WO2016070454A1 - 用于卵细胞体外受精和卵裂培养的自动化装置、载体及方法 - Google Patents

用于卵细胞体外受精和卵裂培养的自动化装置、载体及方法 Download PDF

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Publication number
WO2016070454A1
WO2016070454A1 PCT/CN2014/090998 CN2014090998W WO2016070454A1 WO 2016070454 A1 WO2016070454 A1 WO 2016070454A1 CN 2014090998 W CN2014090998 W CN 2014090998W WO 2016070454 A1 WO2016070454 A1 WO 2016070454A1
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WIPO (PCT)
Prior art keywords
carrier
culture
droplet
cell
egg
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PCT/CN2014/090998
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English (en)
French (fr)
Inventor
徐小杨
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徐小杨
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Priority claimed from CN201420665138.9U external-priority patent/CN204265762U/zh
Priority claimed from CN201420665155.2U external-priority patent/CN204265763U/zh
Priority claimed from CN201410626062.3A external-priority patent/CN104498321B/zh
Priority claimed from CN201410626035.6A external-priority patent/CN104403944B/zh
Priority claimed from CN201410626064.2A external-priority patent/CN104396942B/zh
Priority claimed from CN201410626061.9A external-priority patent/CN104371920A/zh
Application filed by 徐小杨 filed Critical 徐小杨
Publication of WO2016070454A1 publication Critical patent/WO2016070454A1/zh

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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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the invention relates to a device for cell culture, in particular to an automatic device, a carrier and a method for in vitro fertilization and cleavage culture of egg cells.
  • the IVF laboratory sequence culture operation procedure is as follows (take the 3rd day transplantation method as an example): prepare the culture solution in advance according to the surgical plan; surgically aspirate the follicular fluid, and the surgical assistant indicates whether the operator's follicular fluid container (test tube) needs to be replaced.
  • the surgeon continues to pump or stop the suction according to the prompt; after the follicular fluid is filled into the test tube, the surgical assistant is placed in the thermostatic test tube rack, the new test tube container is replaced, and the foregoing process is repeated until the surgeon finishes the suction and completes the operation; laboratory operation
  • the human extracts the follicular fluid containing the cumulus complex and completely transfers it into the sputum egg container, and rapidly recognizes the cumulus cell complex with the assistance of a low-power stereo microscope, and manually transfers it into the sputum.
  • the laboratory operator can also continue to culture until the blastocyst state after transplantation or freeze-thaw; if the operator decides to perform intracytoplasmic sperm injection, the laboratory operator must make a buffer droplet system, prepare the insemination dish, Microinjection insemination is performed under an inverted microscope of the thermostatic platform device, and then the inseminated cells are manually transferred into a culture dish (insemination culture droplets), placed in an incubator, and cultured.
  • the culture droplets are transferred several times in the open environment and the artificial environment in the incubator, and the target cells are also transferred multiple times in different liquid environments.
  • the cell transfer is manually performed by the operator (using a hand-held pipette tool to manually operate the single Cells should be strictly avoided to avoid operational errors such as bubble generation.
  • the culture environment is not continuous, and cell loss and damage are prone to occur.
  • the cell culture environment cannot be standardized, and the operation of any two groups of cells cannot be strictly compared.
  • the training process cannot be strictly compared, the operation process cannot be monitored, and the operation can only be judged from the transplant result.
  • the success rate of clinical reproductive laboratory embryo culture and clinical transplantation is directly related to the proficiency and responsibility of the embryo operator, and is directly related to the management behavior of the laboratory. This is the clinical reproductive medicine unit and operator.
  • the final planting success rate shows an important reason for significant differences. Qualified laboratory operators require rigorous operational training and long-term, extensive practice, which limits the further spread of IVF technology and creates a significant waste of social resources.
  • the object of the present invention is to provide an automated device for in vitro fertilization and cleavage culture of egg cells, simulating the environment of human egg cell insemination and early development of fertilized eggs, which can be automatically obtained from the cumulus complex, and the culture droplets are added to the sperm suspension. Liquid, cumulus complex degranulation, automatic control of droplet composition, controlled culture of blastocysts, and complete transfer of cultured droplets containing embryos.
  • Embodiments of the present invention provide an automated device for in vitro fertilization and cleavage culture of egg cells
  • the utility model comprises an automatic combined type partition combined incubator, a central controller placed outside the incubator and an automatic recognition and sorting device for egg cells;
  • the automatic combined-type partition combination incubator is provided with operation mode devices for different stages in the process of fertilization and cleavage of egg cell droplets in different sections, and each section is connected/separated by an automatic airtight door.
  • the automatic combined-type partition combination incubator is provided with a culture carrier for carrying egg cell droplets and a culture carrier driving device for driving the culture carrier to and from each partition;
  • the egg cell automatic identification sorting device, the operation mode device of different stages, the automatic airtight door and the culture carrier driving device are all connected with the central controller to receive corresponding control commands;
  • the egg cell automatic identification sorting device identifies and sorts the follicular stock solution with the egg cells according to the received control instruction, and then outputs the egg cell droplets to the culture carrier in the automatic combined partition combination incubator;
  • the culture carrier driving device drives the culture carrier to and from the respective sections of the automatic combined partition combination culture according to the control instruction, thereby making the egg cells on the culture carrier
  • the droplets enter each partition to perform the corresponding mode of operation.
  • the automatic combined partitioning combined incubator comprises two partitions, the two partitions passing through the main incubator, the auxiliary incubator, and the main incubator and the auxiliary incubator for realizing the main incubator
  • the automatic airtight door is connected/separated between the bodies.
  • the operation mode device includes, but is not limited to, a direct insemination mode device, a vitrification/recovery mode device, and a transplant mode device.
  • the direct insemination mode device is disposed in the main incubator; the vitrification/recovery mode device and the transplantation mode device are disposed in the sub-incubator.
  • the direct insemination mode device includes a first liquid droplet changing operation device
  • the first liquid droplet changing operation device includes a waste liquid extraction controller, a culture liquid supplement controller, and a droplet output a pipe and a droplet input line, both ends of the droplet output line and the droplet input line are connected to a droplet operation needle to suck/output a droplet
  • the waste liquid extraction controller passes the droplet Output line
  • the waste liquid in the cell droplets on the culture carrier is withdrawn and the extracted waste liquid flow rate is controlled
  • the culture liquid supplementation controller delivers the culture liquid to the cells on the culture carrier through the liquid droplet input line And controlling the flow rate of the delivered culture liquid in the liquid droplet
  • the central controller is respectively connected to the waste liquid extraction controller and the culture liquid supplement controller to control the waste liquid extraction controller and the culture liquid
  • the additional controller works.
  • the waste liquid extraction controller and the culture liquid supplement controller are a peristaltic pump or a stepping motor.
  • the direct insemination mode device further includes a cell image acquiring device connected to the central controller for acquiring cell image information in cell droplets on the culture carrier during direct insemination mode operation .
  • the culture carrier is provided with a plurality of pits for carrying egg cell droplets on the surface, and the pit surface is a superhydrophobic surface.
  • a closed annular restriction portion is provided on the surface of each of the pits on the surface of the culture carrier, and the surface of the restriction portion is an oleophilic surface.
  • the egg cell automatic identification sorting device includes a negative pressure generator, an egg cell image acquiring device, a light source device, a follicular fluid collecting container, and a sorting switch, and the central controller respectively generates the negative pressure , egg cell image acquisition device and sorting switch connection;
  • the sorting switch includes a first valve port, a second valve port and a third valve port, the first valve port is connected to a main input conduit for inputting follicular stock solution with egg cells and for outputting egg cells to the culture carrier Between the main output conduits, the second valve port is connected between the main input conduit and a follicular fluid for outputting egg-removing cells to a secondary output conduit of the follicular fluid collection container, the third valve port being connected for Entering between a secondary input conduit of a particular culture fluid and the primary output conduit; the negative pressure generator being in communication with the follicular fluid collection container through a communication conduit;
  • the light source device is disposed on a side of the main input duct, illuminates the main input duct, and is imaged on the egg cell image acquiring device provided on the other side of the main input duct;
  • the follicular stock solution of the egg cell flows into the main input catheter when the negative pressure generator is started, and the egg cell image information is acquired by the egg cell image acquiring device when flowing through the egg cell image acquiring device, and the central controller according to the egg cell image
  • the information controls opening/closing of the first valve port, the second valve port and the third valve port of the sorting switch, so that egg cells in the follicular fluid with egg cells flow out from the first valve port, and the egg cells are removed.
  • Follicular fluid flows out from the second valve port and flows into the follicular fluid collection container, and causes egg cells flowing out from the first valve port to form an egg cell droplet with a specific culture solution flowing in from the third valve port It flows out to the culture carrier.
  • the operation of the negative pressure generator is controlled by the central controller, thereby controlling the flow rate of the follicular stock solution with egg cells at the main input catheter.
  • the difference between the main input conduit position corresponding to the light source device and the egg cell image acquisition device and the catheter length of the main input catheter position where the first valve port is located is a preset value.
  • the central controller calculates the score according to the egg cell image information and the flow rate of the follicular stock solution with the egg cell at the main input conduit and the difference in the length of the catheter.
  • the vitrification/recovery mode device comprises a carrier mechanical action device, a second liquid droplet changing device, a freezing/recovering carrier, a console carrying the freezing/recovering carrier, and a freezing medium container, among them,
  • the carrier mechanical action device and the second droplet changing device are respectively connected to the central controller to receive a corresponding freeze/recovery control command;
  • the carrier mechanical action device includes a mechanical body and a carrier clamping action device and a droplet pick-up device disposed on the mechanical body; the droplet pick-up device is configured to adsorb and transfer target cell droplets according to a freeze/recovery control command Admitting/transferring target cell droplets from the freeze/recovery carrier to a specific location of the cryo/resuscitation carrier; the carrier gripping action device for freezing the target according to a freeze/recovery control command /Resuscitation carrier on the target cell droplet after completion of the droplet exchange operation Moving into the freezing medium container along with the freezing/recovery carrier to complete freezing/removing the frozen target cell droplets in the freezing medium container from the freezing medium container to the On the console
  • the second droplet changing device is configured to perform a droplet changing operation on a target cell droplet on the freezing/recovering carrier carried by the console according to a freezing/recovery control command.
  • the second liquid droplet changing device is identical in structure to the first liquid dropping device.
  • the vitrification/recovery mode device further includes an image acquisition device connected to the central controller; the image acquisition device is configured to acquire a cell droplet on the freezing/recovering carrier The cell image information is sent to the central controller for processing.
  • the carrier body of the freezing/recovering carrier is a sheet-like structure having an inner surface having a curved surface, and the inner surface is provided with a superhydrophobic surface functional region and a hydrophilic surface functional region, the hydrophilic The surface functional region is surrounded by the superhydrophobic surface functional region, and the hydrophilic surface functional region is provided with a droplet positioning identifier.
  • the superhydrophobic surface functional region is a circular annular region
  • the hydrophilic surface functional region is a circular region
  • the center point of the hydrophilic surface functional region coincides with the lowest point of the carrier body.
  • the transplantation mode device can realize the transplantation function by the vitrification/recovery mode device.
  • the main culture tank/sub-incubator is provided with an environmental management device for controlling the temperature, humidity, pressure, and gas partial pressure in the main culture tank/sub-incubator.
  • the main culture tank/sub-incubation tank is provided with a flow path communicating with the outside, through which the liquid droplets to be cultured sent from the outside are received or sent to the outside for cultivation. Cell droplets.
  • the embodiment of the invention also provides an automated method for in vitro fertilization and cleavage culture of egg cells, which comprises in vitro fertilization and cleavage culture of egg cells by using an automated device, including cumulus compound identification sorting mode, direct insemination mode, glass
  • an automated device including cumulus compound identification sorting mode, direct insemination mode, glass
  • the multiple modes of operation of the defrosting/resuscitation mode and the transfer mode operate on egg cell droplets.
  • the embodiment of the invention further provides a living cell culture carrier, wherein the culture carrier is provided with a plurality of pits for carrying droplets of living cells, and the surface of the pit is a superhydrophobic surface.
  • a closed annular restriction portion is provided on the surface of each of the pits on the surface of the culture carrier, and the surface of the restriction portion is an oleophilic surface.
  • the pit surface is a superhydrophobic surface layer in which a layer of superhydrophobic material is laid or the pit surface is a superhydrophobic surface subjected to superhydrophobic treatment.
  • the embodiment of the invention also discloses an egg cell automatic identification and sorting device, which comprises a negative pressure generator, an egg cell image acquisition device, a light source device, a follicular fluid collection container, a sorting switch, and an image acquisition with the negative pressure generator and the egg cell. a central controller to which the device and the sorting switch are connected;
  • the sorting switch includes a first valve port, a second valve port and a third valve port, the first valve port is connected to a main input conduit for inputting follicular stock solution with egg cells and for outputting egg cells to the culture carrier Between the main output conduits, the second valve port is connected between the main input conduit and a follicular fluid for outputting egg-removing cells to a secondary output conduit of the follicular fluid collection container, the third valve port being connected for Entering between a secondary input conduit of a particular culture fluid and the primary output conduit; the negative pressure generator being in communication with the follicular fluid collection container through a communication conduit;
  • the light source device is disposed on a side of the main input duct, illuminates the main input duct, and is imaged on the egg cell image acquiring device provided on the other side of the main input duct;
  • the follicular stock of the egg cell flows into the main input catheter when the negative pressure generator is started, and flows through the egg Obtaining egg cell image information by the egg cell image acquiring device when the cell image acquiring device, the central controller controlling the first valve port, the second valve port, and the third valve port of the sorting switch according to the egg cell image information Opening/closing, so that egg cells in the follicular stock solution with egg cells flow out from the first valve port, and follicular fluid from which the egg cells are removed flows out from the second valve port and flows into the follicular fluid collection container, and causes The egg cells flowing out of the first valve port and the specific culture liquid flowing in from the third valve port constitute an egg cell droplet and then flow out to the culture carrier.
  • the operation of the negative pressure generator is controlled by the central controller, thereby controlling the flow rate of the follicular stock solution with egg cells at the main input catheter.
  • the difference between the main input conduit position corresponding to the light source device and the egg cell image acquisition device and the catheter length of the main input catheter position where the first valve port is located is a preset value.
  • the central controller calculates the score according to the egg cell image information and the flow rate of the follicular stock solution with the egg cell at the main input conduit and the difference in the length of the catheter.
  • the main input conduit is a transparent conduit, and the diameter of the main input conduit is a fixed value.
  • the sorting switch is a three-way valve, including a valve body having the first valve port, the second valve port and the third valve port, and controlling the first valve port and the second A valve control device for opening/closing the valve port and the third valve port, the central controller being coupled to the valve control device.
  • the embodiment of the invention also discloses a liquid droplet changing operation device for cell culture, comprising a waste liquid extraction controller, a culture liquid supplement controller, a droplet discharge line and a droplet input line, the liquid Both ends of the drop output line and the droplet input line are connected to the droplet operation needle to suck/output the droplet; the waste liquid extraction controller passes the droplet on the culture carrier through the droplet output line The waste liquid is withdrawn and controls the flow rate of the extracted waste liquid, and the culture liquid supplementation controller delivers the culture liquid to the cell droplets on the culture carrier through the liquid droplet input line and controls the transported culture liquid. flow.
  • the liquid droplet changing operation device is placed in an environmentally controllable cell culture incubator.
  • the waste liquid extraction controller and the culture liquid supplement controller are a peristaltic pump or a stepping motor.
  • a cell image acquiring device connected to the central controller is further included for acquiring cell image information in a cell droplet on the culture carrier.
  • a culture liquid selection action device connected to the central controller, configured to receive an instruction of the central controller to move the liquid droplet input line to a container containing different culture liquids In order to obtain the corresponding culture solution.
  • the droplet operation needles connected to the two ends of the droplet output line and the droplet input line are both detachable droplet operation needles
  • the droplet liquid exchange operation device further includes liquid
  • the drip operation needle replacement action device is configured to automatically replace the droplet operation needles connected to the two end terminals of the droplet output line and the droplet input line.
  • a waste liquid collection container for collecting waste liquid in the cell droplets on the culture carrier drawn through the droplet discharge line is further included.
  • the embodiment of the invention further discloses a living cell vitrification/recovery carrier, wherein the carrier body is a transparent sheet-like structure having a curved inner surface, and the inner surface of the carrier body is provided with a superhydrophobic surface functional region and A hydrophilic surface functional region surrounded by the superhydrophobic surface functional region, the hydrophilic surface functional region being provided with a droplet positioning identifier.
  • the superhydrophobic surface functional region is a circular annular region
  • the hydrophilic surface functional region is a circular region
  • the center point of the hydrophilic surface functional region coincides with the lowest point of the carrier body.
  • the superhydrophobic surface functional region is a superhydrophobic surface layer on which an ultrahydrophobic material is laid on the inner surface of the carrier or the superhydrophobic surface functional region is inside the carrier.
  • the hydrophilic surface functional region is a hydrophilic surface layer on which the hydrophilic material is laid on the inner surface of the carrier or the hydrophilic surface functional region is a hydrophilic portion of the inner surface of the carrier.
  • the hydrophilic surface layer obtained after the treatment.
  • the embodiment of the invention further discloses an automatic operation device for vitrification/recovery of living cells, which comprises a central controller, a carrier mechanical action device, a droplet change operation device, a freezing/recovering carrier, and carrying the said a console for freezing/reviving the carrier and a freezing medium container, wherein
  • the carrier mechanical action device and the droplet change operation device are respectively connected to the central controller to receive a corresponding freeze/recovery control command;
  • the carrier mechanical action device includes a mechanical body and a carrier clamping action device and a droplet pick-up device disposed on the mechanical body; the droplet pick-up device is configured to adsorb and transfer target cell droplets according to a freeze/recovery control command Admitting/transferring target cell droplets from the freeze/recovery carrier to a specific location of the cryo/resuscitation carrier; the carrier gripping action device for freezing the target according to a freeze/recovery control command
  • the target cell droplets on the resuscitation carrier after completion of the droplet exchange operation are moved into the freezing medium container together with the freezing/recovery carrier to complete freezing/to freeze the target cell liquid in the freezing medium container Dropping along with the freeze/recovery carrier from the freezing medium container onto the console;
  • the liquid droplet changing operation device is configured to perform a liquid droplet changing operation on a target cell droplet on the freezing/recovering carrier carried by the operation table according to a freezing/recovery control instruction;
  • the freeze/recovery carrier is a viable cell vitrification/recovery carrier according to any one of claims 39 to 42; the specific position is on the hydrophilic surface functional region provided with the droplet positioning marker.
  • an image acquiring device and a display respectively connected to the central controller are further included;
  • the image acquiring device is configured to acquire cell image information in the cell droplets on the freezing/recovering carrier, and send the acquired cell image information to the central controller for processing, and display the image through the display.
  • the image capturing device may be integrated with the liquid droplet changing operation device.
  • the carrier mechanical action device is a movable carrier mechanical action device, and the carrier mechanical action device further includes a moving component connected to the mechanical body, and the carrier mechanical action device passes the movement The part can be moved on the moving rail.
  • the carrier holding action device includes a carrier holding member for holding the freezing/recovering carrier and a fixing bracket for fixing the carrier holding member to the mechanical body.
  • the droplet pick-up device includes a detachable droplet pick-up tube and a detachable vacuum suction device that communicates with the detachable droplet pick-up tube and controls its adsorption.
  • the freezing medium contained in the freezing medium container is liquid nitrogen.
  • the automatic operating system is placed in an environmentally controllable cell culture incubator.
  • the embodiment of the invention also discloses an automatic operation method for vitrification of living cells, comprising the steps of:
  • the target cell droplets are adsorbed and transferred to a specific position of the freezing carrier carried by the console;
  • the frozen carrier is a living cell vitrification/recovery carrier as described above; and the specific position is on the hydrophilic surface functional region provided with a droplet positioning identifier.
  • the embodiment of the invention also discloses an automatic operation method for vitrification of freeze-thaw cells, comprising the steps of:
  • the frozen carrier is a living cell vitrification/recovery carrier as described above; and the specific position is on the hydrophilic surface functional region provided with a droplet positioning identifier.
  • the automatic device for in vitro fertilization and cleavage culture of egg cells disclosed in the present invention simulates the environment of human egg cell insemination and early development of fertilized eggs, and can automatically acquire and culture droplets from the cumulus compound.
  • the apparent culture process is recorded throughout the whole process.
  • the parameters of the culture environment can also be recorded throughout.
  • the epigenetic culture process can be traced completely. After the embryo culture is completed, it can be automatically taken and automatically transplanted according to the transplant requirements.
  • the human can also perform an automatic cryopreservation operation on the target cells or perform an automatic resuscitation operation on the frozen-thawed embryos to complete the workflow.
  • Figure 1 is a block diagram showing the structure of an automated apparatus for in vitro fertilization and cleavage culture of egg cells in an embodiment of the present invention.
  • FIG. 2 is a schematic view showing the structure of a living cell culture incubator in an embodiment of the present invention.
  • FIG. 3 is a schematic view showing the structure of an automatic egg cell sorting and sorting apparatus according to an embodiment of the present invention.
  • Fig. 4 is a block diagram showing the structure of a sorting switch of the automatic egg cell sorting and sorting apparatus shown in Fig. 3.
  • Fig. 5 is a top plan view showing the structure of a living cell culture carrier in an embodiment of the present invention.
  • Figure 6 is a schematic cross-sectional view showing the structure of a living cell culture carrier in an embodiment of the present invention.
  • Fig. 7 is a schematic enlarged view showing the structure of a living cell culture carrier in the embodiment of the present invention.
  • FIG. 8 is a schematic structural diagram of a device for direct insemination mode according to an embodiment of the present invention.
  • Figure 9 is a block diagram showing the structure of a vitrification/recovery mode device in an embodiment of the present invention.
  • Figure 10 is a schematic view showing the structure of a freezing/recovering carrier in the embodiment of the present invention.
  • Figure 11 is a schematic view showing the structure of a console of a vitrification/recovery mode device in an embodiment of the present invention.
  • Figure 12 is a schematic view showing the structure of a carrier mechanical action device of a vitrification/recovery mode device according to an embodiment of the present invention.
  • Figure 13 is a schematic view showing the structure of a second liquid droplet changing device of a vitrification/recovery mode device according to an embodiment of the present invention.
  • Figure 14 is a flow chart showing an automatic operation method of vitrification of living cells in the embodiment of the present invention.
  • Figure 15 is a flow chart showing an automatic operation method for vitrification and resuscitation of living cells in an embodiment of the present invention.
  • an automatic device for in vitro fertilization and cleavage culture of an egg cell comprises an automatic combined partition combination incubator 100, a central controller 9 placed outside the incubator, and automatic identification of egg cells.
  • Sorting device 200 wherein:
  • the automatic combined partition combination incubator 100 includes a main incubator 101, a sub-incubator 102, and an automatic airtight for achieving communication/separation between the main incubator 101 and the sub-culture tank 102.
  • Door 103 The main culture chamber 101 / the auxiliary culture chamber 102 is provided with a culture carrier 1 for carrying egg cell droplets; the main culture chamber/sub-incubator is respectively provided for fertilization and cleavage of egg cell droplets Operating mode devices at different stages of the cultivation process.
  • the inside/outside of the main incubator 101/the auxiliary incubator 102 is configured to drive the culture carrier 1 to and from the main incubator 101 and the sub-incubator when the automatic airtight door 103 is opened.
  • a culture carrier driving device 105 between 102;
  • the egg cell automatic identification sorting device 200, the operation mode device of different stages, the automatic airtight door 103 and the culture carrier driving device 105 are all connected with the central controller to receive corresponding control commands;
  • the egg cell automatic identification sorting device 200 recognizes and sorts the follicular stock solution with the egg cell (cumulus cell complex) according to the received control instruction, and outputs the egg cell droplet to the automatic combined partition combined incubator a culture carrier in 100; when the automatic airtight door is opened according to a control command, the culture carrier driving device 105 drives the culture carrier 1 to and from the device according to a control command
  • the main culture chamber 101 and the sub-incubator 102 are interposed so that the egg cell droplets on the culture carrier 1 enter the main culture cabinet/auxiliary chamber to perform a corresponding operation mode.
  • the automatic combined-type partition combination incubator 100 includes a main incubator 101 and a sub-incubator 102, and an automatic airtight door 103 is disposed between the main incubator 101 and the sub-incubator 102. To achieve communication / separation inside the cabinet.
  • the living cell incubator 100 further includes an automatic airtight door driving device 108 that controls opening/closing of the automatic airtight door 103.
  • the automatic airtight door driving device 108 may be disposed inside or outside the incubator body 101/the auxiliary incubator body 102, and the opening of the automatic airtight door 103 may be controlled by contact/non-contact with the automatic airtight door 103/ shut down.
  • a culture carrier 1 for carrying egg cell droplets is provided in the main culture chamber 101 / sub-incubator 102.
  • the automatic combined partition combination incubator 100 is further provided to drive the culture carrier 1 to and from the main culture cabinet 101 and the auxiliary culture cabinet 102 when the automatic airtight door 103 is opened.
  • the carrier driving device 105 is cultured.
  • the culture carrier driving device 105 may be provided inside or outside the main incubator 101/sub-incubator 102, and the culture carrier 1 may be moved by contact/non-contact with the culture carrier 1.
  • the main culture tank 101 / the sub-culture tank 102 are respectively provided with operation mode means for performing different stages in the process of fertilization and cultivation of the egg cell droplets (supported on the culture carrier 1).
  • the mode of operation includes, but is not limited to, a direct insemination mode device 300, a freeze/recovery mode device 400, and a transplant mode device (not shown).
  • a direct insemination mode device 300 is provided inside the main incubator 101, and a freezing mode device, a freeze-thaw cell resuscitation mode device, and a transplant mode device are provided in the sub-incubator 102.
  • the auxiliary culture chamber 102 (carryed on the culture carrier 1)
  • the culture carrier 1 is moved from the sub-incubator 102 to the inside of the main incubator 101, and the eggs on the culture carrier 1 are passed through the direct insemination mode device 300 inside the main incubator 101. Cell droplets are subjected to fertilization mode operation.
  • the automatic airtight door 103 is controlled to be opened by the central controller 9, and the culture carrier 1 is moved from the main culture chamber 101 to the secondary culture.
  • a corresponding mode operation is performed by a transplant mode device or a freeze/recovery mode device inside the sub-incubator 102.
  • the main culture tank 101 / the sub-culture tank 102 is provided with a flow path 106 that communicates with the outside (in the present embodiment, the flow path 106 is shown on the sub-culture tank 102, but it is understood that the flow
  • the road 106 may also be provided with a main incubator 101 through which the egg cells to be cultured (droplets) fed from the outside or the droplets of the egg cells after the culture are sent to the outside.
  • the egg cell automatic identification sorting device 200 is provided, and the egg cell automatic identification sorting device 200 is used for identifying and acquiring cumulus cells, and adding specific culture to the cumulus cells.
  • the droplets containing the cumulus cells are sent to the culture carrier 1 inside the auxiliary culture chamber 102 through the flow path 106, and then passed through the internal device of the main culture chamber 101/the auxiliary culture chamber 102 to the culture carrier.
  • the egg cell droplets on 1 are subjected to subsequent direct insemination mode, freezing mode, freeze-thaw cell recovery mode, and transplantation mode.
  • Environmental management devices 122a, 122b for controlling the temperature, humidity, pressure, and gas partial pressure in the main culture tank 101 / sub-culture tank 102 are respectively provided in the main culture tank 101 / the sub-culture tank 102. .
  • the environment inside the main incubator 101/auxiliary tank 102 is monitored and controlled by the environmental management devices 122a, 122b, thereby ensuring stability and continuity of operation of each mode.
  • the culture carrier driving device 105, the automatic airtight door driving device 108, the environmental management devices 122a, 122b, and the respective operation mode devices described above are all electrically connected to the central controller 9 to realize one-way/two-way communication, thereby acquiring The information is sent to the central controller 9 for processing, and the control command of the central controller 9 is received to operate accordingly.
  • the automatic device for in vitro fertilization and cleavage culture of the egg cell of the present embodiment is further provided with a display 10 connected to the central controller 9 outside the main culture cabinet 101 / the auxiliary incubator 102.
  • the central controller 109 processes the received mode state image information transmitted by each device, for example, the image information of each marked position is sequentially stored as an image set of a specific location, and is sent to the display 10 for display.
  • the operator can access any of the marker positions through the display 10 at any time.
  • the set of images is set to determine the developmental state of the egg cells and record.
  • the central controller 9 can also be shared with the hospital's integrated information management system, the surgeon can access it at any time, and the patient can also receive it through the public information platform.
  • the automatic combined-type partition combination incubator 100 disclosed in the embodiment realizes the communication/separation inside the box by providing the automatic airtight door 103 between the main incubator body 101 and the auxiliary incubator body 102, and the main The culture chamber 101 / the auxiliary incubator 102 are respectively provided with operation mode means for fertilizing and culturing the egg cell droplets at different stages, and then by the culture carrier driving device 105 when the automatic airtight door is opened Driving the culture carrier to and from the main culture chamber 101 and the auxiliary culture chamber 102, so that the egg cell droplets on the culture carrier enter the main culture cabinet/auxiliary chamber to perform corresponding operation modes. .
  • the entire culture process is carried out in the incubator, and by controlling the cell culture environment between the main culture tank and the auxiliary culture tank, the problems of cell loss and damage can be effectively avoided.
  • the entire operation process can be effectively monitored, which is advantageous for the operation.
  • the egg cell automatic identification sorting device 200 includes a negative pressure generator 201, an egg cell image acquiring device 202, a light source device 203, a follicular fluid collecting container 204, a sorting switch 205, and the negative pressure generator 201 and the egg cell image acquiring device 202.
  • a central controller 9 connected to the sorting switch 205, wherein the negative pressure generator 201, the follicular fluid collecting container 204, and a conduit for sucking and transporting the follicular fluid with egg cells are connected to the negative pressure generator 201.
  • a negative pressure is created on the tube to complete the puncture and aspiration of the follicular stock with egg cells and into the catheter.
  • the egg cell image acquiring device 202 and the light source device 203 are disposed on both sides of the conveying path of the catheter to cooperate with the image information of the egg cell, and send the image information of the obtained egg cell to the central controller 9, and the central controller 9 according to the egg cell
  • the image information controls the opening/closing of the sorting switch 205 provided on the catheter, thereby separating the egg cells and the follicular fluid in the follicular stock solution with the egg cells, thereby obtaining the desired egg cells.
  • the sorting switch 205 includes a first valve port 205a, a second valve port 205b, and a third valve port 205c, wherein:
  • the first valve port 205a is connected between the main input conduit 210 for inputting the follicular stock solution 20 with the egg cells 21 and the main output conduit 211 for outputting the egg cells 21 to the culture carrier 1. That is, communication/isolation is achieved between the main input duct 210 and the main output duct 211 by opening/closing of the first valve port 205a of the sorting switch 205.
  • the second valve port 205b connects the main input conduit 210 and the follicular fluid 22 for outputting the egg cells 21 to the sub-output conduit 212 of the follicular fluid collection container 204. That is, communication/isolation is achieved between the main input duct 210 and the sub-output duct 212 by opening/closing of the second valve port 205b of the sorting switch 205.
  • the third valve port 205c is connected between the auxiliary input conduit 213 for inputting the specific culture liquid 23 and the main output conduit 211; that is, the sub-input conduit 213 and the main output conduit 211 pass the sub-portion
  • the opening/closing of the switch 205 is selected to achieve communication/isolation.
  • the sub-input conduit 213 is provided with a micro-flow pump 207 for controlling the flow rate of the specific culture solution 23 pumped out and flowing into the third valve port 205c.
  • the microfluidic pump 207 is coupled to the central controller 9 to receive control commands.
  • the sorting switch 205 can be implemented by a three-way valve including the first valve port 205a, the second valve port 205b, and a third valve. a valve body 2051 of the port 205c and a valve control device 2052 for controlling opening/closing of the first valve port 205a, the second valve port 205b and the third valve port 205c, the central controller 9 and the valve control device 2052 connection.
  • the valve control device 2052 alternately controls opening/closing of the first valve port 205a, the second valve port 205b, and the third valve port 205c in accordance with an instruction sent from the central controller 9.
  • the negative pressure generator 201 communicates with the follicular fluid collection container 204 through the communication conduit 214, thereby achieving communication with the secondary output conduit 212, and further achieving the opening of the second valve opening 205b.
  • the main input conduit 210 is connected.
  • the operation of the vacuum generator 201 is controlled by the central controller 9, so that the follicular stock solution 20 with the egg cells 21 can be controlled at the main input conduit 210.
  • the flow rate V is controlled by the central controller 9, so that the follicular stock solution 20 with the egg cells 21 can be controlled at the main input conduit 210.
  • the light source device 203 is disposed on a transport path of the main input duct 210 and located on a side outside the main input duct 210, and the egg cell image acquiring device 202 is disposed outside the main input duct The other side is facing the light source device 203.
  • the light source device 20 illuminates the inside of the main input duct 210 and is imaged on the egg cell image acquiring device 202.
  • the light source device 203 is a fiber optic cold light source device
  • the egg cell image acquisition device 202 is a CCD.
  • the image of the cell imaged by the CCD is sent to the central controller 9 for image processing to obtain a more perfect image.
  • the main input conduit 210 is a transparent conduit, and the diameter D of the main input conduit 210 is a fixed value.
  • the device is connected to a puncture needle or the like at the beginning of the main input catheter 210 to perform puncture and aspiration of egg cells.
  • the difference between the main input conduit position corresponding to the light source device 203 and the egg cell image acquisition device 202 and the catheter length difference H at the position of the main input conduit 210 where the first valve port 205a is located is a preset value.
  • the central controller 202 acquires the egg cell image information of the device 202 according to the egg cell image, and combines the flow velocity V of the follicular stock solution 20 with the egg cell 21 at the main input conduit 210 and the catheter length difference H. Calculating an opening/closing time for controlling the first valve port 205a, the second valve port 205b, and the third valve port 205c of the sorting switch 205, thereby causing the egg cells 21 in the follicular stock solution 20 with the egg cells 21 to be
  • the first valve port 205a flows out, and the follicular fluid 22 from which the egg cells 21 are removed flows out from the second valve port 205b and flows into the follicular fluid collection container 204, and the egg cells 21 flowing out from the first valve port 205a are
  • the egg cell droplets 24 are formed from the specific culture solution 23 flowing in from the third valve port 205c, and then flowed out to the culture carrier 1.
  • the second valve port 205b of the sorting switch 205 is controlled to be turned on by the central controller 9 (at this time, the first valve port 205a and the third valve port 205c are closed), and the negative pressure generator 201 is controlled to be started.
  • the follicular stock solution 20 with the egg cells 21 flows into the main input conduit 210 under the action of a negative pressure.
  • the egg cell image acquiring device 202 is coordinated by the light source device 203 to acquire egg cell image information. .
  • the egg cell image acquisition device 202 transmits the acquired egg cell image information to the central controller 9, the central control
  • the device 9 calculates the follicle with the egg cell 21 according to the egg cell image information and combined with the flow velocity V of the follicular stock solution 20 with the egg cell 21 at the main input catheter 210 and the catheter length difference H.
  • the central controller 9 immediately controls the sorting switch 205 Opening the first valve port 205a (when the second valve port 205b and the third valve port 205c are closed), so that the egg cells 21 in the follicular stock solution 20 with the egg cells 21 flow out from the first valve port 205a and enter the place
  • the main output conduit 211 is described.
  • the central controller 9 immediately controls the sorting switch 205 to open the third valve port 205c (at this time, the first The valve port 205a and the second valve port 205b are closed, and the specific culture solution 23 flowing from the sub-input conduit 213 and the egg cell 21 constitute an egg cell droplet 24, and then flow out through the main output conduit 211 to the culture carrier 1.
  • the central controller 9 immediately controls the sorting switch 205 to open the second valve port 205b (at this time, the first valve) The mouth 205a and the third valve port 205c are closed), and the follicular fluid 22 from which the egg cells 21 are removed flows out from the second valve port 205b and flows into the follicular fluid collection container 204.
  • the automatic egg cell sorting and sorting apparatus 200 disclosed in the embodiment can be used for automatically identifying and sorting out ovary cell complexes, egg cells and early fertilized eggs for automatic identification and sorting, thereby improving the accuracy of cell identification and sorting. Reduce operating costs.
  • the cells sorted by the automatic egg cell sorting and sorting apparatus disclosed in the present invention are more suitable for in vitro fertilization and culture.
  • the culture carrier 1 includes a carrier body 11 which is a block structure, preferably square or rectangular.
  • the surface 111 of the carrier body 11 is recessed downwardly with a plurality of dimples 12 for carrying egg cell droplets.
  • the surface 111 of the carrier body 11 and the surface 121 of each of the pits 12 are superhydrophobic surfaces.
  • the surface 111 of the carrier body 11 and the surface 121 of each of the pits 12 are superhydrophobic surfaces on which a layer of superhydrophobic material is laid, or the surface 111 of the carrier body 11 and each of the recesses
  • the surface 121 of the pit 12 is a superhydrophobic surface that has been subjected to superhydrophobic treatment.
  • a closed annular restriction portion 13 is provided on the surface 111 of the carrier body 11 at the edge of each of the pockets 12, and the surface of the restriction portion 13 is an oleophilic surface.
  • the closed annular projection restricting portion 13 having lipophilic surface characteristics can restrict the overflow of the culture oil other than the egg cell droplets, and ensure that the egg cell droplets are always in an independent culture environment.
  • a cell droplet (aqueous solution) 502 containing cells 501 is superhydrophobic surface on the pit surface 121.
  • the upper surface is spherical, and the contact area with the pit surface 121 is small, and does not adhere to the surface thereof (equivalent to a floating state), and can be accurately and accurately positioned to the pit surface 121 in the gravity environment (and is the pit surface 121). The lowest point).
  • a closed annular restriction portion 13 is provided at an edge of each of the dimples 12, and the surface of the restriction portion 13 is an oleophilic surface, and the culture oil 503 other than the cell droplet (aqueous solution) 502 can be restricted.
  • the cell droplets 502 for covering the pit surface 121 and the pit surface 121 are overflowed) to ensure that the cell droplets (aqueous solution) 502 are always in an independent culture environment.
  • the surface 121 of each of the dimples 12 is a curved surface, preferably a semi-spherical surface.
  • the size of each of the dimples 12 is uniform, and the depth and width of the dimples 12 are set differently according to experimental needs.
  • the plurality of pits 12 on the carrier body 11 are arranged in a single line on the surface 111 of the carrier body 11, so that the carrier 1 is a single-row multi-cell culture carrier.
  • the culture carrier 1 used in the present embodiment has a plurality of pits 12 for carrying egg cell droplets on the surface of the carrier body, and the carrier surface 111 and the pit surface 121 are super-hydrophobic treated super a hydrophobic surface, such that when fertilized and cultured on such a culture carrier, since the cell droplets (aqueous solution) are spherical on the superhydrophobic surface of the culture carrier, the contact area with the surface is small, and there is no adhesion to the surface on.
  • the edge of the pit on the surface of the carrier is provided with a closed annular restriction portion, and the restriction portion has an oleophilic surface characteristic, it is possible to limit the overflow of the culture oil other than the cell droplet, and to ensure that the cell droplet is always independent. Cultivate the environment. Therefore, the egg cell droplets (aqueous solution) can be automatically and accurately positioned in the desired environment (fertilization and culture) in a gravity environment, thereby facilitating fertilization and culture operations.
  • the culture carrier 1 provided in the present embodiment can be used as a culture carrier for any living cells, in addition to the egg cell culture vector of the embodiment of the present invention.
  • FIG. 8 is a schematic structural diagram of a direct insemination mode apparatus 300 according to an embodiment of the present invention.
  • the direct insemination mode device 300 includes a first liquid droplet changing operation device, and the first liquid droplet changing operation device includes a waste liquid extraction controller 301, a culture liquid supplement controller 302, and a droplet output tube.
  • the path 303 and the droplet input line 304 and the central controller 9, the both ends of the droplet output line 303 and the droplet input line 304 are connected to the droplet operation needle 305 to suck/output droplets.
  • the waste liquid extraction controller 301 is in communication with the liquid droplet output line 303, and whether to control the cell liquid on the culture carrier 1 by controlling the opening/closing of the liquid droplet output line 303 and controlling the size of the opening.
  • the waste liquid in the drop 24 is withdrawn and the flow rate of the extracted waste liquid is controlled.
  • the culture solution supplement controller 302 is in communication with the droplet input line 304, and realizes whether or not the culture solution (sperm suspension) is realized by the opening/closing of the droplet input line 304 and the size of the control opening. Transfer to the cell droplets on the culture carrier and control the flow of the delivered culture fluid (sperm suspension).
  • the central controller 9 is connected to the waste liquid extraction controller 301 and the culture liquid supplement controller 302, respectively, to control the waste liquid extraction controller 301 and the culture fluid supplement controller 302 to operate. Specifically, the central controller 9 controls the waste liquid extraction controller 301 to control the opening/closing of the liquid droplet output line 303 and the size of the control opening to realize whether the egg cell on the culture carrier 1 will be cultured.
  • the waste liquid in the liquid droplet is withdrawn and the extracted waste liquid flow rate is controlled, and by controlling the culture liquid supplement controller 302 to control the opening/closing of the liquid droplet input line 304 and the size of the control opening to realize whether or not
  • the culture solution (sperm suspension) is delivered to the droplets of egg cells on the culture carrier and the flow rate of the delivered culture solution (sperm suspension) is controlled.
  • the waste liquid extraction controller 301 and the culture liquid supplement controller 302 are a peristaltic pump or a stepping motor.
  • the direct insemination mode device 300 further includes a cell image acquisition device 308 connected to the central controller 9.
  • the cell image acquisition device 308 is configured to acquire cell image information in the cell droplets 24 on the culture carrier 1 and transmit the acquired cell image information to the central control 9.
  • the central controller 9 processes the cell image information sent by the cell image acquisition device 308 (for example, sequentially stores image information of each mark position acquired by the cell image acquisition device 308 as an image set of a specific location). To obtain clearer cell image information, and to display the processed cell image information through the display 10.
  • the cell image information of the operator is used to determine whether the cell droplet 24 needs to perform a liquid exchange operation, so that the waste liquid extraction controller 301 and the culture fluid supplement controller 302 are controlled by the central controller 9 to perform corresponding work. .
  • the cell image acquisition device 308 includes a fiber optic cold light source device and a CCD that are coupled to each other, and the fiber optic cold light source device is disposed on the culture carrier 1 to illuminate the cell droplets 24 on the culture carrier 1 Imaged on a CCD provided on the other side of the culture carrier 1 and facing the cold light source of the optical fiber.
  • the image of the cell imaged by the CCD is sent to the central controller 9 for image processing to obtain a more perfect image.
  • the droplet operation needles 305 connected to the both ends of the droplet output line 303 and the droplet input line 304 are detachable droplets.
  • the needle is operated, and therefore, the first liquid droplet changing operation device of the present embodiment further includes a droplet operation needle replacement action device 306.
  • the droplet operation needle replacement action device 306 is coupled to the central controller 9 to receive a control command.
  • the droplet operation needle replacement action device 306 is for automatically replacing the droplet operation needles connected to the two terminals of the droplet output line 301 and the droplet input line 302.
  • the central controller 9 sends a control command to the droplet operation needle replacement action device 306 before each time the liquid droplet 24 on the culture carrier 1 needs to be subjected to a liquid exchange operation or after the liquid exchange operation is completed, indicating the The droplet operation needle replacement action device 306 performs droplet operation needle 305 replacement for the corresponding droplet output line 303 and/or droplet input line 304.
  • the first liquid droplet changing operation device of the embodiment further includes a culture liquid selection action device 307 connected to the central controller 9.
  • the culture solution selection action device 307 is configured to receive an instruction from the central controller 9 to move the droplet input line 304 into a container 310 containing a different culture solution to obtain a corresponding culture solution (sperm suspension).
  • This embodiment further includes a waste liquid collection container 311 for collecting the waste liquid in the egg cell droplets 24 on the culture carrier 1 drawn through the droplet discharge line 303.
  • the cell image acquiring device 308 can be integrated with the first liquid droplet changing operation device.
  • the direct insemination mode device 300 of the embodiment adopts the above first liquid droplet changing operation device to realize the egg cell fertilization mode, and has the following effects: 1.
  • the automatic cell culture liquid can be continuously replaced during the process of fertilization of the egg cells, and the cell culture liquid (sperm)
  • the flow rate of the suspension can be adjusted according to the experimental requirements; it can also be automatically changed intermittently; 2.
  • the liquid exchange process is fully automated, no manual operation is involved, and the liquid exchange volume control precision is high; 3.
  • the droplet can be completely intact without residual transfer culture method
  • the device capable of directly connecting the additive to manufacture the artificial multi-cell type complex living tissue as a precursor process device for incubating the artificial living tissue in vitro; 4.
  • the first liquid droplet changing operation device used in the embodiment can not only continuously change the automatic cell culture liquid during the process of fertilization and culture of the egg cells, but also can be used for the liquid droplet exchange operation of various living cell cultures. .
  • the freezing/reviving mode device 400 includes a central controller 9, a carrier mechanical action device 4, a second liquid droplet changing device 3, a freezing/recovering carrier 6, a console 7 carrying the freezing/recovering carrier, and a freezing medium container 8. . among them:
  • the freezing/recovering carrier 6 is configured to carry a frozen target cell (fertilized egg cell droplet) for performing a freezing operation, the inner surface of the body has a curved sheet-like structure, and the inner surface of the carrier body is provided with superhydrophobic A surface functional region and a hydrophilic surface functional region are surrounded by the superhydrophobic surface functional region, and the hydrophilic surface functional region is provided with a droplet positioning identifier.
  • the frozen/revived carrier 6 of such a structure is more advantageous for the transfer and localization of egg cell droplets, thereby facilitating the freezing/recovering operation of the egg cell droplets.
  • the specific structure of the carrier will be described in detail later with reference to FIG. among them,
  • the carrier mechanical action device 4 and the second liquid droplet changing operation device 3 are respectively connected to the central controller 9 to receive a corresponding freeze/recovery control command.
  • the carrier mechanical action device 4 includes a mechanical body 401 and a mechanical body 401 disposed on the mechanical body 401 The carrier holding action device 41 and the droplet pick-up device 42 (Fig. 12).
  • the droplet pick-up device 42 is configured to adsorb and transfer a target cell droplet to a specific position of the freeze/recovery carrier 6 according to a freeze/recovery control command/drop the target cell droplet from the freeze/recovery carrier 6 Adsorbed and transferred out.
  • the carrier clamping action device 41 is configured to move the target cell droplets on the freeze/recovery carrier after the completed droplet exchange operation together with the freeze/recovery carrier 6 according to the freeze/recovery control command.
  • the target cell droplets in the freezing medium container 8 to complete the freezing/complete freezing in the freezing medium container 8 are removed from the freezing medium container 8 to the operation table 7 together with the freezing/recovery carrier 6.
  • the second liquid droplet changing operation device 3 is configured to perform a liquid droplet changing operation on a target cell droplet on the freezing/recovery carrier 6 carried on the operation table 7 according to a freezing/recovery control command.
  • the living cell vitrification/recovery carrier 6 of the present embodiment is as shown in Fig. 10.
  • the body 60 of the freeze/recovery carrier 6 has a sheet-like structure in which the inner surface has a curved surface.
  • the inner surface of the body 60 of the freeze/recovery carrier 6 is provided with a superhydrophobic surface functional region 61 and a hydrophilic surface functional region 62 surrounded by the superhydrophobic surface functional region 61, and
  • a droplet location indicator 600 is provided on the hydrophilic surface functional region 62 (to aid in the positioning of cell droplets).
  • the hydrophilic surface functional region 62 is a circular region.
  • the superhydrophobic surface functional region 61 is an annular region surrounding the hydrophilic surface functional region 62.
  • the center point of the hydrophilic surface functional region 62 coincides with the lowest point of the carrier body 60. Thereby it is more conducive to the positioning of cell droplets.
  • the superhydrophobic surface functional region 61 is a superhydrophobic surface layer having a superhydrophobic material laid on the inner surface of the carrier or the superhydrophobic surface functional region is a partial region of the inner surface of the carrier A superhydrophobic surface layer obtained after superhydrophobic treatment.
  • the hydrophilic surface functional region 62 is a hydrophilic surface layer on which the hydrophilic material is laid on the inner surface of the carrier or the hydrophilic surface functional region is a portion of the inner surface of the carrier. Area The hydrophilic surface layer obtained after the hydrophilic treatment of the domain.
  • the vitrification/recovery carrier 6 of the present embodiment When used as a carrier cell droplet for performing a cell vitrification/recovery operation, the hydrophilic surface functional region 62 and the superhydrophobic surface functional region 61 can be combined in a predetermined capacity.
  • the droplets are automatically adsorbed and fixed in the hydrophilic surface functional region 62.
  • the carrier of the regular arcuate structure Under the gravity environment, the carrier of the regular arcuate structure enables the cells contained in the droplet to be automatically positioned on the intersection of the curved surface and the stage.
  • the body surface of the freezing/recovery carrier 6 is a curved surface, and the lowest point of the curved surface coincides with the center point of the hydrophilic surface functional region 62 for locating the position of the cell droplet, the automatic positioning of the cell droplet is facilitated.
  • the region surrounding the hydrophilic surface functional region 62 is the superhydrophobic surface functional region 61, the cell droplets do not adhere to the surface of the superhydrophobic surface functional region 61, thereby further facilitating the positioning of the cell droplets to the droplets. Position the identified hydrophilic surface functional area 62.
  • FIG 11 is a block diagram showing the operation of a console 7 of a vitrification/recovery mode device 400 in accordance with an embodiment of the present invention.
  • the console 7 is a platform with a flat surface and a transparent operating area 71 on the surface.
  • the transparent operating area 71 is used to carry the freeze/recovery carrier 6 described above.
  • the console 7 of the present embodiment is preferably a movable console 7, which realizes the movement of the entire console 7 by providing a connection moving member.
  • the moving part connected to the console 7 is connected to the central controller 9, and is controlled by the central controller 9 to realize the movement of the console 7, so that the console 7 can be controlled to move to a specific position for cell freezing according to the instruction of the central controller 9. / Resuscitation of the liquid exchange operation.
  • FIG 12 is a block diagram showing the structure of the carrier mechanical action device 4 of a freeze/recovery mode device 400 in accordance with an embodiment of the present invention.
  • the carrier mechanical action device 4 specifically includes a mechanical body 401 and a carrier clamping action device 41 and a droplet pick-up device 42 disposed on the mechanical body 401.
  • the droplet pick-up device 42 includes a detachable droplet pick-up tube 421 and a detachable vacuum suction device 422 that communicates with the detachable droplet pick-up tube 421 and controls its adsorption.
  • the droplet pick-up device 42 is fixed to the mechanical body 401 by the detachable vacuum suction device 422.
  • the detachable vacuum suction device 422 correspondingly controls the detachable droplet pick-up tube 421 to adsorb target cell droplets and transfer to a specific position of the freeze/recovery carrier 6 according to the received freeze/recovery control command. / absorbing and transferring the target cell droplets from the freezing/recovering carrier 6 (for example, transferring to the culture carrier 1 Or in other containers).
  • the carrier holding action device 41 includes a carrier holding member 411 for holding a freezing/recovering carrier and a fixing bracket 412 for fixing the carrier holding member 411 to the mechanical body 401.
  • the carrier holding member 411 clamps and moves the target cell droplets on the freezing/recovery carrier 6 after the completed droplet changing operation together with the freezing/recovery carrier 6 according to the freezing/recovery control instruction.
  • the target cell droplets in the freezing medium container 8 to complete freezing/complete freezing in the freezing medium container 8 are taken out from the freezing medium container 8 together with the freezing/recovery carrier 6 and moved onto the operation table 7.
  • the carrier mechanical action device 4 of the present embodiment is a movable carrier mechanical action device 4, and the carrier mechanical action device 4 further includes a moving member 43 coupled to the mechanical body, the carrier mechanical action device 4 is movable on the moving rail 404 by the moving member 43.
  • the moving member 43 connected to the carrier mechanical action device 4 is connected to the central controller 9, and is controlled by the central controller 9 to realize the movement of the carrier mechanical action device 4, so that the carrier mechanical action device 4 can be controlled according to the command of the central controller 9. Operation to a specific location to transfer cell droplets or cryo/recovery carrier 6 is performed.
  • FIG. 13 is a schematic view showing the structure of a second liquid droplet changing operation device 3 of a freezing/restoring mode device in an embodiment of the present invention.
  • the second liquid droplet changing operation device 3 basically conforms to the structure of the first liquid droplet changing device shown in FIG. 8, and includes a waste liquid extraction controller 301, a culture liquid supplement controller 302, and a droplet output line 303.
  • the droplet input line 304, both ends of the droplet output line 303 and the droplet input line 304 are connected to the droplet operation needle 305 to suck/output droplets.
  • the waste liquid extraction controller 301 is in communication with the liquid droplet output line 303, and realizes whether the freeze/recovery carrier 6 is to be controlled by controlling the opening/closing of the liquid droplet output line 303 and the size of the control opening.
  • the waste liquid in the cell droplets is withdrawn and the extracted waste liquid flow is controlled.
  • the culture fluid supplement controller 302 is in communication with the droplet input line 304, and whether the culture fluid is delivered to the liquid through the opening/closing of the droplet input line 304 and the size of the control opening.
  • the egg cell droplets on the carrier 6 are frozen/revived and the delivered culture fluid flow is controlled.
  • the central controller 9 is respectively connected to the waste liquid extraction controller 301 and the culture liquid supplement controller 302 to control the waste liquid extraction controller 301 and the culture fluid supplement controller 302. Work. Specifically, the central controller 9 controls the waste liquid extraction controller 301 to control the opening/closing of the liquid droplet output line 303 and the size of the control opening to realize whether the freezing/recovery carrier 6 is to be The waste liquid in the cell droplets is withdrawn and the extracted waste liquid flow is controlled, and the opening/closing of the liquid droplet input line 304 and the size of the control opening are controlled by controlling the culture liquid supplement controller 302. Whether or not the culture solution is delivered to the cell droplets on the culture carrier and the delivered culture fluid flow rate is controlled.
  • the waste liquid extraction controller 301 and the culture liquid supplement controller 302 are a peristaltic pump or a stepping motor.
  • the droplet operation needle 305 connected to the both ends of the droplet output line 303 and the droplet input line 304 is a detachable liquid.
  • the operation needle is dripped, and therefore, the second liquid droplet changing operation device of the present embodiment further includes a droplet operation needle replacement operation device 306.
  • the droplet operation needle replacement action device 306 is coupled to the central controller 9 to receive a control command.
  • the droplet operation needle replacement action device 306 is for automatically replacing the droplet operation needles connected to the two terminals of the droplet output line 301 and the droplet input line 302.
  • the central controller 9 sends a control command to the droplet operation needle replacement action device 306, each time before the liquid exchange operation on the cell droplets on the freezing/recovery carrier 6 is required or after the liquid exchange operation is completed.
  • the droplet operation needle replacement action device 306 performs droplet operation needle 305 replacement for the corresponding droplet output line 303 and/or droplet input line 304.
  • the second liquid droplet changing operation device 3 of the present embodiment further includes a culture liquid selection action device 307 connected to the central controller 9.
  • the culture fluid selection action device 307 is configured to receive an instruction from the central controller 9 to move the droplet input line 304 into a container 310 containing different culture fluids to obtain a corresponding freeze/recovery liquid (ie, cryoprotection) Agent / resuscitation liquid).
  • the second liquid droplet changing operation device of the present embodiment further includes a waste liquid collecting container 311 for collecting the waste liquid in the egg cell droplets on the freezing/recovering carrier 6 drawn through the liquid droplet output line 303.
  • the freezing medium contained in the freezing medium container 8 of the present embodiment is liquid nitrogen.
  • the autonomic cell vitrification/recovery automatic operation device of the present embodiment further includes an image acquisition device 308b and a display 10 respectively connected to the central controller 9.
  • Image acquisition device 308b is used to acquire egg cell image information in the cell droplets on the freeze/recovery carrier 6, and send the acquired cell image information to the central controller 9 for processing, and display through the display 10. Therefore, the droplet position identification on the hydrophilic surface functional region 62 of the freezing/recovery carrier 6 can be identified by the image information acquired by the image acquisition device 308b, thereby locating the position at which the egg cell droplets are placed.
  • the image acquiring device 308b is configured by the optical fiber cold light source device and the cell image acquiring device CCD.
  • the optical fiber cold light source device is disposed directly above the operation table 7, and faces the egg cell droplet on the freezing/recovery carrier 6 on the transparent operation region 71 of the operation table 7, and the cell image acquisition device CCD is disposed in the operation Directly below the stage 7, and facing the fiber optic cold light source device.
  • the light source device 20 illuminates the cell droplets on the freezing/recovery carrier 6 on the transparent operation region 71 of the console 7, and is imaged on the cell image acquisition device CCD.
  • the image acquiring device 308b of the present embodiment can also be integrated into the second liquid droplet changing operation device 3, and has a unitary structure with the second liquid droplet changing operation device 3.
  • the environmental management device built in the living cell incubator When a plurality of control commands for entering the cell freezing mode are issued by the central controller 9, the environmental management device built in the living cell incubator first receives the control command sent by the central controller 9, correspondingly adjusting the environment of the incubator to the composite cell freezing The status of the operation. Then, the moving member 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position according to the received control command (so that the detachable droplet pick-up tube 421 of the droplet pick-up device 42 faces the target cell droplet) .
  • the droplet pick-up device 42 controls the detachable droplet pick-up tube 421 to adsorb the target cell droplets (for example, from the culture carrier) and transfer to the freeze/recovery carrier on the console 7 according to the received control command.
  • the particular location of 6 i.e., the hydrophilic surface functional region 62 provided with the droplet location identification
  • the moving part of the console 7 moves the console 7 carrying the freeze/recovery carrier 6 (and cell droplets) to the droplet changing operation device operation section (specifically by the image acquisition means 308) in accordance with the received control command.
  • the liquid droplet changing operation device 3 performs a cell freezing operation on the cell droplet at a specific position of the freezing/recovery carrier 6 on the operation table 7 according to the received control command, and changes the liquid according to a predetermined procedure.
  • Drop ingredients eg, cryoprotectants.
  • the recorded cell image information is acquired by the image acquisition device 308b.
  • the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position (the carrier holding member 411 of the carrier holding action device 41 faces the target cell droplet) according to the received control command, and the carrier holding action device 41
  • a freezing medium container 8 for example, liquid nitrogen
  • the environmental management device built in the living cell culture incubator first receives the control command sent by the central controller 9, correspondingly adjusting the environment of the incubator to the composite cell resuscitation. The status of the operation. Then, the moving member 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position according to the received control command (the carrier holding member 411 of the carrier holding action device 41 faces the target cell droplet), Then, the carrier holding action device 41 controls the carrier holding member 411 to discharge the target cell droplets that have been frozen in the freezing medium container 8 together with the freezing/recovery carrier 6 from the freezing medium container 8 in accordance with the received control command. The clip is taken out and moved to the console 7.
  • the moving part of the console 7 moves the console 7 carrying the freeze/recovery carrier 6 (and cell droplets) to the droplet changing operation device operation section according to the received control command (specifically by the image acquisition device 308b)
  • the moving table 7 causes the cell droplets carried thereon to fall on the operation point of the droplet changing operation device), and then, the droplet changing operation device 3 according to the received
  • the control command is a liquid exchange operation for cell resuscitation of the cell droplets at a specific position of the freezing/recovery carrier 6 on the console 7, and the liquid capacity and composition are changed in accordance with a predetermined procedure.
  • the recorded cell image information is acquired by the image acquisition device 308b.
  • the resuscitation is completed until the cells reach the defined equilibrium of the culture medium.
  • the moving member 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position according to the received control command (making the liquid).
  • the detachable droplet pick-up tube 421 of the drip pick-up device 42 faces the target cell droplet, and the detachable droplet pick-up tube 421 exchanges the finished droplet on the freeze/recovery carrier 6 according to the received control command.
  • the target cell droplets after the manipulation are adsorbed and transferred out (for example, transferred to a culture carrier or other container).
  • the cell vitrification/recovery mode device of the present embodiment adopts a transparent plate-like structure carrier having a curved inner surface as a freezing/recovering carrier, and the inner surface of the freezing/reviving carrier is provided with a superhydrophobic surface. a functional region and a hydrophilic surface functional region, the hydrophilic surface functional region being surrounded by the superhydrophobic surface functional region, the hydrophilic surface functional region being provided with a droplet positioning identifier, such that throughout the freezing/recovery
  • the entire freezing/recovering operation process of the frozen target cells can be automatically completed by identifying and locating the droplet positioning marks, without manual intervention, greatly improving work efficiency, ensuring operation stability, timeliness and safety. Sex.
  • an embodiment of the present invention provides an automatic operation method for vitrification of living cells. As shown in FIG. 14, the method includes:
  • Step S101 Send a plurality of control commands into the cell freezing mode to the plurality of executing devices by the central controller;
  • Step S102 by the droplet pick-up device and according to the received control command, the target cell droplet is adsorbed and transferred to a specific position of the freezing carrier carried by the console;
  • Step S103 performing a liquid droplet changing operation on the target cell droplet at a specific position of the freezing carrier carried by the operating table by the liquid droplet changing operation device and according to the received control instruction, and changing the liquid droplet according to a predetermined program. a component, until the target cell reaches a prescribed cryoprotectant equilibrium state, and the droplet capacity reaches a prescribed value range;
  • Step S104 moving the target cell droplets on the frozen carrier after the completed droplet changing operation together with the freezing carrier into the freezing medium container by the carrier clamping action device and according to the received control command. To complete the freezing.
  • the structure of the frozen carrier in the present embodiment employs the structure of the viable cell vitrification/recovery carrier shown in FIG.
  • the invention uses the automatic operation method of the viable cell vitrification of the living cells shown in FIG. 14 instead of the manual operation, and can greatly improve the work efficiency and ensure the stability, timeliness and safety of the operation.
  • 15 is a flow chart of an automatic operation method for vitrification of frozen-thawed cells according to an embodiment of the present invention, the method comprising:
  • Step S201 sending, by the central controller, a plurality of control commands into the cell recovery mode to the plurality of executing devices;
  • Step S202 removing the frozen target cell droplets in the freezing medium container and the freezing carrier from the freezing medium container to the operating table by the carrier clamping action device according to the received control instruction; wherein the target Cell droplets are placed at specific locations on the frozen carrier;
  • Step S203 performing a liquid droplet changing operation on the target cell droplet at a specific position of the freezing carrier carried by the operating table by the liquid droplet changing operation device according to the received control instruction, and changing the liquid capacity according to a predetermined program. And ingredients until the cells reach a defined equilibrium in the culture medium to complete the resuscitation;
  • Step S204 The target cell droplets are adsorbed and transferred out from the freezing carrier by the droplet picking device and according to the received control command.
  • the structure of the resuscitation carrier in this embodiment employs the structure of the viable cell vitrification/recovery carrier shown in FIG.
  • FIG. 14 or FIG. 15 is a flow chart showing an automatic operation method of vitrification freezing/resuscitation of living cells according to an embodiment of the present invention, and it is necessary to understand the steps of the automatic operation method of vitrification freezing/revitalization of living cells proposed by the present invention. Without being limited to the order of execution shown in FIG. 14 or FIG. 15, those skilled in the art can arbitrarily change the automatic operation method steps of rapid vitrification of living cells according to the spirit of the present invention.
  • the graft mode device located within the secondary culture chamber 102 in an automated device for in vitro fertilization and cleavage culture of egg cells can achieve a graft function through the vitrification freeze/recovery mode device 400.
  • the automated device for split culture realizes in vitro fertilization and cleavage culture of egg cells including but not limited to cumulus cell identification sorting mode, direct insemination mode, freezing mode, freeze-thaw cell resuscitation mode and transplantation mode.
  • the operator outputs an instruction to the egg cell automatic identification sorting device 200 to enter the cumulus compound sorting mode through the central controller 9, and the operator inputs the expected number of eggs to be harvested, and the microfluid pump suctions according to the expected egg. A number of culture solutions corresponding to the volume. And the central controller 9 instructs the culture carrier drive unit 105 to operate to bring the loaded culture carrier 1 into a predetermined initial position.
  • the second valve port 205b of the sorting switch 205 is controlled to be turned on by the central controller 209 (when the first valve port 205a and the third valve port 205c are closed), and the negative pressure generator 201 is controlled to be started. . Under the activation of the vacuum generator 201, the follicular stock solution 20 with the egg cells 21 flows into the main input conduit 210 under the action of a negative pressure.
  • the egg cell image acquiring device 202 is coordinated by the light source device 203 to acquire egg cell image information.
  • the egg cell image acquisition device 202 transmits the acquired egg cell image information to the central controller 209, and the central controller 209 combines the follicular stock solution 20 with the egg cells 21 according to the egg cell image information.
  • the flow rate V of the main input catheter 210 and the catheter length difference H are calculated to determine the precise time at which the follicular stock solution 20 with the egg cells 21 reaches the first valve port 205a, and when the egg cell 21 is present
  • the central controller 209 immediately controls the sorting switch 205 to open the first valve port 205a (when the second valve port 205b and the third valve port 205c are closed),
  • the egg cells 21 in the follicular stock solution 20 with the egg cells 21 are caused to flow out from the first valve port 205a and into the main output conduit 211.
  • the central controller 209 immediately controls the sorting switch 205 to open the third valve port 205c (at this time, the first Valve port 205a and second valve port 205b is turned off), the specific culture solution 23 flowing in from the auxiliary input conduit 213 and the egg cells 21 are formed into the egg cell droplets 24, and then flow out through the main output conduit 211 and flow through the flow path 106 into the automatic combined partition combination incubator.
  • the culture carrier 1 was placed in 100.
  • the central controller 209 immediately controls the sorting switch 205 to open the second valve port 205b (at this time, the first valve) The mouth 205a and the third valve port 205c are closed), and the follicular fluid 22 from which the egg cells 21 are removed flows out from the second valve port 205b and flows into the follicular fluid collection container 204.
  • the central controller 9 control device repeats the image recognition, the valve opening and closing of the sorting switch 205, the microfluidic pump 207 pumping out the droplets, and the droplets of the egglets sequentially enter the specific culture position (culture carrier 1), until the negative pressure
  • the generator 201 is turned off and the cumulus cell complex sorting mode is terminated.
  • the central controller 9 controls to open the automatic airtight door 103, and instructs the culture carrier driving device 105 to operate to carry the loaded culture carrier 1 from the auxiliary incubator 102. Transfer to the main incubator 101 (and the central controller 9 controls to close the automatic airtight door 10) to wait for the direct fertilization mode operation.
  • the central controller 9 can control the first liquid droplet changing operation device / the second liquid droplet changing operation device to change the egg cell droplets on the culture carrier 1 before performing the direct fertilization mode operation.
  • the liquid is operated to cover a specific volume of culture oil on the droplets of the egg cells.
  • the central controller 9 controls the environment management device built in the main culture cabinet 101 to adjust the environment in the main culture cabinet 101 so as to conform to the environmental requirements of the egg cell droplet fertilization mode, when the egg cell droplets on the culture carrier 1 enter
  • the central controller 9 controls the direct insemination mode device 300 (first liquid droplet changing operation device) to immediately control the egg cell liquid on the culture carrier 1 when the specific position in the main culture chamber 101 is subjected to the fertilization and culture operation.
  • the droplets are automatically exchanged sequentially, and the image information of the oocytes entering the in vitro culture state is recorded by the cell image acquisition device 308 at a set frequency.
  • the clear image information acquired by the cell image acquisition device 308 is sent to the central controller 9, and the central controller 9 sequentially stores the image information of each marked position as an image set of a specific position; the operator can access the image set of any marked position at any time. , determining the developmental state of the cumulus cell complex and recording;
  • the operator determines the time of adding the sperm suspension according to the state of the egg cell displayed by the image collection, and instructs the central controller; the central controller 9 controls the first droplet changing device to add the egg cell droplets on the culture carrier 1 according to the instruction.
  • the sperm suspension of the volume is completed for insemination.
  • the operator can control the automatic airtight door 103 to be opened by the central controller 9, and instruct the culture carrier driving device 105 to operate to carry the culture carrier 1 from the auxiliary culture cabinet. 102 is transferred to the auxiliary incubator 102 (and the central controller 9 controls to close the automatic airtight door 10) to wait for other mode operations.
  • the environment management device built in the auxiliary incubator 102 When the operator sends a plurality of control commands into the cell freezing mode through the central controller 9, the environment management device built in the auxiliary incubator 102 first receives the control command sent by the central controller 9, and correspondingly adjusts the auxiliary incubator. The environment in 102 to the state of the egg cell freezing operation. Then, the moving member 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position according to the received control command (the detachable droplet pick-up tube 421 of the droplet pick-up device 42 is placed directly on the culture carrier 1 The target cell droplet, that is, the chaotic cell droplet after fertilization).
  • the droplet pick-up device 42 controls the detachable droplet pick-up tube 421 to adsorb the target cell droplets from the culture carrier 1 and transfer to the freeze/recovery carrier 6 on the console 7 in accordance with the received control command.
  • the location i.e., the hydrophilic surface functional area 62 provided with the drop location identification
  • the moving part of the console 7 moves the console 7 carrying the freeze/recovery carrier 6 (and the cell droplets) to the droplet changing device operation section according to the received control command (specifically by the image acquiring device 308b).
  • the second droplet changing operation device 3 is based on the received Control instructions for cell fluid at specific locations on the freeze/recovery carrier 6 on the console 7
  • the droplet is subjected to a liquid exchange operation of cell freezing, and the droplet component (for example, a cryoprotectant) is changed according to a predetermined procedure.
  • the recorded cell image information is acquired by the image acquisition device 308b.
  • the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position (the carrier holding member 411 of the carrier holding action device 41 faces the target cell droplet) according to the received control command, and the carrier holding action device 41
  • a freezing medium container 8 for example, liquid nitrogen
  • the environment management device built in the sub-culture cabinet 102 first receives the control command sent by the central controller 9, and correspondingly adjusts the auxiliary culture cabinet 102. The environment to the state of the composite cell resuscitation operation. Then, the moving member 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position according to the received control command (the carrier holding member 411 of the carrier holding action device 41 faces the target cell droplet), Then, the carrier holding action device 41 controls the carrier holding member 411 to discharge the target cell droplets that have been frozen in the freezing medium container 8 together with the freezing/recovery carrier 6 from the freezing medium container 8 in accordance with the received control command.
  • the clip is taken out and moved to the console 7.
  • the moving part of the console 7 moves the console 7 carrying the freeze/recovery carrier 6 (and the cell droplets) to the droplet changing device operation section according to the received control command (specifically by the image acquiring device 308b).
  • the control command is a liquid exchange operation for cell resuscitation of the cell droplets at a specific position of the freezing/recovery carrier 6 on the console 7, and the liquid capacity and composition are changed in accordance with a predetermined procedure.
  • the recorded cell image information is acquired by the image acquisition device 308b.
  • the moving part 43 of the device 4 moves the carrier mechanical action device 4 to a suitable position in accordance with the received control command (the detachable droplet pick-up tube 421 of the droplet pick-up device 42 faces the target cell droplet), said detachable The droplet pick-up tube 421 adsorbs and transfers the target cell droplets on the freeze/recovery carrier 6 after the completion of the liquid droplet exchange operation according to the received control command (for example, transfer to the culture carrier 1 or other container) .
  • the operator may first control the automatic airtight door 103 to be opened by the central controller 9, and instruct the culture carrier driving device 105 to act to carry the The culture carrier 1 is transferred from the auxiliary incubator 102 to the sub-incubator 102 (and the central controller 9 controls the automatic airtight door 10 to be closed).
  • the second liquid droplet changing operation device 3 performs a liquid exchange operation on the egg cell droplets of the culture carrier 1 according to the received control command, and changes according to a predetermined procedure. Liquid capacity and composition. At this time, the recorded cell image information is acquired by the image acquisition device 308b.
  • the moving member 43 of the carrier mechanical action device 4 moves the carrier mechanical action device 4 to a suitable position according to the received control command (the detachable droplet of the droplet pick-up device 42 is made)
  • the pick-up tube 421 is facing the target cell droplet, and the detachable droplet pick-up tube 421 adsorbs and rapidly drops the egg cell droplet after the completion of the liquid-dropping operation on the freeze/recovery carrier 6 according to the received control command. Load the migration tube to complete the migration mode.
  • Another embodiment of the present invention provides an automated method for in vitro fertilization and cleavage culture of egg cells, which comprises in vitro fertilization and cleavage culture of egg cells, including cumulus compound identification sorting mode and direct insemination mode.
  • the multiple modes of operation of the vitrification/recovery mode and the transfer mode are all operations on egg cell droplets, including but not limited to the identification and sorting of egg cell droplets, the exchange of egg cell droplets, and the transfer of egg cell droplets. transplant.

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Abstract

一种用于卵细胞体外受精和卵裂培养的自动化装置，包括自动联通式分区组合培养箱（100）、置于培养箱外的中央控制器（9）和卵细胞自动识别分选装置（200）；培养箱在不同的分区中设有不同阶段的操作模式装置，且每个分区通过自动气密门（103）来实现连通/隔开；培养箱内设有用于承载卵细胞液滴的培养载体（1）以及带动培养载体往返于各个分区的培养载体驱动装置（105）；卵细胞自动识别分选装置根据接收的控制指令，对带有卵细胞的卵泡原液进行识别和分选后，输出卵细胞液滴至培养箱内的培养载体上；在自动气密门（103）根据控制指令打开时，培养载体驱动装置根据控制指令带动所述培养载体往返于各个分区之间，卵细胞液滴进入各个分区以进行对应的操作模式。该自动化装置可以实现从卵丘复合物自动获取、培养液滴加入精子悬液、卵丘复合物脱颗粒、培养液滴成分自动控制、囊胚的全程受控培养、包含胚胎的培养液滴无残留完整转移。

Description

用于卵细胞体外受精和卵裂培养的自动化装置、载体及方法 技术领域
本发明涉及一种细胞培养的装置，尤其涉及一种用于卵细胞体外受精和卵裂培养的自动化装置、载体及方法。
背景技术
人工辅助生殖技术在20世纪末期开发并应用于临床，21世纪初期在全球范围推广普及，技术开发人获得了全人类的褒扬。这一技术已经在世界范围推广普及，将继续为全体人类造福。
人工辅助生殖，是工程技术和临床医学技术的结合。临床医学的发展解决了生殖细胞的来源(主要是特定发育阶段的人类卵母细胞来源、确认精子体外授精能力)问题，明确了妊娠的生理要素；工程技术解决了体外受精问题和受精卵体外培养过程模拟体内环境的问题。这一技术系列存在体外培养环境对体内环境模拟不完备的技术现实，以及在受精和培养过程中依赖人工操作和体外操作缓冲液体系统的问题，现在通用的IVF操作方法主要是培养箱外开放环境的手工操作，箱外操作脱离了培养环境，依赖缓冲液系统，依赖培养环境外的温度控制装置，而且高度依赖操作人的操作技术和责任心，必须人工操作，操作环境的保持依赖特定的操作规则和设备，工作过程不可追溯，无法标准化。
IVF实验室序列培养操作流程简述如下(以第3日移植方式为例)：依据手术计划提前准备培养液；手术抽吸卵泡液，手术助手指示术者卵泡液容器(试管)是否需要更换，术者根据提示持续抽吸或者停止抽吸；卵泡液装入试管后，手术助手放入恒温试管架，更换新的试管容器，重复前述过程，直到术者结束抽吸，完成手术；实验室操作人将包含卵丘复合物的卵泡液取出，完整转移入捡卵容器，在低倍体视显微镜协助下快速识别卵丘细胞复合物，手工转移入缓 冲液体(缓冲液容器置放在恒温平台之上)，手工冲洗后移入授精培养液(授精培养液容器放置在恒温平台之上)，转移到培养箱环境，在规定的时间加入精子液滴(培养箱外)，培养箱内完成授精后在规定的时间手工脱除颗粒细胞(培养箱外)，在培养箱外快速转移受精卵到卵裂培养液，在规定的时间完成移植或冻融保存，也可以继续培养到囊胚状态后移植或冻融；如果操作人决定进行卵胞浆内单精子显微注射授精，实验室操作人须制作缓冲液液滴系统，准备授精培养皿，在具有恒温平台装置的倒置显微镜下完成显微注射授精，然后将授精后的细胞手工转移入培养皿(授精培养液滴)，置入培养箱内，继续培养。
这一工作体系中，培养液滴在开放环境和培养箱内人工环境多次转移，目标细胞也在不同的液体环境中多次转移，细胞转移依赖操作人手工完成(使用手持吸管工具手工操作单细胞，需严格避免气泡产生等操作失误)，培养环境不连续，也容易出现细胞丢失和损伤；这一工作体系中，细胞培养环境无法标准化，任意两组细胞操作过程不能严格比较，任意两组培养过程不能严格比较，操作过程无法监控，只能从移植结果上判断操作是否成功。
目前的工作规则下，临床生殖实验室胚胎培养和临床移植的成功率与胚胎操作人的熟练程度和责任心直接相关，和实验室的管理行为直接相关，这是各临床生殖医学单位和操作员的最终种植成功率表现出显著差异的重要原因。合格的实验室操作人，需要严格的操作培训和长期、大量的练习，这一要求制约了IVF技术的进一步普及，并造成了社会资源的大量浪费。
发明内容
本发明的目的是提供一种用于卵细胞体外受精和卵裂培养的自动化装置，模拟人类卵细胞授精和受精卵早期发育过程的环境，可以实现从卵丘复合物自动获取、培养液滴加入精子悬液、卵丘复合物脱颗粒、培养液滴成分自动控制、囊胚的全程受控培养、包含胚胎的培养液滴无残留完整转移。
本发明实施例提供了一种用于卵细胞体外受精和卵裂培养的自动化装置，
包括自动联通式分区组合培养箱、置于培养箱外的中央控制器和卵细胞自动识别分选装置；
所述自动联通式分区组合培养箱在不同的分区中设有对卵细胞液滴进行受精及卵裂培养过程中的不同阶段的操作模式装置，且每个分区通过自动气密门来实现连通/隔开；所述自动联通式分区组合培养箱内设有用于承载卵细胞液滴的培养载体以及带动所述培养载体往返于各个分区的培养载体驱动装置；
所述卵细胞自动识别分选装置、不同阶段的操作模式装置、自动气密门以及培养载体驱动装置均与所述中央控制器连接，以接收对应的控制指令；
所述卵细胞自动识别分选装置根据接收的控制指令，对带有卵细胞的卵泡原液进行识别和分选后，输出卵细胞液滴至所述自动联通式分区组合培养箱内的培养载体上；在所述自动气密门根据控制指令打开时，所述培养载体驱动装置根据控制指令带动所述培养载体往返于所述自动联通式分区组合培养的各个分区之间，从而使所述培养载体上的卵细胞液滴进入各个分区以进行对应的操作模式。
作为上述技术方案的改进，所述自动联通式分区组合培养箱包括两个分区，所述两个分区通过主培养箱体、副培养箱体以及用于实现所述主培养箱体和副培养箱体之间的连通/隔开的所述自动气密门构成。
作为上述技术方案的改进，所述操作模式装置包括但不限于直接授精模式装置、玻璃化冷冻/复苏模式装置以及移植模式装置。
作为上述技术方案的改进，所述直接授精模式装置设于所述主培养箱体内；所述玻璃化冷冻/复苏模式装置以及所述移植模式装置设于所述副培养箱体内。
作为上述技术方案的改进，所述直接授精模式装置包括第一液滴换液操作装置，所述第一液滴换液操作装置包括废液抽取控制器、培养液补加控制器、液滴输出管路和液滴输入管路，所述液滴输出管路和液滴输入管路的两终端均连接液滴操作针以吸取/输出液滴；所述废液抽取控制器通过所述液滴输出管路 将培养载体上的细胞液滴中的废液抽出并控制抽出的废液流量，而所述培养液补加控制器通过所述液滴输入管路将培养液输送至所述培养载体上的细胞液滴中并控制输送的培养液流量；所述中央控制器分别与所述废液抽取控制器和所述培养液补加控制器连接，以控制所述废液抽取控制器和所述培养液补加控制器工作。
作为上述技术方案的改进，所述废液抽取控制器和所述培养液补加控制器为蠕动泵或步进电机。
作为上述技术方案的改进，所述直接授精模式装置还包括与所述中央控制器连接的细胞图像获取设备，用于获取直接授精模式操作时所述培养载体上的细胞液滴中的细胞图像信息。
作为上述技术方案的改进，所述培养载体表面上设有多个用于承载卵细胞液滴的凹坑，且所述凹坑表面为超疏水表面。
作为上述技术方案的改进，所述培养载体表面上在每一所述凹坑的边缘凸起设置闭合环状的限制部，且所述限制部的表面为亲油表面。
作为上述技术方案的改进，所述卵细胞自动识别分选装置包括负压发生器、卵细胞图像获取设备、光源设备、卵泡液收集容器以及分选开关，所述中央控制器分别与所述负压发生器、卵细胞图像获取设备和分选开关连接；
所述分选开关包括第一阀口、第二阀口和第三阀口，所述第一阀口连接用于输入带有卵细胞的卵泡原液的主输入导管以及用于输出卵细胞至培养载体的主输出导管之间，所述第二阀口连接所述主输入导管以及用于输出除去卵细胞的卵泡液至所述卵泡液收集容器的副输出导管之间，所述第三阀口连接用于输入特定培养液的副输入导管和所述主输出导管之间；所述负压发生器通过连通导管与所述卵泡液收集容器连通；
所述光源设备设于所述主输入导管外的一侧，对主输入导管内进行照明，并成像在设于所述主输入导管外的另一侧的所述卵细胞图像获取设备上；带有 卵细胞的卵泡原液在负压发生器启动时流入所述主输入导管，并流经所述卵细胞图像获取设备时由所述卵细胞图像获取设备获取卵细胞图像信息，所述中央控制器根据所述卵细胞图像信息控制所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭，从而使带有卵细胞的卵泡原液中的卵细胞从所述第一阀口流出，去除卵细胞的卵泡液从所述第二阀口流出并流入到所述卵泡液收集容器，并使从所述第一阀口流出的卵细胞与从所述第三阀口流入的特定培养液组成卵细胞液滴后流出至所述培养载体。
作为上述技术方案的改进，通过所述中央控制器控制所述负压发生器的工作，从而控制带有卵细胞的卵泡原液在所述主输入导管的流速。
作为上述技术方案的改进，所述光源设备和卵细胞图像获取设备所对应的主输入导管位置与所述第一阀口所在的主输入导管位置的导管长度差值为预设值。
作为上述技术方案的改进，所述中央控制器根据所述卵细胞图像信息，并结合所述带有卵细胞的卵泡原液在所述主输入导管的流速以及所述导管长度差值计算得出所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭时间。
作为上述技术方案的改进，所述玻璃化冷冻/复苏模式装置包括载体机械动作装置、第二液滴换液装置、冷冻/复苏载体、承载所述冷冻/复苏载体的操作台以及冷冻介质容器，其中，
所述载体机械动作装置和所述第二液滴换液装置分别与所述中央控制器连接以接收对应的冷冻/复苏控制指令；
所述载体机械动作装置包括机械本体以及设置在机械本体上的载体夹持动作装置和液滴捡拾装置；所述液滴捡拾装置用于根据冷冻/复苏控制指令对应将目标细胞液滴吸附并转移到所述冷冻/复苏载体的特定位置上/将目标细胞液滴从所述冷冻/复苏载体上吸附并转移出去；所述载体夹持动作装置用于根据冷冻/复苏控制指令，将所述冷冻/复苏载体上的完成液滴换液操作后的目标细胞液滴 连同所述冷冻/复苏载体一起移入到所述冷冻介质容器中以完成冷冻/将所述冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻/复苏载体从所述冷冻介质容器中移出到所述操作台上；
所述第二液滴换液装置用于根据冷冻/复苏控制指令对所述操作台承载的冷冻/复苏载体上的目标细胞液滴进行液滴换液操作。
作为上述技术方案的改进，所述第二液滴换液装置与所述的第一液滴液装置的结构一致。
作为上述技术方案的改进，所述玻璃化冷冻/复苏模式装置还包括与所述中央控制器连接的图像获取装置；所述图像获取装置用于获取所述冷冻/复苏载体上的细胞液滴中的细胞图像信息，并将获取到的细胞图像信息发送给所述中央控制器进行处理。
作为上述技术方案的改进，所述冷冻/复苏载体的载体本体为内表面呈弧面的片状结构，所述内表面上设有超疏水表面功能区域和亲水表面功能区域，所述亲水表面功能区域四周被所述超疏水表面功能区域环绕，所述亲水表面功能区域上设有液滴定位标识。
作为上述技术方案的改进，所述超疏水表面功能区域为圆环形区域，所述亲水表面功能区域为圆形区域。
作为上述技术方案的改进，所述亲水表面功能区域的中心点与所述载体本体的最低点重合。
作为上述技术方案的改进，所述移植模式装置可通过所述玻璃化冷冻/复苏模式装置实现移植功能。
作为上述技术方案的改进，所述主培养箱体/副培养箱体内分别设有用于控制调整主培养箱体/副培养箱体内的温度、湿度、压力和气体分压的环境管理装置。
作为上述技术方案的改进，所述主培养箱体/副培养箱体上设有与外界连通的流路，通过所述流路接收外界送入的待培养细胞液滴或向外界送出培养后的细胞液滴。
本发明实施例还提供了一种用于卵细胞体外受精和卵裂培养的自动化方法，利用自动化装置对卵细胞进行体外受精和卵裂培养的包括卵丘复合物识别分选模式、直接授精模式、玻璃化冷冻/复苏模式以及转移模式的多个操作模式均为对卵细胞液滴进行操作。
本发明实施例还提供了一种活体细胞培养载体，所述培养载体表面上设有多个用于承载活体细胞液滴的凹坑，且所述凹坑表面为超疏水表面。
作为上述技术方案的改进，所述培养载体表面上在每一所述凹坑的边缘凸起设置闭合环状的限制部，且所述限制部的表面为亲油表面。
作为上述技术方案的改进，所述凹坑表面为铺设了一层超疏水材料的超疏水表面层或所述凹坑表面为经过超疏水处理的超疏水表面。
本发明实施例还公开了一种卵细胞自动识别分选装置，包括负压发生器、卵细胞图像获取设备、光源设备、卵泡液收集容器、分选开关以及与所述负压发生器、卵细胞图像获取设备和分选开关连接的中央控制器；
所述分选开关包括第一阀口、第二阀口和第三阀口，所述第一阀口连接用于输入带有卵细胞的卵泡原液的主输入导管以及用于输出卵细胞至培养载体的主输出导管之间，所述第二阀口连接所述主输入导管以及用于输出除去卵细胞的卵泡液至所述卵泡液收集容器的副输出导管之间，所述第三阀口连接用于输入特定培养液的副输入导管和所述主输出导管之间；所述负压发生器通过连通导管与所述卵泡液收集容器连通；
所述光源设备设于所述主输入导管外的一侧，对主输入导管内进行照明，并成像在设于所述主输入导管外的另一侧的所述卵细胞图像获取设备上；带有卵细胞的卵泡原液在负压发生器启动时流入所述主输入导管，并流经所述卵细 胞图像获取设备时由所述卵细胞图像获取设备获取卵细胞图像信息，所述中央控制器根据所述卵细胞图像信息控制所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭，从而使带有卵细胞的卵泡原液中的卵细胞从所述第一阀口流出，去除卵细胞的卵泡液从所述第二阀口流出并流入到所述卵泡液收集容器，并使从所述第一阀口流出的卵细胞与从所述第三阀口流入的特定培养液组成卵细胞液滴后流出至培养载体。
作为上述技术方案的改进，通过所述中央控制器控制所述负压发生器的工作，从而控制带有卵细胞的卵泡原液在所述主输入导管的流速。
作为上述技术方案的改进，所述光源设备和卵细胞图像获取设备所对应的主输入导管位置与所述第一阀口所在的主输入导管位置的导管长度差值为预设值。
作为上述技术方案的改进，所述中央控制器根据所述卵细胞图像信息，并结合所述带有卵细胞的卵泡原液在所述主输入导管的流速以及所述导管长度差值计算得出所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭时间。
作为上述技术方案的改进，所述主输入导管为透明导管，且所述主输入导管的管径为固定值。
作为上述技术方案的改进，所述分选开关为三通阀，包括带有所述第一阀口、第二阀口和第三阀口的阀体以及控制所述第一阀口、第二阀口和第三阀口的开启/闭合的阀门控制装置，所述中央控制器与所述阀门控制装置连接。
本发明实施例还公开了一种用于细胞培养的液滴换液操作装置，包括废液抽取控制器、培养液补加控制器、液滴输出管路和液滴输入管路，所述液滴输出管路和液滴输入管路的两终端均连接液滴操作针以吸取/输出液滴；所述废液抽取控制器通过所述液滴输出管路将培养载体上的细胞液滴中的废液抽出并控制抽出的废液流量，而所述培养液补加控制器通过所述液滴输入管路将培养液输送至所述培养载体上的细胞液滴中并控制输送的培养液流量。
作为上述技术方案的改进，所述液滴换液操作装置置于环境可控的细胞培养箱内。
作为上述技术方案的改进，所述废液抽取控制器和所述培养液补加控制器为蠕动泵或步进电机。
作为上述技术方案的改进，还包括中央控制器，所述中央控制器分别与所述废液抽取控制器和所述培养液补加控制器连接，以控制所述废液抽取控制器和所述培养液补加控制器工作。
作为上述技术方案的改进，还包括与所述中央控制器连接的细胞图像获取设备，用于获取所述培养载体上的细胞液滴中的细胞图像信息。
作为上述技术方案的改进，还包括与所述中央控制器连接的培养液选取动作设备，用于接收所述中央控制器的指令来移动所述液滴输入管路到盛有不同培养液的容器中以获取对应的培养液。
作为上述技术方案的改进，所述液滴输出管路和液滴输入管路的两终端连接的液滴操作针均为可拆卸的液滴操作针，所述液滴换液操作装置还包括液滴操作针更换动作设备，用于对所述液滴输出管路和液滴输入管路的两终端连接的液滴操作针进行自动更换。
作为上述技术方案的改进，还包括用于收集经由所述液滴输出管路抽出的培养载体上的细胞液滴中的废液的废液收集容器。
本发明实施例还公开了一种活体细胞玻璃化冷冻/复苏载体，所述载体本体为内表面呈弧面的透明片状结构，所述载体本体的内表面上设有超疏水表面功能区域和亲水表面功能区域，所述亲水表面功能区域四周被所述超疏水表面功能区域环绕，所述亲水表面功能区域上设有液滴定位标识。
作为上述技术方案的改进，所述超疏水表面功能区域为圆环形区域，所述亲水表面功能区域为圆形区域。
作为上述技术方案的改进，所述亲水表面功能区域的中心点与所述载体本体的最低点重合。
作为上述技术方案的改进，所述超疏水表面功能区域为在所述载体的内表面上铺设了一层超疏水材料的超疏水表面层或所述超疏水表面功能区域为将所述载体的内表面的部分区域经过超疏水处理后所得的超疏水表面层；
所述亲水表面功能区域为在所述载体的内表面上铺设了一层亲水材料的亲水表面层或所述亲水表面功能区域为将所述载体的内表面的部分区域经过亲水处理后所得的亲水表面层。
本发明实施例还公开了一种活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，包括中央控制器、载体机械动作装置、液滴换液操作装置、冷冻/复苏载体、承载所述冷冻/复苏载体的操作台以及冷冻介质容器，其中，
所述载体机械动作装置和所述液滴换液操作装置分别与所述中央控制器连接以接收对应的冷冻/复苏控制指令；
所述载体机械动作装置包括机械本体以及设置在机械本体上的载体夹持动作装置和液滴捡拾装置；所述液滴捡拾装置用于根据冷冻/复苏控制指令对应将目标细胞液滴吸附并转移到所述冷冻/复苏载体的特定位置上/将目标细胞液滴从所述冷冻/复苏载体上吸附并转移出去；所述载体夹持动作装置用于根据冷冻/复苏控制指令，将所述冷冻/复苏载体上的完成液滴换液操作后的目标细胞液滴连同所述冷冻/复苏载体一起移入到所述冷冻介质容器中以完成冷冻/将所述冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻/复苏载体从所述冷冻介质容器中移出到所述操作台上；
所述液滴换液操作装置用于根据冷冻/复苏控制指令对所述操作台承载的冷冻/复苏载体上的目标细胞液滴进行液滴换液操作；
所述冷冻/复苏载体为权利要求39～42中任一项所述的活体细胞玻璃化冷冻/复苏载体；所述特定位置为所述设有液滴定位标识的亲水表面功能区域上。
作为上述技术方案的改进，还包括分别与所述中央控制器连接的图像获取装置和显示器；
所述图像获取装置用于获取所述冷冻/复苏载体上的细胞液滴中的细胞图像信息，并将获取到的细胞图像信息发送给所述中央控制器处理后，通过所述显示器显示。
作为上述技术方案的改进，所述图像获取装置可与所述液滴换液操作装置为一体结构。
作为上述技术方案的改进，所述载体机械动作装置为可移动式载体机械动作装置，所述载体机械动作装置还包括与所述机械本体连接的移动部件，所述载体机械动作装置通过所述移动部件可在运动导轨上移动。
作为上述技术方案的改进，所述载体夹持动作装置包括用于夹持冷冻/复苏载体的载体夹持部件和将所述载体夹持部件固定在所述机械本体上的固定支架。
作为上述技术方案的改进，所述液滴捡拾装置包括可拆卸液滴捡拾管以及与所述可拆卸液滴捡拾管连通且控制其吸附的可拆卸负压抽吸设备。
作为上述技术方案的改进，所述冷冻介质容器内装有的冷冻介质为液氮。
作为上述技术方案的改进，所述自动操作系统置于环境可控的细胞培养箱内。
本发明实施例还公开了一种活体细胞玻璃化冷冻的自动操作方法，包括步骤：
S1、通过中央控制器向多个执行装置发出进入细胞冷冻模式的多个控制指令；
S2、通过液滴捡拾装置并根据接收到的控制指令，将目标细胞液滴吸附转移到操作台承载的冷冻载体的特定位置上；
S3、通过液滴换液操作装置并根据接收到的控制指令，对所述操作台承载的冷冻载体的特定位置上的目标细胞液滴进行液滴换液操作，直至所述冷冻载 体上的目标细胞达到与冷冻保护剂的平衡状态而且液滴容量达到规定数值范围；以及
S4、通过载体夹持动作装置并根据接收到的控制指令，将所述冷冻载体上的完成液滴换液操作后的目标细胞液滴连同所述冷冻载体一起移入到所述冷冻介质容器中以完成冷冻；
其中，所述冷冻载体为以上所述的活体细胞玻璃化冷冻/复苏载体；所述特定位置为设有液滴定位标识的所述亲水表面功能区域上。
本发明实施例还公开了一种玻璃化冻融细胞复苏的自动操作方法，包括步骤：
S1、通过中央控制器向多个执行装置发出进入细胞复苏模式的多个控制指令；
S2、通过载体夹持动作装置并根据接收到的控制指令，将冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻载体从所述冷冻介质容器中移出到操作台上；其中，所述目标细胞液滴置于所述冷冻载体的特定位置上；
S3、通过液滴换液操作装置并根据接收到的控制指令，对所述操作台承载的冷冻载体的特定位置上的目标细胞液滴按照预定的程序进行液滴换液操作，直至所述冷冻载体上的目标细胞达到培养液平衡状态，完成复苏；以及
S4、通过液滴捡拾装置并根据接收到的控制指令，将所述目标细胞液滴从所述冷冻载体上吸附并转移出去；
其中，所述冷冻载体为以上所述的活体细胞玻璃化冷冻/复苏载体；所述特定位置为设有液滴定位标识的所述亲水表面功能区域上。
与现有技术相比，本发明公开的用于卵细胞体外受精和卵裂培养的自动化装置模拟人类卵细胞授精和受精卵早期发育过程的环境，可以实现从卵丘复合物自动获取、培养液滴加入精子悬液、卵丘复合物脱颗粒、培养液滴成分自动 控制、囊胚的全程受控培养、包含胚胎的培养液滴无残留完整转移。正常状态下无需人工干预培养环境，表观培养过程全程记录，培养环境的参数也可以全程记录，表观培养过程可完整追溯，胚胎培养完成后可以自动吸取，并按照移植要求自动装管交付移植人，也可以对目标细胞执行自动冷冻保存操作或者对冻融胚胎执行自动复苏操作，完成工作流程。
附图说明
图1是本发明实施例中一种用于卵细胞体外受精和卵裂培养的自动化装置的结构框图。
图2是本发明实施例中一种活体细胞培养箱的结构示意图。
图3是本发明实施例中一种卵细胞自动识别分选装置的结构示意图。
图4是图3所示的卵细胞自动识别分选装置的分选开关的结构框图。
图5是本实用新型实施例中一种活体细胞培养载体的结构的俯视示意图。
图6是本实用新型实施例中一种活体细胞培养载体的结构的剖面示意图。
图7是本实用新型实施例中一种活体细胞培养载体的放大结构示意图。
图8是本发明实施例中一种直接授精模式装置的结构示意图。
图9是本发明实施例中一种玻璃化冷冻/复苏模式装置的结构框图。
图10是本发明实施例中一种冷冻/复苏载体的结构示意图。
图11是本发明实施例中一种玻璃化冷冻/复苏模式装置的操作台的结构示意图。
图12是本发明实施例中一种玻璃化冷冻/复苏模式装置的载体机械动作装置的结构示意图。
图13是本发明实施例中一种玻璃化冷冻/复苏模式装置的第二液滴换液装置的结构示意图。
图14是本发明实施例中一种活体细胞玻璃化冷冻的自动操作方法的流程图。
图15是本发明实施例中一种活体细胞玻璃化复苏的自动操作方法的流程图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
参见图1，是本发明实施例提供的一种用于卵细胞体外受精和卵裂培养的自动化装置，包括自动联通式分区组合培养箱100、置于培养箱外的中央控制器9和卵细胞自动识别分选装置200；其中：
所述自动联通式分区组合培养箱100包括主培养箱体101、副培养箱体102以及用于实现所述主培养箱体101和副培养箱体102之间的连通/隔开的自动气密门103。所述主培养箱体101/副培养箱体102内设有用于承载卵细胞液滴的培养载体1；所述主培养箱体/副培养箱体内分别设有用于对卵细胞液滴进行受精及卵裂培养过程中的不同阶段的操作模式装置。所述主培养箱体101/副培养箱体102内/外设有用于在所述自动气密门103打开的时候带动所述培养载体1往返于所述主培养箱体101和副培养箱体102之间的培养载体驱动装置105；
所述卵细胞自动识别分选装置200、不同阶段的操作模式装置、自动气密门103以及培养载体驱动装置105均与所述中央控制器连接，以接收对应的控制指令；
所述卵细胞自动识别分选装置200根据接收的控制指令，对带有卵细胞(卵丘细胞复合物)的卵泡原液进行识别和分选后，输出卵细胞液滴至所述自动联通式分区组合培养箱100内的培养载体上；在所述自动气密门根据控制指令打开时，所述培养载体驱动装置105根据控制指令带动所述培养载体1往返于所 述主培养箱体101和副培养箱体102之间，从而使所述培养载体1上的卵细胞液滴进入主培养箱体/副培养箱体内以进行对应的操作模式。
下面，结合图2～图13，详细描述本发明实施例的用于卵细胞体外受精和卵裂培养的自动化装置的各个组成部分的具体结构以及工作原理。
首先，参考图2，该自动联通式分区组合培养箱100包括主培养箱体101和副培养箱体102，所述主培养箱体101和副培养箱体102之间通过设置自动气密门103来实现箱体内部的连通/隔开。所述活体细胞培养箱100还包括自动气密门驱动装置108，该自动气密门驱动装置108控制所述自动气密门103的打开/关闭。该自动气密门驱动装置108可设于培养箱体101/副培养箱体102的内部或外部，可以通过与自动气密门103接触/不接触的方式来控制自动气密门103的打开/关闭。
所述主培养箱体101/副培养箱体102内设有用于承载卵细胞液滴的培养载体1。而所述自动联通式分区组合培养箱100还设有用于在所述自动气密门103打开的时候带动所述培养载体1往返于所述主培养箱体101和副培养箱体102之间的培养载体驱动装置105。该培养载体驱动装置105可设于主培养箱体101/副培养箱体102的内部或外部，可以通过与培养载体1接触/不接触的方式来带动所述培养载体1移动。
所述主培养箱体101/副培养箱体102内分别设有用于对所述卵细胞液滴(承载于培养载体1上)进行受精及培养过程中的不同阶段的操作模式装置。其中，该操作模式装置包括但不限于直接授精模式装置300、冷冻/复苏模式装置400以及移植模式装置(未示)。例如，在主培养箱体101内部设置直接授精模式装置300，而在副培养箱体102内设置冷冻模式装置、冻融细胞复苏模式装置以及移植模式装置。当通过外置开放环境内的卵丘细胞复合物分选装置200获得的卵细胞液滴进入副培养箱体102内时(承载于培养载体1上)后，通过控制自动气密门103打开，将培养载体1从副培养箱体102移动到主培养箱体101内部，并通过主培养箱体101内部的直接授精模式装置300对培养载体1上的卵 细胞液滴进行受精模式操作。待受精模式操作完成后，若需要进行移植模式、冷冻模式或冻融细胞复苏模式，则通过中央控制器9控制自动气密门103打开，将培养载体1从主培养箱体101移动到副培养箱体102内部，并通过副培养箱体102内部的移植模式装置或冷冻/复苏模式装置进行对应的模式操作。
另外，所述主培养箱体101/副培养箱体102上设有与外界连通的流路106(本实施例中显示了流路106设在副培养箱体102上，但可理解，该流路106也可设置)主培养箱体101，通过所述流路106接收外界送入的待培养卵细胞(液滴)或向外界送出培养后的卵细胞液滴。在自动联通式分区组合培养箱的外界环境中，设有所述卵细胞自动识别分选装置200，该卵细胞自动识别分选装置200用于识别和获取卵丘细胞，并往卵丘细胞加入特定培养液后将包含卵丘细胞的液滴通过所述流路106送入到副培养箱体102内部的培养载体1，然后再通过主培养箱体101/副培养箱体102的内部装置对培养载体1上的卵细胞液滴进行后续的直接授精模式、冷冻模式、冻融细胞复苏模式以及移植模式等操作。
在所述主培养箱体101/副培养箱体102内分别设有用于控制调整主培养箱体101/副培养箱体102内的温度、湿度、压力和气体分压的环境管理装置122a、122b。通过所述环境管理装置122a、122b来监测并控制所述主培养箱体101/副培养箱体102内部的环境，从而保证各个模式操作的稳定性和连续性。
以上所述的培养载体驱动装置105、自动气密门驱动装置108、环境管理装置122a、122b以及各个操作模式装置均与中央控制器9电连接，实现单向/双向的通信，从而将获取的信息发送给所述中央控制器9处理，以及接收中央控制器9的控制指令而对应操作。
优选的，本实施例的用于卵细胞体外受精和卵裂培养的自动化装置在所述主培养箱体101/副培养箱体102外还设有与所述中央控制器9连接的显示器10，所述中央控制器109将接收到的各个装置发送的各个模式状态图像信息进行处理后，例如，每一标记位置的图像信息逐次储存为特定位置的图像集合，并发送给所述显示器10进行显示。术者可以随时通过该显示器10调阅任意标记位 置的图像集合，判断卵细胞的发育状态并记录。另外，中央控制器9还可以与医院的综合信息管理系统共享，术者可以随时调阅，病人也可以通过公共信息平台接收。
可见，本实施例公开的自动联通式分区组合培养箱100通过在主培养箱体101和副培养箱体102之间通过设置自动气密门103来实现箱体内部的连通/隔开，且主培养箱体101/副培养箱体102内分别设有用于对卵细胞液滴进行受精及培养过程中的不同阶段的操作模式装置，然后通过培养载体驱动装置105在所述自动气密门打开的时候带动所述培养载体往返于所述主培养箱体101和副培养箱体102之间，从而使所述培养载体上的卵细胞液滴进入主培养箱体/副培养箱体内以进行对应的操作模式。因此，整个培养过程都在培养箱内，通过控制主培养箱体和副培养箱体之间的细胞培养环境连续，从而可以有效避免细胞丢失和损伤等问题。另外，通过标准化主培养箱体和副培养箱体的细胞培养环境，从而可以有效监控整个操作过程，利于操作成功。
参见图3～4，是本发明实施例提供的一种卵细胞自动识别分选装置200的结构示意图。该卵细胞自动识别分选装置200包括负压发生器201、卵细胞图像获取设备202、光源设备203、卵泡液收集容器204、分选开关205以及与所述负压发生器201、卵细胞图像获取设备202和分选开关205分别连接的中央控制器9，其中，负压发生器201、卵泡液收集容器204以及用于抽吸和输送带有卵细胞的卵泡原液的导管连通，该负压发生器201用于对管道形成负压以完成穿刺及抽吸带有卵细胞的卵泡原液并进入导管。该卵细胞图像获取设备202和光源设备203设置于导管的输送路径两侧上，以配合获取卵细胞图像信息，并将获取到的卵细胞图像信息发送给所述中央控制器9，中央控制器9根据卵细胞图像信息控制设置在导管上的分选开关205的开启/关闭，从而实现将带有卵细胞的卵泡原液中的卵细胞和卵泡液分离，从而获取所需的卵细胞。
具体的，如图3所示，所述分选开关205包括第一阀口205a、第二阀口205b和第三阀口205c，其中：
所述第一阀口205a连接用于输入带有卵细胞21的卵泡原液20的主输入导管210以及用于输出卵细胞21至培养载体1的主输出导管211之间。即所述主输入导管210和主输出导管211之间通过所述分选开关205的第一阀口205a的开启/关闭而实现连通/隔离。
所述第二阀口205b连接所述主输入导管210以及用于输出除去卵细胞21的卵泡液22至所述卵泡液收集容器204的副输出导管212之间。即所述主输入导管210和副输出导管212之间通过所述分选开关205的第二阀口205b的开启/关闭而实现连通/隔离。
所述第三阀口205c连接用于输入特定培养液23的副输入导管213和所述主输出导管211之间；即所述副输入导管213和所述主输出导管211之间通过所述分选开关205的开启/关闭而实现连通/隔离。所述副输入导管213上设有微流泵207，用于控制泵出并流入所述第三阀口205c的特定培养液23的流量。所述微流泵207与所述中央控制器9连接以接收控制指令。
如图4所示，在本实施例中，所述分选开关205可采用三通阀来实现，该三通阀包括带有所述第一阀口205a、第二阀口205b和第三阀口205c的阀体2051以及控制所述第一阀口205a、第二阀口205b和第三阀口205c的开启/闭合的阀门控制装置2052，所述中央控制器9与所述阀门控制装置2052连接。所述阀门控制装置2052根据中央控制器9发送的指令，而轮流控制所述第一阀口205a、第二阀口205b和第三阀口205c的开启/闭合。
另外，所述负压发生器201通过连通导管214与所述卵泡液收集容器204连通，从而实现与所述副输出导管212连通，并进一步在所述第二阀口205b的开启下实现与所述主输入导管210连通。在控制所述第二阀口205b开启的情况下，通过所述中央控制器9控制所述负压发生器201的工作，从而可控制带有卵细胞21的卵泡原液20在所述主输入导管210的流速V。
所述光源设备203设于所述主输入导管210的输送路径上，并位于所述主输入导管210外的一侧，所述卵细胞图像获取设备202设于所述主输入导管外 的另一侧并正对所述光源设备203。所述光源设备20对主输入导管210内进行照明，并成像在设于所述卵细胞图像获取设备202上。优选的，所述光源设备203为光纤冷光源设备，所述卵细胞图像获取设备202为CCD。由CCD成像后的细胞图像发送给所述中央控制器9进行图像处理后得到较为完善的图像。
在本实施例中，所述主输入导管210为透明导管，且所述主输入导管210的管径D为固定值。所述主输入导管210的始端连接穿刺针等装置实现穿刺并抽吸卵细胞。
所述光源设备203和卵细胞图像获取设备202所对应的主输入导管位置与所述第一阀口205a所在的主输入导管210位置的导管长度差值H为预设值。
这样，所述中央控制器202根据所述卵细胞图像获取设备202卵细胞图像信息，并结合所述带有卵细胞21的卵泡原液20在所述主输入导管210的流速V以及所述导管长度差值H计算得出控制所述分选开关205的第一阀口205a、第二阀口205b和第三阀口205c的开启/关闭时间，从而使带有卵细胞21的卵泡原液20中的卵细胞21从所述第一阀口205a流出，而去除卵细胞21的卵泡液22从所述第二阀口205b流出并流入到所述卵泡液收集容器204，且从所述第一阀口205a流出的卵细胞21即与从所述第三阀口205c流入的特定培养液23组成卵细胞液滴24后流出至培养载体1。
下面，详细描述本发明实施例提供的一种卵细胞自动识别分选装置的工作过程：
首先，通过中央控制器9控制所述分选开关205的所述第二阀口205b开启(此时第一阀口205a和第三阀口205c关闭)，同时控制所述负压发生器201启动。在所述负压发生器201的启动下，带有卵细胞21的卵泡原液20在负压的作用下流入所述主输入导管210。当所述带有卵细胞21的卵泡原液20在所述主输入导管210流动并流经所述卵细胞图像获取设备202时，由所述光源设备203配合所述卵细胞图像获取设备202以获取卵细胞图像信息。所述卵细胞图像获取设备202将获取到的卵细胞图像信息发送给所述中央控制器9，所述中央控制 器9根据所述卵细胞图像信息，并结合所述带有卵细胞21的卵泡原液20在所述主输入导管210的流速V以及所述导管长度差值H计算得出所述带有卵细胞21的卵泡原液20到达所述第一阀口205a的精确时间，并当所述带有卵细胞21的卵泡原液20到达所述第一阀口205a时，所述中央控制器9立即控制所述分选开关205开启第一阀口205a(此时第二阀口205b和第三阀口205c关闭)，使得所述带有卵细胞21的卵泡原液20中的卵细胞21从所述第一阀口205a流出并进入所述主输出导管211。且当所述卵细胞21从所述第一阀口205a流出并进入所述主输出导管211瞬间，所述中央控制器9立即控制所述分选开关205开启第三阀口205c(此时第一阀口205a和第二阀口205b关闭)，使从所述副输入导管213流入的特定培养液23与所述卵细胞21组成卵细胞液滴24后通过所述主输出导管211流出至培养载体1。而且，当从所述副输入导管213流入的特定培养液23达到预定的容量时，所述中央控制器9立即控制所述分选开关205开启所述第二阀口205b(此时第一阀口205a和第三阀口205c关闭)，使去除卵细胞21的卵泡液22从所述第二阀口205b流出并流入到所述卵泡液收集容器204中。
可见，本实施例公开的卵细胞自动识别分选装置200能适用于自动识别分选出卵丘细胞复合物、卵细胞和早期受精卵的自动识别和分选，提高了细胞识别分选的精确度，减少了操作成本。另外，经过本发明公开的卵细胞自动识别分选装置分选出来的细胞更适于体外授精和培养等。
参见图5～7，是实施例提供的一种培养载体1的结构示意图。该培养载体1包括载体主体11，该载体主体11为块状结构，优选为方形或者长方形。所述载体主体11的表面111向下凹设有多个凹坑12，所述凹坑12用于承载卵细胞液滴。其中，所述载体主体11的表面111以及每一所述凹坑12的表面121均为超疏水表面。例如，所述载体主体11的表面111以及每一所述凹坑12的表面121均为铺设了一层超疏水材料的超疏水表面，或者所述载体主体11的表面111以及每一所述凹坑12的表面121均为经过超疏水处理的超疏水表面。
所述载体主体11的表面111上在每一所述凹坑12的边缘凸起设置闭合环状的限制部13，且所述限制部13的表面为亲油表面。具有亲油表面特性的闭合环状凸起限制部13能够限制卵细胞液滴之外的培养油外溢，保证卵细胞液滴始终处于独立的培养环境。
例如，如图7所示，在所述凹坑表面121均为超疏水表面的培养载体上进行受精和培养时，含有细胞501的细胞液滴(水溶液)502在凹坑表面121的超疏水表面上呈球形，和凹坑表面121的接触面积很小，没有粘附在其表面上(相当于悬浮状态)，在重力环境下可以自动精确定位到凹坑表面121(且是凹坑表面121的最低点)上。而在每一所述凹坑12的边缘凸起设置闭合环状的限制部13，且所述限制部13的表面为亲油表面，能够限制细胞液滴(水溶液)502之外的培养油503(用于覆盖凹坑表面121及置于该凹坑表面121内的细胞液滴502)外溢，从而保证细胞液滴(水溶液)502始终处于独立的培养环境。
在本实施例中，如图6～7所示，每一所述凹坑12的表面121为弧面，优选为半圆球面。每一所述凹坑12的大小一致，凹坑12的深度和宽度根据实验需求而设置不同。
在本实施例中，所述载体主体11上的多个凹坑12呈单行直线排列在所述载体主体11的表面111上，从而是构成的载体1为单列多单元培养载体。
可见，本实施例使用的培养载体1通过在载体主体表面上设有多个用于承载卵细胞液滴的凹坑12，且所述载体表面111以及凹坑表面121均为经过超疏水处理的超疏水表面，这样，当在这样的培养载体上进行受精和培养时，由于细胞液滴(水溶液)在培养载体的超疏水表面上呈球形，和表面的接触面积很小，没有粘附在其表面上。另外，由于载体表面上的凹坑边缘凸起设置闭合环状的限制部，且该限制部具有亲油表面特性，能够限制细胞液滴之外的培养油外溢，保证细胞液滴始终处于独立的培养环境中。因此，卵细胞液滴(水溶液)在重力环境下可以自动精确定位到所需要的(受精和培养)的位置上，从而利于受精和培养操作。
可以理解的，本实施例提供的培养载体1除了可以作为本发明实施例的卵细胞培养载体外，也可以作为任何活体细胞的培养载体。
参见图8，是本发明实施例提供的一种直接授精模式装置300的结构示意图。本实施例中，该直接授精模式装置300包括第一液滴换液操作装置，该第一液滴换液操作装置包括废液抽取控制器301、培养液补加控制器302、液滴输出管路303和液滴输入管路304以及中央控制器9，所述液滴输出管路303和液滴输入管路304的两终端均连接液滴操作针305以吸取/输出液滴。所述废液抽取控制器301与所述液滴输出管路303连通，并通过控制所述液滴输出管路303的开启/关闭以及控制开启的大小来实现是否将培养载体1上的细胞液滴24中的废液抽出及控制抽出的废液流量。而所述培养液补加控制器302与所述液滴输入管路304连通，并通过所述液滴输入管路304的开启/关闭以及控制开启的大小来实现是否将培养液(精子悬液)输送至所述培养载体上的细胞液滴中并控制输送的培养液(精子悬液)流量。
所述中央控制器9分别与所述废液抽取控制器301和所述培养液补加控制器302连接，以控制所述废液抽取控制器301和所述培养液补加控制器302工作。具体的，所述中央控制器9通过控制所述废液抽取控制器301，来实现控制所述液滴输出管路303的开启/关闭以及控制开启的大小来实现是否将培养载体1上的卵细胞液滴中的废液抽出及控制抽出的废液流量，以及通过控制所述培养液补加控制器302，来控制所述液滴输入管路304的开启/关闭以及控制开启的大小来实现是否将培养液(精子悬液)输送至所述培养载体上的卵细胞液滴中并控制输送的培养液(精子悬液)流量。
在本实施例中，所述废液抽取控制器301和所述培养液补加控制器302为蠕动泵或步进电机。
在本实施例中，直接授精模式装置300还包括与所述中央控制器9连接的细胞图像获取设备308。该细胞图像获取设备308用于获取所述培养载体1上的细胞液滴24中的细胞图像信息，并将获取的细胞图像信息发送给所述中央控制 器9。所述中央控制器9根对所述细胞图像获取设备308发送过来的据细胞图像信息进行处理(例如，将细胞图像获取设备308获取的每一标记位置的图像信息逐次储存为特定位置的图像集合)，以获得更清晰的细胞图像信息，并将处理后的细胞图像信息通过所述显示器10显示出来。术者所述细胞图像信息用来判断细胞液滴24是否需要进行换液操作，从而通过中央控制器9控制所述废液抽取控制器301和所述培养液补加控制器302进行相应的工作。
在本实施例中，所述细胞图像获取设备308包括相互配合的光纤冷光源设备和CCD，光纤冷光源设备设于培养载体1上，以照射所述培养载体1上的细胞液滴24，从而成像于设于所述培养载体1的另一侧并正对所述光纤冷光源的CCD上。由CCD成像后的细胞图像发送给所述中央控制器9进行图像处理后得到较为完善的图像。
优选的，在本实施例的第一液滴换液操作装置中，所述液滴输出管路303和液滴输入管路304的两终端连接的液滴操作针305均为可拆卸的液滴操作针，因此，本实施例的第一液滴换液操作装置还包括液滴操作针更换动作设备306。该液滴操作针更换动作设备306与所述中央控制器9连接，以接收控制指令。该液滴操作针更换动作设备306用于对所述液滴输出管路301和液滴输入管路302的两终端连接的液滴操作针进行自动更换。具体的，在每次需要对培养载体1上的细胞液滴24进行换液操作前或者换液操作完成后，所述中央控制器9向液滴操作针更换动作设备306发送控制指令，指示该液滴操作针更换动作设备306对相应的液滴输出管路303和/或液滴输入管路304进行液滴操作针305更换。
优选的，本实施例的第一液滴换液操作装置还包括与所述中央控制器9连接的培养液选取动作设备307。该培养液选取动作设备307用于接收所述中央控制器9的指令来移动所述液滴输入管路304到盛有不同培养液的容器310中以获取对应的培养液(精子悬液)。本实施例还包括用于收集经由所述液滴输出管路303抽出的培养载体1上的卵细胞液滴24中的废液的废液收集容器311。
可以理解的，在本实施例的直接授精模式装置300，所述细胞图像获取设备308可以与所述第一液滴换液操作装置为一体结构。
本实施例的直接授精模式装置300采用上述第一液滴换液操作装置实现卵细胞受精模式，具有以下效果：1、可在卵细胞受精培养过程中实现自动细胞培养液连续更换，细胞培养液(精子悬液)流速可根据实验要求进行调节；也可自动间歇换液；2、换液过程全部自动化，不涉及人工操作，换液量控制精度高；3、液滴可以完整无残留转移的培养方法，可直接连接增材制造人工体外多细胞类型复杂活体组织的装置，作为体外孵育人工活体组织的前驱工序装置；4、可应用于无重力环境(如地外空间)的人工辅助生殖行为和细胞培养。
可以理解的，本实施例所采用的第一液滴换液操作装置除了可以为卵细胞受精培养过程中实现自动细胞培养液连续更换外，还可以用于各种活体细胞培养的液滴换液操作。
参考图9，是本发明实施例中一种玻璃化冷冻/复苏模式装置400的结构框图。该冷冻/复苏模式装置400包括中央控制器9、载体机械动作装置4、第二液滴换液装置3、冷冻/复苏载体6、承载所述冷冻/复苏载体的操作台7以及冷冻介质容器8。其中：
所述冷冻/复苏载体6用于承载冷冻目标细胞(受精后的卵细胞液滴)以进行冷冻操作，其本体内表面呈弧面的片状结构，所述载体本体的内表面上设有超疏水表面功能区域和亲水表面功能区域，所述亲水表面功能区域四周被所述超疏水表面功能区域环绕，所述亲水表面功能区域上设有液滴定位标识。这样结构的冷冻/复苏载体6更有利于卵细胞液滴的转移和定位，从而利于卵细胞液滴的冷冻/复苏操作。关于所述载体的具体结构，在后面会结合图10进行详细描述。其中，
在本实施例中，所述载体机械动作装置4和所述第二液滴换液操作装置3分别与所述中央控制器连接9以接收对应的冷冻/复苏控制指令。
所述载体机械动作装置4包括机械本体401以及设置在机械本体401上的 载体夹持动作装置41和液滴捡拾装置42(图12)。
所述液滴捡拾装置42用于根据冷冻/复苏控制指令对应将目标细胞液滴吸附并转移到所述冷冻/复苏载体6的特定位置上/将目标细胞液滴从所述冷冻/复苏载体6上吸附并转移出去。
所述载体夹持动作装置41用于根据冷冻/复苏控制指令，将所述冷冻/复苏载体上的完成液滴换液操作后的目标细胞液滴连同所述冷冻/复苏载体6一起移入到所述冷冻介质容器8中以完成冷冻/将所述冷冻介质容器8中完成冷冻的目标细胞液滴连同冷冻/复苏载体6从所述冷冻介质容器8中移出到所述操作台7上。
所述第二液滴换液操作装置3用于根据冷冻/复苏控制指令对所述操作台7上承载的冷冻/复苏载体6上的目标细胞液滴进行液滴换液操作。
本实施例的活体细胞玻璃化冷冻/复苏载体6如图10所示，该冷冻/复苏载体6的本体60为内表面呈弧面的片状结构。该冷冻/复苏载体6的本体60内表面上设有超疏水表面功能区域61和亲水表面功能区域62，所述亲水表面功能区域62四周被所述超疏水表面功能区域61环绕，且所述亲水表面功能区域62上设有液滴定位标识600(以助于细胞液滴的定位)。
在本实施例中，所述亲水表面功能区域62为圆形区域。而所述超疏水表面功能区域61为环绕所述亲水表面功能区域62的圆环形区域。而且，该亲水表面功能区域62的中心点与载体本体60的最低点重合。从而更利于细胞液滴的定位。
其中，所述超疏水表面功能区域61为在所述载体的内表面上铺设了一层超疏水材料的超疏水表面层或所述超疏水表面功能区域为将所述载体的内表面的部分区域经过超疏水处理后所得的超疏水表面层。
同样的，所述亲水表面功能区域62为在所述载体的内表面上铺设了一层亲水材料的亲水表面层或所述亲水表面功能区域为将所述载体的内表面的部分区 域经过亲水处理后所得的亲水表面层。
当采用本实施例的玻璃化冷冻/复苏载体6作为承载细胞液滴以进行细胞玻璃化冷冻/复苏操作时，亲水表面功能区域62和超疏水表面功能区域61的结合方式可以使规定容量的液滴自动吸附固定在亲水表面功能区域62，重力环境下，规则弧面结构的载体能使液滴包含的细胞自动定位在弧面与载物台的交线上。由于冷冻/复苏载体6的本体表面为弧面，且该弧面的最低点与用于定位细胞液滴位置的亲水表面功能区域62的中心点重合，从而利于细胞液滴的自动定位。另外，由于环绕亲水表面功能区域62的区域为超疏水表面功能区域61，细胞液滴不会粘附在超疏水表面功能区域61表面上，从而更进一步促使细胞液滴定位到设有液滴定位标识的亲水表面功能区域62上。
图11显示了本发明实施例中一种玻璃化冷冻/复苏模式装置400的操作台7的结构示意图。该操作台7是表面为平面的一个平台，且表面上还是了透明操作区域71。该透明操作区域71用于承载上述冷冻/复苏载体6。本实施例的操作台7优选为可移动式操作台7，其通过设置连接移动部件实现整个操作台7的移动。且操作台7连接的移动部件与中央控制器9连接，受中央控制器9控制实现操作台7的移动，从而可根据中央控制器9的指令控制操作台7移动到特定的位置以进行细胞冷冻/复苏的换液操作。
图12是本发明实施例中一种冷冻/复苏模式装置400的载体机械动作装置4的结构示意图。该载体机械动作装置4具体包括机械本体401以及设置在机械本体401上的载体夹持动作装置41和液滴捡拾装置42。
所述液滴捡拾装置42包括可拆卸液滴捡拾管421以及与所述可拆卸液滴捡拾管421连通且控制其吸附的可拆卸负压抽吸设备422。所述液滴捡拾装置42通过所述可拆卸负压抽吸设备422固定在所述机械本体401上。所述可拆卸负压抽吸设备422根据接收到的冷冻/复苏控制指令，对应控制所述可拆卸液滴捡拾管421吸附目标细胞液滴并转移到所述冷冻/复苏载体6的特定位置上/将目标细胞液滴从所述冷冻/复苏载体6上吸附并转移出去(例如，转移到培养载体1 或者其他容器中)。
所述载体夹持动作装置41包括用于夹持冷冻/复苏载体的载体夹持部件411和将所述载体夹持部件411固定在所述机械本体401上的固定支架412。所述载体夹持部件411根据冷冻/复苏控制指令，将所述冷冻/复苏载体6上的完成液滴换液操作后的目标细胞液滴连同所述冷冻/复苏载体6一起夹持并移入到冷冻介质容器8中以完成冷冻/将所述冷冻介质容器8中完成冷冻的目标细胞液滴连同冷冻/复苏载体6从所述冷冻介质容器8中夹出并移到所述操作台7上。
优选的，本实施例的所述载体机械动作装置4为可移动式载体机械动作装置4，所述载体机械动作装置4还包括与所述机械本体连接的移动部件43，所述载体机械动作装置4通过所述移动部件43可在运动导轨404上移动。所述载体机械动作装置4连接的移动部件43与中央控制器9连接，受中央控制器9控制实现载体机械动作装置4的移动，从而可根据中央控制器9的指令控制载体机械动作装置4移动到特定的位置以对细胞液滴或冷冻/复苏载体6进行转移等操作。
图13是本发明实施例中一种冷冻/复苏模式装置的第二液滴换液操作装置3的结构示意图。该第二液滴换液操作装置3与图8所示的第一液滴换液装置的结构基本一致，包括废液抽取控制器301、培养液补加控制器302、液滴输出管路303和液滴输入管路304，所述液滴输出管路303和液滴输入管路304的两终端均连接液滴操作针305以吸取/输出液滴。所述废液抽取控制器301与所述液滴输出管路303连通，并通过控制所述液滴输出管路303的开启/关闭以及控制开启的大小来实现是否将冷冻/复苏载体6上的细胞液滴中的废液抽出及控制抽出的废液流量。而所述培养液补加控制器302与所述液滴输入管路304连通，并通过所述液滴输入管路304的开启/关闭以及控制开启的大小来实现是否将培养液输送至所述冷冻/复苏载体6上的卵细胞液滴中并控制输送的培养液流量。
所述中央控制器9分别与所述废液抽取控制器301和所述培养液补加控制器302连接，以控制所述废液抽取控制器301和所述培养液补加控制器302工 作。具体的，所述中央控制器9通过控制所述废液抽取控制器301，来实现控制所述液滴输出管路303的开启/关闭以及控制开启的大小来实现是否将冷冻/复苏载体6上的细胞液滴中的废液抽出及控制抽出的废液流量，以及通过控制所述培养液补加控制器302，来控制所述液滴输入管路304的开启/关闭以及控制开启的大小来实现是否将培养液输送至所述培养载体上的细胞液滴中并控制输送的培养液流量。在本实施例中，所述废液抽取控制器301和所述培养液补加控制器302为蠕动泵或步进电机。
优选的，在本实施例的第二液滴换液操作装置3中，所述液滴输出管路303和液滴输入管路304的两终端连接的液滴操作针305均为可拆卸的液滴操作针，因此，本实施例的第二液滴换液操作装置还包括液滴操作针更换动作设备306。该液滴操作针更换动作设备306与所述中央控制器9连接，以接收控制指令。该液滴操作针更换动作设备306用于对所述液滴输出管路301和液滴输入管路302的两终端连接的液滴操作针进行自动更换。具体的，在每次需要对冷冻/复苏载体6上的细胞液滴进行换液操作前或者换液操作完成后，所述中央控制器9向液滴操作针更换动作设备306发送控制指令，指示该液滴操作针更换动作设备306对相应的液滴输出管路303和/或液滴输入管路304进行液滴操作针305更换。
优选的，本实施例的第二液滴换液操作装置3还包括与所述中央控制器9连接的培养液选取动作设备307。该培养液选取动作设备307用于接收所述中央控制器9的指令来移动所述液滴输入管路304到盛有不同培养液的容器310中以获取对应的冷冻/复苏液体(即冷冻保护剂/复苏液体)。本实施例的第二液滴换液操作装置还包括用于收集经由所述液滴输出管路303抽出的冷冻/复苏载体6上的卵细胞液滴中的废液的废液收集容器311。
返回参考图9，本实施例的冷冻介质容器8内装有的冷冻介质为液氮。
另外，本实施例的活体细胞玻璃化冷冻/复苏的自动操作装置还包括分别与所述中央控制器9连接的图像获取装置308b和显示器10。所述图像获取装置 308b用于获取所述冷冻/复苏载体6上的细胞液滴中的卵细胞图像信息，并将获取到的细胞图像信息发送给所述中央控制器9处理后，通过所述显示器10显示。因此，可以通过该图像获取装置308b获取的图像信息来识别冷冻/复苏载体6的亲水表面功能区域62上的液滴定位标识，从而定位卵细胞液滴放置的位置。
在本实施例中，所述图像获取装置308b通过所述光纤冷光源设备和细胞图像获取设备CCD共同构成。其中，光纤冷光源设备设于操作台7的正上方，且正对操作台7的透明操作区域71上的冷冻/复苏载体6上的卵细胞液滴，而所述细胞图像获取设备CCD设于操作台7的正下方，并正对所述光纤冷光源设备。所述光源设备20对操作台7的透明操作区域71上的冷冻/复苏载体6上的细胞液滴进行照明，并成像在设于所述细胞图像获取设备CCD上。可以理解的，本实施例的图像获取装置308b也可以整合到所述第二液滴换液操作装置3中，与所述第二液滴换液操作装置3为一体结构。
下面，详细描述将本实施例的冷冻/复苏模式装置操作任意活体细胞的操作过程：
当通过中央控制器9发出进入细胞冷冻模式的多个控制指令时，活体细胞培养箱内置的环境管理装置首先会接收到中央控制器9发送的控制指令，对应调整培养箱的环境到复合细胞冷冻操作的状态。然后，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使液滴捡拾装置42的可拆卸液滴捡拾管421正对目标细胞液滴)。然后，液滴捡拾装置42根据接收的控制指令，控制所述可拆卸液滴捡拾管421吸附目标细胞液滴(例如，从培养载体上)并转移到所述操作台7上的冷冻/复苏载体6的特定位置(即，设有液滴定位标识的亲水表面功能区域62)上(具体通过图像获取装置308来定位该特定位置)。接着，该操作台7的移动部件根据接收的控制指令，将载有冷冻/复苏载体6(以及细胞液滴)的操作台7移动到液滴换液操作装置操作区间(具体通过图像获取装置308来定位冷冻/复苏载体6上的液滴定位标识，移动操作台7使其承载的细胞液滴落在液滴换液操作装置操作点上)， 然后，液滴换液操作装置3根据接收的控制指令，对所述操作台7上的冷冻/复苏载体6的特定位置上的细胞液滴进行细胞冷冻的换液操作，并按照预定程序改变液滴成分(例如，冷冻保护剂)。此时，通过图像获取装置308b来获取记录细胞图像信息。直到所述冷冻/复苏载体6上的目标细胞达到与冷冻保护剂的平衡状态而且液滴容量达到规定数值范围(即细胞暴露在环境中的表面积达到预设值时)，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使载体夹持动作装置41的载体夹持部件411正对目标细胞液滴)，所述载体夹持动作装置41将所述冷冻/复苏载体6上的完成液滴换液操作后的目标细胞液滴连同所述冷冻/复苏载体6一起夹持并快速移入到冷冻介质容器8(例如，液氮)中以完成冷冻。
当通过中央控制器9发出进入细胞复苏模式的多个控制指令时，活体细胞培养箱内置的环境管理装置首先会接收到中央控制器9发送的控制指令，对应调整培养箱的环境到复合细胞复苏操作的状态。然后，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使载体夹持动作装置41的载体夹持部件411正对目标细胞液滴)，然后，载体夹持动作装置41根据接收的控制指令，控制所述载体夹持部件411将所述冷冻介质容器8中完成冷冻的目标细胞液滴连同冷冻/复苏载体6从所述冷冻介质容器8中夹出并移到所述操作台7上。接着，该操作台7的移动部件根据接收的控制指令，将载有冷冻/复苏载体6(以及细胞液滴)的操作台7移动到液滴换液操作装置操作区间(具体通过图像获取装置308b来定位冷冻/复苏载体6上的液滴定位标识，移动操作台7使其承载的细胞液滴落在液滴换液操作装置操作点上)，然后，液滴换液操作装置3根据接收的控制指令，对所述操作台7上的冷冻/复苏载体6的特定位置上的细胞液滴进行细胞复苏的换液操作，并按照预定程序改变液体容量和成分。此时，通过图像获取装置308b来获取记录细胞图像信息。直至细胞达到规定的培养液平衡状态，完成复苏。载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使液 滴捡拾装置42的可拆卸液滴捡拾管421正对目标细胞液滴)，所述可拆卸液滴捡拾管421根据接收的控制指令，将所述冷冻/复苏载体6上的完成液滴换液操作后的目标细胞液滴吸附并转移出去(例如，转移到培养载体或其他容器中)。
可见，本实施例的细胞玻璃化冷冻/复苏模式装置，通过采用内表面呈弧面的透明片状结构的载体作为冷冻/复苏载体，且该冷冻/复苏载体的内表面上设有超疏水表面功能区域和亲水表面功能区域，所述亲水表面功能区域四周被所述超疏水表面功能区域环绕，所述亲水表面功能区域上设有液滴定位标识，这样，在整个冷冻/复苏的自动操作过程中，通过对液滴定位标识的识别和定位，能够自动完成冷冻目标细胞的整个冷冻/复苏操作过程，无需人工参与，极大地提高工作效率，保证操作的稳定性、及时性和安全性。
参考图14，本发明实施例提供了一种活体细胞玻璃化冷冻的自动操作方法，如图14所示，该方法包括：
步骤S101、通过中央控制器向多个执行装置发出进入细胞冷冻模式的多个控制指令；
步骤S102、通过液滴捡拾装置并根据接收到的控制指令，将目标细胞液滴吸附转移到操作台承载的冷冻载体的特定位置上；
步骤S103、通过液滴换液操作装置并根据接收到的控制指令，对所述操作台承载的冷冻载体的特定位置上的目标细胞液滴进行液滴换液操作，并按照预定程序改变液滴成分，至目标细胞达到规定的冷冻保护剂平衡状态，而且液滴容量达到规定数值范围；以及
步骤S104、通过载体夹持动作装置并根据接收到的控制指令，将所述冷冻载体上的完成液滴换液操作后的目标细胞液滴连同所述冷冻载体一起移入到所述冷冻介质容器中以完成冷冻。
其中，本实施例中的冷冻载体的结构采用图10所示的活体细胞玻璃化冷冻/复苏载体的结构。
本发明使用图14所示的活体细胞玻璃化冷冻的自动操作方法来代替人工作业，能够可以大大的提高工作效率，保证操作的稳定性、及时性和安全性。
参考图15，是本发明实施例中一种玻璃化冻融细胞复苏的自动操作方法的流程图，该方法包括：
步骤S201、通过中央控制器向多个执行装置发出进入细胞复苏模式的多个控制指令；
步骤S202、通过载体夹持动作装置并根据接收到的控制指令，将冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻载体从所述冷冻介质容器中移出到操作台上；其中，所述目标细胞液滴置于所述冷冻载体的特定位置上；
步骤S203、通过液滴换液操作装置并根据接收到的控制指令，对所述操作台承载的冷冻载体的特定位置上的目标细胞液滴进行液滴换液操作，并按照预定程序改变液体容量和成分，直至细胞达到规定的培养液平衡状态，完成复苏；以及
步骤S204、通过液滴捡拾装置并根据接收到的控制指令，将所述目标细胞液滴从所述冷冻载体上吸附并转移出去。
同样的，本实施例中的复苏载体的结构采用图10所示的活体细胞玻璃化冷冻/复苏载体的结构。
图14或图15是根据本发明实施例示出的活体细胞玻璃化冷冻/复苏的自动操作方法的流程图，必须了解的是，本发明所提出的活体细胞玻璃化冷冻/复苏的自动操作方法步骤并不限于图14或图15所示的执行顺序，本领域技术人员可根据本发明的精神任意更动活体细胞快速玻璃化冷冻的自动操作方法步骤。
可以理解的，用于卵细胞体外受精和卵裂培养的自动化装置中的位于副培养箱体102内的移植模式装置可通过所述玻璃化冷冻/复苏模式装置400实现移植功能。
下面，具体描述如何利用本发明实施例提供给的用于卵细胞体外受精和卵 裂培养的自动化装置实现卵细胞体外受精和卵裂培养的包括但不限于卵丘细胞识别分选模式、直接授精模式、冷冻模式、冻融细胞复苏模式以及移植模式等工作过程。
卵丘细胞识别分选模式
结合图1～图3，术者通过中央控制器9输出指令给卵细胞自动识别分选装置200进入卵丘复合物分选模式，术者输入预期获卵数目，微流泵抽吸依据预期获卵数对应容量的培养液。且中央控制器9指令培养载体驱动装置105动作，以将承载的培养载体1进入规定初始位置。
首先，通过中央控制器209控制所述分选开关205的所述第二阀口205b开启(此时第一阀口205a和第三阀口205c关闭)，同时控制所述负压发生器201启动。在所述负压发生器201的启动下，带有卵细胞21的卵泡原液20在负压的作用下流入所述主输入导管210。
当所述带有卵细胞21的卵泡原液20在所述主输入导管210流动并流经所述卵细胞图像获取设备202时，由所述光源设备203配合所述卵细胞图像获取设备202以获取卵细胞图像信息。
所述卵细胞图像获取设备202将获取到的卵细胞图像信息发送给所述中央控制器209，所述中央控制器209根据所述卵细胞图像信息，并结合所述带有卵细胞21的卵泡原液20在所述主输入导管210的流速V以及所述导管长度差值H计算得出所述带有卵细胞21的卵泡原液20到达所述第一阀口205a的精确时间，并当所述带有卵细胞21的卵泡原液20到达所述第一阀口205a时，所述中央控制器209立即控制所述分选开关205开启第一阀口205a(此时第二阀口205b和第三阀口205c关闭)，使得所述带有卵细胞21的卵泡原液20中的卵细胞21从所述第一阀口205a流出并进入所述主输出导管211。且当所述卵细胞21从所述第一阀口205a流出并进入所述主输出导管211瞬间，所述中央控制器209立即控制所述分选开关205开启第三阀口205c(此时第一阀口205a和第二阀口 205b关闭)，使从所述副输入导管213流入的特定培养液23与所述卵细胞21组成卵细胞液滴24后通过所述主输出导管211流出并通过流路106流入自动联通式分区组合培养箱100内的培养载体1上。而且，当从所述副输入导管213流入的特定培养液23达到预定的容量时，所述中央控制器209立即控制所述分选开关205开启所述第二阀口205b(此时第一阀口205a和第三阀口205c关闭)，使去除卵细胞21的卵泡液22从所述第二阀口205b流出并流入到所述卵泡液收集容器204中。
中央控制器9控制装置重复图像识别、分选开关205的阀门开闭、微流泵207泵出液滴、卵细液滴循序进入特定培养位置(培养载体1)这一动作系列，直至负压发生器201关闭，卵丘细胞复合物分选模式终止。
另外，待卵丘细胞复合物分选模式终止后，中央控制器9控制打开所述自动气密门103，并指令培养载体驱动装置105动作，以将承载的培养载体1从副培养箱体102转移到主培养箱体101中(且中央控制器9控制关上自动气密门10)，以等待进行直接受精模式操作。
可以理解的，在进行直接受精模式操作前，所述中央控制器9可控制所述第一液滴换液操作装置/第二液滴换液操作装置对培养载体1上的卵细胞液滴进行换液操作，以在所述卵细胞液滴上覆盖特定容量的培养油。
直接受精模式
所述中央控制器9控制主培养箱体101中内置的环境管理装置调整主培养箱体101中的环境，使符合卵细胞液滴受精模式的环境要求，当培养载体1上的卵细胞液滴进入到主培养箱体101中的特定位置以进行受精和培养操作时，所述中央控制器9控制所述直接授精模式装置300(第一液滴换液操作装置)立即对培养载体1上的卵细胞液滴进行循序自动换液,并通过细胞图像获取设备308以设定的频率记录进入体外培养状态的卵母细胞的图像信息。
细胞图像获取设备308获取的清晰图像信息发送给中央控制器9，中央控制器9将每一标记位置的图像信息逐次储存为特定位置的图像集合；术者可以随时调阅任意标记位置的图像集合，判断卵丘细胞复合物的发育状态并记录；
术者依据图像集合显示的卵细胞状态确定加入精子悬液的时间，并指令中央控制器；中央控制器9依据指令，控制第一液滴换液操作装置向培养载体1上的卵细胞液滴加入规定容量的精子悬液，完成授精。
细胞玻璃化冷冻模式
在所述卵细胞液滴完成受精模式后，术者可通过中央控制器9控制打开所述自动气密门103，并指令培养载体驱动装置105动作，以将承载的培养载体1从副培养箱体102转移到副培养箱体102中(且中央控制器9控制关上自动气密门10)，以等待进行其他模式操作。
当术者通过中央控制器9发出进入细胞冷冻模式的多个控制指令时，副培养箱体102中内置的环境管理装置首先会接收到中央控制器9发送的控制指令，对应调整副培养箱体102中的环境到卵细胞冷冻操作的状态。然后，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使液滴捡拾装置42的可拆卸液滴捡拾管421正对着培养载体1上的目标细胞液滴，即受精后的乱细胞液滴)。然后，液滴捡拾装置42根据接收的控制指令，控制所述可拆卸液滴捡拾管421从培养载体1上吸附目标细胞液滴并转移到所述操作台7上的冷冻/复苏载体6的特定位置(即，设有液滴定位标识的亲水表面功能区域62)上(具体通过图像获取装置308b来定位该特定位置)。接着，该操作台7的移动部件根据接收的控制指令，将载有冷冻/复苏载体6(以及细胞液滴)的操作台7移动到液滴换液装置操作区间(具体通过图像获取装置308b来定位冷冻/复苏载体6上的液滴定位标识，移动操作台7使其承载的细胞液滴落在液滴换液装置操作点上)，然后，第二液滴换液操作装置3根据接收的控制指令，对所述操作台7上的冷冻/复苏载体6的特定位置上的细胞液 滴进行细胞冷冻的换液操作，并按照预定程序改变液滴成分(例如，冷冻保护剂)。此时，通过图像获取装置308b来获取记录细胞图像信息。直到所述冷冻/复苏载体6上的目标细胞达到与冷冻保护剂的平衡状态而且液滴容量达到规定数值范围(即细胞暴露在环境中的表面积达到预设值时)，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使载体夹持动作装置41的载体夹持部件411正对目标细胞液滴)，所述载体夹持动作装置41将所述冷冻/复苏载体6上的完成液滴换液操作后的目标细胞液滴连同所述冷冻/复苏载体6一起夹持并快速移入到冷冻介质容器8(例如，液氮)中以完成冷冻。
细胞玻璃化复苏模式
当通过中央控制器9发出进入细胞复苏模式的多个控制指令时，副培养箱体102中内置的环境管理装置首先会接收到中央控制器9发送的控制指令，对应调整副培养箱体102中的环境到复合细胞复苏操作的状态。然后，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使载体夹持动作装置41的载体夹持部件411正对目标细胞液滴)，然后，载体夹持动作装置41根据接收的控制指令，控制所述载体夹持部件411将所述冷冻介质容器8中完成冷冻的目标细胞液滴连同冷冻/复苏载体6从所述冷冻介质容器8中夹出并移到所述操作台7上。接着，该操作台7的移动部件根据接收的控制指令，将载有冷冻/复苏载体6(以及细胞液滴)的操作台7移动到液滴换液装置操作区间(具体通过图像获取装置308b来定位冷冻/复苏载体6上的液滴定位标识，移动操作台7使其承载的细胞液滴落在液滴换液装置操作点上)，然后，第二液滴换液操作装置3根据接收的控制指令，对所述操作台7上的冷冻/复苏载体6的特定位置上的细胞液滴进行细胞复苏的换液操作，并按照预定程序改变液体容量和成分。此时，通过图像获取装置308b来获取记录细胞图像信息。直至细胞达到规定的培养液平衡状态，完成复苏，载体机械动作 装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使液滴捡拾装置42的可拆卸液滴捡拾管421正对目标细胞液滴)，所述可拆卸液滴捡拾管421根据接收的控制指令，将所述冷冻/复苏载体6上的完成液滴换液操作后的目标细胞液滴吸附并转移出去(例如，转移到培养载体1或其他容器中)。
移植模式
在所述卵细胞液滴完成受精模式后，若术者决定进行移植，术者可先通过中央控制器9控制打开所述自动气密门103，并指令培养载体驱动装置105动作，以将承载的培养载体1从副培养箱体102转移到副培养箱体102中(且中央控制器9控制关上自动气密门10)。
术者可通过中央控制器9指令进入移植模式，首先，第二液滴换液操作装置3根据接收的控制指令，对所述培养载体1的卵细胞液滴进行换液操作，并按照预定程序改变液体容量和成分。此时，通过图像获取装置308b来获取记录细胞图像信息。直至细胞上的液滴达到规定的容积时，载体机械动作装置4的移动部件43根据接收的控制指令，将载体机械动作装置4移动到适合的位置(使液滴捡拾装置42的可拆卸液滴捡拾管421正对目标细胞液滴)，所述可拆卸液滴捡拾管421根据接收的控制指令，将所述冷冻/复苏载体6上的完成液滴换液操作后的卵细胞液滴吸附并快速装入移植管中，从而完成移植模式。
本发明另一实施例提供了一种用于卵细胞体外受精和卵裂培养的自动化方法，利用上述自动化装置对卵细胞进行体外受精和卵裂培养的包括卵丘复合物识别分选模式、直接授精模式、玻璃化冷冻/复苏模式以及转移模式的多个操作模式均为对卵细胞液滴进行操作，包括但不限于卵细胞液滴的识别与分选、卵细胞液滴的换液以及卵细胞液滴的转移及移植。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。

Claims (52)

  1. 一种用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，包括自动联通式分区组合培养箱、置于培养箱外的中央控制器和卵细胞自动识别分选装置；
    所述自动联通式分区组合培养箱在不同的分区中设有对卵细胞液滴进行受精及卵裂培养过程中的不同阶段的操作模式装置，且每个分区通过自动气密门来实现连通/隔开；所述自动联通式分区组合培养箱内设有用于承载卵细胞液滴的培养载体以及带动所述培养载体往返于各个分区的培养载体驱动装置；
    所述卵细胞自动识别分选装置、不同阶段的操作模式装置、自动气密门以及培养载体驱动装置均与所述中央控制器连接，以接收对应的控制指令；
    所述卵细胞自动识别分选装置根据接收的控制指令，对带有卵细胞的卵泡原液进行识别和分选后，输出卵细胞液滴至所述自动联通式分区组合培养箱内的培养载体上；在所述自动气密门根据控制指令打开时，所述培养载体驱动装置根据控制指令带动所述培养载体往返于所述自动联通式分区组合培养的各个分区之间，从而使所述培养载体上的卵细胞液滴进入各个分区以进行对应的操作模式。
  2. 如权利要求1所述的一种用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述自动联通式分区组合培养箱包括两个分区，所述两个分区通过主培养箱体、副培养箱体以及用于实现所述主培养箱体和副培养箱体之间的连通/隔开的所述自动气密门构成。
  3. 如权利要求1所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述操作模式装置包括但不限于直接授精模式装置、玻璃化冷冻/复 苏模式装置以及移植模式装置。
  4. 如权利要求3所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述直接授精模式装置包括第一液滴换液操作装置，所述第一液滴换液操作装置包括废液抽取控制器、培养液补加控制器、液滴输出管路和液滴输入管路，所述液滴输出管路和液滴输入管路的两终端均连接液滴操作针以吸取/输出液滴；所述废液抽取控制器通过所述液滴输出管路将培养载体上的细胞液滴中的废液抽出并控制抽出的废液流量，而所述培养液补加控制器通过所述液滴输入管路将培养液输送至所述培养载体上的细胞液滴中并控制输送的培养液流量；所述中央控制器分别与所述废液抽取控制器和所述培养液补加控制器连接，以控制所述废液抽取控制器和所述培养液补加控制器工作。
  5. 如权利要求4所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述废液抽取控制器和所述培养液补加控制器为蠕动泵或步进电机。
  6. 如权利要求4所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述直接授精模式装置还包括与所述中央控制器连接的细胞图像获取设备，用于获取直接授精模式操作时所述培养载体上的细胞液滴中的细胞图像信息。
  7. 如权利要求1所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述培养载体表面上设有多个用于承载卵细胞液滴的凹坑，且所述凹坑表面为超疏水表面。
  8. 如权利要求7所述的用于卵细胞体外受精和卵裂培养的自动化装置，其 特征在于，所述培养载体表面上在每一所述凹坑的边缘凸起设置闭合环状的限制部，且所述限制部的表面为亲油表面。
  9. 如权利要求1所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述卵细胞自动识别分选装置包括负压发生器、卵细胞图像获取设备、光源设备、卵泡液收集容器以及分选开关，所述中央控制器分别与所述负压发生器、卵细胞图像获取设备和分选开关连接；
    所述分选开关包括第一阀口、第二阀口和第三阀口，所述第一阀口连接用于输入带有卵细胞的卵泡原液的主输入导管以及用于输出卵细胞至培养载体的主输出导管之间，所述第二阀口连接所述主输入导管以及用于输出除去卵细胞的卵泡液至所述卵泡液收集容器的副输出导管之间，所述第三阀口连接用于输入特定培养液的副输入导管和所述主输出导管之间；所述负压发生器通过连通导管与所述卵泡液收集容器连通；
    所述光源设备设于所述主输入导管外的一侧，对主输入导管内进行照明，并成像在设于所述主输入导管外的另一侧的所述卵细胞图像获取设备上；带有卵细胞的卵泡原液在负压发生器启动时流入所述主输入导管，并流经所述卵细胞图像获取设备时由所述卵细胞图像获取设备获取卵细胞图像信息，所述中央控制器根据所述卵细胞图像信息控制所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭，从而使带有卵细胞的卵泡原液中的卵细胞从所述第一阀口流出，去除卵细胞的卵泡液从所述第二阀口流出并流入到所述卵泡液收集容器，并使从所述第一阀口流出的卵细胞与从所述第三阀口流入的特定培养液组成卵细胞液滴后流出至所述培养载体。
  10. 如权利要求9所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，通过所述中央控制器控制所述负压发生器的工作，从而控制带有 卵细胞的卵泡原液在所述主输入导管的流速。
  11. 如权利要求10所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述光源设备和卵细胞图像获取设备所对应的主输入导管位置与所述第一阀口所在的主输入导管位置的导管长度差值为预设值。
  12. 如权利要求11所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述中央控制器根据所述卵细胞图像信息，并结合所述带有卵细胞的卵泡原液在所述主输入导管的流速以及所述导管长度差值计算得出所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭时间。
  13. 如权利要求3所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述玻璃化冷冻/复苏模式装置包括载体机械动作装置、第二液滴换液装置、冷冻/复苏载体、承载所述冷冻/复苏载体的操作台以及冷冻介质容器，其中，
    所述载体机械动作装置和所述第二液滴换液装置分别与所述中央控制器连接以接收对应的冷冻/复苏控制指令；
    所述载体机械动作装置包括机械本体以及设置在机械本体上的载体夹持动作装置和液滴捡拾装置；所述液滴捡拾装置用于根据冷冻/复苏控制指令对应将目标细胞液滴吸附并转移到所述冷冻/复苏载体的特定位置上/将目标细胞液滴从所述冷冻/复苏载体上吸附并转移出去；所述载体夹持动作装置用于根据冷冻/复苏控制指令，将所述冷冻/复苏载体上的完成液滴换液操作后的目标细胞液滴连同所述冷冻/复苏载体一起移入到所述冷冻介质容器中以完成冷冻/将所述冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻/复苏载体从所述冷冻介质容器中移出到所述操作台上；
    所述第二液滴换液装置用于根据冷冻/复苏控制指令对所述操作台承载的冷冻/复苏载体上的目标细胞液滴进行液滴换液操作。
  14. 如权利要求13所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述玻璃化冷冻/复苏模式装置还包括与所述中央控制器连接的图像获取装置；所述图像获取装置用于获取所述冷冻/复苏载体上的细胞液滴中的细胞图像信息，并将获取到的细胞图像信息发送给所述中央控制器进行处理。
  15. 如权利要求13所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述冷冻/复苏载体的载体本体为内表面呈弧面的片状结构，所述内表面上设有超疏水表面功能区域和亲水表面功能区域，所述亲水表面功能区域四周被所述超疏水表面功能区域环绕，所述亲水表面功能区域上设有液滴定位标识。
  16. 如权利要求15所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述亲水表面功能区域的中心点与所述载体本体的最低点重合。
  17. 如权利要求2或3所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述移植模式装置可通过所述玻璃化冷冻/复苏模式装置实现移植功能。
  18. 如权利要求1所述的用于卵细胞体外受精和卵裂培养的自动化装置，其特征在于，所述主培养箱体/副培养箱体内分别设有用于控制调整主培养箱体/副培养箱体内的温度、湿度、压力和气体分压的环境管理装置。
  19. 如权利要求1所述的用于卵细胞体外受精和卵裂培养的自动化装置， 其特征在于，所述主培养箱体/副培养箱体上设有与外界连通的流路，通过所述流路接收外界送入的待培养细胞液滴或向外界送出培养后的细胞液滴。
  20. 一种用于卵细胞体外受精和卵裂培养的自动化方法，其特征在于，利用自动化装置对卵细胞进行体外受精和卵裂培养的包括卵丘复合物识别分选模式、直接授精模式、玻璃化冷冻/复苏模式以及转移模式的多个操作模式均为对卵细胞液滴进行操作。
  21. 如权利要求20所述的用于卵细胞体外受精和卵裂培养的自动化方法，其特征在于，所述对卵细胞液滴进行操作包括但不限于卵细胞液滴的识别与分选、卵细胞液滴的换液以及卵细胞液滴的转移及移植。
  22. 一种活体细胞培养载体，其特征在于，所述培养载体表面上设有多个用于承载活体细胞液滴的凹坑，且所述凹坑表面为超疏水表面。
  23. 如权利要求22所述的活体细胞培养载体，其特征在于，所述培养载体表面上在每一所述凹坑的边缘凸起设置闭合环状的限制部，且所述限制部的表面为亲油表面。
  24. 如权利要求22所述的活体细胞培养载体，其特征在于，所述凹坑表面为铺设了一层超疏水材料的超疏水表面层或所述凹坑表面为经过超疏水处理的超疏水表面。
  25. 一种卵细胞自动识别分选装置，其特征在于，包括负压发生器、卵细胞图像获取设备、光源设备、卵泡液收集容器、分选开关以及与所述负压发生 器、卵细胞图像获取设备和分选开关连接的中央控制器；
    所述分选开关包括第一阀口、第二阀口和第三阀口，所述第一阀口连接用于输入带有卵细胞的卵泡原液的主输入导管以及用于输出卵细胞至培养载体的主输出导管之间，所述第二阀口连接所述主输入导管以及用于输出除去卵细胞的卵泡液至所述卵泡液收集容器的副输出导管之间，所述第三阀口连接用于输入特定培养液的副输入导管和所述主输出导管之间；所述负压发生器通过连通导管与所述卵泡液收集容器连通；
    所述光源设备设于所述主输入导管外的一侧，对主输入导管内进行照明，并成像在设于所述主输入导管外的另一侧的所述卵细胞图像获取设备上；带有卵细胞的卵泡原液在负压发生器启动时流入所述主输入导管，并流经所述卵细胞图像获取设备时由所述卵细胞图像获取设备获取卵细胞图像信息，所述中央控制器根据所述卵细胞图像信息控制所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭，从而使带有卵细胞的卵泡原液中的卵细胞从所述第一阀口流出，去除卵细胞的卵泡液从所述第二阀口流出并流入到所述卵泡液收集容器，并使从所述第一阀口流出的卵细胞与从所述第三阀口流入的特定培养液组成卵细胞液滴后流出至培养载体。
  26. 如权利要求25所述的卵细胞自动识别分选装置，其特征在于，通过所述中央控制器控制所述负压发生器的工作，从而控制带有卵细胞的卵泡原液在所述主输入导管的流速。
  27. 如权利要求26所述的卵细胞自动识别分选装置，其特征在于，所述光源设备和卵细胞图像获取设备所对应的主输入导管位置与所述第一阀口所在的主输入导管位置的导管长度差值为预设值。
  28. 如权利要求27所述的卵细胞自动识别分选装置，其特征在于，所述中央控制器根据所述卵细胞图像信息，并结合所述带有卵细胞的卵泡原液在所述主输入导管的流速以及所述导管长度差值计算得出所述分选开关的第一阀口、第二阀口和第三阀口的开启/关闭时间。
  29. 如权利要求25所述的卵细胞自动识别分选装置，其特征在于，所述主输入导管为透明导管，且所述主输入导管的管径为固定值。
  30. 如权利要求25所述的卵细胞自动识别分选装置，其特征在于，所述分选开关为三通阀，包括带有所述第一阀口、第二阀口和第三阀口的阀体以及控制所述第一阀口、第二阀口和第三阀口的开启/闭合的阀门控制装置，所述中央控制器与所述阀门控制装置连接。
  31. 一种用于细胞培养的液滴换液操作装置，其特征在于，包括废液抽取控制器、培养液补加控制器、液滴输出管路和液滴输入管路，所述液滴输出管路和液滴输入管路的两终端均连接液滴操作针以吸取/输出液滴；所述废液抽取控制器通过所述液滴输出管路将培养载体上的细胞液滴中的废液抽出并控制抽出的废液流量，而所述培养液补加控制器通过所述液滴输入管路将培养液输送至所述培养载体上的细胞液滴中并控制输送的培养液流量。
  32. 如权利要求31所述的用于细胞培养的液滴换液操作装置，其特征在于，所述液滴换液操作装置置于环境可控的细胞培养箱内。
  33. 如权利要求31所述的用于细胞培养的液滴换液操作装置，其特征在于，所述废液抽取控制器和所述培养液补加控制器为蠕动泵或步进电机。
  34. 如权利要求33所述的用于细胞培养的液滴换液操作装置，其特征在于，还包括中央控制器，所述中央控制器分别与所述废液抽取控制器和所述培养液补加控制器连接，以控制所述废液抽取控制器和所述培养液补加控制器工作。
  35. 如权利要求34所述的用于细胞培养的液滴换液操作装置，其特征在于，还包括与所述中央控制器连接的细胞图像获取设备，用于获取所述培养载体上的细胞液滴中的细胞图像信息。
  36. 如权利要求35所述的用于细胞培养的液滴换液操作装置，其特征在于，还包括与所述中央控制器连接的培养液选取动作设备，用于接收所述中央控制器的指令来移动所述液滴输入管路到盛有不同培养液的容器中以获取对应的培养液。
  37. 如权利要求31所述的用于细胞培养的液滴换液操作装置，其特征在于，所述液滴输出管路和液滴输入管路的两终端连接的液滴操作针均为可拆卸的液滴操作针，所述液滴换液操作装置还包括液滴操作针更换动作设备，用于对所述液滴输出管路和液滴输入管路的两终端连接的液滴操作针进行自动更换。
  38. 如权利要求31所述的用于细胞培养的液滴换液操作装置，其特征在于，还包括用于收集经由所述液滴输出管路抽出的培养载体上的细胞液滴中的废液的废液收集容器。
  39. 一种活体细胞玻璃化冷冻/复苏载体，其特征在于，所述载体本体为内表面呈弧面的透明片状结构，所述载体本体的内表面上设有超疏水表面功能区域和亲水表面功能区域，所述亲水表面功能区域四周被所述超疏水表面功能区 域环绕，所述亲水表面功能区域上设有液滴定位标识。
  40. 如权利要求39所述的活体细胞玻璃化冷冻/复苏载体，其特征在于，所述超疏水表面功能区域为圆环形区域，所述亲水表面功能区域为圆形区域。
  41. 如权利要求40所述的活体细胞玻璃化冷冻/复苏载体，其特征在于，所述亲水表面功能区域的中心点与所述载体本体的最低点重合。
  42. 如权利要求39所述的活体细胞玻璃化冷冻/复苏载体，其特征在于，所述超疏水表面功能区域为在所述载体的内表面上铺设了一层超疏水材料的超疏水表面层或所述超疏水表面功能区域为将所述载体的内表面的部分区域经过超疏水处理后所得的超疏水表面层；
    所述亲水表面功能区域为在所述载体的内表面上铺设了一层亲水材料的亲水表面层或所述亲水表面功能区域为将所述载体的内表面的部分区域经过亲水处理后所得的亲水表面层。
  43. 一种活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，包括中央控制器、载体机械动作装置、液滴换液操作装置、冷冻/复苏载体、承载所述冷冻/复苏载体的操作台以及冷冻介质容器，其中，
    所述载体机械动作装置和所述液滴换液操作装置分别与所述中央控制器连接以接收对应的冷冻/复苏控制指令；
    所述载体机械动作装置包括机械本体以及设置在机械本体上的载体夹持动作装置和液滴捡拾装置；所述液滴捡拾装置用于根据冷冻/复苏控制指令对应将目标细胞液滴吸附并转移到所述冷冻/复苏载体的特定位置上/将目标细胞液滴从所述冷冻/复苏载体上吸附并转移出去；所述载体夹持动作装置用于根据冷冻/复苏控制指令，将所述冷冻/复苏载体上的完成液滴换液操作后的目标细胞液滴 连同所述冷冻/复苏载体一起移入到所述冷冻介质容器中以完成冷冻/将所述冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻/复苏载体从所述冷冻介质容器中移出到所述操作台上；
    所述液滴换液操作装置用于根据冷冻/复苏控制指令对所述操作台承载的冷冻/复苏载体上的目标细胞液滴进行液滴换液操作；
    所述冷冻/复苏载体为权利要求39～42中任一项所述的活体细胞玻璃化冷冻/复苏载体；所述特定位置为所述设有液滴定位标识的亲水表面功能区域上。
  44. 如权利要求43所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，还包括分别与所述中央控制器连接的图像获取装置和显示器；
    所述图像获取装置用于获取所述冷冻/复苏载体上的细胞液滴中的细胞图像信息，并将获取到的细胞图像信息发送给所述中央控制器处理后，通过所述显示器显示。
  45. 如权利要求44所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，所述图像获取装置可与所述液滴换液操作装置为一体结构。
  46. 如权利要求43所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，所述载体机械动作装置为可移动式载体机械动作装置，所述载体机械动作装置还包括与所述机械本体连接的移动部件，所述载体机械动作装置通过所述移动部件可在运动导轨上移动。
  47. 如权利要求43所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，所述载体夹持动作装置包括用于夹持冷冻/复苏载体的载体夹持部件和将所述载体夹持部件固定在所述机械本体上的固定支架。
  48. 如权利要求43所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，所述液滴捡拾装置包括可拆卸液滴捡拾管以及与所述可拆卸液滴捡拾管连通且控制其吸附的可拆卸负压抽吸设备。
  49. 如权利要求43所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，所述冷冻介质容器内装有的冷冻介质为液氮。
  50. 如权利要求43所述活体细胞玻璃化冷冻/复苏的自动操作装置，其特征在于，所述自动操作系统置于环境可控的细胞培养箱内。
  51. 一种活体细胞玻璃化冷冻的自动操作方法，其特征在于，包括步骤：
    S1、通过中央控制器向多个执行装置发出进入细胞冷冻模式的多个控制指令；
    S2、通过液滴捡拾装置并根据接收到的控制指令，将目标细胞液滴吸附转移到操作台承载的冷冻载体的特定位置上；
    S3、通过液滴换液操作装置并根据接收到的控制指令，对所述操作台承载的冷冻载体的特定位置上的目标细胞液滴进行液滴换液操作，直至所述冷冻载体上的目标细胞达到与冷冻保护剂的平衡状态而且液滴容量达到规定数值范围；以及
    S4、通过载体夹持动作装置并根据接收到的控制指令，将所述冷冻载体上的完成液滴换液操作后的目标细胞液滴连同所述冷冻载体一起移入到所述冷冻介质容器中以完成冷冻；
    其中，所述冷冻载体为权利要求39～42中任一项所述的活体细胞玻璃化冷冻/复苏载体；所述特定位置为设有液滴定位标识的所述亲水表面功能区域上。
  52. 一种玻璃化冻融细胞复苏的自动操作方法，其特征在于，包括步骤：
    S1、通过中央控制器向多个执行装置发出进入细胞复苏模式的多个控制指令；
    S2、通过载体夹持动作装置并根据接收到的控制指令，将冷冻介质容器中完成冷冻的目标细胞液滴连同冷冻载体从所述冷冻介质容器中移出到操作台上；其中，所述目标细胞液滴置于所述冷冻载体的特定位置上；
    S3、通过液滴换液操作装置并根据接收到的控制指令，对所述操作台承载的冷冻载体的特定位置上的目标细胞液滴按照预定的程序进行液滴换液操作，直至所述冷冻载体上的目标细胞达到培养液平衡状态，完成复苏；以及
    S4、通过液滴捡拾装置并根据接收到的控制指令，将所述目标细胞液滴从所述冷冻载体上吸附并转移；
    其中，所述冷冻载体为权利要求39～42中任一项所述的活体细胞玻璃化冷冻/复苏载体；所述特定位置为设有液滴定位标识的所述亲水表面功能区域上。
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CN201410626062.3A CN104498321B (zh) 2014-11-06 2014-11-06 卵细胞自动识别分选装置
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