WO2014175710A1 - Puce à cellule fluide miniature, procédé de culture de cellule l'employant et appareil pour analyser une image de cellule l'employant - Google Patents

Puce à cellule fluide miniature, procédé de culture de cellule l'employant et appareil pour analyser une image de cellule l'employant Download PDF

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WO2014175710A1
WO2014175710A1 PCT/KR2014/003698 KR2014003698W WO2014175710A1 WO 2014175710 A1 WO2014175710 A1 WO 2014175710A1 KR 2014003698 W KR2014003698 W KR 2014003698W WO 2014175710 A1 WO2014175710 A1 WO 2014175710A1
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cell
cell culture
microfluidic
culture chamber
chip
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PCT/KR2014/003698
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English (en)
Korean (ko)
Inventor
김성우
김덕중
이정환
최용해
김은섭
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나노바이오시스 주식회사
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Publication of WO2014175710A1 publication Critical patent/WO2014175710A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F35/80—Forming a predetermined ratio of the substances to be mixed
    • B01F35/81—Forming mixtures with changing ratios or gradients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/16—Apparatus for enzymology or microbiology containing, or adapted to contain, solid media
    • C12M1/18—Multiple fields or compartments
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • 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
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip

Definitions

  • the present invention relates to a microfluidic cell chip, a cell culture method and a cell image analysis apparatus using the same, and specifically, a microfluidic cell which improves the ability of injecting and culturing cells by separately forming a cell injecting part in addition to an inlet and an outlet.
  • a chip, a cell culture method using the same, and a cell image analyzing apparatus are examples of microfluidic cell chip, a cell culture method using the same, and a cell image analyzing apparatus.
  • the microfluidic cell chip has a function of performing various experiments at once by flowing a fluid through the microfluidic channel. Specifically, micro-channels are made using organs such as plastic, glass, and silicon, and fluids (eg, liquid samples) are transferred through these channels, and then mixed with cells in a plurality of chambers within the microfluidic cell chip. And react. As such, microfluidic cell chips are sometimes referred to as "lab-on-a-chips" in that they are performed in small chips.
  • Microfluidic cell chips can generate cost and time savings in pharmaceuticals, biotechnology and medicine, as well as increase accuracy, efficiency and reliability. For example, the use of microfluidic cell chips can significantly reduce the amount of expensive reagents used for protein and DNA analysis, leading to significant cost savings. In addition, protein samples and cell samples are used in much smaller amounts than conventional methods, thereby reducing sample waste.
  • Microfluidic cell chip 100 is the inlet 110; Diffusion dilution section 120; Cell culture section 130; And a discharge unit 140.
  • the fluid introduced from the inlet 110 flows through the diffusion dilution section 120 of the microfluid and a concentration gradient is formed and flows into the cell culture section 130.
  • the fluid reacts with the cells in the cell culture section 130, which may be injected from the inlet 110.
  • the discharge unit 140 may serve to discharge the fluid, but may also serve as a cell injection unit for injecting cells. That is, cells introduced into the discharge unit 140 may be attached to the cell culture section 130.
  • the conventional microfluidic cell chip can be injected or discharged only through the inlet or outlet.
  • the inlet or outlet is for injecting fluid to react with the cell, or for discharging the fluid or cells after the experiment is completed.
  • the inlet or outlet of the microfluidic cell chip is far from the cell culture chamber in which the cells are located, which makes it difficult to arrange the cells in the desired position.
  • the cells were injected using a relatively close outlet, but in this case, the cells were injected in the direction of the outlet of the microfluidic cell chip to position the cells in the cell culture chamber and then to form a concentration gradient.
  • the fluid is injected, in which the fluid is initially injected at a high speed / high pressure to remove bubbles in the channel, which may cause the cells previously injected to be lost. In addition, this made it difficult to control the exact cell number / seeding density of the cells to be placed in the cell culture chamber.
  • the present invention is to solve the above problems, to provide a microfluidic cell chip to improve the injection and culture ability of the cell by forming a cell injection unit in addition to the inlet and outlet, a cell culture method and a cell image analysis apparatus using the same It aims to do it.
  • a microfluidic cell chip may include a plurality of inflow portions into which a plurality of fluids are respectively injected; A concentration gradient microfluidic channel connected to the plurality of inlets and continuously diluting the concentration of the fluid; A cell culture chamber connected to the concentration gradient microfluidic channel and culturing cells; A cell injection unit formed on the cell culture chamber and into which the cells are injected; And a discharge part connected to the cell culture chamber.
  • the concentration gradient microfluidic channel may be formed in plural.
  • the cell culture chamber is formed in plural, and each of the cell culture chambers may be connected to the concentration gradient microfluidic channel to accommodate fluids of different concentrations.
  • the discharge portion is formed of a plurality, each of the discharge portion may be connected to each of the cell culture chamber.
  • the microfluidic cell chip may further include an opening and closing portion for opening or closing the cell injection portion.
  • a cell culture method using the microfluidic cell chip comprises injecting a plurality of different fluids through an inlet; Continuously diluting the fluid through the concentration gradient microfluidic channel to form a concentration gradient; And culturing the cells by injecting the cells into the cell culture chamber through the cell injection unit.
  • a cell image analysis device may be provided.
  • Cell image analysis device is the microfluidic cell chip; And an optical image analyzing apparatus.
  • the optical image analyzing apparatus may photograph and analyze the cell culture chamber in real time.
  • cells can be injected or discharged directly and accurately through a cell injection unit on a cell culture chamber.
  • the cell injection unit is present on each of the cell culture chambers, it is possible to easily control the exact cell number / density of the cell.
  • the risk of cell loss can be significantly reduced.
  • 1 shows a conventional microfluidic cell chip.
  • Figure 2a shows the front of the microfluidic cell chip according to an embodiment of the present invention.
  • Figure 2b shows the back of the microfluidic cell chip according to an embodiment of the present invention.
  • FIG. 2C shows a side view of a portion of a microfluidic cell chip in accordance with one embodiment of the present invention.
  • FIG. 3 illustrates a cell culture method according to an embodiment of the present invention.
  • Figure 2a shows the front of the microfluidic cell chip according to an embodiment of the present invention.
  • Figure 2b shows the back of the microfluidic cell chip according to an embodiment of the present invention.
  • 2C shows a side view of a portion of a microfluidic cell chip in accordance with one embodiment of the present invention.
  • the microfluidic cell chip 200 includes an inlet 210; Outlet 220; Concentration gradient microfluidic channel 230; Cell culture chamber 240; The cell injector 250 may be included.
  • the fluid that reacts with the cells in the cell culture chamber 240 may be injected through the inlet 210 and discharged through the outlet 220.
  • the inlet 210 may be formed in plural, and each inlet 210 may be injected with a fluid having a different concentration. Different fluids may be mixed with one another to differentiate into fluids having various concentrations and then react with the cells in cell culture chamber 240.
  • a plurality of discharge unit 220 may be formed, each discharge unit 220 may be connected to the cell culture chamber 240.
  • the concentration gradient microfluidic channel 230 may be connected to the inlet 210 and continuously dilute the concentration of the fluid injected through the inlet 210.
  • the concentration gradient microfluidic channel 230 may provide fluid at various concentrations to the cell culture chamber 240 by causing the fluid to be diluted several times while passing through the concentration gradient microfluidic channel 230.
  • the concentration gradient microfluidic channel 230 may be formed in plural.
  • the channels from each inlet 210 branch into one or more channels, where some of the branched channels combine with each other to form one channel and have.
  • Each of the channels is again branched into one or more channels, some of which are combined with one another to form one channel.
  • the concentration gradient microfluidic channel 230 may form various paths through which the fluid can move, and the microfluidic cell chip according to the present invention may implement various conditions of the fluid through various concentration gradients. It is possible to efficiently analyze the sensitivity of the cells.
  • the cell culture chamber 240 is formed in plural, and each cell culture chamber 240 is connected to each of the plural concentration gradient microfluidic channels 230, so that different concentrations are delivered from the concentration gradient microfluidic channel 230.
  • Cells may be injected or discharged into the cell culture chamber 240 through the cell injection unit 250.
  • the cell injection unit 250 may form a passage structure that connects the cell culture chamber 240 to the outside.
  • the cell injection unit 250 may be formed on the cell culture chamber 240, respectively.
  • the opening and closing part 260 is formed on the cell injection part 250 to open or close the cell injection part 250.
  • the cell injection unit 250 located on the cell culture chamber 240 may accurately or easily inject or discharge the cells directly.
  • the cell injection unit 250 is present on each of the cell culture chambers 240, it is possible to easily adjust the exact cell number / seeding density of cells for each cell culture chamber 240.
  • FIG. 3 illustrates a cell culture method according to an embodiment of the present invention.
  • the method 300 according to an embodiment of the present invention may be performed using the microfluidic cell chip 200 according to the embodiment of the present invention described above with reference to FIGS. 2A to 2C.
  • step S310 fluid may be injected through the inlet 210.
  • a plurality of different fluids may be injected into the inlet 210.
  • step S320 the fluid injected through the inlet 210 may be continuously diluted while passing through the concentration gradient microfluidic channel 230 to form a concentration gradient.
  • the cells may be cultured by injecting the cells into the cell culture chamber 240 through the cell injection unit 250.
  • the cells were injected through the outlet of the microfluidic cell chip to place the cells in the cell culture chamber, and then a fluid was injected to form a concentration gradient.
  • the fluid is initially injected at a high speed / high pressure to remove the bubbles in the channel, and thus the first injected cells may be lost due to the injected fluid.
  • the present invention can significantly reduce the risk of cell loss due to fluid injection by injecting cells after generating a concentration gradient first.
  • a cell image analysis device may be provided.
  • Cell image analysis device is a microfluidic cell chip according to an embodiment of the present invention described above with reference to FIG.
  • an optical image analyzing apparatus is to photograph and analyze a cell culture chamber in real time, and various optical image analyzing apparatuses applicable to the art to which the present invention pertains may be used.

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Abstract

La présente invention concerne une puce à cellule fluide miniature, un procédé de culture d'une cellule l'employant et un appareil pour analyser une image de cellule l'employant. La puce à cellule fluide miniature comprend : une pluralité de parties d'entrée pour injecter respectivement une pluralité de fluides ; un canal miniature de gradient de concentration connecté à la pluralité de parties d'entrée pour diluer en continu la concentration du fluide ; une chambre de culture de cellule connectée au canal miniature de gradient de concentration pour cultiver une cellule ; une partie d'injection de cellule formée sur la chambre de culture de cellule pour injecter la cellule ; et une partie d'évacuation connectée à la chambre de culture de cellule. La puce à cellule fluide miniature a la partie d'injection de cellule séparée en plus des parties d'entrée et de la partie d'évacuation, ce qui améliore la capacité d'injection et de culture de la cellule.
PCT/KR2014/003698 2013-04-26 2014-04-28 Puce à cellule fluide miniature, procédé de culture de cellule l'employant et appareil pour analyser une image de cellule l'employant WO2014175710A1 (fr)

Applications Claiming Priority (2)

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KR10-2013-0046904 2013-04-26
KR1020130046904A KR102010268B1 (ko) 2013-04-26 2013-04-26 미세유체 세포칩, 이를 이용한 세포 배양 방법 및 세포 영상 분석 장치

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Cited By (8)

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CN104928178A (zh) * 2015-06-11 2015-09-23 浙江大学 一种三入口浓度梯度发生器及幂函数浓度梯度的产生方法
CN107236668A (zh) * 2017-06-01 2017-10-10 西北工业大学 用于乳腺癌干细胞培养和药物分析的微流控芯片
CN109722387A (zh) * 2019-03-13 2019-05-07 山西农业大学 一种用于药物筛选的集成化微流控芯片及其应用
CN111172025A (zh) * 2020-01-14 2020-05-19 中国科学院烟台海岸带研究所 一种基于细胞的硫化氢活性检测复合微流控芯片装置
CN111876328A (zh) * 2020-07-30 2020-11-03 中国科学院烟台海岸带研究所 一种细胞低氧实验装置及分析方法
CN112300929A (zh) * 2019-07-31 2021-02-02 上海新微技术研发中心有限公司 一种微流控实验板及双面细胞培养方法
CN112375681A (zh) * 2020-11-19 2021-02-19 中国科学院上海微系统与信息技术研究所 一种器官芯片及其应用
CN112608841B (zh) * 2020-12-20 2024-04-02 华中科技大学同济医学院附属协和医院 一种肿瘤类器官培养和药物实验的微流控系统及使用方法

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KR101699232B1 (ko) * 2014-11-12 2017-01-24 고려대학교 산학협력단 주광성 또는 주화성이 우수한 미세조류 균주 선별용 마이크로 장치 및 이를 이용한 미세조류 균주 선별방법
KR102431519B1 (ko) 2015-02-10 2022-08-12 주식회사 미코바이오메드 나노구조물을 포함하는 농도구배 세포칩, 이의 제조 방법 및 이를 이용한 영상 분석 장치
KR101701607B1 (ko) * 2015-05-15 2017-02-13 성균관대학교산학협력단 항암제 내성세포 스크리닝용 미세유체 칩 및 이의 용도
KR101631940B1 (ko) 2015-07-10 2016-06-20 충남대학교산학협력단 이종 세포의 시료감응성 동시 측정을 위한 미세유체칩
US20210348098A1 (en) 2018-09-19 2021-11-11 Cellartgen Inc. Microfluidic device for cerebrovascular simulation and high-efficiency blood-brain barrier simulation system comprising same
CN112300930A (zh) * 2019-07-31 2021-02-02 上海新微技术研发中心有限公司 一种微流控实验板及双面细胞培养方法
KR102475225B1 (ko) * 2020-08-27 2022-12-07 성균관대학교산학협력단 R2r 공정을 이용한 오가노이드 실시간 모니터링 장치 제조

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104928178A (zh) * 2015-06-11 2015-09-23 浙江大学 一种三入口浓度梯度发生器及幂函数浓度梯度的产生方法
CN107236668A (zh) * 2017-06-01 2017-10-10 西北工业大学 用于乳腺癌干细胞培养和药物分析的微流控芯片
CN107236668B (zh) * 2017-06-01 2020-08-07 西北工业大学 用于乳腺癌干细胞培养和药物分析的微流控芯片
CN109722387A (zh) * 2019-03-13 2019-05-07 山西农业大学 一种用于药物筛选的集成化微流控芯片及其应用
CN112300929A (zh) * 2019-07-31 2021-02-02 上海新微技术研发中心有限公司 一种微流控实验板及双面细胞培养方法
CN111172025A (zh) * 2020-01-14 2020-05-19 中国科学院烟台海岸带研究所 一种基于细胞的硫化氢活性检测复合微流控芯片装置
CN111172025B (zh) * 2020-01-14 2022-11-15 中国科学院烟台海岸带研究所 一种基于细胞的硫化氢活性检测复合微流控芯片装置
CN111876328A (zh) * 2020-07-30 2020-11-03 中国科学院烟台海岸带研究所 一种细胞低氧实验装置及分析方法
CN112375681A (zh) * 2020-11-19 2021-02-19 中国科学院上海微系统与信息技术研究所 一种器官芯片及其应用
CN112608841B (zh) * 2020-12-20 2024-04-02 华中科技大学同济医学院附属协和医院 一种肿瘤类器官培养和药物实验的微流控系统及使用方法

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