the amount of sample. On the other hand, when the length of the micro-channel 24 exceeds 1mm, the magnitude of the negative pressure to be applied to the second well 23 increases, while the removal of the sample liquid or the sample slows down, so that the single cell treatment process time This may increase excessively.
  • the heights of the first well 22 and the second well 23 may have the same value H1.
  • the height of the first well 22 and the second well 23 can be variously set, for example 1 mm to 50 mm, 1 mm to 20 mm, for example 1 mm to 10 mm Can be.
  • the amount of the sample liquid or reagent injected into the first well 22 may be insufficient, and the first well 22 and the second well may be insufficient.
  • the height of the well 23 exceeds 50 mm, an unnecessary amount of reagent may be injected into the first well 22, and after the single cell treatment, the sample liquid or reagent may be removed through the second well 23. Is difficult, and the single cell processing process time can be long.
  • the bottom surface of the first well 22 and the bottom surface of the second well 23 may be coplanar, as shown in FIG. 3, that is, in one embodiment, The bottom surface of the first well 22, the bottom surface of the second well 23, and the bottom surface of the microchannel 24 may all be coplanar.
  • the lower surface of the first well 22, the lower surface of the second well 23, and the lower surface of the fine flow path 24 are positioned on the same plane without the step, thereby providing the first well 22 with the first well 22.
  • the sample liquid or reagent stored therein can be easily moved to the fine flow path 24 by the negative pressure applied to the second well 23.
  • the bottom surface diameter D1 of the first well 22 is larger than the bottom surface diameter D3 of the second well 23, but the bottom surface of the first well 22 Diameters of the lower surfaces of the second wells 23 may be the same as or different from each other, and the amount of the sample liquid or reagent injected or discharged, the diameter of the sample liquid or the pipette for injecting the reagent, and the like. It can be set in various ways.
  • the lower surface diameter D1 of the first well 22 and the diameter D3 of the lower surface of the second well 23 are each, for example, 1 mm to 50 mm, 1 mm to 20 mm, for example 1 mm to 10 mm. Can be.
  • the diameters of the lower surfaces of the first well 22 and the second well 23 are less than 1 mm, the amount of the sample liquid or reagent injected into the first well 22 may be somewhat insufficient. It is difficult to induce drag force.
  • the diameter of the bottom surface of the first well 22 and the second well 23 exceeds 50 mm, the amount of reagent to be injected for single cell treatment may be excessively increased.
  • the majority of single cells sink to the lower surface of the first well 22.
  • the flow rate of the sample liquid passing through the micro-channel 24 is rapidly changed by the negative pressure. Will be moved to.
  • the single cells are hardly affected by the negative pressure applied to the second well 23 and show little movement in the lower surface of the first well 22.
  • the behavior of these single cells within the first well 22 is influenced by the ratio of the bottom surface area of the first well 22 to the widthwise cross-sectional area of the microchannel 24.
  • the ratio of the area of the lower surface of the first well 22 to the widthwise cross-sectional area of the microchannel 24 is the width D2 of the microchannel 24 described above and the bottom of the first well 22. It can be variously adjusted according to the surface diameter D1, for example 1000: 1 to 800000000: 1, for example 1000: 1 to 400000000: 1.
  • the single cells are not affected by the negative pressure applied to the second well 23. It may remain on the bottom surface of one well 22.
  • valves formed in a microchannel or multiple pumped devices are used.
  • Such a general microfluidic device is troublesome to precisely control valves or to connect a fluid adapter or a syringe pump each time a specific fluid removal process is performed.
  • the microfluidic device when the microfluidic device is centrifuged by another method for concentrating a single cell, loss of the single cell may occur in the course of aspiration of the sample liquid or reagent after centrifugation.
  • loss of a single cell may occur in the process of washing, and then the single cell may be recovered and further processed. Is required, for example, when a single cell has to be recovered after characterization and extracted from DNA, etc., loss of a single cell may occur during recovery.
  • the body 21 is formed of a hydrophobic material so that a single cell may be formed in the microchannel 24 and the second well in a situation in which no external force is applied. 23) can be prevented from entering.
  • the ratio of the area of the lower surface of the first well 22 to the widthwise cross-sectional area of the microchannel 24 can be controlled.
  • the microfluidic device 100 may more easily remove only a specific fluid from the first well 22 through a simple tool such as a pipette to continuously remove a sample liquid or various reagents. Even after removal into one well 22, no single cell loss occurs.
  • the single cell treatment process may be continuously performed in the microfluidic device 100 while minimizing the loss of the single cell during various treatments of the single cell.
  • the single cell processing unit may be arranged such that two or more microchannels 24 ′ connect the first well 22 and the second well 23 in parallel.
  • the fine flow passage 24 ′ can move the sample liquid or the reagent independently of the fine flow passage 24 of FIGS. 1 to 3 described above.
  • the fine flow passage 24 ′ has a structure in which five flow passages are arranged in parallel to each other, but are not necessarily limited thereto, and the magnitude of the hydraulic resistance that varies according to the quantity of the fine flow passage 24 ′,
  • the diameter of the first well 22, the cross-sectional area, and the magnitude of the sound pressure applied to the second well 23 may be set in various ways.
  • the single cell processing unit may further include a first reservoir 25 formed between two or more microchannels 24 and the second well 23. That is, the first reservoir 25 having a larger area as compared with FIGS. 1 to 3 described above, so that the sample liquid or reagents may be temporarily stored and finally discharged through the second well 23. May be formed.
  • the length of the microchannel 24 is reduced to correspond to the area occupied by the first reservoir 25, but is not necessarily limited thereto, and the specific shape of the first reservoir 25, an acceptable volume, and the like are not limited thereto.
  • the interval between the first well 22 and the second well 23, the sample solution that can be accommodated by the first well 22 and the second well 23, or the amount of the reagent may be variously set.
  • the single cell processing unit may further include a second reservoir 26 formed in each of two or more microchannels 24. That is, as compared with the above-described FIGS. 1 to 3, the second reservoir 26 having a width wider than the width of the fine passage 24 may be formed at one side of the fine passage 24. That is, it provides a space in which the sample liquid or reagents that are moving inside the microchannel 24 may be temporarily stored.
  • the second reservoir 26 is formed independently for each of the fine flow paths 24, but is not necessarily limited thereto, and the second reservoirs 26 may be formed to be connected to each other.
  • the specific shape of the reservoir 26, the acceptable volume, etc. are the width and length of the micro-channel 24, the distance between the first well 22 and the second well 23, the first well 22 and the second
  • the amount of the sample liquid or reagent that can be accommodated in the well 23, and the flow rate of the fluid flowing through the microchannel 24 in the presence of the second reservoir 26 may be set in various ways.
  • the microfluidic device 100 may be a single cell, even more specifically, a single cell processing unit 20, more specifically, the micro-channel 24 is designed in various ways. While minimizing the loss, the single cell treatment process may be continuously performed in the microfluidic device 100.
  • FIG. 7 is an image showing a microfluidic device including two or more single cell processing units according to another embodiment.
  • the microfluidic device 200 includes two or more single cell processing units 20, and may be arranged in a row between neighboring single cell processing units 20. That is, the two or more single cell processing units 20 may be arranged to have a matrix form.
  • single cell processing of each of the single cell processing units 20 may be performed independently.
  • the microfluidic device 200 may be, for example, a well-plate having a plurality of single cell processing units 20, but is not limited thereto.
  • the single cell processing unit 20 can also be applied to various experimental tools.
  • the microfluidic device 200 may include two or more single cell processing units 20 to enable independent single cell processing, thereby improving single cell processing efficiency.
  • Single cell treatment method the step of injecting a sample solution containing a single cell into the first well 22, the step of fixing the single cell by injecting a fixed solution into the first well 22, Injecting the permeate solution into the first well 22 to pretreat the fixed single cell, and injecting the dye solution into the first well 22 to stain the pretreated single cell.
  • the sample liquid 3 including the single cell 2 is injected into and received in the first well 22.
  • the accommodated sample liquid 3 does not move inside the microchannel 24 due to the hydraulic resistance of the microchannel 24 and remains in the first well 22.
  • the single cells 2 mostly sink to the bottom surface of the first well 22.
  • the first well 22 when a negative pressure greater than or equal to the threshold pressure due to the hydraulic resistance of the microchannel 24 is applied to the second well 23 using a pipette or the like, the first well 22 may be accommodated.
  • the sample liquid 3 flows along the fine flow path 24 toward the second well 23 at the first speed V1.
  • the single cells 2 that have sunk on the lower surface of the first well 22 are induced by the negative pressure, and move toward the micro-channel 24 at the second speed V2.
  • the widthwise length of the microchannel 24 is formed to be smaller than the size of the single cell 2, so that the single cells 2 do not pass through the microchannel 24 and enter the first well 22. Will remain. Further, by adjusting the cross-sectional area of the micro channel 24 in the width direction cross-sectional area and a first well 22 of the second speed V2 than the first speed V1 at least 10 - it shows a low speed of about six times. Accordingly, even if the sample liquid 3 is removed from the first well 22 as shown in FIG. 10, the single cells 2 still remain in the first well 22.
  • it may further comprise the step of washing the first well 22 by injecting a washing solution into the first well 22, the washing step is the sample solution injection step, fixing step, infiltration
  • the washing step is the sample solution injection step, fixing step, infiltration
  • the removal of the sample solution, the fixed solution, the penetrating solution and the staining solution except for the single cell 2 may be performed.
  • the washing liquid 4 in the washing step, when the washing liquid 4 is injected into the first well 22 of FIG. 10, the washing liquid 4 is also formed by the hydraulic resistance of the micro-channel 24. (22) It exists inside. Subsequently, when a negative pressure is applied to the second well 23, as shown in FIG. 9 described above, the washing liquid 4 passes through the microchannel 24 and is received in the second well 23, whereas a single cell is used. (2) hardly move inside the first well 22.
  • the fixing solution 5 is injected into the first well 22 of FIG. 12 to fix the single cell 2 to the lower surface of the first well 22.
  • the single cells 2 fixed to the lower surface of the first well 22 by the fixation solution 5 are removed together with a small amount of fixation solution 5 as shown in FIG. 14 even if the fixation solution 5 is removed by negative pressure. It remains inside the one well 22.
  • the inside of the first well 22 is cleaned by performing the washing process as illustrated in FIG. 11 once, and then a negative pressure is applied to the second well 23 to remain in the first well 22. Remove the fixed solution (5) and wash solution. If some of the fixed single cells 2 are detached from the lower surface of the first well 22 in the course of performing the washing process, the width of the microchannel 24, the hydraulic resistance, and the single cells 2 are described above. Due to the drag induced, the single cell 2 remains inside the first well 22.
  • the permeate solution 6 is injected into the first well 22 to pass through the cell membrane of the single cell 2 fixed to the lower surface of the first well 22.
  • a negative pressure is applied to the second well 23 to remove the permeate solution 6 as shown in FIG. 16.
  • the inside of the first well 22 is cleaned by performing the washing process as illustrated in FIG. 11 once, and then a negative pressure is applied to the second well 23 to remain in the first well 22. Remove the permeate (6) and wash solution.
  • the dye solution 7 is injected into the first well 22 to perform staining of the infiltrated single cell 2, and then the second well 23. Negative pressure is applied to remove the dye solution 7 as shown in FIG.
  • the inside of the first well 22 is cleaned by performing the washing process as illustrated in FIG. 11 once, and then a negative pressure is applied to the second well 23 to remain in the first well 22. Remove the dye solution (7) and wash solution.
  • a sample cell injected into the first well 22 of the microfluidic device 100, or a cell treatment process for continuously exchanging various reagents is performed, It may remain in the first well 22 without being lost.
  • one embodiment may provide a single cell processing method capable of continuously processing a single cell 2 through the microfluidic device 100.
  • a predetermined groove pattern is formed on the surface of the upper surface of a PDMS (Dow Corning Co., Ltd.) substrate having a width of 75 mm, a length of 25 mm, and a thickness of 5 mm using a soft lithography method.
  • the depth of the formed groove pattern may be 10 ⁇ m, and the line width may be 6 ⁇ m.
  • the PDMS substrate is punched to form a first open portion and a second open portion that open the PDMS substrate in the vertical direction.
  • the first open portion and the second open portion may have a circular cross-sectional shape, the cross-sectional diameter of the first open portion may be 4 mm, and the cross-sectional diameter of the second open portion may be 0.3 mm.
  • the formed first open portion and the second open portion are connected to the groove pattern, respectively.
  • the slide glass is attached to the upper surface of the PDMS substrate on which the groove pattern is formed, and then the upper and lower sides of the PDMS substrate to which the slide glass is attached are inverted, thereby manufacturing the microfluidic device as illustrated in FIG. 1.
  • the space formed by the first open portion and the slide glass is the first well
  • the space formed by the second open portion and the slide glass is the second well
  • the space formed by the groove and the slide glass is the micro flow path.
  • MCF7 breast cancer cells
  • PBS phosphate buffered saline
  • PFA paraformaldehyde
  • the first and the Hoechst blue
  • FITC green
  • PE red
  • 0.1% saponin 0.1%
  • saponin 0.1%
  • a mixed solution 20 ⁇ L of PBS to the staining solution to the first well injection, and performs MCF7 dyeing of over 90 minutes.
  • the stained MCF7 is observed through a fluorescence microscope.
  • the nuclear nucleus portion of MCF7 is well colored by Hoechst
  • the cytokeratin portion of MCF7 is green by FITC
  • the EpCAM portion of MCF7 is red by PE.
  • prostate cancer cells PC3
  • melanoma M14
  • colorectal cancer cells HT29
  • gastric cancer cells HGC27, NUGC2, MKN7, MKN28
  • ovary MCF7 as a single cell Cancer cells (OVCA433) or the like
  • PC3 prostate cancer cells
  • M14 melanoma
  • HT29 colorectal cancer cells
  • HGC27, NUGC2, MKN7, MKN28 gastric cancer cells
  • OVCA433 ovary MCF7 as a single cell Cancer cells
  • the cytokeratin portion may be dyed red using PE, and the CD45 portion may be dyed green using Alexa488.
  • microfluidic device As such, using the microfluidic device according to the embodiment, it is possible to continuously perform various cell treatments of a single cell in the microfluidic device.
  • MCF7 as a single cell, about 10 to 40 pre-fixed MCF7s are injected into the first well of the microfluidic device, 20 ⁇ L of PBS is injected into the first well, and the pipette is placed in the second well. The process of removing the PBS remaining in the first well by applying negative pressure is repeated a total of 32 times. The quantity of MCF7 remaining in the first well is recorded for each iteration step, and the number of times the MCF7 is lost is determined. The results are shown in Table 1 below.
  • the number of times MCF7 loss occurred was only 2 out of 32 times. You can check it. That is, by using the microfluidic device according to the embodiment, it is possible to minimize the loss of a single cell even when undergoing repeated cell treatment step unlike the general centrifugation method.

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Abstract

L'invention concerne : un élément microfluidique comprenant un premier puits dans lequel une solution d'échantillon contenant une cellule unique, en tant que cellule cible, ou un réactif est logée, un second puits disposé de façon à être espacé du premier puits, et au moins deux microcanaux pour relier le premier puits et le second puits en s'étendant de la surface inférieure du premier puits à la surface inférieure du second puits, la largeur des microcanaux étant plus petite que la taille de la cellule unique ; et un procédé de traitement de cellule unique l'utilisant.
PCT/KR2016/009420 2016-03-10 2016-08-25 Élément microfluidique et procédé de traitement de cellule unique l'utilisant WO2017155170A1 (fr)

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KR1020160028958A KR101970646B1 (ko) 2016-03-10 2016-03-10 미세 유체 소자, 및 이를 이용한 단일 세포 처리 방법
KR10-2016-0028958 2016-03-10

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KR102013996B1 (ko) * 2017-12-01 2019-10-21 (주) 비비비 미세유체분석칩 제조 방법
KR102013997B1 (ko) * 2017-12-04 2019-08-29 (주) 비비비 미세 주입기를 가진 미세유체분석칩 및 그 제조 방법 및 그 사용 방법
KR102556859B1 (ko) 2022-12-15 2023-07-20 (주)제이에이치에너지 건축물 에너지 진단 시스템 및 방법

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