WO2012032844A1 - 細胞培養容器及びその容器を用いた細胞培養方法 - Google Patents
細胞培養容器及びその容器を用いた細胞培養方法 Download PDFInfo
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- 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
- C12M23/24—Gas permeable parts
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- 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
- C12M23/04—Flat or tray type, drawers
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- 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
- C12M23/12—Well or multiwell plates
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- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
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- the present invention relates to a cell culture container having a well for culturing cells and forming an environment suitable for cell culture in the well, and a cell culture method using the container.
- a cell culture container is used to analyze the function of a cell and the utility of a drug on the cell, for example, by simulating a microenvironment in a living body.
- Non-Patent Document 1 In normal tissues in the body, sufficient oxygen and nutrients are supplied from the blood vessels. On the other hand, in the tumor tissue, there is no neovascularization according to the growth of cancer cells, and the individual blood vessel structure is disordered and fragile. It is said that there is. The relationship between cancer and low concentrations of oxygen and nutrients has been debated in the past, but research is particularly actively conducted recently. As knowledge obtained so far, for example, it is known that some cancer cells have acquired resistance in a low oxygen concentration region (see, for example, Non-Patent Document 1).
- Non-Patent Document 1 mentions low oxygen concentration as one of the important elements of the stem cell niche, and it is shown that cancer cells become cancer stem cell “like” by low oxygen concentration. As one of the approaches for examining the function of cancer in detail, culture of cancer cells in a low oxygen concentration state has become important, and it is necessary to reproduce the environment in which cancer cells exist .
- cell culture is generally performed in an incubator capable of maintaining a temperature of 37 ° C., water vapor saturation, and about 5% CO 2 .
- the oxygen concentration in the incubator is about 20%, which is almost the same as in the atmosphere.
- a special incubator capable of maintaining a low oxygen concentration state such as 0.7% or about 5% is used.
- ⁇ TAS micro Total Analysis system
- microfluidics a micro container (for example, a volume of 1 ⁇ L or less) manufactured using microfabrication technology is used. If the volume in the container is made minute, the liquid is confined in a closed system in a minute space, and the influence of the surrounding wall becomes large and it is considered that the liquid becomes difficult to flow, and even if there is a heat source, the occurrence of convection is suppressed.
- Patent Document 1 As a method for creating a concentration gradient of a specific component using a micro container as described above, for example, as disclosed in Patent Document 1, a method of bringing a plurality of liquids having different concentrations into contact with each other is generally used. However, when such a method is diverted to cell culture, the cell culture medium always flows, so the problem is that the cell is subjected to some stress due to the flow of the cell culture medium, and the liquid factor of the microenvironment around the cell Is caused to flow away by the flow of the cell culture solution, resulting in an environment different from the actual environment surrounding the cells.
- the upper surface of the well of the micro container is often formed of PDMS (polydimethylsiloxane). Since PDMS is gas permeable, it is effective for maintaining the same gas partial pressure in the well as in the incubator in normal cell culture. However, since the inside of the well is in equilibrium with the inside of the incubator via the PDMS film, it is difficult to form a concentration gradient such as an oxygen concentration gradient in a low oxygen concentration region.
- PDMS polydimethylsiloxane
- an object of the present invention is to stably create an environment having a concentration range suitable for cell culture of gases typified by oxygen and carbon dioxide.
- the cell culture container of the present invention is formed inside a substrate, has a space for containing cells, is connected to a flow path through which the cell culture solution is circulated, and one of a pair of opposite side surfaces of the space transmits gas and allows liquid to pass through.
- a gas comprising a first gas permeable membrane that is not allowed to pass, a well comprising a second gas permeable membrane that does not allow gas to pass through the other of the pair of side surfaces, and a well that is in contact with the well through the first gas permeable membrane.
- a second flow channel that is in contact with the well through the second gas permeable membrane and that flows a gas that does not contain the specific component or that contains a specific component at a lower concentration than the gas that flows through the first flow channel. And a flow path.
- the cell is considered as the minimum unit for measuring the function of the cell or tissue. If a cell is approximately regarded as a sphere having a diameter R, in order to observe the interaction between two cells, it is necessary that two cells be arranged on the bottom surface of the well. It becomes the size of. If the cell diameter is 10 ⁇ m, a circle having a minimum diameter of 20 ⁇ m is required. If the height is twice that of the cells, the minimum volume is 6.3 ⁇ 10 ⁇ 6 mm 3 .
- the liquid is confined in a closed system in a minute space, and the influence of the surrounding walls becomes large, making it difficult to flow and suppressing the occurrence of convection.
- the flow rate at the time of exchanging the culture medium is at most about 10 ⁇ m / sec.
- the Reynolds number (Re) is sufficiently smaller than 2300, which transitions from laminar flow to turbulent flow, and becomes laminar flow. Is considered to be controlled by diffusion. That is, the environment formed in the well can be stably maintained.
- At least one of the upper and lower surface sealing portions sealing the upper and lower surfaces of the well is a transparent window that allows the inside of the well to be visually recognized from the outside. Then, it becomes possible to confirm the position and state of the cultured cells from the outside using a microscope or the like.
- an oxygen monitor substance that changes optical properties according to the oxygen concentration in the contact liquid can be fixed to the inner surface of the transparent window. Then, the oxygen concentration distribution formed in the well can be optically measured.
- a cell culture container can also be used as a test chip for use in verification of an oxygen concentration gradient in a well.
- oxygen monitor substance for example, a fluorescent dye such as platinum porphyrin can be used.
- a fluorescent dye such as platinum porphyrin
- the measurement of oxygen concentration distribution using platinum porphyrin or the like will be described in detail in Examples.
- At least one of the upper and lower surface sealing portions that seal the upper and lower surfaces of the well is a transparent window through which the inside of the well can be visually recognized.
- Oxygen can be mentioned as a specific component that is a target of the concentration gradient formed in the well.
- the oxygen concentration in the well is preferably less than 21%. Then, an oxygen concentration gradient close to the in vivo environment can be created in the well.
- Examples of cells cultured by the cell culture method of the present invention include iPS cells. In that case, it is preferable to adjust the flow rates of the high-concentration gas and the low-concentration gas so that the oxygen concentration at the cell stop position is about 5%.
- the low oxygen concentration state is a state where the oxygen concentration is approximately 0 to 5%.
- the reason is as follows.
- the “low oxygen concentration state” for culturing cancer cells simulating a living body is appropriately set to an oxygen concentration of about 0 to 5%.
- the cell culture container includes a first gas permeable membrane and a second gas permeable membrane in which a pair of opposing side surfaces of the space inside the well each transmit gas and do not allow liquid to pass therethrough, and flow a gas containing a specific component.
- the concentration gradient of the specific component formed in the cell culture solution in the well can be adjusted by the specific component concentration of the gas flowing through the first channel and the second channel and the flow rate of each gas.
- the cell culture method according to the present invention uses the cell culture container of the present invention configured so that the inside of the well can be visually recognized from the outside, recognizes the position of the cell introduced into the well, and Since the specific component concentration is adjusted to a predetermined concentration, it is possible to make the environment surrounding the cell an environment suitable for culture or an environment close to the in vivo environment.
- high-concentration gas and low-concentration gas are always circulated in the first flow path and the second flow path, but the cell culture solution is not circulated in the well, so that the cells can be cultured without applying unnecessary stress to the cells. It is possible to stably create a suitable environment.
- FIG. 1A It is a top view which shows one Example of a cell culture container. It is sectional drawing in the AA position of FIG. 1A. It is a perspective view of the Example. It is a principal part perspective view of the container for the simulation of the oxygen concentration distribution in a well. It is a density
- the cell culture container 1 of this embodiment includes a well 5, a first channel 10 and a second channel 12 inside.
- the well 5 is a rectangular parallelepiped space.
- a part of the first flow channel 10 is in contact with one side surface of the pair of side surfaces facing each other through the gas permeable membrane 14 and the second flow channel 12 is disposed on the opposite side surface.
- the gas permeable membranes 14 and 16 are membranes that allow gas to pass but not liquid.
- One of the other pair of side surfaces of the well 5 is provided with a cell culture fluid introduction channel 6 and the other side surface with a discharge channel 8.
- the cell culture container 1 is sandwiched between two transparent substrates 2 and 4 as sealing substrates, and a flow path forming sheet 3 having a well 5, a first flow path 10 and a through groove that forms a second flow path 12. These are integrated by thermocompression bonding and configured as one chip.
- the transparent substrates 2 and 4 may be flat plates that are transparent enough to allow the inside of the well 5 to be visually recognized from the outside, such as a glass substrate or a quartz glass substrate.
- the transparent substrate 4 preferably has a thickness of about 0.5 to 1.0 mm in order to maintain strength. For example, when it is assumed that the inside of the well 5 is observed using an inverted microscope, the transparent substrate 2 has a thickness of about 0.17 mm.
- NEOFRON registered trademark
- EFEP ethylene-perfluoroethylene propene copolymer
- the thickness of the flow path forming sheet 3 is suitably 20 ⁇ m to 1000 ⁇ m.
- Neoflon can be patterned by cutting.
- Poeflon registered trademark: Sumitomo
- PTFE polytetrafluoroethylene
- the transparent substrate 4 sealing the upper surfaces of the well 5, the first flow path 10 and the second flow path 12 includes an introduction flow path 6, a discharge flow path 8, a first flow path 10 and a second flow path.
- Through holes 18, 20, 22, 24, 26, and 28 serving as inlets or outlets of the respective flow paths 6, 8, 10, 12 are opened at positions of 12 end portions.
- the well 5 is filled with the cell culture solution, the cells are accommodated in the cell culture solution, and the cell culture solution in the well 5 is kept stationary, and the first flow path 10 and the second flow
- a concentration distribution of the specific component can be formed in the well 5.
- a gas having a higher concentration of the specific component to be contained is a high concentration gas, and a gas having a lower concentration is a low concentration gas.
- the specific component is, for example, oxygen or carbon dioxide.
- the through hole 22 is used as a high concentration gas inlet to the first flow path 10
- the through hole 24 is used as the discharge port
- the through hole 26 is connected to the second flow path 12.
- the low concentration gas inlet and the through hole 28 are used as the outlet.
- a test chip for measuring the oxygen concentration distribution can be obtained.
- a pressure-sensitive coating method has been known for some time. This utilizes the fact that a fluorescent dye such as platinum porphyrin is quenched by oxygen and the amount of luminescence is a function of oxygen partial pressure.
- a reagent is prepared by dissolving the fluorescent dye in a solvent together with the matrix polymer, and this is applied to the inner surface of the transparent substrate 2 or 4 with a thickness of, for example, 3 ⁇ m.
- P-TMSP poly 1-trimethylsilyl 1-propyne
- the fluorescence intensity is measured by using, for example, a 405 nm violet laser as excitation light, making it uniform with a diffusion plate and irradiating the upper surface of the oxygen concentration visualizing test chip, and measuring 650 nm fluorescence emitted from the fluorescent dye with a CCD camera.
- the oxygen partial pressure at each position where the fluorescent dye is applied can be obtained.
- a fluorescent reagent such as platinum porphyrin is quenched by oxygen according to the Stern-Bolmer formula shown in the following formula (1).
- I is the emission intensity at the oxygen partial pressure p
- I ref is the emission intensity at the oxygen partial pressure p ref (usually set to atmospheric pressure 21 kPa)
- a 0 to A 3 are fittings. It is a coefficient.
- FIG. 6 shows the result of plotting the oxygen partial pressure and the measured fluorescence amount according to the above equation (1).
- the vertical axis represents the reciprocal of the normalized fluorescence amount
- the horizontal axis represents the normalized oxygen partial pressure. From this figure, the amount of fluorescence is several tens of times larger than the normal pressure near the oxygen partial pressure, and the amount of luminescence changes greatly with respect to the oxygen partial pressure.
- the oxygen concentration gradient tip well according to the present invention It turns out that it has sufficient performance to measure the concentration.
- the oxygen concentration distribution in the test chip well can be visualized. This makes it possible to observe and analyze the oxygen concentration distribution in the test chip well, to control the oxygen concentration gradient in the well, and to control any position in the well to a desired concentration. is there.
- FIG. 4 shows data obtained by simulating a concentration gradient formed in the well 5 using fluid analysis software.
- CoventorWare® (Coventor, Inc.), which performs analysis by the finite element method, was used as fluid analysis software for simulation.
- the fluid analysis software other analysis software using a finite element method can be used.
- a model shown in FIG. 3 simulating the cell culture vessel 1 was used.
- a region (concentration gradient forming region) 58 filled with water is disposed between two flow paths 54 and 56 surrounded by porous membranes 50 and 52, respectively.
- the width of the porous membranes 50 and 52 between the flow paths 54 and 56 and the concentration gradient forming region 58 is 100 ⁇ m, and the planar shape of the concentration gradient forming region 58 is a square of 500 ⁇ m ⁇ 500 ⁇ m.
- FIG. 4A represents the oxygen concentration distribution in the model of FIG.
- the result shown in FIG. 4B is at the center of the concentration gradient formation region (the position on the line indicated by the arrow in FIG. 4A).
- the concentration gradient formation region has a linear concentration. A gradient is formed, and the concentration gradient increases as the gas flow rate increases, and decreases as the gas flow rate decreases.
- the concentration and flow rate of the high concentration gas flowing through the first flow path 10 and the concentration and flow rate of the low concentration gas flowing through the second flow path 12 in the cell culture container 1 are controlled to form in the well 5.
- the concentration gradient of the specific component at any position in the well 5 can be controlled to a desired concentration.
- the high concentration gas flowing in the first flow path 10 is a gas containing about 5% oxygen
- the low concentration gas flowing in the second flow path 12 is a gas containing 0.1% oxygen.
- the relationship between the flow rate of the high-concentration gas and the low-concentration gas and the concentration distribution formed in the well 5 when the two gases are allowed to flow and reach an equilibrium state is measured or The simulation is performed, and the measurement data or simulation data is held in a storage medium or the like. Based on the measurement data or simulation data held, the flow rates of both gases for setting the concentration at an arbitrary position in the well 5 to a desired concentration can be set.
- the measurement data of the relationship between the flow rates of the high concentration gas and the low concentration gas and the concentration distribution formed in the well 5 can be obtained using, for example, a test chip coated with platinum morphylin. It can be obtained by the simulation described with reference to FIG.
- the culture medium is supplied from the through-hole 18 which is the culture medium inlet, and the well 5 is filled.
- the cancer cells are introduced into the well 5 together with the culture medium.
- the supply flow rate of the culture solution is preferably controlled so that the cells remain stopped near the center of the well 5. After filling the well 5 with the culture solution, the supply of the culture solution is stopped, and the culture solution in the well 5 is brought into a stationary state.
- the position of the cell introduced into the well 5 is recognized.
- Examples of the cell position recognition method include image recognition using a microscope image. Conditions are selected from the measurement data or simulation data prepared in advance so that the oxygen concentration at the recognized cell position and the surrounding environment are suitable for culture.
- the oxygen concentration in the well 5 between the first flow path 10 and the second flow path 12 reaches an equilibrium state after a predetermined time has passed, and the well 5 A stable oxygen concentration gradient is formed therein, and the cancer cells introduced into the well 5 can be cultured under suitable conditions. Since there is no flow of the culture solution in the well 5, the surrounding environment can be maintained in a stable state without the cancer cells being stressed. Since the well 5 has a size of 1 mm 3 or less, convection of the culture solution does not occur, and an environment suitable for cell culture can be stably maintained.
- a gas containing about 5% oxygen is used as the high concentration gas.
- a gas containing 21% oxygen is used as the high concentration gas. Since a living body has an oxygen concentration gradient of 0 to 21%, any environment in the living body can be reproduced in the well 5 by using a gas containing 21% oxygen as a high concentration gas.
- osteoblasts When it is desired to simulate the bone marrow in the well 5, osteoblasts, bone marrow cells, osteoclasts, etc. may be used as the cells to be placed in the well 5.
- a piece may be arranged.
- the arrangement of the bone fragments can be performed by placing the bone fragments in a well in a state where the upper surface portion in the middle of chip manufacture is opened, for example.
- the bone fragment is cut into small pieces to fit into the well using a scalpel or the like, and placed at a desired position on the bottom surface of the well using tweezers or the like. Thereafter, a process of bonding the upper member is performed.
- an oxygen concentration gradient exists in the same direction. There is an oxygen concentration gradient in the direction through which the blood vessel passes, and the oxygen concentration is maximum in the blood vessel.
- the oxygen concentration in the end organs away from the lung is considered to be about 5%, unlike 20% in the atmosphere.
- cancer cells When investigating the behavior of leukemia cancer cells, introduce cancer cells into the well and observe. When simulating other microenvironments around the tumor, it can be performed by introducing one or more cancer cells into the low concentration side of the well 5.
- cancer cells that behave like cancer stem cells can be identified by confirming that certain cells are in the resting phase of the cell cycle or have drug resistance to anticancer agents. If cancer stem cell-like cells can be detected, the cells can be removed using, for example, optical tweezers.
- iPS cells may be cultured in the well 5. iPS cells are said to be activated in a region having an oxygen concentration of about 5%, and can be cultured by forming a region having an oxygen concentration of about 5% at the position of iPS cells in well 5.
- various cells can be cultured by the cell culture method using the cell culture vessel 1.
- Cells that have been cultured and taken out can be subjected to analysis by various conventional techniques such as gene analysis.
- the present invention can be used for culturing cells for various analyzes such as gene analysis.
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Abstract
Description
この実施例の細胞培養容器1はウエル5、第1流路10及び第2流路12を内部に備えている。ウエル5は直方体形状の空間であり、その対向する一対の側面の一方の側面に第1流路10の一部区間がガス透過膜14を介して接し、反対側の側面に第2流路12の一部区間がガス透過膜16を介して接している。ガス透過膜14,16は、ガスは通すが液体を通さない膜である。ウエル5の他の一対の側面の一方の側面には細胞培養液の導入用流路6、他方の側面には排出用流路8を備えている。
2,4 透明基板
3 流路形成シート
5 ウエル
6 導入用流路
8 排出用流路
10 第1流路
12 第2流路
14,16 ガス透過膜(多孔質膜)
18,20,22,24,26,28 貫通孔
Claims (8)
- 基体内部に形成され、細胞を収容する空間をもち、細胞培養液を流通させる流路に繋がり、前記空間の対向する一対の側面の一方が気体を透過させ液体を透過させない第1ガス透過膜からなり、前記一対の側面の他方が気体を透過させ液体を透過させない第2ガス透過膜からなるウエルと、
前記第1ガス透過膜を介して前記ウエルと接し、特定成分を含むガスを流通させる第1流路と、
前記第2ガス透過膜を介して前記ウエルと接し、特定成分を含まないか又は前記第1流路に流すガスよりも特定成分を低濃度に含むガスを流通させる第2流路と、を備えた細胞培養容器。 - 前記ウエルは6.3×10-6mm3から1mm3の間の容積を有する請求項1に記載の細胞培養容器。
- 前記ウエルの上下面を封止している上下面封止部の少なくとも一方が外部から前記ウエル内部を視認可能な透明窓となっている請求項1又は2に記載の細胞培養容器。
- 一方の前記透明窓の内面に、接触液中の酸素濃度に応じて光学的性質を変化させる酸素モニタ物質が固定されている請求項3に記載の細胞培養容器。
- 請求項3に記載の細胞培養容器のウエルに細胞培養液を充填するとともに細胞を導入するステップと、
前記ウエルに導入された細胞のウエル内における停止位置を前記透明窓を介して認識するステップと、
前記第1流路及び第2流路のガス流量とウエル内に形成される特定成分の濃度分布との予め求められた関係に基づいて、細胞の停止位置における前記特定成分の濃度を所定濃度にするために第1流路及び第2流路のガス流量を設定するステップと、
前記ウエル内の細胞培養液は静止させたままで、前記第1流路及び第2流路に前記ガス流量設定ステップで設定したガス流量でガスを流すステップと、
を備えて細胞の培養を行なう細胞培養方法。 - 前記特定成分は酸素である請求項5に記載の細胞培養方法。
- 前記ウエル内の酸素濃度は21%未満である請求項6に記載の細胞培養方法。
- 前記細胞はiPS細胞であり、細胞の停止位置における酸素濃度を約5%にする請求項7に記載の細胞培養方法。
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CN201180041898.1A CN103080294B (zh) | 2010-09-08 | 2011-07-08 | 细胞培养容器及使用该容器的细胞培养方法 |
US13/821,063 US20130164848A1 (en) | 2010-09-08 | 2011-07-08 | Cell culture container and cell culture method using the container |
US16/235,432 US20190136171A1 (en) | 2010-09-08 | 2018-12-28 | Cell culture container and cell culture method using the container |
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WO2013046980A1 (ja) * | 2011-09-27 | 2013-04-04 | 株式会社島津製作所 | 細胞選別器及び細胞選別方法 |
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CN111575184B (zh) * | 2020-05-21 | 2022-11-15 | 上海理工大学 | 可以控制细胞培养环境溶氧水平的微流控芯片及制作方法 |
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CN104854230A (zh) * | 2012-12-12 | 2015-08-19 | 日立化成株式会社 | 癌细胞分离装置及癌细胞分离方法 |
JP2019000120A (ja) * | 2012-12-12 | 2019-01-10 | 日立化成株式会社 | がん細胞単離方法 |
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JPWO2012032844A1 (ja) | 2014-01-20 |
US20130164848A1 (en) | 2013-06-27 |
JP5703302B2 (ja) | 2015-04-15 |
CN103080294A (zh) | 2013-05-01 |
CN103080294B (zh) | 2017-02-15 |
US20190136171A1 (en) | 2019-05-09 |
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