WO2017007278A1 - Dispositif de culture cellulaire automatisé, et procédé de fonctionnement de dispositif de culture - Google Patents

Dispositif de culture cellulaire automatisé, et procédé de fonctionnement de dispositif de culture Download PDF

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Publication number
WO2017007278A1
WO2017007278A1 PCT/KR2016/007437 KR2016007437W WO2017007278A1 WO 2017007278 A1 WO2017007278 A1 WO 2017007278A1 KR 2016007437 W KR2016007437 W KR 2016007437W WO 2017007278 A1 WO2017007278 A1 WO 2017007278A1
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WO
WIPO (PCT)
Prior art keywords
container
incubator
protocol
microscope
automatic cell
Prior art date
Application number
PCT/KR2016/007437
Other languages
English (en)
Korean (ko)
Inventor
남도현
유규하
이진구
Original Assignee
사회복지법인 삼성생명공익재단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020160037323A external-priority patent/KR101767336B1/ko
Application filed by 사회복지법인 삼성생명공익재단 filed Critical 사회복지법인 삼성생명공익재단
Priority to US15/569,873 priority Critical patent/US20180127695A1/en
Priority to EP16821684.4A priority patent/EP3284815B1/fr
Priority to CN201680024532.6A priority patent/CN107636142A/zh
Publication of WO2017007278A1 publication Critical patent/WO2017007278A1/fr
Priority to HK18110679.7A priority patent/HK1251248A1/zh

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B25—HAND TOOLS; PORTABLE POWER-DRIVEN TOOLS; MANIPULATORS
    • B25J—MANIPULATORS; CHAMBERS PROVIDED WITH MANIPULATION DEVICES
    • B25J9/00—Programme-controlled manipulators
    • B25J9/02—Programme-controlled manipulators characterised by movement of the arms, e.g. cartesian coordinate type
    • 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
    • C12M1/26—Inoculator or sampler
    • 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
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

Definitions

  • the present invention relates to an automatic cell incubator and a method of operating the incubator, and more particularly, to an automatic cell incubator including at least one of an incubator, a microscope, a robot arm, a reservoir, a liquid handler, a centrifuge, and a control device. It relates to a method of operation.
  • Cell culture generally refers to a process of separating and culturing cells separated from an organism. Aseptically select biological tissues, treat them with digestive enzymes such as trypsin or pronase, separate them into single cells, perform primary cultures, and transplant single cells obtained by dispersing passaged cell lines or cell lines with the same enzyme treatment to the growth medium. Inoculate the following passages. As such, a method of dispersing and culturing single cells by treatment with proteolytic enzymes is referred to as cell culture.
  • Cell culture includes monolayer culture in which cells attach to the incubator and proliferate, and suspension culture in which cells proliferate in a suspended state without attachment / extension.
  • monolayer culture in which cells attach to the incubator and proliferate
  • suspension culture in which cells proliferate in a suspended state without attachment / extension.
  • single cell cultures in which single cells are cultured to form colonies, and conversely, bulk cultures in which a large number of cells are cultured.
  • the present invention seeks to provide an automatic cell incubator comprising at least one of an incubator, microscope, robotic arm, reservoir, liquid handler, centrifuge and control device, and to provide a method for the operation of the incubator.
  • an automatic cell incubator may be provided.
  • an embodiment of the present invention may be provided a method of operating an automatic cell incubator.
  • An automatic cell incubator containing at least one container for culturing cells, a microscope for observing the cell state in the container, a robot arm for moving the location of the container, liquid into the container And a control device for controlling the operation of at least one of a liquid handler and an incubator, a microscope, a robotic arm, and a liquid handler for introducing or withdrawing liquid from the vessel.
  • the automatic cell incubator according to an embodiment of the present invention further comprises a reservoir for storing the container to be moved by the robot arm and a centrifuge for separating particles of the material contained in the container by using centrifugal force.
  • a reservoir for storing the container to be moved by the robot arm and a centrifuge for separating particles of the material contained in the container by using centrifugal force.
  • Method for operating an automatic cell incubator the step of extracting at least one container containing the cells cultured for a predetermined time in the incubator from the incubator using a robot arm, the cell state in the extracted container Observing using a microscope, selecting an operating protocol for the container based on the results of the observation, and driving the robot arm according to the selected operating protocol.
  • the operation protocol for the container may include at least one of a protocol for an operation for re-injecting the container into the incubator, a protocol for an operation for injecting media into the container, and a protocol for a subculture operation using the container. Can be.
  • driving the robot arm may cause the robot arm to move the container extracted from the incubator or reservoir to the loader.
  • the method of operating an automatic cell incubator includes: moving a container moved to a loader to a first working part using a grip part included in a liquid handler, inhaling the medium provided in the medium storage using the liquid handler, Dispensing the medium into a container located in the first working portion, moving the container dispensed with the medium to the loader using the grip portion, and re-injecting the container moved to the loader into the incubator.
  • driving the robot arm causes the robot arm to move the container extracted from the incubator or reservoir to the loader.
  • the method of operating an automatic cell incubator includes the steps of: moving a container moved to the loader to a first working part using a grip part included in the liquid handler, and a second operation of a material contained in the container moved to the first working part.
  • Transferring the vessel having a predetermined shape located in the portion performing centrifugation by moving the vessel having a predetermined shape to a centrifuge, processing the separated particles through centrifugation, and processing the processed particles using a microscope Observing, transferring the processed particles to a new vessel, and reloading the new vessel into the incubator. It may further include.
  • the container having a predetermined shape is a tubular container having a stopper, and may be, for example, an E-tube (Eppendorf-Tube) or a C-tube (Centrifuge-Tube).
  • the step of transferring the material contained in the container moved to the first working portion to a container of a predetermined shape located in the second working portion, after dispensing and mixing a predetermined solution into the container moved to the first working portion The method may further include transferring the obtained mixed liquid to a container having a predetermined shape.
  • Processing the separated particles through centrifugation removing the supernatant generated in the container through centrifugation, injecting a predetermined material into a container of a predetermined shape and mixing ( mixing and suspension separation, centrifuging the vessel containing the suspended separated material, removing the supernatant generated in the vessel by centrifugation, injecting the medium into the vessel, and mixing ( mixing) and mixing the predetermined material and the cell solution using another container having a predetermined shape.
  • the predetermined material may be an enzyme or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Observing the processed particles according to an embodiment of the present invention by using a microscope further includes transferring the material contained in the container having a predetermined shape to the observation aid before the microscope observes the observation particle.
  • Transferring the processed particles into a new container further includes dispensing the medium into a new container prior to the transfer of the particles, wherein the material containing the processed particles is dispensed with the medium. Can be transferred to a new container.
  • a computer-readable recording medium having recorded thereon a program for executing the above method on a computer may be provided.
  • culturing the cells stably and precisely or separating the cells eg, centrifugation, etc.
  • processing eg, addition of enzyme, pipetting, medium addition, etc.
  • an automatic cell incubator according to an embodiment of the present invention can be performed quickly and efficiently Trypan blue aliquots, cell identification (eg counting, etc.) that can determine the percentage of dead cells.
  • FIG. 1A is a block diagram of an automatic cell incubator in accordance with one embodiment of the present invention.
  • FIG. 1B is a block diagram of an automatic cell incubator according to another embodiment of the present invention.
  • 1C is a cross-sectional view of a centrifuge according to an embodiment of the present invention.
  • Figure 2 is an embodiment of an automatic cell incubator according to an embodiment of the present invention.
  • FIG 3 is a plan view of a work space of an automatic cell incubator according to an embodiment of the present invention.
  • FIG. 4 is a flowchart illustrating a method of operating an automatic cell incubator according to an embodiment of the present invention.
  • FIG. 5 is a flowchart illustrating a method of operating an automatic cell incubator based on a protocol selected according to an embodiment of the present invention.
  • 6A-6C are flowcharts illustrating a method of operating an automatic cell incubator according to a selected protocol.
  • FIG. 7 is a plan view of an automatic cell incubator without the robot arm according to another embodiment of the present invention.
  • FIG 8 is an embodiment of a cooling unit according to an embodiment of the present invention.
  • FIG 9 is an embodiment of a heating unit according to an embodiment of the present invention.
  • FIG. 10 is an embodiment of a work unit according to an embodiment of the present invention.
  • FIG 11 is an embodiment of a grip unit according to an embodiment of the present invention.
  • FIG 13 is an embodiment of a microscope according to an embodiment of the present invention.
  • FIG 14 is an embodiment of an incubator and incubator loader in accordance with one embodiment of the present invention.
  • 15 is an example of (a) an implementation of an image acquisition unit and (b) an acquired image screen according to an embodiment of the present invention.
  • an automatic cell incubator may be provided.
  • an embodiment of the present invention may be provided a method of operating an automatic cell incubator.
  • An automatic cell incubator containing at least one container for culturing cells, a microscope for observing the cell state in the container, a robot arm for moving the location of the container, liquid into the container And a control device for controlling the operation of at least one of a liquid handler and an incubator, a microscope, a robotic arm, and a liquid handler for introducing or withdrawing liquid from the vessel.
  • the automatic cell incubator according to an embodiment of the present invention further comprises a reservoir for storing the container to be moved by the robot arm and a centrifuge for separating particles of the material contained in the container by using centrifugal force.
  • a reservoir for storing the container to be moved by the robot arm and a centrifuge for separating particles of the material contained in the container by using centrifugal force.
  • Method for operating an automatic cell incubator the step of extracting at least one container containing the cells cultured for a predetermined time in the incubator from the incubator using a robot arm, the cell state in the extracted container Observing using a microscope, selecting an operating protocol for the container based on the results of the observation, and driving the robot arm according to the selected operating protocol.
  • the operation protocol for the container may include at least one of a protocol for an operation for re-injecting the container into the incubator, a protocol for an operation for injecting media into the container, and a protocol for a subculture operation using the container. Can be.
  • driving the robot arm may cause the robot arm to move the container extracted from the incubator or reservoir to the loader.
  • the method of operating an automatic cell incubator includes: moving a container moved to a loader to a first working part using a grip part included in a liquid handler, inhaling the medium provided in the medium storage using the liquid handler, Dispensing the medium into a container located in the first working portion, moving the container dispensed with the medium to the loader using the grip portion, and re-injecting the container moved to the loader into the incubator.
  • driving the robot arm causes the robot arm to move the container extracted from the incubator or reservoir to the loader.
  • the method of operating an automatic cell incubator includes the steps of: moving a container moved to the loader to a first working part using a grip part included in the liquid handler, and a second operation of a material contained in the container moved to the first working part.
  • Transferring the vessel having a predetermined shape located in the portion performing centrifugation by moving the vessel having a predetermined shape to a centrifuge, processing the separated particles through centrifugation, and processing the processed particles using a microscope Observing, transferring the processed particles to a new vessel, and reloading the new vessel into the incubator. It may further include.
  • the container having a predetermined shape is a tubular container having a stopper, and may be, for example, an E-tube (Eppendorf-Tube) or a C-tube (Centrifuge-Tube).
  • the step of transferring the material contained in the container moved to the first working portion to a container of a predetermined shape located in the second working portion, after dispensing and mixing a predetermined solution into the container moved to the first working portion The method may further include transferring the obtained mixed liquid to a container having a predetermined shape.
  • Processing the separated particles through centrifugation removing the supernatant generated in the container through centrifugation, injecting a predetermined material into a container of a predetermined shape and mixing ( mixing and suspension separation, centrifuging the vessel containing the suspended separated material, removing the supernatant generated in the vessel by centrifugation, injecting the medium into the vessel, and mixing ( mixing) and mixing the predetermined material and the cell solution using another container having a predetermined shape.
  • the predetermined material may be an enzyme or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Observing the processed particles according to an embodiment of the present invention by using a microscope further includes transferring the material contained in the container having a predetermined shape to the observation aid before the microscope observes the observation particle.
  • Transferring the processed particles into a new container further includes dispensing the medium into a new container prior to the transfer of the particles, wherein the material containing the processed particles is dispensed with the medium. Can be transferred to a new container.
  • a computer-readable recording medium having recorded thereon a program for executing the above method on a computer may be provided.
  • any part of the specification is to “include” any component, this means that it may further include other components, except to exclude other components unless otherwise stated.
  • the terms “... unit”, “module”, etc. described in the specification mean a unit for processing at least one function or operation, which may be implemented in hardware or software or a combination of hardware and software. .
  • a part of the specification is “connected” to another part, this includes not only “directly connected”, but also “connected with other elements in the middle”.
  • FIG. 1A is a block diagram of an automatic cell incubator in accordance with one embodiment of the present invention.
  • Automated cell incubator 1000 for receiving at least one container for culturing cells, the microscope 200 for observing the state of the cells in the container, the position of the container Robot arm 300 for movement, liquid handler 400 and incubator 100, microscope 200, robot arm 300, and liquid handler for introducing liquid into or out of the container.
  • Control device 500 for controlling the operation of at least one of 400.
  • the incubator 100 of the automatic cell incubator 1000 there may be at least one container containing cells.
  • the cells may be cultured in the incubator 100 for a predetermined time.
  • the predetermined time may be 1 day to 1 week, and the time the cells should be present in the incubator may be increased or decreased to 2 days or 3 days depending on the culture conditions of the cells.
  • the container in which these cells are housed may be, for example, a 6 well plate, but is not limited thereto. Any container that can contain at least one of a cell and a cell fluid can be referred to herein as a "container".
  • the microscope 200 is an inverted microscope, the lens can be replaced automatically.
  • the lens may be replaced with a lens having a magnification of x1.25, x4, x10 or x20 based on the lens replacement signal applied from the control device 500.
  • the microscope 200 may be used to observe the color, area or size of the medium precisely and accurately. It is also possible to observe the culture state of the cells.
  • the robot arm 300 includes a rod part for gripping various containers at one end thereof, and may be implemented in a multi-joint form operated by at least one motor. Therefore, the robot arm 300 may maintain the horizontal or vertical state of the rod portion and move up, down, left, right, or rotationally.
  • the robot arm 300 may be controlled by the control device 500 described above.
  • the robot arm 300 may be disposed at the center of the automatic cell incubator 1000 to be used for movement of various containers, or may be additionally disposed at one side to be used for processing a cell.
  • the liquid handler 400 may transport a container or a solution.
  • Pipettes Pierette
  • Pipettes that can be included in the liquid handler 400 may be used to introduce the liquid into or out of the container.
  • the control device 500 may be a wearable device such as a personal computer, a laptop, a tablet PC, a smartphone, a PDA, or a smart watch.
  • the control device 500 may refer to a device capable of transmitting and receiving data to or from another device in a wired or wireless manner.
  • the control device 500 is provided with a display function, it is possible to provide the operation state information, cell information and the like of the automatic cell incubator 1000 to the user in real time.
  • FIG. 1B is a block diagram of an automatic cell incubator according to another embodiment of the present invention.
  • the automatic cell incubator 1000 uses the reservoir 600 and the centrifugal force for storing the container to be moved by the robot arm 300 to collect particles of the material contained in the container. It may further include a centrifuge 700 for separation.
  • the reservoir 600 may include at least one mounting bracket for temporarily or short-term storage of various containers.
  • the reservoir 600 may be capable of rotational movement based on a signal applied from the control device 500.
  • the robot arm 300 may not only transport various containers to the storage 600 but also freely change the positions of the various containers such as extracting the various containers from the storage 600 and re-injecting the containers into the incubator 100. Can be.
  • the centrifuge 700 is for separating cells contained in a container (eg, C-tube, etc.), and may separate the cell layer using centrifugal force.
  • a container eg, C-tube, etc.
  • 1C is a cross-sectional view of a centrifuge according to an embodiment of the present invention.
  • Centrifugal separator 700 transmits the rotational force from the bucket 710 for receiving the container, a high speed rotation motor 720 for rotating the bucket 710 at high speed, high speed rotation motor 720
  • the high speed rotation unit 730 which is a high speed rotation space including at least one rod for rotating the container, and detects the rotation speed of the high speed rotation motor 720 when the speed is sufficiently reduced (for example, 1 r / min).
  • the electromagnet clutch 740 for stopping the rotation of the bucket 710, the high-speed rotary motor 720 and the electromagnet clutch 740 are rotated at a low speed in the existing rotation direction or the opposite direction bucket 710 It may include a position control motor 750 for rotating the position of the user's desired position.
  • the desired location of the user may be the location (eg, location at start) when inserting the container into the bucket 710 prior to centrifugation.
  • the centrifuge 700 measures the solution amount of the container containing the solution to be centrifuged by using a camera unit or the like, and then, on the opposite bucket of the bucket containing the container containing the solution to be centrifuged.
  • the principle of stably grasping the center of gravity that may be required during centrifugation may be applied by automatically filling the container with the same amount of water.
  • Figure 2 is an embodiment of an automatic cell incubator according to an embodiment of the present invention.
  • the automatic cell incubator 1000 according to an embodiment of the present invention may be implemented in various structures and forms.
  • the automatic cell incubator 1000 includes an incubator 100, a microscope 200, a robot arm 300, a liquid handler 400, and a control device 500. can do. At least one of the incubator, microscope, robotic arm and liquid handler can be connected to the control device 500 and can be manipulated by the control device 500.
  • the automatic cell incubator 1000 may further include a reservoir 600 and a centrifuge 700.
  • the automatic cell incubator 1000 is provided with a work space (eg, a circular dotted line region of FIG. 2) for observation, measurement, processing, etc. of cells, such as the first working part, the second working part, and the like. This will be described later with reference to FIG. 3.
  • FIG 3 is a plan view of a work space of an automatic cell incubator according to an embodiment of the present invention.
  • the workspace has at least one pipette compartment (eg 1, 2), a C-tube compartment (eg 3), a buffer zone (eg 4, 14), a loader (5) may be included.
  • the loader 5 is movable in a predetermined direction and angle.
  • the automatic cell incubator 1000 may further include a waste storage 6, a first working part 7, a second working part 8, an image acquisition part 9, a medium container 10, and the like.
  • the image acquisition unit 9 may include at least one camera unit and an illumination unit.
  • the camera unit may acquire an image of a container including cells, and determine a cell layer or the like based on the luminance value, chroma value, pixel value, etc. of the acquired image.
  • a general image processing technique for the obtained image it is possible to determine the boundary of the image, and to estimate the cell layer or the like relatively accurately according to the determined boundary.
  • the automatic cell incubator 1000 includes a first medium (material) storage 11, a second medium (material) storage 12, a T-flask working part 13, a decapper (eg, 16, 17), the liquid handler 400, the centrifuge 700 may be further included.
  • the T-flask working part 13 may move the T-flask in a single or multiple times in the up and down directions based on a signal applied from the control device 500.
  • the decapper may be a T-flask decapper 16, a C-tube decapper 17, or the like.
  • the liquid handler 400 is movable in the front and rear directions along the longitudinal rod. In addition, the liquid handler 400 is movable in the left and right directions along the horizontal rod. Operation of the automatic cell incubator 1000 according to an embodiment of the present invention will be described below with reference to FIGS. 4 to 6C.
  • FIG. 4 is a flowchart illustrating a method of operating an automatic cell incubator according to an embodiment of the present invention.
  • the incubator 100 using the robot arm 300 at least one container containing the cells cultured for a predetermined time in the incubator 100. Extracting from step (S100), observing the cell state in the extracted container by using a microscope (S200), selecting an operation protocol for the container based on the result of the observation (S300) and the selected In operation S400, the robot arm 300 may be driven according to an operation protocol.
  • cells may be cultured for 3 days to 4 days.
  • the container containing the cells needs to be moved from the incubator 100 to the work space for observation, measurement or processing on the cultured cells.
  • At least one container containing the cells cultured in the incubator 100 for a predetermined time may be extracted from the incubator 100 by the robot arm 300 (S100).
  • the cell state in the extracted container can be observed using the microscope 200 (S200). In other words, the cell state can be photographed through the microscope 200. Microscopic images of cells obtained through imaging can be transmitted to and displayed on the control device 500.
  • the user may observe or analyze a microscope image of the cell to select an operation protocol for the container in which the cell is accommodated (S300).
  • the operation of the automatic cell incubator 1000 may be controlled according to the operation protocol selected by the user through the control device 500 (S400).
  • the robotic arm may be driven in accordance with the selected operating protocol to change the position of the vessel, the position may be changed and various operations on the vessel (eg, observation, measurement or processing of cultured cells, etc.) may be performed.
  • the operating protocol for the container may be at least one of the protocols for the culture operation.
  • FIG. 5 is a flowchart illustrating a method of operating an automatic cell incubator based on a protocol selected according to an embodiment of the present invention.
  • At least one container containing cells cultured for a predetermined time in the incubator 100 is robotized. Extracting from the incubator 100 using the arm 300 (S100), observing the cell state in the extracted container using the microscope 200 (S200), operation for the container based on the results of the observation
  • the method may include selecting a protocol (S300), and the robot arm 300 may be driven according to the selected operating protocol. In other words, when the first protocol is selected as shown in FIG. 5, the operation of the robot arm 300 may proceed to step A.
  • the process may proceed to step B with respect to the driving of the robot arm 300
  • the third protocol when the third protocol is selected, the process may proceed to step C with respect to the driving of the robot arm 300.
  • the first protocol may be a protocol for operation for re-charging the container into incubator 100.
  • the second protocol may be a protocol for operation for injecting the medium into the container
  • the third protocol may be a protocol for subculture operation with the container.
  • 6A-6C are flowcharts illustrating a method of operating an automatic cell incubator according to a selected protocol.
  • the container when the first protocol is selected, which may be a protocol for operation to reload the container into the incubator 100, the container may be reloaded into the incubator 100 by the robot arm 300. (S410).
  • driving the robot arm 300 according to the selected operation protocol eg, the first protocol
  • S400 may include re-injecting the container into the incubator 100 using the robot arm 300 (S410). It may include.
  • driving the robot arm 300 is robot arm It may include a step (S420) to cause 300 to move the container extracted from the incubator 100 or the reservoir 600 to the loader (5).
  • the operation method of the automatic cell incubator 1000 the step of moving the container moved to the loader (5) to the first working part 7 using the grip part included in the liquid handler 400 (S510), Aspirating the medium provided in the medium storage using the liquid handler 400, and dispensing the sucked medium to a container located in the first working part (S520), the container dispensed with the medium using the grip unit
  • the method may further include the step S530 of moving to the loader 5 and the step S540 of re-injecting the container moved to the loader 5 into the incubator 100.
  • the robot arm 300 may move the container extracted from the incubator 100 or the reservoir 600 to the loader 5.
  • the container may be a cell container such as a 6well plate or the like.
  • the container moved to the loader 5 may be moved to the first working part 7 by the grip part included in the liquid handler 400.
  • the lid of the 6well plate moved to the first working part 7 may be opened.
  • the medium provided in the second medium (material) storage 12 may be sucked using the liquid handler 400, and the sucked medium may be dispensed into a container located in the first working part 7 (S520).
  • the lid of the 6well plate may be closed after dispensing the medium.
  • the container dispensed with the medium may be moved to the loader 5 by the grip part.
  • the container moved to the loader 5 may be reloaded into the incubator 100.
  • the step of driving the robot arm 300 may include the step (S430) to cause the arm 300 to move the container extracted from the incubator 100 or reservoir 600 to the loader (5).
  • the operation method of the automatic cell incubator 1000 the step of moving the container moved to the loader (5) to the first working part 7 using the grip part included in the liquid handler 400 (S610), Transferring the material contained in the container moved to the first working part 7 to the container of the predetermined shape located in the second working part (S620), the container of the predetermined shape to the centrifuge 700 Performing centrifugation by moving (S630), processing the separated particles through centrifugation (S640), observing the processed particles using a microscope 200 (S650), new processed particles
  • the method may further include transferring the container (S660) and re-injecting the new container into the incubator 100 (S670).
  • the container having a predetermined shape is a tubular container having a stopper, and may be, for example, an E-tube (Eppendorf-Tube) or a C-tube (Centrifuge-Tube).
  • the material eg, cells, cell fluid, etc.
  • the 6well plate cell container may be transferred to a container of a predetermined shape (eg, C-tube) located in the second working part 8.
  • the liquid handler 400 can move the cell container of the loader 5 to the first working part 7.
  • the liquid handler 400 also moves the C-tube of the C-tube reservoir (eg, 3) to a decapper 17 and causes the decapper 17 to open the lid of the C-tube. It can be done.
  • the unopened C-tube may be moved to the second working part 8 by the liquid handler 400.
  • the lid of the 6well plate cell container moved to the first working part 7 may be opened by the liquid handler 400.
  • the first working part 7 can be tilted and the cell fluid in the 6 well plate cell container tilted by the liquid handler 400 can be transferred to the C-tube of the second working part 8. After transfer of the cell fluid, the lid of the 6 well plate cell container may be closed by the liquid handler 400.
  • the step (S620) of transferring the material contained in the container moved to the first work part 7 to a container having a predetermined shape located on the second work part 8 is moved to the first work part 7.
  • the method may further include transferring a mixed solution obtained after mixing and shaking a predetermined solution in the prepared container to a container having a predetermined shape (eg, a C-tube).
  • the supernatant generated in the vessel may then be removed by centrifugation.
  • a predetermined material may be injected into the C-tube to mix and wait for a predetermined time.
  • the first material may be an enzyme or the like, and the predetermined time may be 3 minutes, but is not necessarily limited thereto.
  • the liquid handler 400 may inhale the PBS solution of the second medium (material) storage 12 or the like and dispense the 6 well plate cell container of the first working part 7. As described above, since the first working part 7 may be tilted, the first working part 7 may be repeatedly inclined and moved to perform a shaking process. The liquid handler 400 may inhale the PBS solution from the 6 well plate cell container of the first working part 7 and dispense it into the C-tube provided in the second working part 8.
  • step S640 of processing the separated particles through centrifugation removing the supernatant generated in the container through centrifugation, injecting a predetermined material into a container having a predetermined shape. Mixing and suspending the mixture, centrifuging the vessel containing the suspended separated material, removing the supernatant generated in the vessel through centrifugation, and injecting the medium into the vessel. Mixing (mixing) and mixing the predetermined material and the cell solution using another container having a predetermined shape.
  • the predetermined material may be an enzyme or Phosphate Buffer Saline (PBS).
  • PBS Phosphate Buffer Saline
  • a predetermined material eg, PBS, etc.
  • the centrifugal separation may be performed by moving the C-tube containing the suspended separated material to the centrifuge 700.
  • the supernatant generated in the vessel (C-tube) is removed by centrifugation, and the medium may be injected into the vessel and mixed.
  • the liquid handler 400 may move the C-tube from which the supernatant has been removed to the second working portion 8.
  • the liquid handler 400 sucks the enzyme of the second medium (material) storage 12, dispenses the C-tube of the second working part 8, and repeatedly sucks and discharges the C-tube. Mixing can be performed. After mixing, it may wait for a predetermined time.
  • the liquid handler 400 may suck the PBS solution of the second medium (material) storage 12 and discharge it to the C-tube provided in the second working part 8.
  • the liquid handler 400 may perform floating separation by repeating suction and discharge in the C-tube provided in the second working part 8.
  • the C-tube containing the suspended separated material is moved from the second working part 8 to the decapper 17 by the liquid handler 400, and the cap of the C-tube is closed by the decapper 17. Can be.
  • the closed C-tube may be moved to the centrifuge 700 to be centrifuged.
  • the supernatant generated in the vessel (C-tube) through centrifugation may be removed by the liquid handler (400).
  • the centrifuged and supernatant-removed vessel may be moved to the second working portion 8 by the liquid handler 400.
  • the liquid handler 400 may suck the medium of the second medium (material) storage 12 and dispense the C-tube moved to the second working part 8.
  • the liquid handler 400 may perform mixing by repeating suction and discharge in the C-tube.
  • the liquid handler 400 may dispense the PBS into an E-tube provided in the second working part 8.
  • the liquid handler 400 may inhale the cell solution from the C-tube provided in the second working part 8 and dispense it into the E-tube. Mixing can be performed by repeating suction and discharge in the E-tube.
  • Observing the processed particles according to an embodiment of the present invention by using the microscope 200 further includes transferring the material contained in the container having a predetermined shape to the observation aid before observing the microscope 200.
  • the observation aid may include a microscope chip (eg, a C-chip, etc.).
  • the liquid handler 400 may inhale trypan blue and dispense it to an observation aid (eg, a C-chip) located in the first working part 7.
  • an observation aid eg, a C-chip
  • the cell fluid may be sucked from the C-tube of the second working part 8 and dispensed into the C-chip, and the suction and discharge may be repeatedly performed in the C-chip to perform mixing.
  • the mixed solution can be transferred to another C-chip.
  • the liquid handler 400 may move the C-chip to the loader 5.
  • the C-chip moved to the loader 5 may be moved to the microscope 200 by the robot arm 300, and the cell state may be observed through the microscope.
  • the control device 500 may perform cell counting through analysis of the microscopic image of the cells obtained through the microscope.
  • Transferring the processed particles to a new container according to an embodiment of the present invention further comprises dispensing the medium into a new container prior to the transfer of the particles, the material containing the processed particles is a medium Can be transferred to a new dispensed container.
  • the robot arm 300 may move the new container (eg, 6 well plate) stored in the reservoir 600 to the loader 5.
  • the new container moved to the loader 5 may be moved to the first working part 7 by the liquid handler 400.
  • the lid of the new container can be opened.
  • the liquid handler 400 may dispense the medium of the second medium (material) reservoir 12 into a new container located in the first work part 7.
  • the liquid handler 400 may suck the cell fluid from the C-tube of the second working part 8 and dispense it into a new container of the first working part 7. The lid of the new container can then be closed.
  • the liquid handler 400 may move the new container of the first working part 7 to the loader 5.
  • the robot arm 300 may reload the new container of the loader 5 into the incubator 100.
  • FIG. 7 is a plan view of an automatic cell incubator omitted robot arm according to another embodiment of the present invention
  • Figure 8 is an embodiment of the cooling unit according to an embodiment of the present invention
  • Figure 9 is an embodiment of the present invention According to an embodiment of the heating unit.
  • FIG. 10 is an embodiment of a work part according to an embodiment of the present invention
  • FIG. 11 is an embodiment of a grip part according to an embodiment of the present invention
  • FIG. 12 is a decapper according to an embodiment of the present invention.
  • 13 is an embodiment of a microscope according to an embodiment of the present invention
  • Figure 14 is an embodiment of an incubator and incubator loader according to an embodiment of the present invention.
  • FIGS. 8 to 10 (a) and (b) are views showing different view points.
  • the robot arm 300 may be omitted in the automatic cell incubator 2000 according to another embodiment of the present invention.
  • the size of the automatic cell incubator 2000 may be further reduced than that of the automatic cell incubator 1000 described above.
  • the role of the robot arm 300, which may be omitted, may be performed by the grip unit 410. Therefore, even if the robot arm 300 is omitted, a similar function to that of the automatic cell incubator 1000 may be stably performed by the grip part 410.
  • the automatic cell incubator 2000 includes a waste storage 6, an image acquisition unit 9, a decapper 16, a 6 well container cover deck 32, a work unit 30, and an enzyme tube deck. 40, a heating unit 50, a microscope 60, a cooling unit 70, an incubator 100, an incubator loader 110, a grip unit 410, a reservoir 600, a centrifuge 700, etc. may be included.
  • the automatic cell incubator 2000 may be connected to the control device 500 by wire or wirelessly.
  • the cooling unit 70 includes a peltier element, a heat sink, a cooling fan, and a temperature inside the cooling unit 70 is constant through a sliding door. Can be maintained.
  • the cooling unit 70 may provide a refrigeration function for maintaining the medium or PBS solution in the vessel 10 at 4 degrees Celsius. Temperature control may be possible through current supply to the Peltier device.
  • the medium or PBS solution cooled by the cooling unit 70 may be heated by the heating unit 50 having the electric heater and the temperature sensor. For example, the medium or the PBS solution may be heated to 37 degrees Celsius by the heating unit 50.
  • At least one of rotation, shaking, and tilting of the transferred container 10 may be performed in the work part 30.
  • various movements can be performed for 6 well containers, 75 T-Flask, and the like.
  • At least one vacuum pad may be mounted at a portion where the working part 30 and the container 10 contact each other to secure the container 10.
  • a rotating shaft for rotating the container 10 may exist at an upper end of the working part 30, and a tilting shaft for tilting the container 10 may exist at a stop portion.
  • the grip part 410 may be a motor grip part capable of gripping and transporting a container 10 such as a 6 well plate, a T-Flask, and a C-tube.
  • the grip part 410 may be a vacuum grip part in which a vacuum pad is mounted at one end as shown in FIG. 11.
  • the vacuum grip unit can transfer a lid of a 6 well plate using a vacuum pad or the like.
  • the decapper 16 may perform the function of opening and closing the caps of the C-Tube and the T-Flask. For example, after the container 10 is fixed with the container fixing part, the decapper 16 moves to the lid opening position, the lid fixing part is lowered, and then rotates in the lid opening direction to open the lid of the container 10. Can be. In addition, similarly, after the container 10 is fixed to the container fixing part, the decapper 16 may move to the lid closing position, and the lid fixing part may be lowered and then rotated in the direction of closing the lid to close the lid. Using the lid detection sensor included in the decapper 16 can check the status of the lid open / close in real time.
  • the automatic cell incubator 2000 may include a microscope 60 for observing a cell culture state.
  • the microscope 60 may acquire an image while moving the container up, down, left and right by using the container loader provided inside the microscope 60.
  • the microscope 60 can change the lens magnification of the microscope 60 using an electric turret.
  • the automatic cell incubator 2000 may include an incubator 100 and an incubator loader 110 for transporting the container 10 into and out of the incubator 100.
  • the incubator loader 110 may be used to carry a container 10 that may be inflow or outflow through the door of the incubator 100.
  • the container loader part takes over the container 10 to position the container 10 in the plate hotel.
  • the plate hotel can be rotated using a motor mounted on the bottom center.
  • 15 is an example of (a) an implementation of an image acquisition unit and (b) an acquired image screen according to an embodiment of the present invention.
  • the liquid level detection function may be performed using the image acquisition unit 9 including the vision camera.
  • the location of the cell pellet may be detected based on the acquired image, and the remaining solution except for the cell pellet may be sucked out.
  • the user can extract the extraction object precisely.
  • Computer readable media can be any available media that can be accessed by a computer and includes both volatile and nonvolatile media, removable and non-removable media.
  • Computer readable media may include both computer storage media and communication media.
  • Computer storage media includes both volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data.
  • Communication media typically includes computer readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave, or other transmission mechanism, and includes any information delivery media.

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Abstract

La présente invention concerne, selon un mode de réalisation, un dispositif de culture cellulaire automatisé comprenant : un incubateur destiné à recevoir au moins un récipient de culture de cellules; un microscope pour observer l'état des cellules dans le récipient; un bras de robot pour déplacer l'emplacement du récipient; un manipulateur de liquide pour permettre à un liquide de s'écouler dans le récipient ou évacuer le liquide du récipient; et un dispositif de commande pour commander le fonctionnement d'au moins un élément parmi l'incubateur, le microscope, le bras de robot et le manipulateur de liquide.
PCT/KR2016/007437 2015-07-09 2016-07-08 Dispositif de culture cellulaire automatisé, et procédé de fonctionnement de dispositif de culture WO2017007278A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US15/569,873 US20180127695A1 (en) 2015-07-09 2016-07-08 Automatic device for culturing cell and operating method thereof
EP16821684.4A EP3284815B1 (fr) 2015-07-09 2016-07-08 Dispositif de culture cellulaire automatisé, et procédé de fonctionnement de dispositif de culture
CN201680024532.6A CN107636142A (zh) 2015-07-09 2016-07-08 自动细胞培养器及其培养器的操作方法
HK18110679.7A HK1251248A1 (zh) 2015-07-09 2018-08-20 自動細胞培養器及其培養器的操作方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20150098009 2015-07-09
KR10-2015-0098009 2015-07-09
KR1020160037323A KR101767336B1 (ko) 2015-07-09 2016-03-29 자동 세포 배양기 및 그 배양기의 동작 방법
KR10-2016-0037323 2016-03-29

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CN108504565A (zh) * 2017-02-27 2018-09-07 厦门鹭港兆康生物科技有限公司 一种自动定位播种机
CN108728333A (zh) * 2018-04-28 2018-11-02 广州滨鑫生物科技有限公司 一种生物技术用细胞培养箱及其培养方法
CN114525205A (zh) * 2021-11-22 2022-05-24 浙江泰林医学工程有限公司 用于细胞培养的自动换液装置

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JP2007312668A (ja) * 2006-05-25 2007-12-06 Hitachi Medical Corp 自動培養装置
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JP2005304303A (ja) * 2004-04-16 2005-11-04 Olympus Corp 給排ロボットおよび自動培養装置
JP2006055027A (ja) * 2004-08-18 2006-03-02 Nikon Corp 自動培養装置、および自動培養システム
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Publication number Priority date Publication date Assignee Title
CN108504565A (zh) * 2017-02-27 2018-09-07 厦门鹭港兆康生物科技有限公司 一种自动定位播种机
CN108728333A (zh) * 2018-04-28 2018-11-02 广州滨鑫生物科技有限公司 一种生物技术用细胞培养箱及其培养方法
CN114525205A (zh) * 2021-11-22 2022-05-24 浙江泰林医学工程有限公司 用于细胞培养的自动换液装置

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