WO2017107837A1 - 全自动细胞连续培养系统 - Google Patents

全自动细胞连续培养系统 Download PDF

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Publication number
WO2017107837A1
WO2017107837A1 PCT/CN2016/110035 CN2016110035W WO2017107837A1 WO 2017107837 A1 WO2017107837 A1 WO 2017107837A1 CN 2016110035 W CN2016110035 W CN 2016110035W WO 2017107837 A1 WO2017107837 A1 WO 2017107837A1
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Prior art keywords
cell culture
bag
base
shaker
unit
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Legal status (The legal status 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 status listed.)
Ceased
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PCT/CN2016/110035
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English (en)
French (fr)
Chinese (zh)
Inventor
余学军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Ji Kai Gene Technology Co Ltd
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Shanghai Ji Kai Gene Technology Co Ltd
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.)
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Publication date
Priority claimed from CN201510981653.7A external-priority patent/CN105385587B/zh
Priority claimed from CN201510981648.6A external-priority patent/CN105385597B/zh
Priority claimed from CN201610131141.6A external-priority patent/CN105567565B/zh
Priority claimed from CN201610131142.0A external-priority patent/CN105567566B/zh
Priority claimed from CN201610325563.7A external-priority patent/CN105785560B/zh
Priority claimed from CN201610325576.4A external-priority patent/CN105925481A/zh
Application filed by Shanghai Ji Kai Gene Technology Co Ltd filed Critical Shanghai Ji Kai Gene Technology Co Ltd
Priority to US16/064,414 priority Critical patent/US10934513B2/en
Priority to JP2018533724A priority patent/JP6889723B2/ja
Publication of WO2017107837A1 publication Critical patent/WO2017107837A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16—Vibrating; Shaking; Tilting
    • 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
    • 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
    • C12M23/14—Bags
    • 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
    • C12M23/20—Material Coatings
    • 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
    • C12M23/22—Transparent or translucent parts
    • 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
    • C12M23/48—Holding appliances; Racks; Supports
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
    • C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/10—Condensers affording dark-field illumination
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/26—Stages; Adjusting means therefor

Definitions

  • the invention relates to a fully automatic cell continuous culture system, which is mainly used for culturing and observing cells.
  • the immune cell culture for human treatment is mainly cultured in a cell culture dish or a culture flask under a laboratory clean bench, and is continuously bottle-cultured according to the density of cell growth.
  • This cell culture mode is not only It is time-consuming and labor-intensive, and it is more likely to cause pollution due to the need for different bottle separation and sample loading.
  • ZL2007200130617 discloses a microbial culture incubator comprising a shaker, a motor and a speed reduction mechanism.
  • the shaker is assembled on the guide rail by a roller, the shaker hinges one end of the rocker arm, and the other end of the rocker arm is hinged on the eccentric shaft of the disc.
  • the central axis of the disc is coupled to the power take-off shaft of the reduction mechanism.
  • the disc design of the shaker is not suitable for continuous culture of cells.
  • ZL2013102727942 discloses a variable amplitude convoluted cell culture shaker comprising: an eccentric device, a tray, a receiving vessel, a base, a motor, a counterweight, a controller, the eccentric device being mounted on the base, the eccentric device comprising a main eccentric a secondary eccentric wheel, a main eccentric wheel and a secondary eccentric wheel are provided with a counterweight, and a tray is arranged above the secondary eccentric wheel, and the motor is connected to one of the eccentric devices via a transmission device, and the set of eccentric devices comprises: a main eccentric wheel a variator, a horn, an amplitude locating plate, the amplitude locating plate is disposed on the main eccentric wheel, the horn is connected to the amplitude locating plate through the horn, and the upper end of the variator is connected to the tray, and the motor is connected There is a controller.
  • the shaker can realize the luffing operation, its tray design is not suitable for continuous culture of cells.
  • the invention CN2014103149910 discloses an oscillating shaker comprising: a frame, an oscillating box, a motor, a belt, a pulley, a synchronous wheel, a timing belt, an eccentric shaft, an oscillating plate, a pulley, a guide rail and a main shaft, and an oscillating box is arranged under the frame.
  • the motor is disposed on one side of the oscillating box, the main shaft is disposed at the center of the gantry, and a pulley and a synchronous wheel are disposed under the main shaft, the belt is disposed on the pulley and connected to the motor, and the timing belt is disposed on the synchronous wheel, the timing belt
  • the other end is connected with an eccentric shaft, the upper end of the main shaft is connected with an eccentric wheel, an oscillating plate is fixed on the eccentric wheel, an oscillating box is fixed under the oscillating plate, and a guide rail and a pulley are arranged under the oscillating box.
  • the traditional cell culture is cultured in a CO2 incubator in a petri dish or a culture flask.
  • the culture conditions are relatively high, and it needs to be operated under a clean bench, then placed in a carbon dioxide incubator for cultivation, and taken out at the appropriate time to cause The state of cell growth was observed under a microscope.
  • the dark field microscope mainly uses a special dark field condenser to deflect the illumination light without entering the objective lens, and only the scattered light of the sample enters the objective lens. Thus, a bright image is obtained on a dark background. Contrary to the dark field illumination, the illuminated light directly reaches the imaging plane, which is called the field of view illumination.
  • the Tyndall phenomenon means that when the concentrated beam is incident on the sol, a bright column of light can be seen from the side of the beam. During the propagation of the light, when the light is irradiated to the particle, the particle diameter is slightly smaller than the incident. At the wavelength of light, light is scattered, and each particle becomes a light-emitting point, and a light column can be seen from the side.
  • the dark field microscope makes use of the above principles.
  • the special optical configuration of the dark field microscope allows the illumination incident light to be intruded into the objective, but the sample is illuminated and emits reflected light.
  • the plane field of view perpendicular to the optical axis is dark, and the bright object image formed by the scattered light and the reflected light can be clearly seen on the dark background, the object image and the background.
  • the sample in the field of view illuminated by oblique light, scatters and reflects light from various structural surfaces of the sample, seeing the bright contours of many organelles, such as the nucleus, mitochondria, vacuoles, and certain inclusions. If it is a dividing cell, its various spindles and chromosomes can also be seen.
  • a culture bag holder for a culture shaker which can be used for continuous cell culture according to the requirements of different cell cultures.
  • the culture bag holder of the present invention can be used in combination with a curved 3D culture bag to meet various needs of continuous cell culture.
  • a second object of the present invention is to develop a culture shaker that can be used for continuous cell culture according to the requirements of different cell cultures.
  • the shaker of the present invention can be used in conjunction with a curved 3D culture bag to meet the various needs of continuous cell culture.
  • the third object of the present invention is to provide a non-contact sensor connector and application from the various risk factors existing in the cell culture process, combined with various conditions of cell culture, and can monitor cells in real time without contacting the sample. Relevant indicators of culture fluid.
  • a fourth object of the present invention is to provide a fully enclosed 3D cell which can be exposed from the initial culture of the cell to the end of the culture without considering the various risk factors present in the cell culture process, in combination with various conditions of cell culture. Culture the bag.
  • a fifth object of the present invention is to provide an efficient dark field microscope for on-line observation of cell culture that enables real-time observation.
  • a sixth object of the present invention is to provide a continuous cell continuous culture system based on the above, which can realize continuous culture and observation of cells, and determination of various parameters of the culture solution.
  • the invention firstly provides a culture bag holder for a continuous cell culture shaker, comprising a curved bottom plate and a cell bag fixing member, wherein the curved bottom plate is provided with one or more fitting fixing holes.
  • the fitting fixing hole is provided with an internal thread to better fix the external fitting.
  • the curved bottom plate is provided with an objective lens insertion notch.
  • the edge of the curved bottom plate is provided with a baffle.
  • the curved bottom plate is further provided with a heating plate.
  • the heating plate is a temperature-controlled heating plate, and the temperature-controlled heating plate is generally provided with a heating element and a temperature measuring element. Temperature controlled heating is a prior art.
  • the cell bag securing members may be disposed on either side of the curved bottom plate and may include a platform extending from both side edges of the curved bottom plate to both sides, and having components matching the fixing members on the cell bag.
  • the continuous cell culture shaker culture bag holder of the invention can be used together with the stereo cell culture bag, and can meet the requirements of heating, sample loading and other cell culture detection equipment installation in continuous cell culture, and realize continuous culture of cells, and Cell damage is less in cell culture.
  • a continuous cell culture shaker is provided.
  • the continuous cell culture shaker of the present invention comprises a culture bag holder, a rocking frame and a rocking device, wherein the culture bag holder is provided with a curved bottom plate and a cell bag fixing member, the rocking frame includes a rocking base, and the rocking base A support arm is disposed thereon, the culture bag bracket is fixedly connected to the swing frame via a support arm, and the rocking device comprises a base, and the base is provided with a rocking assembly, and the rocking base is connected with the rocking assembly.
  • One or more fitting fixing holes may be disposed on the curved bottom plate.
  • the fixing hole is provided with an internal thread to better fix the external fitting. Space for mounting or inserting external accessories should be left under the mounting holes.
  • the curved bottom plate is provided with a heating plate.
  • the curved bottom plate is further provided with an objective lens insertion gap.
  • the edge of the curved bottom plate is provided with a baffle.
  • the rocking assembly can be any mechanism that can effect a rocking motion.
  • the rocking assembly may include a hinge seat and at least one upper and lower reciprocating mechanism, and the hinge seat is provided at the base
  • the swinging base of the rocking frame is hinged with the hinged seat, and the upper and lower reciprocating mechanism is connected or pressed against the rocking base.
  • the up and down reciprocating mechanism may be various existing reciprocating mechanisms, such as an electric push rod, a linear motor, a crank linkage mechanism, and the like.
  • the shaker of the invention can satisfy the heating, shaking, loading and other cell culture testing equipment installation in the cell culture, and realize continuous culture of the cells.
  • a contactless sensor connector includes a base plate and an open sensor probe accommodating chamber, wherein the base plate is provided with a transparent partition, and the sensor probe accommodating the chamber and the The base plates are connected and located on one side of the transparent partition, and the other side of the transparent partition is provided with a conversion film.
  • the transparent partition is colorless and transparent, and the sides are smooth.
  • the sensor probe receiving chamber can be plugged or screwed to the sensor probe.
  • the sensor probe accommodating chamber is provided with internal threads.
  • the conversion film is a pH-induced color-changing film or a dissolved oxygen-induced color-changing film.
  • the outer edge of the transparent partition provided with the side surface of the conversion film is provided with a flange projecting from the base plate 1.
  • the sensor probe accommodating chamber may be fixedly connected to the base plate or may be detachably connected.
  • Removable connections include, but are not limited to, plugs, threaded connections, and the like.
  • the sensor probe accommodates a smooth transition between the chamber and the base.
  • the non-contact sensor connector of the present invention can be used in a closed cell culture device.
  • the cell culture device includes, but is not limited to, a cell culture bag.
  • the design of the non-contact sensor joint of the invention can make the sensor used together without sterilization and sterilization, not only is the operation more convenient, but also can better prevent pollution.
  • a cell culture bag includes: a closed bag body and a fixing member disposed on two sides of the bag body, the top surface of the closed bag body is provided with an external liquid sample port, and an external liquid injection a mouth, a gas inlet and a gas outlet, wherein the bottom surface of the closed bag body is provided with a liquid recovery port and at least one sensor joint, and the external liquid sample port, the external liquid injection port, the gas inlet, the gas outlet and the liquid recovery port are provided There are sterile quick connectors.
  • the closed bag body is made of a multi-layer composite structure.
  • the closed bag body is surrounded by an upper bag piece, a lower bag piece, a left side bag piece and a right side bag piece to form a three-dimensional pocket.
  • the lower bottom edge of the left side pocket piece and the right side pocket piece are curved.
  • the left side panel and the right side panel are oval in shape, and the upper and lower pockets are rectangular.
  • a transparent observation area is disposed in a middle portion of the bottom of the bag body.
  • the transparent observation zone is located at the bottom of the cell culture bag.
  • the cells in the cell culture bag can be directly observed from the bottom by providing a cell morphology observation device on the culture platform.
  • the sensor connector may be provided with one or more.
  • two sensor connectors are provided, one of which is a sensor connector for accessing the pH sensor probe and the other is a sensor connector for accessing a dissolved oxygen (DO) sensor probe.
  • DO dissolved oxygen
  • the sensor connector can be a contact sensor connector or a non-contact sensor connector.
  • the sensor connector is a non-contact sensor connector, i.e., the sensor probe is not in contact with the culture fluid in the bag after insertion of the sensor connector.
  • the cell culture bag of the present invention can be used for cell culture.
  • the cell culture bag of the invention has a unique structure and can be continuously cultured under a small amount of culture liquid until the culture requirement is reached.
  • the 3D shaped cell culture bag designed by the invention has the smallest shear force generated by the shaking of the culture platform during the cell culture process, and the shear damage caused to the cells is also minimal.
  • All the joints of the cell culture bag of the invention adopt the aseptic quick connection method, thereby minimizing the risk of cell contamination caused by the culture operation.
  • the volume of the cell culture bag of the present invention can be infinitely expanded according to the amount of culture required.
  • a dark field microscope for online observation of cell culture comprising at least: a lighting element, a movable bracket, a support base, an objective lens, a photosensitive element, a pipe diameter unit, and A/ a D conversion unit, a power module, and a control system, the lighting element being disposed on a top of the movable bracket, the movable bracket being disposed on the support base, the pipe unit being coupled to a lower portion of the movable bracket,
  • the objective lens is disposed on the pipe diameter unit and corresponding to the lower side of the lighting element, the photosensitive element is disposed in the pipe diameter unit for collecting the light source signal on the objective lens, the A/D conversion unit is connected to the photosensitive element, and the control system is connected An A/D conversion unit that supplies power to a dark field microscope for on-line observation of cell culture.
  • the lighting element comprises a circular lamp holder, and an illumination lamp arranged on the lamp holder and arranged in a plurality of concentric circles, the center of the concentric circle being coincident with the center position of the lamp holder.
  • the distance from the socket to the objective lens is 10-30 cm.
  • the light source of the illumination lamp forms a dark field with the objective lens to meet the needs of dark field illumination.
  • the illuminating lamp is an LED lamp.
  • the support base comprises a stepping motor, a sliding mechanism and a base
  • the sliding mechanism comprises a slider and a slide rail
  • the control system is connected and controls a stepping motor
  • the stepping motor is connected to the slider
  • the slider The slide rail is slidably coupled to the base and horizontally movable, and the movable bracket is disposed on the slider.
  • the pipe diameter unit is slidably coupled to the movable bracket, and the pipe diameter unit is connected to the movable bracket to be provided with a sliding slot, and the pipe diameter unit can slide up and down along the sliding groove.
  • control system comprises:
  • a movable bracket control unit for controlling the movement of the movable bracket
  • a lighting element control unit for controlling a switch of the lighting element
  • View unit for viewing what is observed by the objective lens in the current state
  • a storage unit for storing the observed content that needs to be saved is
  • the movable bracket has a hollow structure, and the movable bracket control unit is placed in the hollow structure.
  • the photosensitive element is a CCD sensor.
  • the dark field microscope for online observation of cell culture of the present invention can be used to observe cells in a cell culture bag in cell culture.
  • the device can realize continuous and repeated observation of the sample, but it is not necessary to repeatedly sample.
  • the sample is observed, it is more convenient and quick to move the sample between the photosensitive element and the objective lens only by moving the movable bracket through the control system;
  • the destruction of the sample during the production of the microscopic sample makes the test result more objective.
  • a fully automated cell culture system comprising at least: a control module, a control platform, a dark field microscope for online observation of cell culture, a cell culture shaker, and a cell culture bag. And power module;
  • the cell culture shaker comprises a culture bag holder, a rocking frame and a rocking device, wherein the culture bag holder is provided with a curved bottom plate and a cell culture bag fixing member, the rocking frame includes a rocking base, and the swing base is provided a support arm, the culture bag bracket is fixedly connected to the swing frame via a support arm, the rocking device comprises a base, and the base is provided with a rocking assembly, the rocking base is connected with the rocking assembly, the arc An objective lens insertion notch is arranged on the bottom plate;
  • the cell culture bag includes a closed bag body, and a transparent observation area is disposed in a middle portion of the bottom surface of the bag body; the bag body is placed on a cell culture shaker, and the transparent observation area is consistent with the position of the objective lens gap;
  • the dark field microscope for online observation of cell culture comprising an objective lens, which can be inserted into an incision insertion gap of a cell culture shaker;
  • the control platform is respectively connected to a dark field microscope and a cell culture shaker for online observation of cell culture;
  • the control module is connected to the control platform
  • the power module supplies power to the entire system.
  • the dark field microscope for online observation of cell culture includes a lighting element, a movable bracket, a support base, a photosensitive element, a pipe diameter unit, an A/D conversion unit, a power module, and a control module.
  • the lighting element is disposed on a top of the movable bracket
  • the movable bracket is disposed on the support base
  • the pipe diameter unit is connected to a lower portion of the movable bracket
  • the objective lens is disposed on the pipe diameter unit and corresponds to Below the illumination element
  • the photosensitive element is disposed in the tube diameter unit for collecting a light source signal on the objective lens
  • the A/D conversion unit is connected to the photosensitive element
  • the control module is connected to the A/D conversion unit.
  • the lighting element comprises a circular lamp holder, and an illumination lamp arranged on the lamp holder and arranged in a plurality of concentric circles.
  • the center of the concentric circle coincides with the center position of the lamp holder.
  • the distance from the socket to the objective lens is 10-30 cm.
  • the light source of the illumination lamp forms a dark field with the objective lens to meet the needs of dark field illumination.
  • the illuminating lamp is an LED lamp.
  • the support base comprises a stepping motor, a sliding mechanism and a base
  • the sliding mechanism comprises a slider and a slide rail
  • the control module is connected to and controls a stepping motor
  • the stepping motor is connected to the slider
  • the slider is slidably coupled to the base via a slide rail and is horizontally movable, and the movable bracket is disposed on the slider.
  • one or more fitting fixing holes are arranged on the curved bottom plate, and a space for mounting or inserting the external fitting is left under the fixing hole position of the fitting.
  • the curved bottom plate is provided with a heating plate.
  • the edge of the curved bottom plate is provided with a baffle.
  • the cell culture bag fixing member is disposed on both sides of the curved bottom plate, and includes a platform extending from both sides of the curved bottom plate to both sides, and the platform is provided with a fixing rod clamp and/or a fixing rod insertion mechanism.
  • the rocking base is a flat plate, and both ends are provided with support arms, and the culture bag bracket is not in contact with the rocking base, and a gap is left to install or insert the external fitting.
  • the rocking assembly comprises a hinge seat and at least one upper and lower reciprocating mechanism, the hinge seat is disposed on the base, the swing base is hinged with the hinge seat, and the upper and lower reciprocating mechanism is connected or pressed against the rocking base .
  • the cell culture bag comprises: a fixing member on both sides of the bag body, the top surface of the closed bag body is provided with an external liquid loading port, an external liquid injection port, a gas inlet and a gas outlet, The bottom surface of the closed bag body is provided with a liquid recovery port and at least one sensor connector, and the external liquid sample port, the external liquid injection port, the gas inlet, the gas outlet and the liquid recovery port are provided with a sterile quick connector.
  • the closed bag body is surrounded by an upper bag piece, a lower bag piece, a left side bag piece and a right side bag piece to form a three-dimensional bag.
  • a cavity, the lower left side of the left side pocket piece and the right side pocket piece are curved, and the left side pocket piece and the right side pocket piece are both elliptical, the upper bag piece and the lower bag piece It is a rectangle.
  • the innermost layer of the closed bag is a polyethylene layer.
  • the closed bag body is made of a five-layer composite structure, and the polyethylene layer, the adhesive layer, the ethylene propylene alcohol layer, the adhesive layer and the polyethylene layer are sequentially arranged from the inside to the outside.
  • the sensor joint is provided with one or more.
  • the sensor connector is a non-contact sensor connector or a contact sensor connector.
  • the non-contact sensor connector comprises a base plate and a sensor probe receiving cavity
  • the base plate is provided with a transparent partition
  • the sensor probe receiving cavity is connected to the base plate and located at the transparent partition
  • the other side of the transparent partition is provided with a conversion film.
  • the conversion film is a pH-induced color-changing film or a dissolved oxygen-induced color-changing film.
  • the control platform comprises a control cabinet, and an automatic heating unit, a preheating bag and a peristaltic pump arranged on the control cabinet;
  • the automatic heating unit setting comprises a preheating plate, and the preheating bag is set in the preheating On the hot plate
  • the peristaltic pump comprises a first peristaltic pump and a second peristaltic pump, the automatic heating unit further comprising a cell culture fluid tank, the tank communicating with the inlet of the preheating bag through the first conduit, The outlet of the preheating bag is connected to the cell culture bag through a second pipe, the first peristaltic pump is connected to the first pipe, the second peristaltic pump is connected to the second pipe, the automatic heating unit, the first peristaltic pump and the second The peristaltic pump is connected to the control module.
  • a gas mixing unit is further disposed inside the control cabinet, and an air outlet of the gas mixing unit is disposed on the control cabinet, and is connected to the cell culture bag through a third conduit, and the air inlet of the gas mixing unit is connected An external air source, the gas mixing unit is connected to the control module.
  • control cabinet is further provided with a pH detecting joint and a dissolved oxygen detecting joint, the pH detecting probe and the dissolved oxygen detecting probe.
  • control module includes:
  • a movable bracket control unit for controlling the movement of the movable bracket
  • a lighting element control unit for controlling a switch of the lighting element
  • a viewing unit for viewing what is observed by the objective lens in the current state, and various parameters of the cell growth environment in the current state;
  • a storage unit for storing the observed content to be saved and recording various parameters of the cell growth environment
  • a gas control unit for controlling gas in the cell culture bag
  • a cell culture fluid control unit for controlling the temperature and supply of the cell culture fluid.
  • the fully automated cell continuous culture system of the present invention can be used for continuous cell culture.
  • the user can adjust the swing frequency and angle according to the state of the cell culture to ensure that the cells are in an optimal growth environment
  • the probes of various detectors can be implemented to detect various parameters in the cell culture bag
  • the cell culture bag has a unique structure and can be continuously cultured under a small amount of culture medium until the culture requirement is reached;
  • the 3D shaped cell culture bag designed by the invention has the least shear force generated by the shaking of the culture platform during the cell culture process, and the shear damage caused to the cells is also minimal;
  • the cell culture bag joint adopts a sterile quick connection method, which can minimize the risk of cell contamination caused by the culture operation;
  • the volume of the cell culture bag can be infinitely expanded according to the amount of culture required
  • FIG. 1 is a schematic view showing a culture bag holder according to an embodiment of the present invention.
  • FIG. 2 is a schematic view showing a culture bag holder according to another embodiment of the present invention.
  • Fig. 3 is a schematic view showing a culture bag holder according to still another embodiment of the present invention.
  • FIG 4, 5 and 6 show schematic views of a cell culture shaker according to an embodiment of the present invention.
  • FIG. 7, 8, and 9 show schematic views of a sensor connector according to an embodiment of the present invention.
  • Figure 10 is a schematic top plan view of a cell culture bag according to an embodiment of the present invention.
  • Figure 11 is a schematic view showing the underside of a cell culture bag according to an embodiment of the present invention.
  • Figure 12 is a schematic view showing the structure of a cell culture bag body according to a preferred embodiment of the present invention.
  • the dashed line points to the pocket that illustrates the angle theory being occluded.
  • Figure 13 is a schematic view showing the connection line of the top surface of the bag body in the state in which the cell culture bag is used in the embodiment of the present invention.
  • Fig. 14 is a schematic view showing the connection line of the bottom surface of the bag body in the state in which the cell culture bag is used according to the embodiment of the present invention.
  • Figure 15 is a schematic view showing the structure of a dark field microscope for on-line observation of cell culture in the present invention.
  • Figure 16 is a schematic view showing the principle of dark field illumination in the present invention.
  • Figure 17 shows a schematic diagram of a dark field microscope control system
  • Figure 18 shows a schematic of a fully automated cell culture system.
  • FIG. 19 shows a schematic diagram of a control module according to an embodiment of the invention.
  • FIG. 20 is a schematic diagram of a control module according to another embodiment of the present invention.
  • 21 is a schematic diagram of a control module according to still another embodiment of the present invention.
  • FIG. 22 shows a schematic diagram of a control module according to still another embodiment of the present invention.
  • the continuous cell culture shaker culture bag holder 1 shown in FIG. 1 includes a curved bottom plate 11 and a cell bag fixing member 12, and the curved bottom plate 11 is provided with one or a plurality of fitting fixing holes 111.
  • the curved bottom plate 11 is for receiving a main portion of the curved 3D cell culture bag.
  • the curved bottom plate is designed so that the curved 3D cell culture bag can maintain the curved shape at the bottom during the cell culture process, so that during the cell culture process, the shaking force of the culture platform produces the minimum shear force and the shear damage to the cells. Also the smallest.
  • the two sides of the curved bottom plate are curved.
  • the fitting fixing hole 111 is provided with an internal thread to better fix the external fitting.
  • External accessories can be probes for various detectors, such as pH and dissolved oxygen detection probes.
  • the external fittings may also be various external liquid inlet pipes, external liquid injection components, liquid outlet pipes, external gas delivery pipes, and the like for continuous cell culture.
  • the curved bottom plate 11 is provided with an objective lens insertion notch 112.
  • a transparent observation zone can be arranged at the bottom of the culture bag, and the objective lens insertion notch 112 matches the transparent observation zone, and the objective lens can be inserted, thereby observing the cells in the cell bag directly from the bottom by using the objective lens.
  • the curved bottom plate has an arc shape that is recessed downward in use. Further, the objective lens insertion notch 112 is disposed in a concave bottom region of the curved bottom plate. Further, the objective lens is inserted into the edge of the notch 112 near the curved bottom plate to facilitate the entrance and exit of the objective lens.
  • the edge of the curved bottom plate 11 is provided with a baffle 113.
  • the baffle prevents the cell culture bag from slipping and shifting out of the culture bag holder.
  • a heating plate 114 is disposed on the curved bottom plate 11.
  • the heating plate can be heated by cell culture to provide a suitable temperature for cell culture.
  • the cell bag fixing member 12 may be disposed on both sides of the curved bottom plate, and may include a platform 121 extending from both side edges of the curved bottom plate to both sides, and a fixing member on the cell bag is disposed thereon.
  • Matching parts may be a member that is fixed by mechanical force, adhesion, magnetic force, or the like.
  • the member matching the fixing member on the cell bag may be a fixed rod clamp and/or a fixed rod insertion mechanism. In a preferred embodiment as shown in FIG.
  • a fixing rod is arranged on both sides of the cell bag, and a fixed rod clamp 122 and a fixed rod insertion mechanism 123 are arranged on the platform, and the combination of the two can be easily realized not only with the cell bag fixing rod but also The detachable connection and the cell culture bag are well fixed to prevent it from coming off the tray during shaking.
  • the lower portion of the culture bag holder for the continuous cell culture shaker is further provided with a support bracket 124 so that an external fitting insertion space is left under the curved bottom plate.
  • the bracket 124 can be used to erect the curved bottom plate such that the fitting fixing hole 111 of the curved bottom plate or the objective lens insertion notch 112 has a space for the external fitting to be installed.
  • the culture cell holder for a continuous cell culture shaker of the present invention is preferably used in a continuous cell culture shaker as shown in Figs. 4-6 and used in combination with the illustrated stereo cell culture bag 4.
  • the continuous cell culture shaker comprises the culture bag holder 1, the rocking frame 2 and the rocking device 3 of the present invention, the rocking frame 2 comprising a rocking base, the rocking base when the bracket does not have the support bracket 124 A support arm (corresponding to the support bracket 124) should be provided on the support, and the culture bag bracket is fixed on the support arm.
  • the shaking device 3 is not limited to the illustration, and may be any mechanism that can realize a rocking motion.
  • the rocking device 3 pushes the rocking base 21 to perform a rocking motion, and then drives the culture bag bracket fixed on the support arm to drive the cell culture bag 4 to shake.
  • the tray is heated by the heating plate, and various monitoring probes can be connected to the cell culture bag through the accessory fixing hole 111 provided at the bottom of the tray to detect the cell culture state.
  • the shaking can also be stopped, and the objective lens is inserted into the notch 112 of the bracket to insert the objective lens, and the cells in the transparent cell culture bag can be directly observed without sampling.
  • the continuous cell culture shaker shown in FIG. 4-6 includes a culture bag holder 1, a rocking frame 2, and a rocking device 3, wherein the culture bag holder 1 is provided with a curved bottom plate 11 and a cell bag fixing member 12,
  • the rocking frame 2 includes a rocking base 21 on which a support arm 211 is disposed.
  • the culture bag bracket 1 is fixedly coupled to the rocking frame 2 via a support arm 211, and the rocking device 3 includes a base 32.
  • the base is provided with a rocking assembly 31 that is coupled to the rocking assembly 31.
  • the curved bottom plate 11 is for accommodating a main portion of the curved 3D cell culture bag 4.
  • the curved bottom plate is designed so that the curved 3D cell culture bag can maintain the curved shape at the bottom during the cell culture process, so that during the cell culture process, the shaking force of the culture platform produces the minimum shear force and the shear damage to the cells. Also the smallest.
  • the two sides of the curved bottom plate are curved.
  • one or more accessory fixing holes 111 may be disposed on the curved bottom plate 11.
  • the fixing hole 111 is provided with an internal thread to better fix the external fitting.
  • External accessories can be probes for various detectors, such as pH and dissolved oxygen detection probes.
  • the external fittings may also be various external liquid inlet pipes, injection parts, liquid outlet pipes, external gas delivery pipes, and the like which are convenient for continuous cell culture.
  • the curved bottom plate 11 is provided with an objective lens insertion notch 112.
  • a transparent observation zone can be arranged at the bottom of the culture bag, and the objective lens insertion notch 112 matches the transparent observation zone, and the objective lens can be inserted, thereby observing the cells in the cell bag directly from the bottom by using the objective lens.
  • the curved bottom plate has an arc shape that is recessed downward in use. Further, the objective lens insertion notch 112 is disposed in a concave bottom region of the curved bottom plate.
  • the edge of the curved bottom plate 11 is provided with a baffle 113.
  • the baffle prevents the cell culture bag from slipping and shifting out of the culture bag holder.
  • a heating plate 114 is disposed on the curved bottom plate 11.
  • the heating plate can be heated during cell culture to provide a suitable temperature for cell culture.
  • the cell bag fixing member 12 may be disposed on two sides of the curved bottom plate, and may be a platform 121 extending from both side edges of the curved bottom plate to both sides, and the fixing member on the cell bag is disposed thereon.
  • Matching parts may be a member that is fixed by mechanical force, adhesion, magnetic force, or the like.
  • a fixing rod is arranged on both sides of the cell bag, and at this time, a fixing rod clamp 122 and/or a fixing rod insertion mechanism 123 may be provided on the platform, thereby realizing the fixing rod with the cell bag. Removable connection.
  • the design of the support arm 211 in the rocking frame 2 can serve to support the culture bag carrier 1.
  • a space for mounting or inserting an external fitting should be left under the fitting fixing hole 111 of the culture bag holder 1.
  • the support arm may be integrally formed with the rocking base as part of the rocking base 21, or may be mounted as a separate component on the rocking base, or It can also be integrally formed with the culture bag holder 1.
  • the support arm can reserve space for the connection between the cell culture bag and the external accessory.
  • the culture bag holder 1 since the culture bag holder 1 is elevated, only a small swing force is required to the swing base 21, and the culture bag holder 1 is provided. It can swing in a wide range.
  • the rocking base 21 is a flat plate, and both ends are provided with support arms 211.
  • the culture bag carrier 1 is not in contact with the rocking base 21, and a gap is left to be installed or inserted into the outside.
  • Accessories including various probes and objectives.
  • the rocking assembly 31 can be any existing mechanism that can effect a rocking motion.
  • the rocking assembly 31 includes a hinged seat 311 and at least one upper and lower reciprocating mechanism 312.
  • the hinged seat 311 is disposed on the base 32, and the rocking base 21 of the rocking frame 2 is The hinged seat 311 is hinged to provide a rocking center that is coupled or pressed against the rocking base 21 such that the upper and lower reciprocating mechanisms 312 can push the rocking base 21 to reciprocate about the center of the swing.
  • the hinge seat 311 may have one or more. In the example shown in FIG. 6, in the middle of the base 32, two hinge seats 311 are symmetrically disposed to make the rocking more stable. Based on the operation of the upper and lower reciprocating mechanisms 312, the rocking frame can be tilted left and right.
  • the upper and lower reciprocating mechanisms 312 may be various existing reciprocating mechanisms, such as an electric push rod, a linear motor, a crank linkage mechanism, and the like.
  • the upper and lower reciprocating mechanism 312 may include a motor and a cam, and the motor cam is fixed on an output shaft of the motor, and the cam directly presses against the rocking base 21 or pushes up and down in the guide groove. The rods are pressed or hinged.
  • the motor of the upper and lower reciprocating mechanism 312 can be placed in the base 32.
  • the upper and lower reciprocating mechanisms 312 may be one or more. Normally, an up and down reciprocating mechanism 312 can satisfy the swinging demand. Based on such a swing design, the swing amplitude and frequency of the shaker can be easily adjusted by adjusting the up and down reciprocating frequency of the upper and lower reciprocating mechanisms, or adjusting the distance between the upper and lower reciprocating mechanisms and the hinged seat.
  • the upper and lower reciprocating mechanism 312 pushes the rocking base 21 to swing around the hinge seat, thereby driving the culture bag bracket 1 fixed on the support arm 211 to drive the cell culture bag 4 to shake.
  • the tray is heated by the heating plate, and various monitoring probes can be connected to the cell culture bag through the accessory fixing hole 111 provided at the bottom of the tray to detect the cell culture state.
  • the shaking can also be stopped, and the objective lens can be directly observed by inserting the objective lens into the notch 112 of the bracket.
  • the non-contact sensor connector shown in FIG. 7-9 includes a base plate 41701 and an open sensor probe accommodating chamber 41702.
  • the base plate 41701 is provided with a transparent partition 41703.
  • the sensor probe accommodating chamber 41702 With the base 41701 is connected to one side of the transparent partition 41703, and the other side of the transparent partition 41703 is provided with a conversion film.
  • the open sensor probe housing chamber means that the chamber is provided with an opening that can be used to facilitate insertion of the sensor probe.
  • the conversion film and the sensor probe accommodating chamber 41702 are respectively disposed at two sides of the transparent partition 41703.
  • the arrow shown in Fig. 8 points to the side on which the conversion film is provided.
  • the conversion film is in contact with the liquid to be tested, and the sensor probe is fixed in the sensor probe accommodating chamber, opposite to the conversion film and isolated from the liquid to be tested by transparent partition.
  • the base plate is engageable with a liquid mounting container to be tested.
  • a liquid mounting container to be tested Taking a cell culture bag as an example, one side or edge of the substrate 41701 can be welded or welded to the cell culture bag, and the cell culture bag is provided with an opening at a position corresponding to the transparent partition 41703, and the opening makes the transparent partition
  • the conversion membrane can be in contact with the culture liquid phase in the cell culture bag, and the sensor probe inserted into the sensor probe accommodation chamber 41702 is isolated from the culture liquid phase in the cell culture bag due to the presence of the transparent partition 41703.
  • the transparent partition 41703 is colorless and transparent, and has smooth sides.
  • the smooth transparent partition is more conducive to the color of the conversion film.
  • the transparent partition 41703 is not limited to being disposed only in the middle of the base plate 41701, and only a base having a certain width around the transparent partition is sufficient to be combined with the cell culture bag.
  • the sensor probe accommodating chamber 41702 can be plugged or screwed with the sensor probe.
  • the sensor probe receiving chamber 41702 is provided with internal threads 41704. Matched, the sensor probe is provided with an external thread. The threaded connection further ensures the stability of the sensor probe detection.
  • the conversion film is a pH-induced color-changing film or a dissolved oxygen-induced color-changing film.
  • the pH-induced color-changing film means that the light-transmitting color can be changed according to the pH value of the contact liquid.
  • the pH-induced color-changing film can be obtained by mixing a pH indicator with a film-forming material and then forming a film.
  • pH-induced color-changing film belongs to the prior art, such as a pH sensing film used for a pH electrode.
  • the sensor module of the conversion film is a pH-induced color-sensitive film that can be matched with an RGB sensor and a white light source.
  • the mechanism for detecting the pH value is: the pH-induced color-changing film changes color according to the pH value of the liquid contacted, thereby affecting the color of the white light source penetrating the film, and the white light source and the pH-induced color-changing film can convert the pH signal into a color signal, RGB
  • the sensor detects the color signal of the transmissive conversion film and the transparent partition, and obtains the pH value of the liquid to be tested in the installation container of the liquid to be tested according to the relationship between the color of the conversion film and the pH value, and the relationship between the color of the conversion film and the pH value can be utilized in advance. The value of the standard solution was obtained.
  • the dissolved oxygen-induced color-changing film can change the light-transmitting color according to the dissolved oxygen value of the contact liquid.
  • the dissolved oxygen-induced color-changing film can be obtained by mixing a dissolved oxygen indicator with a film-forming material and then forming a film.
  • Dissolved Oxygen-Induced Discoloration Membrane The dissolved oxygen-chromic film is of the prior art.
  • the conversion film is a sensor connector that dissolves the oxygen-induced color-changing film and can be matched with an RGB color sensor and a white light source.
  • the mechanism for detecting the dissolved oxygen value is: dissolving the oxygen-induced color changing film to change the color according to the dissolved oxygen concentration of the contact liquid, thereby affecting the white light.
  • the source penetrates the color of the film, the white light source is combined with the dissolved oxygen-induced color-changing film to convert the dissolved oxygen signal into a color signal, and the RGB color sensor detects the color signal of the transmissive conversion film and the transparent partition, and is known according to the relationship between the color and the dissolved oxygen concentration.
  • the dissolved oxygen value of the liquid to be tested in the liquid to be tested is measured, and the relationship between the color of the conversion film and dissolved oxygen can be obtained by using a standard solution of a known dissolved oxygen concentration in advance.
  • the outer edge of the transparent partition 41703 provided with the conversion film is provided with a flange 41705 protruding from the base plate 41701.
  • the flange can alleviate the impact of the culture fluid on the conversion membrane to further ensure the stability of the sensor probe detection.
  • the sensor probe accommodating chamber 41702 can be fixedly connected to the base plate 41701 or can be detachably connected.
  • Removable connections include, but are not limited to, plugs, threaded connections, and the like.
  • the sensor probe accommodating chamber 41702 and the base plate 41701 are smoothly transitioned at the junction.
  • the non-contact sensor connector of the present invention is particularly suitable for use in cell culture bags.
  • the pH and dissolved oxygen of the liquid to be tested in the liquid to be tested can be converted into a color signal, and the color can be obtained by inserting the RGB sensor probe into the sensor probe housing chamber.
  • the signal is converted into a level signal or a square wave signal, and the controller of the RGB sensor reads the level signal or the square wave signal, and obtains the cell culture liquid according to the conversion relationship between the level signal corresponding to the color of the conversion film and the pH value and dissolved oxygen. pH and dissolved oxygen value.
  • the conversion relationship between the level signal corresponding to the color of the conversion film and the pH value and dissolved oxygen can be obtained by a conventional technique, for example, by plotting a curve after detecting the standard solution of a known pH value or a dissolved oxygen value.
  • the cell culture bag shown in FIG. 10-11 includes a closed bag body 41 and fixing members 42 disposed on both sides of the bag body.
  • the top surface of the closed bag body 41 is provided with an external liquid loading port 411 and an outer portion.
  • the bottom surface of the closed bag body 41 is provided with a liquid recovery port 415 and at least one sensor joint 417, the external liquid sample port 411, the external liquid injection port 412, and the gas.
  • a sterile quick joint 418 (shown in Figures 13 and 14) is provided on the inlet 413, the gas outlet 414, and the liquid recovery port 415.
  • the top surface of the cell culture bag is provided with an external liquid sample port, an external liquid injection port, a gas inlet, and a gas outlet to satisfy the process of adding the cell culture solution and the outside even if the culture device is not removed during the cell culture process.
  • Liquids such as penicillin solution, streptomycin solution, inoculum, etc.
  • a liquid recovery port is provided on the bottom surface of the cell culture bag, and the culture solution can be replaced in whole or in part without removing the culture device.
  • a sensor connector is provided on the bottom surface of the cell culture bag, which can be used to turn on the sensor to monitor relevant indicators in the cell culture fluid bag.
  • a quick connector is a connector that allows a pipe to be turned on or off without the need for a tool.
  • the sterile quick connector used is a conventional sterile quick connector and is commercially available. Aseptic quick connectors can be welded or welded to the bag.
  • the enclosed bag body may be made of a flexible material.
  • the flexible material refers to a material that can be deformed after being stressed.
  • the material of the closed bag body is made of PE and EVOH.
  • the closed bag body is made of a multi-layer composite structure.
  • the innermost layer of the closed bag body is a polyethylene (PE) layer.
  • PE polyethylene
  • the innermost layer refers to the layer that contacts the culture solution, and the contact with the culture solution is a stable PE material, a low dissolution product, and no animal source.
  • the closed bag body is made of a five-layer composite structure, which is a polyethylene (PE) layer, an adhesive layer, an ethylene propylene alcohol (EVOH) layer, and a bonding layer from the inside to the outside.
  • PE polyethylene
  • EVOH ethylene propylene alcohol
  • Layer and polyethylene (PE) The material of such a five-layer composite structure is prior art and is commercially available.
  • the thickness of the pouch constituting the sealed pouch can be conventional, and in the preferred embodiment 0.325 mm is employed.
  • the cell culture bag of the invention can be sterilized by gamma rays, and the irradiation dose is ⁇ 25 Gy, ⁇ 50 Gy. It can be sealed by heating.
  • the closed bag body is surrounded by an upper bag piece 4161, a lower bag piece 4162, a left side bag piece 4163 and a right side bag piece 4164. Cavity. More preferably, the lower bottom edge of the left side pocket piece 4163 and the right side pocket piece 4164 are curved. The bottom curved design allows the 3D shape of the cell culture bag to form a smaller shear force during shaking of the culture platform and minimizes shear damage to the cells.
  • the left side pocket piece 4163 and the right side pocket piece 4164 are each elliptical, and the upper pocket piece 4161 and the lower pocket piece 4162 are rectangular.
  • a transparent viewing zone 419 is provided in the middle of the bottom of the bag body 41.
  • the transparent observation zone is provided at the bottom of the cell culture bag, and the cells in the cell culture bag can be directly observed from the bottom by setting a cell morphology observation device on the culture platform.
  • the sensor connector may be provided with one or more.
  • two sensor connectors are provided, one of which is a sensor connector 4171 for accessing the pH sensor probe and the other is for access to dissolved oxygen ( DO) Sensor connector 4172 of the sensor probe.
  • DO dissolved oxygen
  • the sensor connector can be a contact sensor connector or a non-contact sensor connector.
  • the sensor connector is a non-contact sensor connector, i.e., the sensor probe is not in contact with the culture fluid in the bag after insertion of the sensor connector.
  • the non-contact sensor connector includes a base plate 41701 and a sensor probe receiving cavity 41702.
  • the middle portion of the base plate is provided with a transparent partition 41703.
  • the chamber 41702 is connected to the base plate 41701 and is located on one side of the transparent partition 41703, and the other side of the transparent partition (see the illustrated arrow) Pointing) is provided with a conversion film.
  • the conversion membrane is a pH-induced film.
  • the pH-induced color-changing film can be obtained by mixing a pH indicator with a film-forming material and then forming a film.
  • the pH-induced color-changing film belongs to the prior art, such as a pH sensing film used for a pH electrode.
  • the conversion membrane is a dissolved oxygen color-changing membrane.
  • the dissolved oxygen-induced color-changing film can be obtained by mixing a dissolved oxygen indicator with a film-forming material and then forming a film.
  • the dissolved oxygen color-changing film belongs to the prior art.
  • the pH and dissolved oxygen of the culture solution in the cell culture bag are converted into a color signal, and the color signal can be read by inserting the RGB sensor into the sensor connector, thereby depending on the color and pH of the conversion film.
  • the relationship between the value and dissolved oxygen is obtained, and the pH value and dissolved oxygen value of the cell culture solution are obtained.
  • the RGB color sensor used in the non-contact sensor probe does not require sterilization, which is not only easy to operate, but also better to prevent contamination.
  • the contact sensor connector can be a conventional aseptic quick connector that can be directly plugged or screwed into a sterile pH sensor or a probe for a dissolved oxygen sensor.
  • the aforementioned non-contact sensor connector can also be used as a contact sensor connector after removing the transparent partition and the conversion film.
  • the fixing member 4 is used for fixing and fixing the cell culture bag in the culture platform, and the fixing of the cell culture bag can be realized by using various existing components capable of fixing the liquid bag body, for example, it can be used alone.
  • a fixed component such as a viscous component or the like may be realized, or a component that needs to be fixed in cooperation with a matching device provided on the culture platform, such as a member fixed by mechanical force, adhesive force, or magnetic force.
  • the fixing member is a fixing rod, and the fixing rod can be detachably connected with a fixing rod clamp or a fixing rod insertion mechanism added on the culture platform, so that the fixed cell culture bag can be achieved. purpose.
  • the two ends of the bag body 41 are provided with a sealing area 44, and the fixing rod is placed in the sealing area.
  • the cell culture bag of the present invention When the cell culture bag of the present invention is used, it can be fixed to the culture platform by the fixing member 42, and the external liquid sample port 411, the external liquid injection port 412, the gas inlet 413, the gas outlet 414, and the liquid recovery port of the cell culture bag.
  • the 415 is connected through a pipeline to a device for supplying an external liquid, a gas, or a liquid or a gas, through a sterile quick connector, in which the probe of the corresponding sensor is inserted.
  • the external liquid is added as needed, the culture solution is changed, an appropriate cell culture atmosphere is provided, and the condition of the cell culture medium and the growth of the cells are monitored.
  • the external liquid loading port 411 can quickly connect the external pipeline through the aseptic quick joint 418 as shown in FIG. 13 , and the external pipeline can sequentially include a silicone tube 431 and a Luer connector 432, and then connected to An external liquid supply device can be further connected to the tee 433 to connect the plurality of external liquid storage units.
  • the external liquid injection port 412 can be quickly butted to the external pipeline through the aseptic quick joint 418 as shown in FIG. 13 , and the external pipeline can sequentially include a silicone tube 431 , a luer connector 432 , and a needleless sampler 434 . Connect external liquid supply Set.
  • the gas inlet 413 can be quickly docked to the external pipeline via the aseptic adapter 418 as shown in FIG. 13, and the external conduit can include a silicone tube 431 and an air filter 435 in sequence to connect the external gas supply device.
  • the gas outlet 414 can be quickly connected to the external pipeline through the aseptic quick joint 418 as shown in FIG. 13 , and the external pipeline can include a silicone tube 431 , an air filter 435 , a silicone tube 431 , and an air shutoff valve 436 . Furthermore, it communicates with the outside world or the gas collecting device.
  • the liquid recovery port 415 can be quickly docked to the external pipeline via the aseptic adapter 418 as shown in FIG. 14.
  • the external conduit can include a silicone tube 431 and a Luer connector 432 in sequence to connect the external liquid collection device.
  • a sheet clamp 437 may be disposed on the silicone tube of the pipeline to control the ingress and egress, and the components of the pipeline may be sealedly connected by the tie 438.
  • the cell culture bag of the invention can meet the following cell culture requirements:
  • the continuous culture condition is not replaced: the initial culture of the cells is small, and the cell culture medium is continuously increased as the cell culture cell density increases; the cell culture bag of the present invention can satisfy the initial cell culture amount. In rare cases, it can also be cultured in a cell culture bag;
  • the cell culture bag of the present invention can be used for culturing, and the cell culture bag can be heated;
  • the design of the 3D culture bag can be connected to the gas device necessary for cell culture, and the pH value and dissolved oxygen of the culture solution can be revised by adjusting the gas environment;
  • the cells in the culture bag are subjected to a small shear force, which is advantageous for cell growth.
  • a dark field microscope for on-line observation of cell culture including but not limited to: illumination element, movable holder 9, support base 5, objective lens 61, photosensitive element 10, diameter unit 6, A/ D conversion unit, control system and power module.
  • the lighting element is disposed on the top of the movable bracket 9, the movable bracket 9 is disposed on the support base 5, the pipe diameter unit 6 is disposed at the lower portion of the movable bracket 9, and the objective lens 61 is disposed on the pipe diameter unit 6 and corresponds to the lighting component
  • the photosensitive element 10 is disposed in the pipe diameter unit 6 for collecting the light source signal on the objective lens 61
  • the A/D conversion unit is connected to the photosensitive element 10
  • the control system is connected to the A/D conversion unit
  • the power supply module is used for cell culture online. Observed by a dark field microscope powered.
  • the photosensitive element uses a CCD sensor
  • the A/D conversion unit transmits the image signal obtained by the CCD sensor to the control system.
  • the control system further includes a viewing unit disposed in the computer for viewing the observed content of the objective lens in the current state, and a storage unit for storing the observed content to be saved.
  • the lighting element is disposed on the top of the movable bracket 9, and the lighting elements include, but are not limited to, a circular socket 8 and LEDs arranged on the circular socket 8 to form a plurality of concentric circles.
  • the lamp is preferably 5 concentric circles.
  • the distance from the objective lens to the lamp holder is 25 cm, and it can also be set to 10-30 cm.
  • Each concentric circle includes ten to twenty LED lights to provide a light source for the entire device. The lights on the upper circumference of each concentric circle can be turned on or off at the same time.
  • the lighting element further includes a lens 82 disposed under the LED lamp such that the light emitted by the LED lamp is refracted after passing through the lens, thereby satisfying the need for dark field illumination.
  • the angle between the light emitted by the illumination element and the horizontal plane is not greater than the angle A, which is the angle between the most central light 83 emitted by the illumination element and the horizontal axis of the objective lens 61.
  • the lens is used to change the direction of the light.
  • the light emitted by the illumination element is refracted by the lens and is mostly incident on the objective lens.
  • the support base 5, as shown in FIG. 15, includes a stepping motor 501, a sliding mechanism and a base 503.
  • the sliding mechanism includes a slider 502 and a slide rail.
  • the slider 502 is connected to the base 503 via a slide rail.
  • the input motor 501 is connected to the control system and is disposed at one end of the base 503.
  • the slider 502 is connected to the stepping motor 501, and the movable bracket 9 is disposed on the slider 502.
  • the stepping motor 501 can be implemented by using a conventional stepping motor on the market.
  • the stepping motor 501 can drive the movement of the slider 502 to change the relative position of the movable bracket 2 of the entire microscope. .
  • the pipe diameter unit 6 is slidably coupled to the movable bracket 9, and the pipe diameter unit 6 is connected to the movable bracket 9 at a chute, and the pipe diameter unit 6 can slide up and down along the chute.
  • a through hole is arranged on opposite sides of the movable bracket 9 , and screws are respectively disposed on the two through holes.
  • the control system 7 includes, but is not limited to, a dark field microscope illumination element control unit 701; a dark field microscope movable carriage control unit 702, such as a user can control by entering a specific value
  • the stepping motor further controls the moving distance of the movable bracket 2;
  • the dark field microscope viewing unit 703, the main It is used for the user to view the content that can be observed through the objective lens in the current state;
  • the dark field microscope storage unit 704 is mainly used to store the observed content that the user thinks needs to be saved, so as to be viewed again.
  • the movable bracket 9 has a hollow structure, and the lighting element control unit can be disposed within the hollow structure.
  • control system for the process of controlling the lighting elements can all utilize the computers and integrated circuits of the prior art. Modules, programmable logic devices, other hardware or existing software modules are implemented.
  • the use of the device has great advantages when observing samples, especially cells cultured in cell bags or cell factories.
  • the movable support 9 is moved by the control system 7 so that the entire culture container is placed between the illumination element and the objective lens 61.
  • the movable support 9 is moved by the control system 7 to leave the culture container when needed.
  • the position of the movable bracket 9 is moved by the control system.
  • a control module at least: a control module, a control platform, a dark field microscope for cell culture online observation, a cell culture shaker, a cell culture bag, and a power module
  • the control module is connected to the control platform to control
  • the platform respectively connects and controls a cell culture shaker and a dark field microscope for on-line observation of cell culture, and a dark field microscope for cell culture observation on the line for observing cells cultured on a cell culture shaker, the cell culture bag setting On a cell culture shaker, the power module supplies power to the entire system.
  • the cell culture shaker in the automatic cell culture system is shown in Fig. 4-6, and comprises a culture bag holder 1, a rocking frame 2, and a shaking device 3, wherein the culture bag holder 1 is provided with a curved bottom plate 11 and cell culture.
  • a bag fixing member 12 the rocking frame 2 includes a rocking base 21, and the rocking base 21 is provided with a support arm 211, and the culture bag bracket 1 is fixedly connected to the rocking frame 2 via a support arm 211, and the rocking
  • the device 3 includes a base 32 on which is mounted a rocking assembly 31 that is coupled to the rocking assembly 31.
  • the curved bottom plate 11 is as shown in FIG. 1 and is provided with an objective lens insertion notch 112.
  • a transparent observation zone is arranged at the bottom of the culture bag, and the objective lens insertion notch 112 is matched with the transparent observation zone, and the objective lens can be inserted, thereby observing the cells in the cell culture bag directly from the bottom by using the objective lens.
  • the curved bottom plate 11 is for accommodating a main portion of the curved 3D cell culture bag 4.
  • the curved bottom plate is designed to make the curved 3D cell culture bag maintain the arc at the bottom during the cell culture process, so that the culture platform is shaken during the cell culture process. The shear force generated by the movement is minimal, and the shear damage to the cells is also minimized.
  • the sides of the curved bottom plate are curved.
  • one or more accessory fixing holes 111 may be disposed on the curved bottom plate 11.
  • the fixing hole 111 is provided with an internal thread to better fix the external fitting.
  • External accessories can be probes for various detectors, such as pH and dissolved oxygen detection probes.
  • the external fittings may also be various external liquid inlet pipes, injection parts, liquid outlet pipes, external gas delivery pipes, and the like which are convenient for continuous cell culture.
  • the curved bottom plate has an arc shape that is recessed downward in use. Further, the objective lens insertion notch 112 is disposed in a concave bottom region of the curved bottom plate.
  • the edge of the curved bottom plate 11 is provided with a baffle 113.
  • the baffle prevents the cell culture bag from slipping and shifting out of the culture bag holder.
  • a heating plate 114 is disposed on the curved bottom plate 11.
  • the heating plate can be heated during cell culture to provide a suitable temperature for cell culture.
  • the control module can set the temperature required for the cell culture bag 4 and control the heating of the heating plate.
  • the cell culture bag fixing member 12 may be disposed on two sides of the curved bottom plate, and may be a platform 121 extending from both side edges of the curved bottom plate to both sides, and is disposed on the cell culture bag.
  • a component that matches the fixed part may be a member fixed by mechanical force, adhesion, magnetic force, or the like, or a screw hole and a nut may be provided on the platform 121 to fix the cell culture bag 4 to the platform.
  • the cell culture bag 4 is provided with fixing rods on both sides thereof. At this time, the fixing rod clamp 122 and/or the fixing rod insertion mechanism 123 may be provided on the platform, thereby realizing the cell culture bag. 4 detachable connection of the fixed rod.
  • the design of the support arm 211 in the rocking frame 2 can serve to support the culture bag carrier 1.
  • a space for mounting or inserting an external fitting should be left under the fitting fixing hole 111 of the culture bag holder 1.
  • the support arm may be integrally formed with the rocking base as a part of the rocking base 21, or may be mounted as a separate component on the rocking base, or may be integrally formed with the culture bag bracket 1.
  • the support arm can reserve space for the connection between the cell culture bag and the external accessory.
  • the culture bag holder 1 since the culture bag holder 1 is elevated, only a small swing force is required to the swing base 21, and the culture bag holder 1 is provided. It can swing in a wide range.
  • the rocking base 21 is a flat plate, and both ends are provided with support arms 211.
  • the culture bag carrier 1 is not in contact with the rocking base 21, and a gap is left to be installed or inserted into the outside.
  • Accessories including various probes and objectives.
  • the rocking assembly 31 can be any existing mechanism that can effect a rocking motion.
  • the rocking assembly 31 includes a hinged seat 311 and at least one upper and lower reciprocating mechanism 312.
  • the hinged seat 311 is disposed on the base 32, and the rocking base 21 of the rocking frame 2 is The hinged seat 311 is hinged, Thereby, a swing center is provided, which is connected or pressed against the rocking base 21, so that the up and down reciprocating mechanism 312 can push the rocking base 21 to reciprocate around the swing center.
  • the hinge seat 311 may have one or more. In the example shown in FIG. 6, in the middle of the base 32, two hinge seats 311 are symmetrically disposed to make the rocking more stable. Based on the operation of the upper and lower reciprocating mechanisms 312, the rocking frame can be tilted left and right.
  • the upper and lower reciprocating mechanisms 312 may be various existing reciprocating mechanisms, such as an electric push rod, a linear motor, a crank linkage mechanism, and the like.
  • the upper and lower reciprocating mechanism 312 may include a motor and a cam, and the motor cam is fixed on an output shaft of the motor, and the cam directly presses against the rocking base 21 or pushes up and down in the guide groove. The rods are pressed or hinged.
  • the motor of the upper and lower reciprocating mechanism 312 can be placed in the base 32.
  • the upper and lower reciprocating mechanisms 312 may be one or more. Normally, an up and down reciprocating mechanism 312 can satisfy the swinging demand. Based on such a swing design, the swing amplitude and frequency of the shaker can be easily adjusted by adjusting the up and down reciprocating frequency of the upper and lower reciprocating mechanisms, or adjusting the distance between the upper and lower reciprocating mechanisms and the hinged seat.
  • the upper and lower reciprocating mechanism 312 pushes the rocking base 21 to swing around the hinge seat, thereby driving the culture bag bracket 1 fixed on the support arm 211 to drive the cell culture bag 4 to shake.
  • the tray is heated by the heating plate, and various monitoring probes can be connected to the cell culture bag through the accessory fixing hole 111 provided at the bottom of the tray to detect the cell culture state.
  • the shaking can also be stopped, and the objective lens can be directly observed by inserting the objective lens into the notch 112 of the bracket.
  • FIG. 15 A dark field microscope for online observation of cell culture in an automated cell culture system is shown in Fig. 15, including but not limited to: illumination element, movable holder 9, support base 5, objective lens 61, photosensitive element 10, and diameter unit 6, A / D conversion unit.
  • the lighting element is disposed on the top of the movable bracket 9, the movable bracket 9 is disposed on the support base 5, the pipe diameter unit 6 is disposed at the lower portion of the movable bracket 9, and the objective lens 101 is disposed on the pipe diameter unit 6 and corresponds to the lighting component
  • the photosensitive element 10 is disposed in the pipe diameter unit 6 for collecting the light source signal on the objective lens 61
  • the A/D conversion unit is connected to the photosensitive element 10 and the support base 5
  • the control module is connected to the A/D conversion unit.
  • the photosensitive element adopts a CCD sensor
  • the A/D conversion unit transmits the image signal obtained by the CCD sensor to the control module.
  • the control module includes a viewing unit disposed in the computer for viewing the content observed by the objective lens in the current state, and a storage unit for storing the observed content to be saved.
  • the lighting elements are disposed on top of the movable bracket 9, and the lighting elements include, but are not limited to, circular
  • the lamp holder 8 and the circular lamp holder 8 are arranged in a plurality of concentrically arranged LED lamps, for example, five concentric circles.
  • the distance between the lamp holder and the objective lens is 18 cm, and the general distance can also be set to 10-30 cm.
  • Ten to twenty LED lights are included on each concentric circle to provide a light source for the entire unit. The lights on the upper circumference of each concentric circle can be turned on or off at the same time.
  • the angle between the light emitted by the illumination element and the horizontal plane is not greater than the angle A, which is the most central light 83 emitted by the illumination element.
  • the lens is used to change the direction of the light.
  • the light emitted by the illumination element is refracted by the lens and is mostly incident on the objective lens.
  • the support base 5 includes a stepping motor 501, a sliding mechanism and a base 503.
  • the sliding mechanism includes a slider 502 and a slide rail.
  • the slider 502 is connected to the base 503 via a slide rail, and the stepping motor 501 is connected to the control module.
  • the slider 502 is connected to one end of the base 503, the slider 502 is connected to the stepping motor 501, and the movable bracket 2 is disposed on the slider 502.
  • the stepping motor 501 can be implemented by using a conventional stepping motor 501 on the market, and the stepping motor 501 can drive the movement of the slider 502 to make the relative position of the movable bracket 9 of the entire microscope. change.
  • the pipe diameter unit 6 is slidably coupled to the movable bracket 9 , and the pipe diameter unit 6 is connected to the movable bracket 9 and provided with a sliding slot.
  • the pipe diameter unit 6 can slide up and down along the sliding slot.
  • a through hole is arranged on opposite sides of the movable bracket 9 , and screws are respectively disposed on the two through holes.
  • the movable bracket 9 has a hollow structure, and the lighting element control unit can be disposed within the hollow structure.
  • the use of the device has great advantages in observing samples.
  • the movement of the movable holder 9 is controlled by the control module, and the entire culture container is placed between the illumination element and the objective lens 61.
  • the objective lens 61 is inserted into the notch 112 by the cell culture shaker objective lens to observe the cells, and the control module can be moved.
  • the mobile stand 9 is moved away from the culture container, and the position of the movable stand 9 is moved by the control module when it is necessary to observe again.
  • the cell culture bag used in the automatic cell culture system is shown in Figs. 10 and 11, and the cell culture bag 4 includes: a closed bag body 41 and fixing members 42 provided on both sides of the bag body, and the closed bag body 41 is topped.
  • the surface is provided with an external liquid loading port 411, an external liquid injection port 412, a gas inlet 413 and a gas outlet 414.
  • the bottom surface of the closed bag body 41 is provided with a liquid recovery port 415 and at least one sensor joint 417.
  • Sample port 411, external liquid injection port 412, gas inlet A sterile quick connector 418 is provided on the port 413, the gas outlet 414, and the liquid recovery port 415.
  • the top surface of the cell culture bag 4 is provided with an external liquid sample port, an external liquid injection port, a gas inlet, and a gas outlet in order to satisfy the process of adding the cell culture solution, even if the culture device is not removed during the cell culture process.
  • External liquids such as penicillin solution, streptomycin solution, inoculum, etc.
  • a liquid recovery port is provided on the bottom surface of the cell culture bag, and the culture solution can be replaced in whole or in part without removing the culture device.
  • a sensor connector is provided on the bottom surface of the cell culture bag, which can be used to turn on the sensor to monitor relevant indicators in the cell culture fluid bag.
  • a quick connector is a connector that allows a pipe to be turned on or off without the need for a tool.
  • the sterile quick connector used is a conventional sterile quick connector and is commercially available. Aseptic quick connectors can be welded or welded to the bag.
  • the enclosed bag body may be made of a flexible material.
  • the flexible material refers to a material that can be deformed after being stressed.
  • the material of the closed bag body is made of PE and EVOH.
  • the closed bag body is made of a multi-layer composite structure.
  • the innermost layer of the closed bag body is a polyethylene (PE) layer.
  • PE polyethylene
  • the innermost layer refers to the layer that contacts the culture solution, and the contact with the culture solution is a stable PE material, a low dissolution product, and no animal source.
  • the closed bag body is made of a five-layer composite structure, which is a polyethylene (PE) layer, an adhesive layer, an ethylene propylene alcohol (EVOH) layer, and a bonding layer from the inside to the outside.
  • PE polyethylene
  • EVOH ethylene propylene alcohol
  • Layer and polyethylene (PE) The material of such a five-layer composite structure is prior art and is commercially available.
  • the thickness of the pouch constituting the sealed pouch can be conventional, and in the preferred embodiment 0.325 mm is employed.
  • the cell culture bag of the invention can be sterilized by gamma rays, and the irradiation dose is ⁇ 25 Gy, ⁇ 50 Gy. It can be sealed by heating.
  • the closed bag body is surrounded by an upper bag piece 4161, a lower bag piece 4162, a left side bag piece 4163 and a right side bag piece 4164. . More preferably, the lower bottom edge of the left side pocket piece 4163 and the right side pocket piece 4164 are curved.
  • the bottom curved design allows the 3D shape of the cell culture bag to form a smaller shear force during shaking of the culture platform and minimizes shear damage to the cells.
  • the left side pocket piece 4163 and the right side pocket piece 4164 are each elliptical, and the upper pocket piece 4161 and the lower pocket piece 4162 are rectangular.
  • a transparent viewing zone 419 is provided in the middle of the bottom of the bag body 41.
  • the transparent observation zone is provided at the bottom of the cell culture bag, and the cells in the cell culture bag can be directly observed from the bottom by setting a cell morphology observation device on the culture platform.
  • the sensor connector may be provided with one or more.
  • two sensor connectors are provided, one of which is a sensor connector 4171 for accessing the pH sensor probe and the other is Sensor connector 4172 for accessing a dissolved oxygen (DO) sensor probe.
  • DO dissolved oxygen
  • the sensor connector can be a contact sensor connector or a non-contact sensor connector.
  • the sensor connector is a non-contact sensor connector, i.e., the sensor probe is not in contact with the culture fluid in the bag after insertion of the sensor connector.
  • the non-contact sensor connector includes a base plate 41701 and a sensor probe receiving cavity 41702 as shown in FIGS. 7-9, and a transparent partition 1703 is disposed in a middle portion of the base plate.
  • the sensor probe receiving cavity 1702 is connected to the base plate 41701 and is located on one side of the transparent partition 41703, and the other side of the transparent partition (see the arrow pointing in the figure) is provided with a conversion film.
  • the conversion membrane is a pH-induced film.
  • the pH-induced color-changing film can be obtained by mixing a pH indicator with a film-forming material and then forming a film.
  • the pH-induced color-changing film belongs to the prior art, such as a pH sensing film used for a pH electrode.
  • the conversion membrane is a dissolved oxygen color-changing membrane.
  • the dissolved oxygen-induced color-changing film can be obtained by mixing a dissolved oxygen indicator with a film-forming material and then forming a film.
  • the dissolved oxygen color-changing film belongs to the prior art.
  • the pH and dissolved oxygen of the culture solution in the cell culture bag are converted into a color signal, and the color signal can be read by inserting the RGB sensor into the sensor connector, thereby depending on the color and pH of the conversion film.
  • the relationship between the value and dissolved oxygen is obtained, and the pH value and dissolved oxygen value of the cell culture solution are obtained.
  • the pH and dissolved oxygen results obtained from the signal from the sensor connector can ultimately be passed to the control module for recording and viewing.
  • the RGB color sensor used in the non-contact sensor probe does not require sterilization, which is not only easy to operate, but also better to prevent contamination.
  • the contact sensor connector can be a conventional aseptic quick connector that can be directly plugged or screwed into a sterile pH sensor or a probe for a dissolved oxygen sensor.
  • the aforementioned non-contact sensor connector can also be used as a contact sensor connector after removing the transparent partition and the conversion film.
  • the fixing member 42 is configured to fit and fix the cell culture bag in the culture platform, and the fixing of the cell culture bag can be realized by using various existing components capable of fixing the liquid bag body, for example, it can be used alone.
  • a fixed component such as a viscous component or the like may be realized, or a component that needs to be fixed in cooperation with a matching device provided on the culture platform, such as a member fixed by mechanical force, adhesive force, or magnetic force.
  • the fixing member is a fixing rod, and the fixing rod can be detachably connected with a fixing rod clamp or a fixing rod insertion mechanism added on the culture platform, so that the fixed cell culture bag can be achieved. purpose.
  • the two ends of the bag body 41 are provided with a sealing area 44, and the fixing rod is placed in the sealing area.
  • the cell culture bag of the present invention When the cell culture bag of the present invention is used, it can be fixed to the culture platform by the fixing member 42, and the external liquid sample port 411, the external liquid injection port 412, the gas inlet 413, the gas outlet 414, and the liquid recovery port of the cell culture bag. 415
  • the probes of the corresponding sensors are inserted into the sensor joints through the pipelines by means of a sterile quick joint, respectively, via a pipeline to a device for supplying an external liquid, a gas or a liquid or a gas.
  • the external liquid is added as needed, the culture solution is changed, an appropriate cell culture atmosphere is provided, and the condition of the cell culture medium and the growth of the cells are monitored.
  • the external liquid loading port 411 can quickly connect the external pipeline through the aseptic quick joint 418 as shown in FIG. 13 , and the external pipeline can sequentially include a silicone tube 431 and a Luer connector 432, and then connected to An external liquid supply device can be further connected to the tee 433 to connect the plurality of external liquid storage units.
  • the external liquid injection port 412 can quickly connect the external pipeline through the aseptic quick connector 18 as shown in FIG. 13 , and the external pipeline can include a silicone tube 431 , a Luer connector 432 , and a needleless sampler 434 in sequence. Connect an external liquid supply device.
  • the gas inlet 413 can be quickly docked to the external pipeline via the aseptic adapter 418 as shown in FIG. 13, and the external conduit can include a silicone tube 431 and an air filter 435 in sequence to connect the external gas supply device.
  • the gas outlet 414 can be quickly connected to the external pipeline through the aseptic quick joint 418 as shown in FIG. 13 , and the external pipeline can include a silicone tube 431 , an air filter 435 , a silicone tube 431 , and an air shutoff valve 436 . Furthermore, it communicates with the outside world or the gas collecting device.
  • the liquid recovery port 415 can be quickly docked to the external pipeline via the aseptic adapter 418 as shown in FIG. 14.
  • the external conduit can include a silicone tube 431 and a Luer connector 432 in sequence to connect the external liquid collection device.
  • a sheet clamp 437 may be disposed on the silicone tube of the pipeline to control the ingress and egress, and the components of the pipeline may be sealedly connected by the tie 438.
  • the continuous culture condition is not replaced: the initial culture of the cells is small, and the cell culture medium is continuously increased as the cell culture cell density increases; the cell culture bag of the present invention can satisfy the initial cell culture amount. In rare cases, it can also be cultured in a cell culture bag;
  • the cell culture bag of the present invention can be used for culturing, and the cell culture bag can be heated;
  • the design of the 3D culture bag can be connected to the gas device necessary for cell culture, and the pH value and dissolved oxygen of the culture solution can be revised by adjusting the gas environment;
  • the cells in the culture bag are subjected to a small shear force, which is advantageous for cell growth.
  • the control platform in the fully automated cell culture system is provided with an integrated circuit board.
  • the integrated circuit boards are connected to at least a dark field microscope and a cell culture shaker, respectively, and the integrated circuit board is connected to the control module.
  • the integrated circuit board controls the dark field microscope switch and/or illumination components, as well as controls the cell culture shaker frequency, amplitude, and heating temperature.
  • the integrated circuit board is connected to the shaking device of the cell culture shaker to control the swing frequency and amplitude of the shaker.
  • the integrated circuit board is also connected to the temperature control heating element on the culture bag holder, and the temperature control heating element can be controlled to heat the cell culture bag at the set temperature.
  • the control platform can also include a flow control element that provides a cell culture fluid conduit for the cell culture fluid to the cell culture bag.
  • a flow control element that provides a cell culture fluid conduit for the cell culture fluid to the cell culture bag.
  • the integrated circuit board is further connected to a cell culture fluid line flow control element to control the flow rate of the cell culture fluid line.
  • the control platform can also include a gas mixing unit that supplies gas to the cell culture bag.
  • a flow control element is provided in the gas mixing unit.
  • a solenoid valve or the like is suitable for controlling the flow of gas.
  • the integrated circuit board is further connected to a gas mixing unit flow control element to control gas flow in the gas line.
  • control platform may also include an automatic heating unit for preheating the cell culture fluid.
  • the automatic heating unit includes heating elements such as a preheating plate, a heating pad, a heating tube, a heating wire, and the like.
  • the automatic heating unit also includes a temperature measuring element.
  • the temperature measuring element can be disposed on the preheating plate for sensing the temperature of the preheating plate.
  • Various temperature measuring probes, sensors, etc. can also be used for the temperature measuring component.
  • Automatic heating technology belongs to the prior art.
  • the integrated circuit board is connected to the automatic heating unit, and the automatic heating unit can be controlled to preheat the cell culture liquid at a preset temperature.
  • the control platform can be equipped with a preheating bag with an automatic heating unit that can be connected to the cell culture fluid line and contacted with the heating element of the automatic heating unit to preheat the cell culture fluid.
  • the control platform can also include a pH detector and a dissolved oxygen detector.
  • the pH detector and the dissolved oxygen detector are respectively connected to the corresponding sensor connectors on the cell culture bag via the pH sensor probe and the dissolved oxygen sensor probe.
  • the integrated circuit board is connected with the pH detector to control the switch of the pH detector; and the integrated circuit board is connected with the dissolved oxygen detector to control the switch of the dissolved oxygen detector.
  • the control platform may further be provided with a pH detecting joint and a dissolved oxygen detecting joint for transmitting the signal of the measured pH value and the dissolved oxygen amount in the cell culture bag to the control module.
  • the pH detection connector and the dissolved oxygen detection connector are respectively connected to the corresponding sensor connector on the cell culture bag through the pH sensor probe and the dissolved oxygen sensor probe, and the information from the cell culture bag is transmitted to the pH detector and the dissolved oxygen detector to obtain the pH.
  • the dissolved oxygen signal on the other hand, respectively connected to the control mode Block to pass the pH and dissolved oxygen signals to the control module.
  • the control platform can also further include a display screen.
  • the integrated circuit board is connected to the display screen to control the display switch.
  • the display can be further connected to the control module.
  • the display can be used to display the setup parameters or system immediate parameters that need to be understood, including but not limited to: culture solution temperature, incubation time, shaker speed, swing amplitude, gas ratio, dissolved oxygen value, pH value, and the like.
  • control platform further includes a control cabinet 13, an automatic heating unit, a preheating bag 1309, and a peristaltic pump disposed on the control cabinet 13;
  • the automatic heating unit includes a preheating plate, and the preheating bag 1309 Disposed on the preheating plate, the peristaltic pump includes a first peristaltic pump 1301 and a second peristaltic pump 1302.
  • the inlet of the preheating bag 1309 is connected to the external cell culture fluid 1306 through the first conduit 1308, and the outlet of the preheating bag 1309 is passed through the second conduit.
  • the first peristaltic pump 1301 is connected to the first pipe 1308, the second peristaltic pump 1302 is connected to the second pipe 1307, and the automatic heating unit, the first peristaltic pump 1301 and the second peristaltic pump 1302 are connected to the control module.
  • the first peristaltic pump 1301 and the second peristaltic pump 1302 can be connected to the control module via the integrated circuit board, so that the peristaltic pump can be controlled to add the cell culture fluid according to the flow rate set by the control module.
  • the control cabinet 13 is a carrier, the shape of which is not specifically required, and is illustrated as a regular cubic structure in which various working units and integrated circuits can be accommodated.
  • the preheating plate is disposed on the top of the control cabinet 13, and a preheating bag 1309 is disposed thereon.
  • the role of the preheating plate is to heat the cell culture medium in the preheating bag 1309.
  • the cell culture medium is commercially available and is suitable for the type of cell culture required.
  • the volume of the preheating bag 1309 is generally set to 500 ml, which may also vary depending on design or culture scale requirements.
  • the automatic heating unit can be connected to the control module.
  • the control module can be connected to the automatic heating unit via the integrated circuit board, so that the automatic heating unit can be controlled according to the temperature set by the control module.
  • the specific temperature is set by the control module.
  • the first peristaltic pump 1301 pumps the external cell culture fluid into the preheating bag 1309, and the cell culture fluid reaching a certain temperature is pumped into the cell culture bag through the second peristaltic pump 1302.
  • a gas mixing unit is further disposed inside the control cabinet 13, and the gas mixing unit is disposed on the gas pipeline, and the gas mixing unit is connected to an external air source, mainly oxygen, air and carbon dioxide.
  • the external gas entering gas mixing unit is mixed, passes through the gas outlet 1305, flows through the third conduit (not shown), and enters the cell culture bag.
  • the gas mixing unit includes a plurality of intake ports and a mixed gas outlet pipe.
  • the air inlets are not less than three, and at least oxygen, air and carbon dioxide can be separately transported.
  • Each gas inlet may be provided with a gas flow control element.
  • the components of the prior art that can realize gas flow control can be used for the air inlet, such as a solenoid valve, etc., which can realize gas flow adjustment. door.
  • the gas mixing unit may further comprise a gas mixing chamber, which may open the gas inlet to the gas mixing chamber and then output the gas through the mixed gas outlet tube.
  • the air inlets may be directly led to the mixed gas outlet pipe, and naturally mixed in the mixed gas outlet pipe to be output.
  • the gas mixing unit is connected to the control module, and the control module sets the supply of gas, such as the flow rate of the gas, the time of delivery, and the composition ratio of various gases.
  • the control module can be connected to the flow control component of the gas mixing unit via the integrated circuit board, so that the gas delivery ratio can be controlled according to the gas mixing ratio, the flow rate, the delivery time, and the like set by the control module.
  • control cabinet is further provided with a pH detector and a dissolved oxygen detector.
  • the pH detector and the dissolved oxygen detector are connected to the control module and the sensor connector on the cell culture bag, and the measured pH value and dissolved oxygen signal in the cell culture bag are transmitted to the control module.
  • the control cabinet is provided with a pH detecting connector 1303 and a dissolved oxygen detecting connector 1304.
  • the pH detector and the dissolved oxygen detector are respectively connected to the corresponding sensor connector and the control module on the cell culture bag through the connector.
  • the pH detecting connector and the dissolved oxygen detecting connector are respectively connected to the corresponding sensor connector on the cell culture bag via the pH sensor probe and the dissolved oxygen sensor probe.
  • the pH detecting connector 1303 and the dissolved oxygen detecting connector 1304 can transmit signals from the pH sensor probe and the dissolved oxygen sensor probe to the pH detector and the dissolved oxygen detector via a signal conducting member (not shown).
  • the pH detecting connector 1303 and the dissolved oxygen detecting connector 1304 can also transmit a signal of a pH value and a dissolved oxygen amount obtained by the pH detector and the dissolved oxygen detector to the control module.
  • the integrated circuit board is connected to a pH detector and a dissolved oxygen detector to control the switching of the pH detector and the dissolved oxygen detector.
  • Both the pH sensing connector 1303 and the dissolved oxygen detecting connector 1304 employ signal transponders commonly used in the market, such as SMA 905 plugs. Signaling members are, for example, fiber optic connectors that are common on the market.
  • control module 400 is coupled to the control platform 300.
  • the user can control the system through the control module 400 and view and record various parameters in various cell cultures as well as the structure of the cells and the like.
  • the control module can be placed on a computer.
  • Control module 400 includes, but is not limited to:
  • the movable bracket control unit 410 is configured to control the movement of the movable bracket of the dark field microscope 200.
  • a movable bracket connection control unit is coupled to the motor of the movable bracket.
  • the user can control the stepping motor to control the moving distance of the movable bracket 9 by inputting a specific value; the movable bracket control unit can directly connect the motor of the movable bracket (as shown in FIG. 19), or The motor of the movable bracket is connected to the control unit of the control platform via the integrated circuit board of the control platform (as shown in FIG. 20).
  • the lighting element control unit 420 is configured to control the lighting elements of the dark field microscope 200.
  • the lighting component control unit can set at least control parameters of the lighting component, such as a switch, a brightness, an opening and closing time interval, and the like.
  • the control is flat
  • the integrated circuit board of the stage can control the illumination elements of the dark field microscope.
  • the illumination element control unit is connected to the illumination elements of the dark field microscope via the integrated circuit board of the control platform.
  • the viewing unit 430 is mainly used to view the contents of the object and/or the cell growth environment, such as the pH value and the dissolved oxygen value, which can be observed by the objective lens of the dark field microscope 200 in the current state.
  • the storage unit is coupled to a dark field microscope, a pH detector, and a dissolved oxygen detector.
  • the storage unit 450 is mainly used for storing contents observed by a dark field microscope that the user thinks need to be preserved, and environmental parameters during cell growth, such as pH value and dissolved oxygen, for review; viewing unit and dark field
  • the microscope, pH detector and dissolved oxygen detector are connected.
  • the gas control unit 460 is mainly used to control the gas mixing unit.
  • the gas control unit may set at least one or more of a gas mixing ratio, a flow rate, and a delivery time.
  • the gas control unit can be connected to the flow control element of the gas mixing unit through the integrated circuit board of the control platform, so that the gas delivery can be set and controlled so that when the cell culture bag needs to be ventilated, the external gas is set to a predetermined ratio. After mixing, enter the cell culture bag;
  • the cell culture liquid control unit 460 mainly controls the automatic heating unit to heat the external cell culture medium when the cell culture medium in the cell culture bag needs to be replaced, and controls the cell culture liquid to be input into the cell culture bag.
  • the culture liquid control unit can set at least the heating temperature of the automatic heating unit and the conveying speed of the cell culture liquid.
  • the culture fluid control unit is connected to the control platform.
  • the culture fluid control unit can be connected to the automatic heating unit and the peristaltic pump via the integrated circuit board of the control platform, so that the cell culture fluid is heated to a set temperature and then input into the cell culture bag.
  • control module further includes one or more of the following:
  • the cell culture shaker control unit 470 is configured to set the frequency of the cell culture shaker 100, the swing amplitude, the heating temperature to the cell culture solution, and the like.
  • the cell shaker control unit is connected to the cell shaker.
  • the cell shaker control unit can be connected to the cell shaker through the integrated circuit board of the control platform, so that the cell shaker can operate according to the set frequency, swing and temperature.
  • the control platform display unit 480 is connected to the display screen of the control platform to set the display content.
  • control module for the control process of the movable bracket, the control process of the lighting component, the process of viewing the contents of the cell culture bag and various parameters, controlling the external air source and the cell culture.
  • the process of entering the cell culture bag and controlling the cell culture shaker can be realized by using a computer, an integrated circuit module, a programmable logic device, other hardware or an existing software module in the prior art.

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CN201510981653.7A CN105385587B (zh) 2015-12-23 2015-12-23 一种非接触式传感器接头及其应用
CN2015109816537 2015-12-23
CN201510981648.6A CN105385597B (zh) 2015-12-23 2015-12-23 一种细胞培养袋及其应用
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CN201610131141.6A CN105567565B (zh) 2016-03-08 2016-03-08 一种连续细胞培养摇床
CN201610131142.0A CN105567566B (zh) 2016-03-08 2016-03-08 一种连续细胞培养摇床用培养袋托架
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